v F. u M 5....fiimmm In“... .gflfléfic “will: Humane. a 9" 21:3. 5.. .. .m, . r. . ..‘ . I91! r itatthvl fl. :2 «burr! .5;L\. .. .4- wv ‘ News 1 - 02“ IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 31293 017721279 This is to certify that the dissertation entitled Studies of C/EBP transcription factors in myelomonocytic cell lineages presented by Hsien-Ming Hu has been accepted towards fulfillment of the requirements for Ph . D . Microbiology degree in / t / I - t ”I L All"! _’ 1 Major p ofessor Date /O/8/98 / / MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINE retum on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1M WWW“ STUDIES OF C/EBP TRANSCRIPTION FACTORS IN MYELOMONOCYTIC CELL LINEAGES By Hsien—Ming Hu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1 998 ABSTRACT STUDIES OF CIEBP TRANSCRIPTION FACTORS IN MYELOMONOCYTIC CELL LINEAGES By Hsien-Ming Hu CIEBP-related proteins comprise a family of basic-region leucine zipper (bZIP) transcription factors. These proteins dimerize through a leucine zipper and bind to DNA through an adjacent basic region. Previous in vitro studies have implicated CIEBPB in the regulation of many inflammation-associated genes, including proinflammatory cytokines. Recently, it was found that LPS stimulation of peritoneal macrophages from C/EBPB-deficient mice led to a normal induction of a number of proinflammatory cytokines. Thus it is hypothesized that other CIEBP family members can support the expression of IL-6 and other proinflammatory cytokines. In the first part of this study, experiments have been conducted to show that ClEBPa, CIEBPB, and CIEBPS are expressed in bone marrow-derived macrophages, and that all of them are available to support LPS-induced cytokine expression. When ectopically expressed in P388 B Iymphoblasts, which normally lack the ability to express cytokines upon LPS stimulation, each of these CIEBP isoforms is capable of conferring LPS-inducible expression of lL-6 and MCP-l. These results demonstrate the redundancy of CIEBPa, CIEBPB and CIEBP6 in supporting the LPS induction of lL-6 and MOP-1. In the second part of this study, we have sought to identify the structural basis for this apparent redundancy. Surprisingly, we have found that P388 stably expressing truncated forms of CIEBPB, that lack all regulatory domains and retain only the bZlP regions, are capable of inducing lL-6 and MCP-1 transcription in response to LPS. In contrast, transfectants expressing a CIEBP chimera, in which the leucine zipper of C/EBPB is replaced with that of yeast transcription factor GCN4, have a reduced ability to induce lL-6 and MCP-1. Furthermore, a truncated form of CIEBP6 and, to a lesser extent, a truncated form of CIEBPa have both been shown to support LPS activation of the IL~6 promoter in transient transfection assays. Together, these results have implicated the leucine zipper domain in some function other than dimerization to known CIEBP isoforms, and have suggested that CIEBP redundancy in regulating cytokine expression may result from their highly related bZIP domains. In chapter 4, the capabilities of CIEBP isoforms to induce myeloid-specific genes are investigated. Individual ClEBPs are ectopically-expressed in a pre- granulocytic cell line, 320cl3. The results demonstrate that a ClEBPa-C/EBPB heterodimer is the most likely effector among other forms of CIEBP, in inducing the transcription of several primary granule product-encoding genes including myeloperoxidase, cathepsin G, and lysozyme. Dedicated to my beloved family, my wife, Yen-Hsueh Su my lovely daughter, Alyssa for their love and support iv ACKNOWLEDGMENTS I would like to express the deepest appreciation to my mentor, Dr. Richard Schwartz, for his guidance and encouragement. I am grateful to our collaborator Dr. Peter Johnson and his lab in National Cancer Institute for supplying reagents and for technical advice. I would also like to thank members of my guidance committee, Drs. Jerry Dodgson, Donald Jump, Water Esselman and Donna Koslowskyfor their precious time and suggestions. And for the friendship and helpful discussions from my colleague Oiang Tian. TABLE OF CONTENTS ListofTables......... List of Figures........................ ListcfAbbreviations........................................................... Chapter 1: Literature Reviews... . .CIEBP related transcription factors 1.1 CIEBPa... 1.2 CIEBPB 1.3 CIEBP6 1.4 CIEBPe viii .xi 1.5 ClEBPy 2. Hematopoiesis.................................................................. 2.1 Overview... 2. 2 Growth factors 2. 3 Myelomonocytic dIfferentIatIon 2. 4 CIEBP proteins and myelomonocytic differentiation 3. Inflammation... 3.1 Introduction 3. 2 Macrophage activatich and acute phase response ... H 3. 3 Proinflammatory cytokines... 3. 3. 2 Interleukin-1.. 3 3 3 Tumor necrosis £333.32... ....III II. III III III III III III III II. 3. 3. 4 Monccyte chemoattractant protein-1... .. 3. 4 CIEBP proteins and the expression of proinflammatory cytokines... . 3. 5 Other cooperating transcription factors... 4. Questionsaddressedinthisthesis.......................................... References Chapter 2: Redundancy of CIEBPa, -B, and —6 in supporting the lipopolysaccharide-induced transcription of IL-6 and monocytem chemoattractant protein-1... Abstract... Introduction... .. Materials and methods vi .3. .. 35. OCOCDQGALAA ...11 ......11 ......13 ......14 .....16 ......18 ......18 ......19 3.3.1 Interleukin-6 ......24 .....25 ...25 .....26 ...27 .30 32 Results......... 48 DIscussmn .............65 References 71 Chapter 3: Conventional activation domains are dispensable for the role of ClEBPs in LPS induction of lL-6 and MCP-1 expression............ .......75 Abstract... .....76 Introduction 77 Materials and methods 79 Results 84 Discussion... ....100 References............ .....106 Chapter 4. Regulation of the expression of primary granule proteins byCIEBPtranscnptlonfactorfamrly 110 Abstract... .. . .. 111 lntroduction 112 Materialsandmethcds 114 Results119 DIscussmn133 References138 ConcluSIonsl41 vii LIST OF TABLES Chapterl Table 1. Proinflammatory cytokines produced by activated macrophages..............................................20 Chapter2 Table 1. Comparison of LPS induction of lL-6 and MOP-1 mRNA between P388-CBIPuro and P388—CB/661 Chapter4 Table 1. Induction of MPO, Cat G and L2 transcriptions in 320 cl3 transfectants... viii Chapter 1 Chapter 2 Chapter 3 Figure 1. Figure 2. Figure 3. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 1. Figure 2. Figure 3. Figure 4. LIST OF FIGURES Structure of the CIEBP transcription factor... Origin of the hematopoietic cell lineages... Cis—acting elements of the lL-6 promoter... .. EMSA of CIEBPor, CIEBPB, and CIEBP6 DNA binding activity in bone marrow-derived macrophages........................... .. Analyses of P388 cells stably transfected with CIEBPa, ...CIEBIDB... or CIEBP6 expression vectors... . Northern analyses of lL-6 and MCP- 1 expression in P388transfectants... Analyses of P388 cells stably transfected for dual expression of CIEBPB and CIEBPS... EMSA of NF-KB DNA binding activity in P388 transfectants... Structures of the various altered CIEBP isoforms used in the studies described in this paper... Northern analyses of lL-6 and MCP-l expression in P388transfectants.. EMSA of CIEBP DNA binding activities in P388 cells stably transfected with C/EBPB, LIP, C/EIBIDBM... 273, and CIEBPB: GL2 expression vectors... EMSA of CIEBP binding activities in P388 transfectants in the presence of unlabeled competing oligonucleotide binding sites... ix ..12 .23 .50 ...56 ...60 ....64 85 ...87 ...88 89 Chapter 4 Figure 5. Western analysis Of LIP, CIEBPB192.276 (3192-275) and ClEBPB:G.1(GLZ) expression compared to that of CIEBPB (B) in the P388 transfectants... Figure 6. EMSA of CIEBP binding activities in P388 transfectants in the presence of unlabeled competing oligonucleotide binding sites... .... Figure 7. CIEBPB192-276 (8192-276) and LIP can support the LPS induced activation of the lL-6 promoter in transient transfections of P388 cells... .. Figure 8. CIEBPB192-276 (13192-278) and LIP fail to activate an albumin DEI site-reporter in transient transfections of P388 cells with and without LPS stimulation... Figure 9. CIEBP6131-272 (6181-272) can support the LPS induced activation of the lL-6 promoter in transient transfections of P388 cells Figure 1. Structure of the retroviral vector pSV(X)Neo containing the CIEBP cDNA inserted at a BamH1 site... Figure 2. Northern blot analysis of 320 transfectants... .. Figure 3. Induction of granule proteins myeloperoxidase (MPO), cathepsin G (Cat G), and lysozyme ”(L2)” in 320 cl3 transfectants... .. Figure 4. EMSA of CIEBP DNA binding activities in 320 cl3 cells stably transfected with CIEBPor, CIEBPB, CIEBP6 and ClEBPe expression vectors... Figure 5. Western blot analyses of CIEBP proteins derived from nuclear extracts of the transfectants... Figure 6. Northern blot analysis of CIEBPor expression in 32D transfectants... ..91 ..93 ..95 ...97 ..99 .116 .121 ...123 128 ..129 .131 CIEBP LTR G-CSF M-CSF GM-CSF MCP-1 LPS LIP bZIP FACS TNF-cr lFN-ry mos EMSA LIST OF ABBREVIATIONS CCAATIenhancer binding protein long terminal repeat interleukin granulocyte-colony stimulating factor macrophage-colony stimulating factor granulocytelmacrophage-colony stimulating factor monccyte chemoattractant protein-1 lipopolysaccharide liver inhibitory protein basic region-leucine zipper fluorescence-activated cell sorting tumor necrosis factor-or interferonay inducible nitric oxide synthase electrophoretic mobility shift assays xi Chapter 1 Literature Review 1. CIEBP-related transcription factors: CIEBP-related proteins comprise a family of basic-region leucine zipper (bZIP) transcription factors (Johnson et al. 1994). Members of the CIEBP family are highly homologous in their C-terminal dimerization and DNA binding domains, but are more divergent in the N-terminal transactivation domain. Homo— and heterodimers can be formed between any pair of the family members and bind to a similar DNA sequence (see Figure 1). Dimerization is a prerequisite for DNA-binding activity. The domain responsible for dimerization is a leucine zipper which is an alpha helix with a leucine residue every seven amino acids (Landschulz et al. 1988). The paired a-helices of the dimer associate in a parallel orientation through their hydrophobic surfaces to create a coiled—coil structure (O’shea et al. 1989). Not only can dimerization occur within the family, but heterodimerization with other activator protein families has also been demonstrated (Hsu et al. 1994). In addition, protein-protein interactions other than heterodimerization have been shown to occur between CIEBPs and many transcription factors. This phenomenon is postulated to be the basis of the synergistic effect between ClEBPs and these factors in many CIEBP-regulated promoters. A detailed review of this aspect of CIEBP function will be given in a later section. Located immediately to the N-terminal side of the zipper ——NH2 MONA-”mm . 7 COOH w I l L . OOOH 331...... Ann: / Figure 1. Structure of the CIEBP transcription factor. region is a positively charged domain known as the basic region that functions as the DNA contact surface (Vinson et al. 1992). Dimerization juxtaposes the two basic regions which make specific contacts with DNA along the major grooves of the DNA duplex. The most conserved region between the family members is the basic-region. In fact, many residues which are critical in making contact with DNA are identical. Thus, it is not surprising to find that the DNA binding specificities of CIEBPs are very similar (Williams et al. 1991). The deduced consensus binding site for CIEBPs is : 5’-T(T/G)NNGNAA(TIG)—3’ (Johnson et al. 1993). The CIEBP family is capable of activating in vivo transcription from promoters that contain such a consensus sequence. This sequence is found in the promoters of a number of genes that fall into five categories: inflammation-associated genes (including cytokines), liver-specific genes, adipocyte-speciflc genes, myelcid- specific genes and immunoglobulin genes (Akita et al. 1990, Brooks et al. 1992, Christy et al. 1989, Zhang et al. 1994). The regulatory domain of C/EBPs is present at the N-terminal end of the proteins. Although this domain is not as conserved as the bZlP domain among family members, several clusters of sequence similarity have been identified in this region (Johnson et al. 1994). Mutations in these clusters abolish transactivation ability without affecting DNA-binding and dimerization (Friedman et al. 1990). In addition to these activation domains, a negatively-acting element has also been identified in the regulatory domain of CIEBPa, C/EBPB and C/EBPe (Pei et al. 1991, Williams et al. 1995, Williamson et al. 1998). It is suggested that this element may function as an attenuator to keep the activation domains in check. This block could be removed by extracellular signals which modify the attenuator by a post-translational mechanism, probably via phosphorylation. Since the discovery of C/EBPor in 1988, five CIEBP-related proteins with physical and functional homology to C/EBPa have been identified. These CIEBPs are differentially expressed in many cell types. The variety of CIEBP family members and their potential for heterodimer formation could provide a large repertoire of transcription factors with complex in viva regulatory features. In the following sections, I will review the identification, tissue distribution and regulatory functions of each member of the CIEBP family. The nomenclature used in this thesis for each CIEBP member is adopted from the one proposed by McKnight and colleagues (Cao et al. 1991). According to the order of discovery, they are designated as CIEBPcr, CIEBPB, CIEBPS, CIEBPs, CIEBPy and CIEBPC. 1.1 CIEBPcr CIEBPa, the founding member of the family, was first identified in crude nuclear extract from adult rat liver (Johnson et al. 1987). It was discovered in a search for DNA-binding factors that recognize viral gene regulatory sequences that function ubiquitously, such as the CCAAT box and “enhancer core” elements of retroviral LTRs. Subsequently, it was found that these two elements have very weak homology to the CIEBP consensus, and that C/EBPor does not bind to these elements in vivo (Vinson et al. 1992). The fact that it’s expression level is high in liver cells, and that many liver-specific genes contain the CIEBP binding site in their cis-regulatory sequences, led to the realization that ClEBPa is an important transcription factor regulating liver-specific genes (Birkenmeier et al. 1989, Williams et al. 1991 ). CIEBPor is also very abundant in fat cells where it was found to be important for terminal differentiation (Christy et al. 1989, Kaestner et al. 1990). Several genes whose transcription is specific to differentiated adipocytes are regulated by ClEBPa. When the preadipocytic cell line 3T3-L1 is induced to differentiate by growth to confluency and exposure to hormones, the expression of ClEBPor is increased markedly (Birkenmeier et al. 1989, Christy et al. 1989). This finding implies that ClEBPor is an important regulator in the process of adipocyte differentiation. A proof that the induction of C/EBPa is essential for adipocyte differentiation comes from a study in which the expression of ClEBPa is suppressed by overexpression of antisense RNA for ClEBPor (Lin et al. 1992). 3T3-L1 cells expressing ClEBPor antisense RNA fail to undergo morphological differentiation and fat-specific genes are not induced. C/EBPa is also shown to be expressed in early myeloid cells in the hematopoietic system. in addition, many myeloid-specific genes, such as granulocyte colony-stimulating factor (G-CSF) receptor (Smith et al. 1996), neutrophil elastase (Oelgeschlager et al. 1996) and myeloperoxidase (Ford et al. 1996), contain CIEBP binding sites in their promoters. Recent studies have shown that CIEBPa plays a critical role in granulocytic differentiation. A gene disruption experiment has revealed a lack of mature granulocytes in the blood of CIEBPa knockout mice, while other blood cell types are not affected (Zhang et al. 1997). 1.2 C/EBPB CIEBPB has also been reported as NF-lL6 (Akira et al. 1990), AGP/EBP (Chang et al. 1990), LAP (Desccmbes et al. 1990a), lL-608P (Poli et al. 1990) and CRP2 (Williams et al. 1991). It was discovered from a human cDNA library by its ability to bind the IL-1 responsive element in the lL-6 gene promoter (lsshiki et al. 1990). Other CIEBPB homologs were later isolated from a variety of species, including mouse (Chang et al. 1990), rat (Desccmbes et al. 1990) and chicken (Katz et al. 1993). The rat C/EBPB was cloned from a liver cDNA expression library screened with oligonucleotides containing lL-6 responsive cis- regulating elements from liver-specific acute phase response genes (Poli et al. 1990). Tissue distribution studies show that CIEBPB is present at high level in the liver (Alam et al. 1992, Birkenmeier et al. 1989) and in the myelomonocytic lineages (including monocyte/macrophages and granulocytes) of the hematopoietic system (Scott et al. 1992, Katz et al. 1993). These results are consistent with a primary role for CIEBPB in the regulation of acute phase proteins and inflammation-associated genes. CIEBPB is also expressed in many other tissues, although at a lower level. The list includes ovarian granulosa cells (Sirois et al. 1993), differentiating adipocytes (Cac et al. 1991), pituitary cells (Wegner et al. 1992) and mammary epithelial cells (Robinson et al. 1998). CIEBPB has been closely linked to lL-6 expression and signaling. Its trans- activating potential is enhanced by lL-6 in transfected hepatoma cells, where it acts as an inducer of acute phase response genes (Akira et al. 1990). The induction of acute phase protein genes by lL-6 probably involves the activation of C/EBPB, which binds to CIEBP recognition sites in the promoters of these genes. Although the primary mechanism of CIEBPB regulation within the acute phase response appears to be post-transcriptional (Ramji et al. 1993), C/EBPB mRNA levels are also induced by lL-6 (Akira et al. 1990). CIEBPB is also an important component in the regulation of genes specifically induced in activated macrophages by proinflammatory stimulants such as LPS, IL-6 and lL-1 (Natsuka et al. 1992). The regulation of inflammation-associated genes in activated macrophages will be discussed in detail in later sections. A naturally existing truncated form of CIEBPB, known as LIP, was reported by Descombes and Schibler (1990b). LIP lacks the first 131 amino acid residues in the N-tenninal region of CIEBPB, but retains the bZlP domain. LlP cannot activate transcription from CIEBP-dependent promoters because it lacks the transactivation domain. When cotransfected with CIEBPB, LIP inhibits CIEBPB- mediated transactivation of a target promoter, presumably, by competition for the DNA-binding site. Additionally, the LlP-CIEBPB heterodimer is inactive or less active in comparison to the CIEBPB homodimer. It was proposed that UP is produced by a leaky ribosome scanning mechanism that occurs because initiation at the first AUG codon is inefficient. On the other hand LIP may be an artifact of proteolysis in some systems (Baer et al. 1998). The biological significance and mechanisms regulating this CIEBPB isoform remain elusive. In this thesis, we have discovered that LIP is capable of activating lL-6 and MCP-1 transcription under conditions of LPS stimulation. This unexpected observation has led to the discovery that the leucine zipper region of CIEBPs may have a role other than simply serving as a dimerization domain. These studies will be described in detail in the third chapter. 1.3 CIEBP6 The gene for CIEBPS, alternatively known as NF-IL6B (Kinoshita et al. 1993) and CRP3 (Williams et al. 1991), was obtained by cross-hybridization using the CIEBP bZIP domain. Present at a very low level in many tissues, CIEBP5 mRNA can be increased dramatically by proinflammatory stimulants such as LPS and lL-6, suggesting a role in the regulation of the acute phase response and inflammation. Unlike CIEBPB, the activation of CIEBP5 during the acute phase response occurs predominantly via increased transcription of the gene, rather than by post-translational modification of preexisting protein molecules (Ramji et al. 1993). In addition to the liver, CIEBP6 is expressed transiently in differentiating adipocytes (Cao et al. 1991), and is also present in the myelomonocytic lineages of the hematopoietic system (Scott et al. 1992). 1.4C/EBPa The gene for CIEBPe (originally named CRP1) was first cloned from a rat genomic DNA library by hybridization to ClEBPor. Because neither mRNA nor protein could initially be detected from a variety of tissues, the function and regulation of ClEBPe remained uncertain until recently. The gene for human (Chumakov et al. 1997) and murine (Antonson et al. 1996) CIEBPe were cloned and found to be expressed exclusively in cells of hematopoietic origin. It is proposed that CIEBPe may play a role in regulating myeloid differentiation because C/EBPe is found to be expressed only in early myeloid cells. Additionally, C/EBPe can transactivate reporter constructs containing myeloid- specific c—mim or human myeloperoxidase promoters (Chumakov et al. 1997). Most convincing in regard to its role in myeloid differentiation is the fact that CIEBPe-deficient mice fail to develop functional mature granulocytes (Yamanaka et al. 1997b). Consistent with this result, ClEBPs is up-regulated during granulocytic differentiation (Yamanaka et al. 1997a). 1.5 ClEBPy C/EBPy (also known as lglEBP) was originally identified by its ability to bind to the lg heavy chain promoter and lg heavy chain enhancer (Roman et al. 1990). ClEBPy is the only family member to be ubiquitously expressed. ClEBPy can form heterodimers with other CIEBP family members and exhibits similar DNA- binding properties. However, because CIEBPy lacks the N-ten'ninal transactivation domain found in other CIEBP activators, it alone neither activates 1.4C/EBP8 The gene for ClEBPs (originally named CRP1) was first cloned from a rat genomic DNA library by hybridization to CIEBPa. Because neither mRNA nor protein could initially be detected from a variety of tissues, the function and regulation of ClEBPe remained uncertain until recently. The gene for human (Chumakov et al. 1997) and murine (Antonson et al. 1996) ClEBPa were cloned and found to be expressed exclusively in cells of hematopoietic origin. It is proposed that ClEBPe may play a role in regulating myeloid differentiation because CIEBPe is found to be expressed only in early myeloid cells. Additionally, C/EBPe can transactivate reporter constructs containing myeloid- specific c-mim or human myeloperoxidase promoters (Chumakov et al. 1997). Most convincing in regard to its role in myeloid differentiation is the fact that C/EBPe-deficient mice fail to develop functional mature granulocytes (Yamanaka et al. 1997b). Consistent with this result, CIEBPs is up-regulated during granulocytic differentiation (Yamanaka et al. 1997a). 1.5 CIEBPy CIEBPy (also known as lglEBP) was originally identified by its ability to bind to the lg heavy chain promoter and lg heavy chain enhancer (Roman et al. 1990). ClEBPy is the only family member to be ubiquitously expressed. CIEBPy can form heterodimers with other CIEBP family members and exhibits similar DNA- binding properties. However, because CIEBPy lacks the N-terminal transactivation domain found in other CIEBP activators, it alone neither activates nor represses transcription. Instead, CIEBPy is found to act as a dominant negative inhibitor of the CIEBP activators. Thus, the structure and the function of ClEBPy is very similar to that of LIP. The ubiquitous expression of CIEBPy appears to suggest that CIEBPy simply acts as a buffer for CIEBP activators in many tissues, insuring that CIEBP activator activity is suppressed until its buffer capacity is exceeded by increasing CIEBP activator concentration. 1.6 CIEBPC CIEBPC (also known as CHOP) was originally cloned based on its induction by DNA damaging agents and concomitant growth arrest (Fomace et al. 1989). Subsequently, it was found to exhibit homology to the other CIEBP proteins in the leucine zipper region. Considered a distantly related CIEBP protein, CIEBPQ not only lacks the activation domain found in other CIEBP activators, but is also devoid of the DNA binding domain. As a consequence, CIEBPI; by itself cannot form a homodimer and bind to the CIEBP consensus. But it can form heterodimers with other CIEBP family members and prevent them from binding to DNA, thus acting as a trans-dominant negative regulator of the CIEBP family (Ron et al. 1992). Although it is implicated in mediating the cell’s response to environmental stress, the physiological function of CIEBPC remains obscure. In summary, CIEBP family members are versatile transcription factors expressed in a variety of tissues. They have been shown to regulate target genes that are critical for cell type-specific functions. In addition, accumulating evidence has also suggested their involvement in regulating growth/differentiation of many 10 different tissues. This thesis will focus on elucidating the role of various CIEBP proteins in the myelomonocytic lineages of the hematopoietic system. Thus, in the next two sections I will review the current literature on hematopoiesis and inflammation, the two processes in which ClEBPs have been implicated to play an important role. 2 Hematopoiesis: 2.1 Overview Hematopoiesis is the process in which different types of blood cells arise from a common pluripotent stem cell. During this process, stem cells either renew themselves or differentiate along a number of pathways and generate mature blood cells with specialized functions. Two major groups of blood cells generated in the process are lymphoid cells, which include 8 and T lymphocytes, and myeloid cells, which include erythrocytes, megakaryocytes, mast cells, granulocytes, and macrophages. The structure of the hematopoietic system is shown in Figure 2. The stem cells are relatively few in number but can persist throughout life by undergoing proliferation to produce daughter stem cells. Early in hematopoiesis, a stem cell differentiates to either a lymphoid stem cell or a myeloid stem cell. Subsequent differentiation of lymphoid and myeloid stem cells generates committed progenitor cells for each cell type. Progenitor cells are restricted in their ability to produce a single type of mature blood cell. During terminal differentiation, changes in cell morphology and cell surface markers 11 Erythroid progcm'tor Erythl'ocyte Figure 2. Origin of the hematopoietic cell lineages. become evident and maturing cells also start to express genes critical to the functions of a specific lineage. These changes have allowed the development of such techniques as fluorescence-activated cell sorting (FACS) and histochemical staining to monitor and dissect the process of cell differentiation. 2.2 Growth factors Bone marrow is the major hematopoietic organ in adults. In bone marrow, non- hematopoietic cells, known as stromal cells, support the growth and differentiation of the hematopoietic cells by providing a hematopoietic-inducing microenvironment consisting of a cellular matrix and either membrane-bound or secreted growth factors. Among the various growth factors, four colony stimulating factors (CSF), named for their ability to stimulate the formation of hematopoietic cell colonies in bone marrow culture, are the major regulators to promote survival, proliferation, differentiation, and maturation of myeloid cells. The list includes Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF. In addition to bone-marrow stromal cells, other leukocytes such as activated T helper cells and activated macrophages, also produce hematopoietic growth factors known as cytokines. For example, lL-4, lL-5, lL—6, lL-7, lL-8, and lL-9 are also involved in hematopoiesis. Growth factors exert their function by binding to their cognate receptors on the cell surface and activating a series of downstream signal transducing events. Eventually, the signals result in the activation or repression of transcription of a 13 selected group of genes necessary for lineage commitment and hematopoietic differentiation. 2.3 Myelomonocytic differentiation Monocyte/macrophages and granulocytic neutrophils are two closely related cell lineages in the hematopoietic system. They share the same early developmental pathway from a pluripotent stem cell to a bipotential granulocyte- monocyte progenitor cell. Depending on the type and concentration of growth factors present in the microenvironment, the bipotential precursor cells can be induced to differentiate into either macrophages or neutrophils. M-CSF and GM- CSF are the major growth factors that act on the monocyte precursor, while G- CSF and GM-CSF are the inducers for the granulocyte progenitor. During granulocytic differentiation, there are easily identifiable morphological changes in the nucleus and cytoplasm, which can be assessed by Wright-Giemsa staining of cytospin slides. The morphology of a cell changes from a round and compact nucleus with relatively small cytoplasm to nucleus ratio (myeloblasts), to a bent or horseshoe-shaped nucleus (promyelocytes), and then to a segmented polymorphic nucleus with large cytoplasm to nucleus ratio (mature granulocytes). Other functional assays used to characterize granulocyte differentiation include histochemical staining for leukocyte alkaline phosphatase (LAP) and nitroblue tetrazolium (NBT). In addition, Northern blot analyses for the mRNA of primary granule myeloperoxidase is often used as an indication of the onset of 14 granulopoiesis, while the appearance of lactoferrin mRNA indicates a mature stage of the process. The maturation of monocyte/macrophages can also be divided into several stages based on morphological and functional characteristics. In the bone marrow, early progenitor cells called monoblasts (compact and round nucleus with relatively small cytoplasm to nucleus ratio) divide and differentiate into promonocytes (relatively larger cytoplasm to nucleus ratio), which eventually become monocytes (round cytoplasm with irregular-shaped nucleus). Mature monocytes then enter and remain in the circulation for several hours before extravasating to various tissues to become differentiated macrophages (large irregular-shaped cytoplasm containing many vacuoles). Functional assays for lysozyme and naphthylaoetate esterase, along with Northern analysis for the mRNA of c-fms (a proto-oncogene coding for M-CSF receptor), are frequently used for the evaluation of monocytic differentiation. Due to difficulties in the isolation and purification of a large number of hematopoietic progenitor cells from bone marrow cells, the study of myeloid differentiation has mainly relied on the use of established cell lines which can be induced to differentiate by growth factors or chemical agents. For example, human cell lines HL60 (Collins et al. 1987) and U937 (Oberg et al. 1993), isolated from patients with acute myeloid leukemia, appear to be frozen at an immature stage of myeloid differentiation. They can be induced to differentiate either along the monocytic pathway with TPA, or along the granulocytic pathway with retinoic acid or DMSO. 320d3 is another murine cell line commonly used to 15 study granulocytic differentiation (Valtieri et al. 1987). Isolated from mice infected by Friend virus, 32Dcl3 cells are immature pre-granulocytic cells. They require IL- 3 for continuous proliferation without undergoing differentiation. Upon the replacement of lL-3 with G-CSF, a majority of the cell population can be induced to differentiate into mature granulocytes over a period of 10-14 days. Such inducible models have facilitated the study of mechanisms of myelomonocytic differentiation. 2.4 CIEBP proteins and myelomonocytic differentiation Transcription factors play a major role in differentiation in a number of cell types, including the various hematopoietic lineages. To understand the process of myeloid differentiation, it is important to identify and characterize the transcription factors that activate target genes in the myeloid lineages. Several lines of evidence have suggested that members of the CIEBP family may play an important role in the regulation of myelomonocytic differentiation. First, in the hematopoietic system, CIEBPa, -B and —6 are highly expressed in myeloid, but not in erythroid and lymphoid cells (Scott et al. 1992). Recently, the human CIEBPe gene was cloned and found to be expressed exclusively in immature myeloid cells (Chumakov et al. 1997). Second, a unique temporal expression pattern of CIEBP isoforms has been observed in differentiating myelomonocytic cells (Scott et al. 1992). When murine 320 cells were induced to differentiate along the granulocytic pathway, it was found that CIEBPa was highly expressed in the early stage and downregulated with maturation, whereas CIEBPB and 16 CIEBP5 were upregulated. Meanwhile, C/EBPa mRNA was found to be greatly induced during in vivo granulocytic differentiation of human primary 0034+ cells (Yamanaka et al. 1997a). Third, binding sites for CIEBPs have been shown to be critical for the activity of a number of myeloid-specific promoters, including myeloperoxidase (Ford et al. 1996), neutrophil elastase (Oelgeschlager et al. 1996), M-CSF receptor (Zhang et al. 1994), GM-CSF receptor (Hohaus et al. 1995) and G-CSF receptor (Smith et al. 1996). Although CIEBPa has been proposed to be the major regulator for these promoters, in many cases other CIEBP family members ( CIEBPB, -6 and —e) have also been shown to be active. Gene disruption experiments have provided more insights into the functions of CIEBP proteins in myeloid differentiation. In CIEBPa knock-out mice, analysis of the hematopoietic system has demonstrated a specific defect in production of granulocytic cells (Zhang et al. 1997) . CIEBPa null mice (-l-) do not produce any mature neutrophils, while other cell lineages including monocyte/macrophages, red blood cells and lymphoid cells are not affected. FACS analysis in embryonic and newborn animals confirmed that myeloid markers (Mac-1 and Gr-1) are greatly reduced, with normal 8- and T-cell subsets. Expression of G-CSF receptor mRNA was also profoundly and selectively reduced. These data strongly suggest that CIEBPa has a critical role in granulocyte differentiation. This conclusion was further confirmed in a study in which ClEBPa was conditionally expressed in bipotential progenitor cells, HL-60 and U937 (Radomska et al. 1998). It was shown that conditional expression of ClEBPa in these cell lines was sufficient to induce granulocytic differentiation but not 17 f! monocytic differentiation. Moreover, induced expression of ClEBPa in bipotential precursors blocked their monocytic differentiation program. These results indicate that CIEBPa serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes. Another CIEBP family member found to be involved in regulating myeloid differentiation is C/EBPe. ClEBPa-deficient mice have an increased number of granulocytic precursors in the blood and bone marrow. However, the number of Gr—1-positive mature granulocytes is markedly decreased (Yamanaka et al. 1997b). Thus, CIEBPe-deflcient mice have a similar phenotype to their CIEBPa- deficient counterparts. During myeloid differentiation, ClEBPe is expressed slightly after CIEBPa, suggesting a sequential manner of regulation between these two activators. Indeed, ectopic expression of ClEBPa in myeloid progenitors induces the expression of CIEBPs mRNA and granulocytic differentiation (Radomska et al. 1998). In contrast to ClEBPa and -e, targeted disruption of CIEBPB or —6 does not seem to cause adverse effects on myelopoiesis, consistent with a role of CIEBPB and -6 in the inflammatory response rather than lineage commitment. 3. Inflammation 3.1 Introduction Inflammation, which is initiated by tissue injury (usually caused by trauma or infection), comprises a series of adaptive responses which ultimately facilitate the clearance of infectious agents and the removal of injured tissues. Thus, 18 inflammatory responses serve as an important mechanism for the host to defend against the invasion of pathogenic microorganisms. The responses involved can be both localized and systemic. Three major events accompanying a local inflammatory response are (1) vasodilation, (2) increased capillary permeability, and (3) influx of phagocytic cells. On the other hand, a systemic response known as the acute phase response, includes the induction of fever, increased synthesis of hormones such as ACTH and hydrocortisone, increased production of a large number of hepatocyte—derived acute phase proteins, and increased production of white blood cells. The systemic response involves many different organs throughout the body. The key mediators that orchestrate these activities and link all these different cell types and organs together in a inflammatory response are cytokines. These cytokines are also termed proinflammatory cytokines due to their ability to promote an inflammatory response. Among the important proinflammatory cytokines are lL-1, TNF-a, lFN-y, lL-6, and a group of polypeptides collectively known as chemokines. 3.2 Macrophage activation and acute phase response The acute inflammatory response is initiated following activation of tissue macrophages. Macrophages become activated when stimulated by bacterial Products such as lipopolysaccharides (LPS), or by cytokines produced by activated T cells such as IFN-y and lL-1. Activated macrophages have increased phagocytic activity, increased microbicidal activity, and most importantly, i“creased secretion of proinflammatory cytokines. A list of these cytokines is 19 Table 1. Proinflammatory cytokines produced by activated macrophages. Interleukin-1a (IL-1a) Interleukin-1 B (IL-1B) Interleukin-6 (IL-6) Interleukin-8 (IL-8) Interleukin—11 (IL-11) Interleukin-12 (IL-12) Interferon-a (lFN-a) Tumor necrosis factor-a (TNF-a) Monocyte chemoattractant protein-1 (MOP-1) Macrophage inflammatory protein-1a (MIP-1a) 20 given in Table 1. Proinflammatory cytokines produced by activated macrophages serve as the mediators of the inflammatory response by acting on many different tissues and organs. In a local inflammatory response, both IL-1 and TNF-a induce increased expression of cell-adhesion molecules (CAMs) on endothelial cells, resulting in increased adhesion of circulating white blood cells to vascular endothelial cells and their extravasation into the site of inflammation. lL-8 is noted by its ability to act as a potent chemotactic factor to attract neutrophils to vascular endothelial cells. lFN-a has also been shown to chemotactically attract macrophages to a site where antigen is localized. Several proinflammatory cytokines are also responsible for many systemic responses that occur in an acute inflammatory response. For example, lL-1, TNF-a and lL-6 can each act on the hypothalamus to induce a fever. And lL-6 along with lL-1 and TNF-a can induce production of acute phase proteins by the liver. Acute phase proteins (APPs) are a family of approximately 30 plasma proteins produced in increased amounts by the liver in inflammation. Their concentrations in the serum can be raised by varying magnitudes, ranging from several to 1000—fold. The APPs serve multiple functions in a immune response. They have a role as immune mediators and inhibitors, and as scavengers of cell- derived products released from damaged tissues and macrophages. In addition, they are also involved in the healing process of the injured tissues. Studies of APP production by lL-6 stimulation in hepatocytes have revealed the important role of CIEBP transcription factors. It is well-established that the lL-6 signaling pathway greatly enhances the transactivation potential of CIEBPB mainly by a 21 v' post-translational mechanism (Ramji et al. 1993). In the meantime, the expression of CIEBP5 mRNA is increased dramatically in hepatocytes stimulated by lL-6 (Akira et al. 1990). It has been shown that the promoters of APPs contain binding sites for CIEBP proteins, and that CIEBPs can transactivate the promoters from these genes (Ramji et al. 1993). Thus, it is believed that both CIEBPB and CIEBP5 are the major regulators of the acute phase proteins produced by the liver in response to lL-6, and other cytokines as well. 3.3 Proinflammatory cytokines Proinflammatory cytokines produced by activated macrophages, endothelial cells and fibroblasts are responsible for both local and systemic responses that occur during an acute immune response. Here I will summarize the general properties of several important proinflammatory cytokines. 3.3.1 Interleukin-6 (IL-6) lL—6 is a polypeptide mediator with important roles in a wide variety of systems including regulation of the immune response, the acute phase response and hematopoiesis. Major cellular sources for lL-6 include monocyte/macrophages, endothelial cells and fibroblasts (Akira et al. 1992). They can be induced to produce lL-6 in response to a variety of stimuli such as LPS, virus infection, lL-1 and TNF-a. Detailed studies of the promoter region of the lL-6 gene have revealed several important cis-acting regulatory elements (Dendorfer et al. 1994). As shown in Figure 3, a c-foslserum response element (SRE) contains two overlapping transcription control elements, NF-IL6 (CIEBPB) 22 3M" m one AP-t (>0ng In W um TATA «W 5’ ./ . l [—— 1 J .145 .73 a mandatory m Mfrs muffs NFfB W Serum IL 1 lL1 W «.1 we TNF TNF TNF m LPS Forakolln Figure 3. Cis-acting elements of the IL-6 promoter. 23 And the multiple response element (MRE). One NF-xB binding site is located on the 3’ side of CIEBPB binding element. Together, these elements account for the inducibility of lL-6 production by multiple stimuli. Other regulatory sequences located further upstream are two glucocorticoid response elements and one AP-1 binding site. The major functions of lL-6 that are relevant to an acute immune response include acting as a main regulator for acute phase protein synthesis, and as a mediator of fever. 3.3.2 Interleukin-1 (IL-1) lL-1 activity is actually composed of two polypeptide mediators of similar molecular size (17 kD) (Scales 1992). The IL-1 receptor binds both lL-1a and IL- [3 with a similar affinity. Many cell types have been shown to produce lL-1, the most studied being peripheral monocyte/macrophages. Other cell types capable of producing IL-1 include T and B lymphocytes, smooth muscle, endothelial and various brain cells. A variety of stimuli can induce lL-1 synthesis such as microbial products and cytokines. Major cis-regulatory elements identified in the IL-1B promoter region are the binding sites for CIEBP, NF-KB, and AP-1, as well as the serum response element (SRE) (Zhang et al. 1993). Like lL-6, lL-1 can act on the hypothalamus to induce fever, and induce synthesis of acute phase proteins in hepatocytes. It also has a function in attracting phagocytic cells to the site of inflammation by inducing the expression of adhesion molecules in vascular endothelial cells. 24 3.3.3 Tumor necrosis factor-a (TNF-or) TNF-a was originally identified as an endotoxin-induced serum factor that causes necrosis of tumors in vivo and tumor cell cytotoxicity in vitro. It was later shown to be a pleiotropic mediator involved in many immune responses. lts diverse actions in inflammation, in addition to cytotoxicity, include chemotactic activity for influx of phagocytic cells to inflammation sites, activation of endothelial cells and fibroblasts to produce various cytokines, and induction of fever (T suji et al. 1992). The major source for TNF-a is macrophages. Like other proinflammatory cytokines, TNF-a can be induced by microbial products and cytokines. A number of transcription factors contribute to the regulation of the TNF-a gene. CIEBPB has been shown to be important in TNF-a gene activation in myelomonocytic cells (Pope at al. 1994). A CIEBP binding site is located between 74 and 100 bp upstream of the transcription start site. The TNF-a promoter also contains several potential binding sites for additional transcription factors that may work in concert with CIEBPB. They include binding sites for NF-xB, NF-AT, AP-1, AP-2, Ets, and Sp-1, as well as a cyclic AMP response element (CRE) (Zagariya at al. 1998) 3.3.4 Monocyte chemoattractant protein-1 (MCP-1) MCP-1 was first discovered in murine fibroblasts as a platelet-derived growth factor (PDGF)-induced immediate early response gene and designated as JE (Cocharin et al. 1983).lt was later found by homology to be related to a number of cytokines and identical to the human monocyte chemoattractant protein-1 25 Ira- flBujfzti‘iFi—‘VW ma (MCP-1) (Rollins et al. 1989). MCP-1 belongs to a large family of cytokines with chemotactic activity, known as chemokines. These chemotactic cytokines are produced by macrophages, endothelial cells, and lymphocytes upon LPS, lL-1 or TNF stimulation. They are responsible for the influx of phagocytic cells from blood to tissue during inflammation. MCP-1 is chemotactic for monocyte/macrophages and T cells, with no activity on neutrophils. Studies of the MCP-1 promoter have identified a binding site for NF-KB, which becomes occupied upon stimulation with lL-1B, TNF-a or TPA (Ueda et al. 1994). Induction by lL-1B also requires an AP-1 site for maximal activity (Martin et al. 1997). Although no LPS-responsive element has been identified in the MOP-1 promoter, our study has added CIEBPs to the list of transcription factors that are critical for MOP-1 expression (Bretz et al. 1994, Hu et al. 1998). 3.4 CIEBP proteins and the expression of proinflammatory cytokines A CIEBP activity has been implicated in the regulation of many proinflammatory cytokine genes. The promoters for lL-6, lL-1a, lL—1B, lL-8, TNF- or, and G—CSF contain known or predicted CIEBP binding sites (Akira et al. 1990, Furutani et al. 1986, Shirakawa et al. 1993, Zhang et al. 1993). The LPS-induced expression of lL-1B and G-CSF requires one or more elements that bind a CIEBP-like activity (Shirakawa et al. 1993, Nishizawa et al. 1990). Furthermore, both CIEBPB and C/EBP6 can transactivate a reporter gene regulated by the lL-6 promoter in transient expression assays. Indeed, we have previously shown that 26 W._...-,_. overexpression of CIEBPB confers upon a B-lymphoblastic cell line the ability to induce lL-6 and MCP-1 in response to LPS (Bretz et al. 1994). In order to ascertain the specific role of C/EBPB, knock-out mice were generated by gene targeting in two independent studies (Tanaka et al. 1995, Screpanti et al. 1995). CIEBPB null mice appear to develop normally. An inspection of the ability of peritoneal macrophages derived from these animals to produce proinflammatory cytokines has shown that LPS stimulation leads to a normal induction of a number of proinflammatory cytokines, except for G-CSF. In fact, the expression level of IL-6 is increased. These results suggest that another CIEBP family member(s) can substitute for CIEBPB. We have shown that this, indeed, is the case. ClEBPor and -6 are also expressed by macrophages, and either can activate the expression of lL-6 and MCP-1 in LPS-treated P388 lymphoblasts. These results will be presented in Chapter 2. 3.5 Other cooperating transcription factors Many studies have demonstrated that CIEBP proteins can interact with a number of transcription factors bound to nearby binding sites on target promoters. A synergistic effect is often seen as a result of such interactions. Although the molecular mechanism underlying this synergism is not well understood, it is believed that protein-protein interactions between these transcription factors play a major role. C/EBPa has been shown to interact with a similar set of transcription factors in many myeloid-specific promoters. For example, two transcription factors, PU-1 and AML-1, can interact with C/EBPa 27 and synergistically activate the M-CSF (Zhang et al. 1994), G-CSF (Smith et al. 1996) and GM-CSF (Hohaus et al. 1995) receptor promoters. A similar regulatory scheme is observed in the promoters of neutrophil elastase (Oelgeschlager et al. 1996) and myeloperoxidase (Ford et al. 1996). A direct physical interaction between ClEBPa and AML-1 has also been demonstrated (Zhang et al. 1996). Several transcription factors have been reported to have functional and/or physical interactions with CIEBPB. The chicken homolog of CIEBPB, NF-M collaborates with c-Myb in the combinatorial activation of two myeloid-specific genes, mim-1 and lysozyme (Ness et al. 1993). C/EBPB also works in conjunction with Sp—1 to activate the rat CYPZD5 gene that encodes a cytochrome P450 (Lee et al. 1994). Interestingly, CIEBPa cannot cooperatively activate the promoter with Sp-1. This is one of very few examples showing promoter specificity between CIEBP family members. It is shown that the specificity is determined by the leucine zipper and activation domains of CIEBPB. The most well-documented examples of interactions involving CIEBPB come from studies of the Rel/NF-KB transcription factor family. NF-KB transcription factors are active as homo— and heterodimers composed of a variety of family members including NF-xB1(p50), NF-x82(p52), c-rel, RelA(p65), and RelB (Baeuerle et al. 1994). The p50Ip65 heterodimer is the most common form of NF- KB. NF-KB activity is regulated by nuclear and cytoplasmic partitioning. Several extracellular signals, including LPS and lL-1 and TNFa, induce NF-KB activity by causing the dissociation of NF-xB from an inhibitory subunit, 1K3, whose function is to retain NF-KB protein in the cytoplasm. The release of NF-xB from le allows 28 the transcriptionally active NF-KB to translocate to the nucleus. Binding sites for NF-xB have been identified in the promoters of many inflammation-associated and acute phase genes. Thus, NF-xB and CIEBPs are often co-induced and regulate many of the same target genes including lL-1B (Shirakawa et al. 1993), TNF-a (Natsuka et al. 1992), lL-6 (lsshiki et al. 1990), inducible nitric oxide synthase (iNOS) (Lowenstein et al. 1993) , and lL-8 (Mukaida et al. 1990). In an effort to identify nuclear factors that interact with NF-ICB, LeClair et al. (1992) screened a 1. cDNA expression library with a radiolabeled NF-KB polypeptide. One of the genes identified was found to be CIEBPB. A direct interaction between NF-KB and CIEBPB was proposed to involve, respectively, the Rel homology and the leucine zipper domains. This result was confirmed by studies of the regulation of lL-8 (Stein et al. 1993b, Kunsch et al. 1994) and lL-6 genes (Matsusaka et al. 1993). In the lL-8 promoter, which contains adjacent binding sites for NF-KB and CIEBP, cooperative binding and synergy by NF-KB and CIEBP family members (including CIEBPa, -B and -6) were demonstrated (Stein et al. 1993a). These effects are likely based on direct protein-protein interactions. Using transient transfection with a reporter construct driven by the lL-6 promoter containing binding sites for NF-xB and CIEBP, it was shown that NF-xB and CIEBPB cooperatively transactivate the lL-6 promoter and that this synergy requires intact binding sites for both activators, as mutation in either site abolished the synergistic effect (Matsusaka et al. 1993). Together, these results indicate that NF-KB is an important partner of CIEBP proteins in regulating many inflammation-associated and acute phase genes. 29 Tm... ......._,,_,___,__7 We have shown that in our model system, the murine lymphoblast P388, NF- KB activity is induced by LPS stimulation (Hu et al. 1998), and have confirmed that NF-KB p65 cooperates with CIEBP family members in transactivating the IL- 6 promoter (Tian and Schwartz, unpublished). Surprisingly, we have found that truncated forms of C/EBPB, -6 and, to a lesser extent, -a which lack the activation domains but retain the basic region and leucine zipper are capable of transactivating the lL-6 promoter under conditions of LPS stimulation. The activation domains of CIEBP isoforms are apparently unnecessary for activation of the lL-6 promoter in response to LPS. These and other results that are presented in Chapter 3 show that at least some structural determinants for CIEBP activity reside in the leucine zipper domain. The leucine zipper region is thus implicated in some function other than dimerization to other known CIEBP family members. A model is proposed that the leucine zipper region, conserved among CIEBP family members, not only serves as a site for dimerization but also a site for physical interaction with NF-xB and/or additional transcription factors to form a higher-order ternary structure necessary for robust activation of the lL-6 promoter. Such a model may also explain the redundancy of CIEBPa, -B and -5 observed in the LPS-induction of lL-6 and MCP-1 that is described in Chapter 2. 4. Questions addressed in this thesis CIEBPB has long been considered a key regulator of genes associated with the inflammation and acute phase response, such as proinflammatory cytokines. 30 However, targeted gene disruption has shown that the macrophages from CIEBPB knock-out mice are still capable of producing many proinflammatory cytokines, except for G-CSF, in response to LPS stimulation. This observation has raised the hypothesis that other CIEBP member(s) are functionally redundant to CIEBPB and can replace the activity of CIEBPB when it’s not available. This question has been addressed and detailed in Chapter 2. During the course of study of various truncated forms of CIEBPB, we discovered unexpectedly that truncated forms of CIEBPB which lack any conventional activation domain but retain the bZlP domain are capable of supporting LPS induction of lL-6 and MCP-1. This observation has led us to hypothesize that the redundancy of CIEBPs in regulating proinflammatory cytokine expression may result from their highly related bZlP domains. This part of the study has been described in Chapter 3. Functional redundancy of CIEBP family members has also been observed in the regulation of other myeloid-specific genes such as those encoding the primary granule proteins expressed by the granulocytes. Multiple CIEBP family members are expressed by the granulocytes and their precursors in an overlapping manner. How the different forms of CIEBP dimers regulate these myeloid-specific genes is not well understood. 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M. Torti. 1992. Tumor necrosis factor: Structure and function. In Cytokine in health and disease. ed. S. L. Kunkel and D. G. Remick. Marcel Dekker, Inc. New York Ueda, A., K. Okuda, S. Ohno, A. Shirai, T. lgarashi, K. Matsunaga, J. Fukushima, S. Kawamoto, Y. lshigatsubo and T. Okubo. 1994. NF-xB and Sp1 regulate 37 transcription of the human monocyte chemoattractant protein-1. J. Immunol. 153: 2052 Valtieri, M., D. J. Tweardy, D. Caracciolo, K. Johnson, F. Marilio, S. Altman, D. Snatoli and G. Rovera. 1987. Regulation of proliferative and differentiative responses in a murine progenitor cell line. J. Immunol. 138: 3829 Vinson, C. R., P. B. Sigler and S. L. McKnight. 1992. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science 246: 911 Wegner, M., Z. Cao and M. G. Rosenfeld. 1992. Calcium-regulated phosphorylation within the leucine zipper of CIEBPB. Science 256: 370 Williams, S. C., C. A. Cantwell and P. F. Johnson. 1991. A family of CIEBP- related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 5: 1553 Williams, S. C., M. Baer, A. J. Dillner and P. F. Johnson. 1995. CRP2 (CIEBPB) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J. 14: 3170 Williamson E. A., H. N. Xu, A. F. Gombart, W. Verbeek, A. K, Chumakov, A. D. Friedman and H. P. Koeffler. 1998. Identification of transcriptional activation and repression domains in human CCAATIenhancer-binding protein 6. J. Biol. Chem. 273: 14796 Yamanaka, R., G. Kim, H. S. Radomska, J. Lekstrom-Himes, L. T. Smith, P. Antonson, D. G. Tenen and K. G. Xanthopoulos. 1997a. CCAAT/enhancer binding protein 8 is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing. Proc. Natl. Acad. Sci. USA. 94: 6462 Yamanaka, R., C. Barlow, J. Lekstrom-Himes, L. H. Castilia, P. P. Liu, M. Eckhaus, T. Decker, A. Wynshaw-Boris and K. G. Xanthopoulos. 1997b. Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein e—deficient mice. Proc. Natl. Acad. Sci. USA. 94: 13187 Zagariya, A., S. Mungre, R. Lovis, M. Birrer, S. Ness, B. Thimmapaya and R. Pope. 1998. Tumor necrosis factor alpha gene regulation: enhancement of ClEBPB-induced activation by c-Jun. Mol. Cell. Biol. 18: 2815 Zhang, Y. and W. N. Rom. 1993. Regulation of the interleukin-1B (IL-1B) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs. Mol. Cell. Biol. 13: 3831 38 Zhang, D-E., C. J. Hetherington, H. M. Chen and D. G. Tenen. 1994. The macrophage transcription factor PU.1 directs tissue specific expression of the macrophage colony-stimulating factor receptor. Mol. Cell. Biol. 14: 373 Zhang, D-E., J. Hetherington, S. Meyers, K. L. Rhoades, C. J. Larson, H. M. Chen, S. W. Hiebert and D. G. Tenen. 1996. CCAAT enhancer-binding protein (CIEBP) and AML1 (CBFa2) synergistiwlly activate the macrophage colony- stimulating factor receptor promoter. Mol. Cell. Biol. 16: 1231 Zhang, D-E., P. Zhang, N. D. Wang, C. J. Hetherington, G. J. Darlington and D. G. Tenen. 1997. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAATIenhancer-binding protein a—deficient mice. Proc. Natl. Acad. Sci. USA. 94: 569 39 Chapter 2 Redundancy of CIEBPa, -B, and -6 in Supporting the Lipopolysaccharide- Induced Transcription of lL-6 and Monocyte Chemoattractant Protein-1 Hsien-Ming Hu, Mark Baer, Simon C. Williams, Peter F. Johnson, and Richard C. Schwartz 40 ABSTRACT CIEBPoi, -B and —6 are members of the CCAAT/enhancer binding protein family of transcriptional regulators. All three of these factors are expressed by bone marrow-derived macrophages, with the DNA binding activity of CIEBPB and -8 increased by treatment with LPS while that of CIEBPa is decreased. We have ectopically expressed each CIEBP protein in P388 lymphoblasts. The expression of any of these transcription factor is sufficient to confer the LPS-inducible expression of IL-6 and monocyte chemoattractant protein-1 to Iymphoblasts, which normally lack CIEBP factors and do not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPa, -B and —6 are redundant in regard to the expression of lL-6 and monocyte chemoattractant protein-1. Since ClEBPB-deficient mice have been reported to be largely normal in their expression of proinflammatory cytokines, it is likely that the lack of CIEBPB is compensated for by the induction of C/EBP5 upon LPS treatment. 41 INTRODUCTION CIEBP-related proteins comprise a family of basic region-leucine zipper transcription factors (reviewed in Johnson et. al. 1994). These proteins dimerize through a leucine zipper and bind to a consensus DNA motif through an adjacent basic region. CIEBP-related transcription factors have been implicated in the regulation of a number of proinflammatory cytokines as well as other gene products associated with the activation of macrophages by microbial products and cytokines. For example, the promoter regions of the genes for IL-6, lL-1a, IL- 18, lL-8, TNF-a, G-CSF, nitric oxide synthase, and lysozyme (Akira et al. 1990, Furutani et al. 1986, Lowenstein et al. 1993, Natsuka et al. 1992, Shirakawa et al. 1993, Zhang et al. 1993) contain CIEBP binding motifs. Furthermore, both CIEBPB and -6 can trans-activate a reporter gene regulated by the IL-6 promoter in transient expression assays (Akira et al. 1990, Kinoshita et al. 1992). We have previously shown that the stable expression of CIEBPB in a murine B lymphoblast cell line can confer the ability to induce lL-6 and MCP-1 expression with LPS (Bretz et al. 1994). Two groups of investigators have recently generated mice deficient for C/EBPB expression (Screpanti et al. 1995, Tanaka et al. 1995). Tanaka et al. (1995) found that LPS stimulation of peritoneal macrophages from such animals led to a normal induction of a number of proinflammatory cytokines, including lL-6. Basal levels of lL-6 mRNA were, in fact, elevated. These animals’ macrophages, however, failed to express G-CSF mRNA in response to LPS stimulation. 42 Screpanti et al. (1995) found CIEBPB deficient mice to have elevated levels of IL- 6 expression, but did not otherwise report the ability of macrophages from those mice to produce proinflammatory cytokines. Consistent with the findings of Tanaka et al. (1995), ablation of CIEBPB expression in human fibroblasts with either antisense- or ribozyme-mediated elimination of CIEBPB mRNA blocked TNF-a induction of G-CSF, but not lL-6 expression (Kiehntopf et al. 1995). The above results indicate that CIEBPB is not necessary for the induction of IL- 6 in the inflammatory response. However, the requirement of a CIEBP activity for LPS induction of lL-6 is very likely, since we have previously demonstrated a critical role for C/EBPB in this process (Bretz et al. 1994). Several monocyte and macrophage cell lines have been reported to express both CIEBPB and ClEBPtS (Bretz et al. 1994, Kinoshita et al. 1992), and immature myelomonocytic cell lines have also been reported to express C/EBPa (Scott et al. 1992). It is thus reasonable to propose that the expression of lL-6 and other proinflammatory cytokines by the macrophages of ClEBPB-deficient mice is supported by CIEBP6 or, perhaps, CIEBPa. C/EBPor, CIEBPB and CIEBP6 have all been reported to be functional in a heterologous transgenic rescue assay for a Drosophila CIEBP mutant, slow border cells (Rorth et al. 1994), but the functional redundance of ClEBPs in cytokine expression in mammalian cells has not been demonstrated. In this report we have directly compared the capacities of C/EBPa, CIEBPB, and CIEBP6 to confer LPS-induced cytokine expression to a lymphoblastic cell line normally lacking this capability. Using stable transfection and endogenous cytokine genes containing a full complement of regulatory sequences, we show 43 that any of these CIEBPs can confer LPS-inducible expression of the genes encoding lL-6 and MCP-1. These results demonstrate the redundance of CIEBPa, CIEBPB and CIEBP6 in supporting the LPS induction of lL-6 and MCP- 1. MATERIALS AND METHODS Cells and cell culture: Bone marrow-derived macrophages were obtained from C57 Black/6 mice. Bone marrow was explanted from femurs into DMEM supplemented with 10% FCS, 10% heat-inactivated horse serum, and 20% L cell-conditioned medium at a density of 107 cells/ml in 25 ml on 150-mm tissue culture plates. After 48 h, the nonadherent cells were removed and replated at a density of 3 x 105 cells/ml in 10 ml on 100—mm tissue culture plates. Culture continued for 7 days, with a change of medium every 3 days. P388 cells are murine B lymphoblasts (Bauer et al. 1986) (American Type Culture Collection, Rockville, MD; CCL46). P388-CB cells are P388-CZ cells previously described by Bretz et al. (1994). Cells were cultured in RPMI 1640 medium supplemented with 5% FCS and 50 uM 2-ME. lnductions were conducted with LPS derived from Escherichia coli serotype 0552B5 (Sigma Chemical Co., St. Louis, MO) added to 10 ug/ml. I——£ (”—3 (I; Transfections: Transfections of G418-resistant vectors were conducted with 103 cells, 5 ug of DNA, and 40 pg of lipofectin (Life Technologies, Grand Island, NY) in 3 ml of Opti-MEM l medium (Life Technologies). Cells were incubated in the transfection mixture for 16 hr followed by the addition of RPMI 1640 supplemented with 20% FCS. After 72 h, the medium was replaced with the standard growth medium supplemented with G418 (Life Technologies) at 0.67 mglml. Transfections of puromycin-resistant vectors were conducted similarly with a selective concentration of puromycin (Boerhinger Mannheim, Indianapolis, IN) at 7 jig/ml. Expression vectors: pSV(X)Neo is leP-NEO SV(x)1 (Cepko at al. 1984) and uses the promoter of Moloney murine leukemia virus. pSV(x)ClEBPor was constructed by insertion of the BamHl/Kpnl fragment encoding rat CIEBPor from pMEXC/EBP (Williams et al. 1991) into the BamHI site of pSV(X)Neo with BamHI linkers. pSV(x)C/EBPB was constructed by insertion of the BamHI fragment encoding rat CIEBPB from pMEXCRP2 (Williams et al. 1991) into the BamHI site of pSV(X)Neo. To construct an expression for CIEBPB, the sequences encoding murine CIEBP8 (Williams et al. 1991) were first inserted into the Sphl and Hindlll sites of pMEX (Vlfilliams et al. 1991) by a three-part ligation: one inserted fragment extended from a PCR-introduced Sphl site 40 bp upstream of the CIEBP8 initiation codon to an Apal site approximately 100 bp into the coding sequence, and the other fragment extended from the Apal site to a PCR- introduced Hindlll site just downstream of termination codon. The Sphl/Hindlll 45 fragment was then inserted with BamHI linkers into the BamHI site of pSV(X)Neo to produce pSV(x)CIEBP6. The same BamHI fragment was inserted into the BamHI site of pBABE-Puro (Morgenstem et al. 1990) to construct pBABE- CIEBPS. Nucleic acid isolation and analysis: Total RNA isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s directions. RNAs were electrophoresed through 1% agaroselformaldehyde gels. Transfer to membranes were hybridized and washed to a stringency of 0.1% SSPE at 65°C. Hybridization probes were prepared with random priming kit (Life Technologies) with the incorporation of 5’-[or-32P]dATP (3000 Cilmmol; DuPont-New England Nuclear, Newton, CT). The lL-6 probe was a 0.65-kb murine cDNA (from N. Jenkins and N. Copeland, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD). The MCP-1 probe was a 0.58-kb murine cDNA (Rollins et al. 1988). The GAPDH probe was a 1.3-kb rat cDNA (Fort et al. 1985). Western analysis: Nuclear extracts were prepared as described below. The extracts (20 ug) were adjusted to 1x Laemmli sample buffer (Laemmli 1970) and processed on a 12% PAGE gel. The gel was transferred to a Protran membrane (Schleicher and Schuell, Keene, NH), and Ag-Ab complexes were visualized with the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). 46 _ ' "an s.‘ mar ' V j P GAT SPA; homi TCG TCG. 1 00: com; anne sequ. Al! bi 0‘: nu HEPI ug of 5.5% Fer 5 4‘“ Electrophoretic mobility shift assay (EMSA): Nuclear extracts were prepared as described by Lee et al. (Lee et al. 1988), except that the samples were not dialyzed into buffer D. Protein was incubated with a double-stranded oligonucleotide probe containing an optimal CIEBP binding site (5’- GATCCTAGATATCCCTGA'I'I'GCGCAATAGGCTCAAAGCTG-3’ annealed with 5’-AA‘I‘I'CAGCTTI'GAGCCTATTGCGCAATCAGGGATATCTAG-3’) or to a probe homologous to the NF-KB binding site of the Ig K light chain enhancei (5’- TCGACTCCCTGGGGACTI'TCCAGGCTCC-3’ annealed with 5’- TCGAGGAGCCTGGAAAGTCCCCAGGGAG-3’). A probe containing a CTF/NF- 1 consensus binding site (Landschulz et al. 1988) was used as a nonspecific competitor in some assays (5’-GATCC'ITTGGCATGCTGCCAATATG-3’ annealed with 5’-AATI'CATATTGGCAGCATGCCAAAG-3’). Underlined sequences correspond to the binding motifs of the specified transcription factors. All binding reactions were performed at 23°C in a 25—pl mixture containing 6 pg of nuclear extract (1 mglml in buffer C), 6% (v/v) glycerol, 4% (wlv) Ficoll, 10mM HEPES (pH 7.9), 10 mM DTT, 0.25 pg of BSA, 0.06% (wlv) bromophenol blue, 1 pg of poly(dl-dC), and 1.25 ng of probe. Samples were electrophoresed through 5.5% polyacrylamide gels in 1x Tris-Borate (pH 8.3) and 0.5 mM EDTA at 150 V. For supershifts, nuclear extracts were preincubated with antisera for 30 min at 4°C before the binding reaction. Antisera: Rabbit anti-CIEBPor was generated by immunization with a peptide corresponding to amino acids 253 to 268 of rat CIEBPor (Landschulz et al. 1988). 47 w“ Rabbit anti-CIEBPB was generated by immunization with a peptide corresponding to amino acids 1 to 12 of CIEBPB (Williams et al. 1991) or was purchased from Santa Cruz Biotechnology (Santa Cruz, CA; CIEBPB;C-19). Rabbit anti-CIEBPS was obtained from M. Hannink (University of Missouri- Columbia) or was purchased from Santa Cruz Biotechnology (CIEBPS; C-22). Rabbit anti-ClEBPe was purchased from Santa Cruz Biotechnology (CRP-1; C- 22). Rabbit panCRP antiserum was generated by immunization with a peptide corresponding to a conserved motif within the basic region of CIEBP family members (Williams et al. 1995). Rabbit anti-p50 and anti-p65 were obtained from N. Rice (National Cancer Institute-Frederick Cancer Research and Development Center). RESULTS ClEBPa CIEBPQ and CIEBP6 are all expresseg in primagy bone marrow-derived macrophages. To determine which C/EBPs are expressed in primary macrophages, EMSAs were performed on the nuclear extracts of bone marrow— derived macrophages. Supershifts with specific antisera revealed both C/EBPa and CIEBPB DNA binding activities before LPS stimulation (Fig. 1A). CIEBPB became the predominant binding species after treatment with LPS for 4h; however, C/EBPa binding species were still present at a low level, and CIEBP6 binding species were induced (Fig. 1B). Thus, ClEBPa C/EBPB and CIEBPtS are all potentially available to support the expression of inflammatory cytokines in 48 Figure 1. EMSA of CIEBPa, CIEBPB, and CIEBP6 DNA binding activity in bone marrow-derived macrophages. A, No LPS treatment. B, Four-hour LPS treatment. Binding reactions included normal rabbit serum (N), ClEBPa antiserum (or), CIEBPB antiserum (B), or CIEBPa antiserum (6). Some binding reactions, in addition to specific antisera, included 10-fold (10), 30-fold (30), and 100-fold (100) excess quantities of unlabeled CIEBP or CTFINF1 binding oligonucleotides. The bar to the right indicates the positions of supershifted EMSA species. ‘ 49 C/EBPG ClEBPfi C/EBPS (7.." (19".1 (VIZ-P ('Il-Al‘l (7"? (TD/I‘ll A A .41 A 4 A x l iii mum in 3" It!) \ ,i iii 10 ion IO 30 im \ .- in Jump in Jlt m. “ C .- ll um I,“ 'I III CIEBPrx C/EBPB CIEBPB (‘lli' ( 'I’I'Al‘l ( If." (TFI‘ Fl (31:. ( TFI'.‘ I1 AA 44 N u I. IIIIIIOJOIG \ [I lumlmlolfllfl) NI .- Figure 1. EMSA of ClEBPa, CIEBPB, and CIEBP8 DNA binding activity in bone marrow—derived macrophages. 4 A \ 5 H‘JONIIIOIIIID ‘ 50 macrophages. To further ensure the specificity of our assay, competitions were performed with the unlabeled CIEBP binding site and an unlabeled CTF/NF-1 binding site. Both with (Fig. 1B) and without (Fig. 1A) LPS treatment, a I00-fold excess of the CIEBP binding site almost completely eliminated detectable CIEBP binding species, while a 100-fold excess of the CTFINF-1 binding site barely reduced the abundance of such species. Since CRP-1 (ClEBPe) (Williams et al. 1991) has recently been reported to be a myeloid-specific transcription factor (Chumakov et al. 1997), we also examined whether this CIEBP family member was present in macrophages. EMSAs did not reveal CRP-1 (CIEBPe) binding activity in bone marrow-derived macrophages either before or after LPS stimulation (data not shown). Ectopic expression of CIEBPa, CIEBPfi and CIEBPS in P388 B lymphoblasts. We previously produced two transfectant populations of P388 cells that express CIEBPB through a murine retroviral vector (P388-02 and P388-C2-2) as well as a control population transfected with the same vector lacking an expressed insert (P388-N60) (Bretz et al. 1994). P388 is a murine B lymphoblastic cell line (Bauer et al. 1986) that lacks CIEBPa, CIEBPB and CIEBP8 expression (Bretz et al. 1994). To study the capacities of CIEBPa and C/EBP6 to support the expression of proinflammatory cytokines in comparison to C/EBPB populations of P388 cells were transfected with pSV(X)C/EBPor or pSV(X)C/EBP6. Pools of stably transfected cells were obtained after selection with G418. Cells transfected with 51 pSV(X)C/EBPa were designated P388-Ca, and cells transfected with pSV(X)C/EBP5 were designated P388CB. For consistency, the previous P388-CZ cells were designated P388-CB. CIEBP expression in the transfected populations was initially characterized by EMSA (Fig. 2A). In comparison to nuclear extracts from P388-N90, nuclear extracts from P388-CB, P388-CS, and P388-Ca yielded supershifted protein-DNA complexes upon incubation with antisera specific to CIEBPB, CIEBPS, and C/EBPa, respectively. The EMSA species that gave rise to the supershifts were also evident in the samples incubated with normal rabbit serum. This analysis did not reveal DNA binding activity for any CIEBP family members that had not been transfected into these populations in either the absence or the presence of LPS treatment. Supershift species for CIEBPa, -B, and —6 were only observed in cells transfected for their expression. Additionally, supershift species for CRP-1 (C/EBPe) were not observed in any of the transfectants. As in the assays using extracts from bone marrow-derived macrophages, competitions were performed with the unlabeled CIEBP binding site and an unlabeled CTFINF-1 binding site (Fig. 2B). All the supershifted protein-DNA complexes observed upon incubation with antisera specific to CIEBPB, CIEBPB, and C/EBPa were effectively competed by a 100-fold excess of the CIEBP binding site, while a 100-fold excess of the CTFINF-1 binding site had little effect. The competition revealed a prominent protein-DNA complex that was not supershifted by specific antisera. but was effectively competed by the unlabeled CIEBP binding site. This species probably represents lglEBP (CIEBPy), which is 52 TI ‘3‘ n '5'. Figure 2. Analyses of P388 cells stably transfected with ClEBPa, CIEBPB or CIEBP6 expression vectors. Cell line nomenclature is described in Results. A, EMSA of CIEBP DNA binding activities in P388 transfectants with and without 4-h LPS treatment. Reactions included normal rabbit serum (N), CIEBPa antiserum (a), CIEBPB antiserum (B), CIEBP6 antiserum (6), or CRP-1 (ClEBPe) antiserum (e). The positions of CIEBP-specific Ab supershifted species are indicated by arrowheads on the right. B, EMSA of CIEBP DNA binding activities in P388 transfectants in the presence of unlabeled competing oligonucleotide binding sites. Binding reactions included normal rabbit serum (N), CIEBPa antiserum (a), CIEBPB antiserum (B), or CIEBP6 antiserum (6). Some binding reactions, in addition to specific antisera, included 10-fold (10), 30-fold (30), and 100-fold (100) excess quantities of unlabeled CIEBP or CTFINF1 binding oligonucleotides. The bar to the right indicates the positions of supershifted EMSA species. The asterisk marks the position of the likely lglEBP (CIEBPy) EMSA species. C. Western blot analysis of CIEBP proteins derived from nuclear extracts of the transfectants. The positions of CIEBPa (a), CIEBPB (B), CIEBP6 (6), and cross-reactive material (CRM) are indicated by arrows on the right. The positions of m.w. markers are indicated on the left. 53 vm .2202, :23an Emma 3 $36 gamma 5;, 3855.138 3.8 88 3° 832‘. .N 2:3 Iain Ion on I. - .3 III 1| '8 i0 '1‘ I: ...: 5 ... ...—.3 :— .. I “1 V q “1 —.—l\..ui v LI”: w ...-(Br. ...-3v T-lrfih v LI“: v ...: .x :— 3: .3 ... a I U £9-32 2.0.53... 6943»... m IIE DE be hig sin SUI blo LPS 909 exa (leis CSF highly expressed in P388 cells (data not shown) and other immature B cells (Roman at al. 1990). Western blot analysis of nuclear extracts from the same transfected populations using panCRP antiserum confirmed expression of the ClEBPs from the transfected vectors (Fig. 2C). The immunogenic peptide used in generating panCRP antiserum is completely conserved among CIEBP family members (24); thus, this antiserum can be used for quantitative comparisons of protein levels between different CIEBP family members. CIEBPB protein levels were much higher than those of CIEBP6 and CIEBPor (Fig. 2C) even though the abundance of EMSA species among the transfectants, particularly C/EBPB and CIEBP6 was similar (Fig. 2A). This suggests a higher specific DNA binding activity for CIEBP6. Successful transfection of the P388 populations was also confirmed by Southern blot and Northern blot analyses (data not shown). LPS-induced cflokine expression is supported by QIEBPa and CIEBP6 as well as CIEBPQ. Cultures of P388-CB, P388-C6, and P388-Ca cells were treated with LPS over a time course of 0, 2, 4, 8, and 24 h, and RNA was isolated. A control population of P388 lymphoblasts transfected with pSV(X)Neo was also examined. Northern analyses and RNase protection assays were performed to detect transcripts encoding lL-6, MOP-1, lL- I a, IL- 10, TNF-a, MlP-1a, and G- CSF. Transcripts encoding GAPDH were also examined as a normalization control. LPS was found to induce transcripts for lL—6 and MCP-1 in P388-CB, P388-C6, and P388-Ca cells (Fig. 3). All three CIEBPs were quite effective in 55 cu:- m mes run-cs nun-ca mascouczoeu zoos-zoom u r. ....I O...) ...... i ...... - no~| m. ...... Figure 3. Northem analyses of lL-6 and MCP-1 expression in P388 transfectants. Total RNA was isolated over time course of LPS treatment as indicated. Ten micrograms of RNA was analyzed on Northern blots that were successively hybridized to probes for IL-6 and MCP-1, and GAPDH. 56 CE inducing lL-6 and MCP-1 RNAs. Induction was evident by 2 h of LPS treatment. with a decline by 24 h. The family members differed in the time required to reach peak levels of RNA: CIEBP6 transfectants required 2 h, and C/EBPB and CIEBPa transfectants required as much as 8 h to reach peak levels. CIEBP6 may also be the most effective family member considering its relatively low abundance in P388-C6 (Fig. 2B). CIEBPa may be the least effective, as P388-Ca cells show lower peak levels of lL-6 and MCP-1 RNAs. Also note that CIEBPB expression is associated with significantly higher basal levels of MOP-1 transcripts than those seen with either CIEBPa or CIEBP6 expression. Transcripts encoding IL-1a, IL-1B, and G-CSF were not induced by LPS (data not shown), and weak LPS inductions of TNF-a and MlP-1a were not augmented in any of the CIEBP transfectants compared with those in P388-N90 cells (data not shown). These results were reproducible in similar independeme transfected populations (data not shown). The various CIEBP family members thus differ subtly in their ability to support cytokine expression. Coexpression of CIEBP6 with CIEBPQ augments the expression of lL—6 an_d MCP-1, but does not support the expression of additional proinfla_mmgtorv McKines. Since authentic macrophages were demonstrated to express multiple CIEBPs (Fig. 1), we sought to produce transfectants expressing multiple ClEBPs to test whether combinatorial expression confers augmented capacities to transcribe proinflammatory cytokine genes. In particular, we sought to produce cells coexpressing CIEBPB and CIEBP6 because these DNA binding activities 57 were enhanced upon LPS treatment of bone marrow-derived macrophages (Fig. 1). To produce cells expressing both CIEBPB and CIEBP6, CIEBP6 was introduced into P388-CB cells with the murine retroviral vector pBABE-CIEBP6. P388-CB cells were transfected with either pBABE-CIEBP6 or the parental vector lacking an expressed insert, pBABE-Puro. Pools of stably transfected cells were obtained after selection with puromycin. Cells doubly transfected with pSV(X)C/EBPB and pBABE-CIEBP6 were designated P388-CBI6 and cells doubly transfected with pSV(X)C/EBPB and pBABE-Puro were designated P388- CB/Puro. Supershifting of EMSA species with specific antisera verified the expression of C/EBPB and CIEBP6 in the doubly transfected population, while the control transfection population expressed only C/EBPB (Fig. 4A). Successful transfection was also confirmed by Southern and Northern blot analyses (data not shown). When the LPS induction of lL-6 and MCP-1 RNAs was examined in these transfected populations. the level of expression was augmented in cells expressing both CIEBPB and CIEBP6 compared with that in cells expressing only CIEBPB (Fig. 4B). Densitometry revealed peak inductions of 2.3-fold for IL-6 and MCP-1 in cells expressing CIEBPB, and peak inductions of 3.5-fold for lL-6 and 3.8-fold for MCP-1 were observed in cells coexpressing CIEBP6 and CIEBPB (Table 1). Whether the coexpression of CIEBP6 and CIEBPB augmented the LPS induction of IL-6 and MOP-1 in an additive or a synergistic manner is unclear. Since the previous data (Figs. 2C and 3) suggest that CIEBP6 may be more effective than C/EBPB in supporting transcription of lL-6 and MCP-1 RNAs, the 58 Figure 4. Analyses of P388 cells stably transfected for dual expression of CIEBPB and CIEBP6. Cell line nomenclature is described in Results. A, EMSA of C/EBP DNA binding activities in P388 transfectants with and without 4-h LPS treatment. Reactions included normal rabbit serum (N), ClEBPa antiserum (a), CIEBPB antiserum (B), CIEBP6 antiserum (6), or CRP-1 (CIEBPe) antiserum (a). The positions of CIEBP supershift species are indicated by arrowheads on the right. B, Northern analyses of lL-6 and MCP-1 expression. Total RNA was isolated over time course of LPS treatment and analyzed as described in Figure 3. 59 Figure 4. Analyses of P388 cells stably transfected for dual expression of C/EBPB and C/EBP6. 60 Ia Table I. Comparison of LPS induction of lL—6 and MCP-I mRNA between P388- CB/Puro and P388- CB/B“ LPS, 0 2 4 s 24 P388-calm 1.0 1.6 2.3 1.9 0.8 IL-6 RNA P388-cells 1.0 2.8 3.5 3.3 1.5 lL-6 RNA P388-CW 1.0 1.7 2.1 2.3 1.1 MCP-I RNA P388-cars 1.0 2.4 3.7 3.8 2.2 MCP-l RNA “ The autoradiograms presented in Figure 48 were analyzed by densitometer. and raw values for IL-6 and MCP-l were normalized to values for GAPDH. The expres- sion levels for each cell line were set at 1.0 at *0 h. LPS treatment and the tabulated values represent fold increases in expression. 61 augr depi TNF Ci'E not inde ShO‘ Iran ham augmented expression of these mRNAs upon LPS induction may be solely dependent upon the added expression of CIEBP6. Examination of IL-1a, lL-1B, TNF-or. MlP-1ci. and G-CSF expression showed no effect of coexpression of CIEBPB and CIEBP6 on the induction of RNAs encoding these cytokines (data not shown). These results were reproducible in a similar population of P388 cells independently transfected for coexpression of CIEBPB and CIEBP6 (data not shown). Unexpectedly, in repeated attempts we were unable to obtain transfectants coexpressing CIEBPa and CIEBPB or CIEBPa and CIEBP6. NF-KB @MSI DNA binding activity is induced by LPS in the P388 transfectants. NF-xB has been implicated in the regulation of numerous cytokines that are expressed by macrophages in response to LPS (reviewed in Baeuerle et al. 1994, Grilli et al. 1993). In particular, mutation of an NF-icB binding site in the human lL-6 promoter completely abolished responsiveness to LPS (Dendorfer et al. 1994). Additionally, the lL-6 promoter (Matsusaka et al. 1993) and the lL-8 promoter (Matsusaka et al. 1993, Stein et al. 1993) are activated synergistically by CIEBPB and NF-xB. The Importance of NF-KB in the expression of proinflammatory cytokines led us to determine whether NF-KB was indeed activated upon LPS treatment of P388 cells. The lack of cytokine induction in P388-Neo cells could be caused by an absence of NF-icB expression or activation. The inability of CIEBP transfectants of P388 cells to induce cytokines other than lL-6 and MCP-1 could be similarly explained. On the other hand, the ability of CIEBPB to mediate a higher basal level of MCP-1 expression 62' than other CIEBPs could be caused by constitutive NF-xB activity in P388-CB cells. To address these issues, EMSAs were performed using a probe for NF-xB binding. As shown in Figure 5A, an LPS-induced EMSA species was observed in all transfectants, including the P388-Neo control. Formation of this LPS-induced species could be quantitatively blocked by either p50 or p65-specific antisera (Fig. 53), showing that the major species induced is a p50/p65 heterodimer. Thus, NF-icB (p50p65) is translocated to the nucleus of P388 cells and is probably available to support the LPS-induced expression of proinflammatory cytokines. The inability of P388 cells to induce lL-6 and MCP-1 can be specifically attributed to the absence of CIEBP family members. 63 Figu WEI Posi Dani IN). MI: ‘.0|.§4|.- Figure 5. EMSA of NF-icB DNA binding activity in P388 transfectants. A. Cells were grown in the absence of LPS (-) or 4 h in the presence of LPS (+). The position of NF-xB EMSA species is indicated by arrowheads on the right of each panel. 8. Nuclear extract of P388-Neo cells was treated with normal rabbit serum (N), p50 antiserum (p50), or p65 antiserum (p65). The arrowhead on the right indicates the position of NF-KB EMSA species. DISCUSSION The data presented in this paper demonstrate that CIEBPa. CIEBPB, and CIEBP6 are each sufficient to confer LPS-inducible expression of lL-6 and MCP- 1 to P388 B lymphoblasts. We have shown that CIEBPa and CIEBPB are expressed in unstimulated bone marrow-derived macrophages, while LPS stimulation downregulates CIEBPa expression and up-regulates expression of CIEBP6. Thus, all three of these ClEBPs are expressed in bone marrow-derived macrophages and could participate in the LPS induction of lL-6 and MCP-1. The observation of a largely normal cytokine response to LPS treatment in the macrophages of CIEBPB-deficient mice (T anaka et al. 1995) can be explained by the availability of CIEBPa and/or CIEBP6. The induction of CIEBP6 by LPS in bone marrow-derived macrophages makes it a particularly attractive candidate for replacing CIEBPB activity. In fact, CIEBP6 may be more effective than CIEBPB in supporting the transcription of lL-6 and MCP-1 genes, since a relatively low level of its expression in P388-C6 transfectants allows a quite vigorous induction of lL-6 and MCP-1. This induction is at least equal to that observed in P388-CB cells, which express a much higher level of CIEBPB, and is more rapid. P388-C6 cells also display a level of DNA binding similar to that of P388-CB cells, suggesting a higher sp. act. for DNA binding. CIEBP6 has previously been reported to be a stronger trans-activator than CIEBPB using the human IL-6 promoter in a reporter construct (Kinoshita et al. 1992). The presence of a regulatory domain (RD2) in CIEBPB that represses DNA binding 65 activity may explain its lower activity (Williams et al. 1995). On the other hand, CIEBPa appears less effective than CIEBP6 in inducing lL-6 and MCP-1 while being expressed at a similar level to CIEBP6 in transfectants. Additionally, ClEBPa DNA binding activity is reduced upon LPS treatment of bone marrow- derived macrophages, making it a less likely candidate to replace CIEBPB activity in ClEBPB-deficient mice. Collectively, the data suggest a prominent role for CIEBP6 in the LPS induction of inflammatory cytokines and implicate CIEBP6 as the most plausible activity to compensate for the lack of C/EBPB in CIEBPB- deficient animals. The kinetics of LPS induction of lL-6 and MCP-1 mRNAs are generally similar among transfectants for the various CIEBP family members. Induction is evident by 2 h and declines by 24 h. There may be differences, however, in the time required to attain peak RNA levels among CIEBP family members. The CIEBP6 transfectants reached peak levels at 2 h compared with 4 or 8 h for CIEBPB and ClEBPa transfectants. and the ClEBPB/6 transfectants showed a dramatic induction by 2 h. Our previous studies (Bretz et al. 1994) found that the kinetics of proinflammatory cytokine mRNA production in a macrophage cell line, P388D1 (IL1), also reached peak RNA levels by 2 h. This may suggest the importance of CIEBP6 expression in vivo. Indeed, we have shown in this study that CIEBP6 is induced in LPS stimulation of bone marrow-derived macrophages. The delay in reaching peak RNA levels for C/EBPB and CIEBPa transfectants may indicate a requirement for the induction of other factors for optimal expression with these CIEBPs. The delay may reflect the time required to induce and synthesize these 66 factors, or, on the other hand, the delay may simply indicate a lower rate of transcription requiring longer times to attain peak levels. It is clear that LPS induction of lL-6 and MCP-1 mRNAs in our system operates through either the post-transcriptional activation of ClEBPs or the induction of a necessary cooperating transcription factor. EMSA analysis demonstrated CIEBP binding activity for the transfected genes before LPS treatment, and LPS treatment neither induced CIEBP family members other than those transfected nor increased the binding activity of the transfected CIEBPs. If LPS treatment is modulating the activity of CIEBPs in our system, it must be in a manner not evident in EMSA analysis. Other investigators have found in transient transfection studies of the lL-6 promoter that coexpression of CIEBPB and NF-icB synergistically activates the lL-6 promoter (Matsusaka et al. 1993), and mutation of an NF-icB binding site in the human lL-6 promoter completely abolished responsiveness to LPS (Dendorfer et al. 1994). We have found that LPS induces NF-KB (p50/p65) in the P388 transfectants, and it is likely that this is the primary role of LPS in our system. A synergism between the activities of CIEBPB and CIEBP6 has been reported for the transient trans-activation of the human lL-6 promoter (Kinoshita et al. 1992). and we did observe that coexpression of CIEBP6 with CIEBPB augments the LPS induction of lL-6 and MOP-1 mRNAs over that observed for CIEBPB alone. It is unclear from our data whether that augmentation is synergistic or additive, since CIEBP6 by itself appears more active than CIEBPB in supporting LPS induction of lL-6 and MOP-1. Interestingly, despite repeated attempts we 67 _ -..__-_' were unable to obtain transfectants doubly expressing CIEBPa and CIEBPB or CIEBPa and CIEBP6. Although bone marrow-derived macrophages coexpress these ClEBPs, we have not detected CIEBPa expression in any mature macrophage cell lines (our unpublished observation) (Bretz et al. 1994, Scott et al. 1992). These observations may indicate that CIEBPa expression is incompatible with the immortalization of mature macrophage cell lines. Consistent with this idea, CIEBPa has previously been shown to inhibit proliferation in adipocytes (Umek et al. 1991 ), hepatocytes (Hendricks-Taylor et al. 1995), and other cell types (Hendricks-Taylor et al. 1995). Among the several cytokine mRNAs examined, only lL-6 and MCP-1 displayed robust LPS inductions. The lack of induction of other cytokines may reflect the requirement of transcription factors in addition to the CIEBP family for a full cytokine response. We have found that NF-icB (p50p65) is induced by LPS in the P388 transfectants, so such a deficiency must be attributed to other transcription factors. For instance, the murine lL—10 gene requires a novel 6-bp sequence (- 2280 to -2275) in addition to CIEBP and NF-KB binding sites (Godambe et al. 1995). Furthermore, previous investigators have noted differences in the regulation of MOP-1, lL-1a, and lL-1B (Ohmori et al. 1990, Tannenbaum et al. 1989). For example, agents that elevate intracellular levels of cAMP suppress the LPS induction of MCP-1, but do not affect the induction of lL-la, and actually enhance lL-1B induction. Stimuli other than LPS. such as IFN-y, lL-1, or TNF, might also provide a more complete cytokine response through their ability to activate other transcription factors. 68 An alternative explanation for the lack of induction of cytokines other than lL-6 and MOP-1 may be the expression of lglEBP (Cl EBPy) in P388 lymphoblasts. lglEBP has been reported to be a trans-dominant inhibitor of CIEBP family members (Cooper et al. 1995). It may block CIEBP activity on the promoters of those cytokine genes for which we do not observe activation. It will be of interest to assess the ability of lglEBP to inhibit CIEBP activation of the promoters for IL- 6 and other proinflammatory cytokines in a transient expression system lacking endogenous lglEBP expression. The expression of IL-6 and MCP-1, while both showing strong inductions with LPS, differ in regard to the basal levels of their mRNAs among the various CIEBP transfectants. In particular, MCP-1 displays an appreciable level of RNA in P388-CB in the absence of LPS. Since we do not observe NF-KB activity in the absence of LPS, it appears that MCP-1 does not require NF-KB for significant basal expression of its RNA. It will be of interest to compare the structure of the MCP-1 promoter to that of lL-6. The data presented here lead us to predict that CIEBP6 expression may be crucial to supporting proinflammatory cytokine expression in vivo. Tanaka et al. (1995) did not find severe impairment of proinflammatory cytokine expression in ClEBPB-deficient animals. We have now shown that while both ClEBPa and CIEBP6 can support the LPS activation of endogenous lL-6 and MCP-1 genes, the LPS activation of bone marrow-derived macrophages down-regulates C/EBPa activity and up-regulates CIEBP6 activity. CIEBP6 is thus the best candidate for the factor allowing ClEBPB-deficient mice to display a largely 69 normal cytokine expression in response to LPS stimulation. A lack of CIEBP6 expression would be expected to reduce and/or delay peak expression of lL-6 and MCP-1 mRNAs. The development of knockout mice deficient in CIEBP6 expression and mice deficient in both C/EBPB and CIEBP6 expression should provide the ultimate test of this issue. Finally. why are there multiple CIEBP family members with seemingly redundant function within the inflammatory response? First, one should recognize that our system has only allowed examination of lL-6 and MCP-1 expression; promoter-specific functions of CIEBP family members are certainly possible for other genes. More significantly, differential function of C/EBP family members may become apparent under the influence of inflammatory stimuli other than LPS. 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Suematsu, N. Yoshida, and T. Kishimoto. 1995. Targeted disruption 0f the NF-IL6 gene discloses its essential role in bacteria killing and tumor cYtotoxicity by macrophages. Cell 80: 353. 73 Tannenbaum. C. S., and T. A. Hamilton. 1989. Lipopolysaccharide-induced gene expression in murine peritoneal macrophages in selectively suppressed by agents that elevate intracellular CAMP. J. Immunol. 142: 1274. Umek, R. M., A. D. Friedman, and S. L. McKnight. 1991. CCAAT-enhancer binding protein: a component of a differentiation switch. Science 251: 288. Williams. S. C., C. A. Cantwell, and P. F. Johnson. 1991. A family of CIEBP- related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 5: 1553. Williams. S. C., M. Baer, A. J. Dillner, and P. F. Johnson. 1995. CRP2 (CIEBPB) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J. 14: 3170. Zhang, Y., and W. N. Rom. 1993. Regulation of the interleukin- 1B (IL-1B) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs. Mol. Cell. Biol. 13: 3831. 74 Chapter 3 Conventional Activation Domains are Dispensable for the Role of CIEBPs in LPS Induction of lL-6 and MOP-1 Expression Hsien-Ming Hu, Qiang Tian, Mark Baer, Simon C. Williams, Peter F. Johnson, and Richard C. Schwartz 75 ABSTRACT CIEBP or, B, and 6 are all expressed by bone marrow-derived macrophages. Ectopic expression of any of these transcription factors is sufficient to confer LPS-inducible expression of lL-6 and monocyte chemoattractant protein 1 (MCP- 1) to lymphoblasts, which normally lack CIEBP factors and do not display LPS induction of proinflammatory cytokines. Thus, the activities of CIEBPa, B, and 6 are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, we have found that the bZlP regions of CIEBPB and CIEBP6 are of themselves also capable of supporting LPS induction of IL-6 and MCP-1. The bZIP region of C/EBPa also shows modest activity. Furthermore, the naturally occurring transdominant negative inhibitor LIP is capable of supporting the LPS induction of IL—6 and MCP-1. Replacement of the leucine zipper of CIEBPB with that of Yeast GCN4 yields a chimeric protein that can dimerize and specifically bind to a CIEBP consensus sequence, but shows a markedly reduced ability to activate IL- 5 and MCP-1. These results implicate the leucine zipper region in some function other than dimerization with known CIEBP family members in the activation of IL- 6 anal MCP-1 transcription, and suggest that CIEBP redundancy in regulating cYtOkine expression may result from their highly related bZlP domains. 76 INTRODUCTION CIEBPa, B, and 6 are members of the CCAAT/enhancer binding protein family of transcription factors (reviewed in Johnson and Williams, 1994). These proteins are basic region-leucine zipper transcriptional regulators that dimerize through their leucine zippers and bind to a consensus DNA motif through their adjacent basic regions. CIEBPB and CIEBP6 have been implicated in the regulation of proinflammatory cytokines as well as other gene products associated with the activation of macrophages and the acute phase inflammatory response. For example the promoter regions of the genes for lL-6, lL-1a, lL-1B, lL-8, TNFor, G- CSF, nitric oxide synthase, and lysozyme (Akira et al., 1990; Furutani et al., 1986; Lowenstein et al., 1993; Natsuka et al., 1992; Shirakawa et al., 1993; Zhang and Rom, 1993) contain CIEBP binding sites. Furthermore, CIEBPB and CIEBP6 have both been shown to activate a reporter gene controlled by the IL-6 Promoter in transient expression assays (Akira et al., 1990; Kinoshita et al., 1992). We have previously demonstrated that the stable expression of CIEBPB in a murine B lymphoblast cell line was sufficient to confer LPS-inducibility of lL—6 and monocyte chemoattractant protein 1 (MCP-1) expression (Bretz et al., 1994). There have been a limited number of reports demonstrating specificity of a par‘ticular CIEBP family member for a given promoter. One example is the rat CYP2D5 gene that encodes a cytochrome P450. It is transactivated °°°Deratively by CIEBPB and SP1, but not ClEBPa (Lee et al, 1994). The specificity of this cooperativity is determined by the leucine zipper and activation 77 it domains of CIEBPB (Lee et al, 1997) Another case is promoter P1 of the proprotein processing enzyme furin (Ayoubi et al., 1994). It could be transactivated by CIEBPB but not CIEBPa or CIEBP6. Recently, it has been reported that CIEBP6 but not CIEBPB, can transactivate the promoter for nerve growth factor (Colangelo et al., 1998). On the other hand, CIEBPa, B and 6 are all functional in a heterologous transgenic rescue assay for a Drosophila CIEBP mutant, slow border cells (Rorth, 1994). Our own studies have demonstrated redundancy in the abilities of C/EBPa, B and 6 to support LPS-induction of lL—6 and MOP-1 transcription (Hu et al., 1998). A simple hypothesis for the redundancy of CIEBPs that we have observed is that their highly homologous bZlP domains (\Mlliams et al., 1991) are all that is truly required of CIEBPs for activation of the genes for IL-6 and MOP-1. Against this hypothesis stand results obtained with a truncated form of CIEBPB that initiates at Met 132 and lacks activation domains. This protein, referred to as LIP, Could not activate transcription and, in fact, inhibited CIEBPB-mediated transcriptional activation of a promoter derived from the DE-l site of albumin (Descombes and Schibler, 1991). In this report, we have surprisingly found that trurHeated forms of CIEBPB that lack known activation domains, including the naturally occurring transdominant negative inhibitor LIP, are also capable of s'UDIDorting LPS induction of lL-6 and MCP-1. Furthermore, a truncated form of C’EBP6 and, to a lesser extent, a truncated form of CIEBPa that similarly lack conventional activation domains have transcriptional activity on the IL-6 promoter. Replacement of the leucine zipper of CIEBPb with that of yeast GCN4 78 yields a chimeric protein that can dimerize and specifically bind to a CIEBP consensus sequence, but has reduced ability to activate IL-6 and MCP-1. These results implicate the leucine zipper domain in some function other than dimerization to known CIEBP family members in the activation of lL-6 and MCP-1 transcription, and suggest that CIEBP redundancy in regulating cytokine expression may result from their highly related bZlP domains. MATERIAL AND METHODS Cells and cell culture. P388 are murine B lymphoblasts (Bauer et al., 1986) (American Type Culture Collection; CCL46). P388-CB cells have been described Previously by Hu et al. (1998). Cells were cultured in RPMI 1640 medium SuPplemented with 5% FCS and 50 pM 2-ME. lnductions were conducted with LPS derived from Escherichia coli serotype 055:35 (Sigma) added to 10 pglml. Lransfections. Stable transfections were conducted with 2x106 cells, 5 pg of DNA, and 10 pl of DMRlE-C reagent (Life Technologies) in 1.2 ml of Opti-MEM l medium (Life Technologies). Cells were incubated in the transfection mixture for 16 h followed by the addition of 2.8 ml of the standard growth medium. After 24 hOlJrs, the medium was replaced with the standard growth medium suPplemented with G418 (Life Technologies) at 0.67 mglml. 79 Transient transfections were conducted with 2x106 cells, 4 pg of DNA, and 8 pl of DMRlE-C reagent (Life Technologies) in 1.2 ml of Opti-MEM I medium (Life Technologies). The DNA was comprised of either 1 pg of the IL-6 promoter- reporter or the albumin DEI promoter-reporter, 1 pg of the SV40 early promoter- reporter, 0.1 pg of CIEBP expression vector, and pMEX plasmid to total 4 pg. Cells were incubated in the transfection mixture for 5 h followed by the addition of RPMI 1640 medium supplemented to 15% with FCS. After 24 h, the medium of certain transfections was supplemented with 10 pglml LPS. After 4 h in the presence or absence of LPS transfected cells were harvested, lysed, and analyzed for luciferase activity by using the Luciferase Reporter Gene Assay Kit (Boehringer Mannheim) and for B-galactosidase activity by using the Luminescent B-Galactosidase Genetic Reporter System ll (Clontech). Mion vectors ampromoter-reportfi. For stable transfections, C/EBPs were expressed from leP-NEO SV(X)1 constructs (Cepko et al., 1984). CIEBP sequences were inserted into the BamHI site of the vector. Inserted sequences were transcribed from the Moloney murine leukemia virus promoter and the gene conferring G418-resistance was expressed from a subgenomic splicing product from the same promoter. For transient transfections, CIEBPs were expressed from pMEX (Williams et al., 1991), which also utilizes the Moloney murine 'eukemia virus promoter. The construction of CIEBP deletions and CIEBPBzGLz have been described previously (Williams et al., 1991 ). 8O The lL-6 promoter-reporter consists of the murine lL-6 promoter (T anabe et al., 1 988) (-250 to +1) inserted into the luciferase vector, pXP2 (Nordeen , 1988). The albumin DE-I promoter-reporter is (DEI)4(-35alb)LUC (Williams et al. 1995) which is derived from pXP2 (Nordeen , 1988) and contains four copies of the DEI element upstream of the albumin minimal promoter. The SV40 early promoter- reporter is a commercial product, pBgal-Control (Clontech), where the SV40 early promoter and enhancer sequences are cloned upstream and downstream , respectively, of the IacZ gene. RNA isolation and analysis. Total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s directions. RNAs were electrophoresed through 1% agarosel‘formaldehyde gels. Transfers to membranes were hybridized and washed to a stringency of 0.1x SSPE at 65°C. Hybridization probes were prepared with a random priming kit (Life Technologies) with the incorporation of 5’-[or-32P]dATP (3000 Cilmmol; DuPont- New England Nuclear). The lL-6 probe was a 0.65 kb murine cDNA (from N. Jenkins and N. Copeland, National Cancer Institute-Frederick Cancer Research and Development Center). The MCP-1 probe was a 0.58 kb murine cDNA (Rollins et al., 1988). The GAPDH probe was a 1.3 kb rat cDNA (Fort et al., 1985) Western anal sis. Nuclear extracts were prepared as described below. The extracts (50 pg) were adjusted to 1x Laemmli sample buffer (Laemmli, 1970) and 81 processed on a 12% PAGE gel. The gel was transferred to Protran membrane (Schleicher and Schuell), and Ag-Ab complexes were visualized with the Enhanced Chemiluminescence Kit (Amersham). Electrophoretic mobilig shift assay (EMSA). Nuclear extracts were prepared as follows. Cells were washed in phosphate-buffered saline and lysed in 15 mM KCI, 10 mM HEPES [pH 7.6], 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0. 1 % [vol/vol] NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 pglml leupeptin, 5 14ng antipain, and 5 pglml aprotinin for 10 min on ice. Nuclei were pelleted by centrifugation at 14,000 x g for 20 sec at 4°C. Proteins were extracted from nuclei by incubation at 4°C with vigorous vortexing in buffer C (420 mM NaCl, 20 mM HEPES [pH 7.9], 0.2 mM EDTA, 25% [vol/vol] glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 pglml leupeptin, 5 pglml antipain, and 5 pglml aprotinin). Nuclear debris was pelleted by centrifugation at 14,000 x g for 15 min at 4°C and the supernatant extract was collected and stored at -70°C. The EMSA probe was a double-stranded oligonucleotide containing an optimal CIEBP binding site (5’-GATCCTAGATATCCCTGATTGCGCAATAGGC- TCAAAGCTG-3’ annealed with 5’-AATTCAGCTTTGAGCCTATTGCGCAATC- AG GGATATCTAG-3’) labeled with the incorporation of 5’-[a-32P]dATP (3000 Cilmmol; DuPont-New England Nuclear) and Klenow DNA polymerase. A probe containing a CTFINF-1 biding site (Landschulz et al., 1988) (5’-GATCC‘ITI'_& GCATGCTGCCAATATG-3’ annealed with 5’-AATTCATA'ITGGCAGCATGCC- 82 MAG-3’) was used as a nonspecific competitor in some assays. Underlined sequences correspond to the binding motifs of the specified transcription factors. DNA binding reactions were performed at room temperature in a 25 pl reaction mixture containing 6 pl of nuclear extract (1mglml in buffer C)] and 5 pl of 5 x binding buffer (20% [wt/vol] Ficoll, 50 mM HEPES [pH 7.9], 5mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained 1 pg poly(dl- dC), 1.25 ng of probe, bromophenol blue to a final concentration of 0.06% [wt/vol], and water to volume. For supershifts, nuclear extracts were preincubated with antibodies for 30 min at 4°C prior to the binding reaction. Samples were electrophoresed through 5.5% polyacrylamide gels in 1x TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) at 160 V. Antibodies. Rabbit anti-CIEBPB specific to the carboxyl terminus (product C-19) and normal rabbit lgG were purchased from Santa Cruz Biotechnology. Rabbit anti-CIEBPB specific to the amino terminus was generated by immunization with a peptide corresponding to amino acids 1 to 12 of CIEBPB (Williams et al., 1991). 83 RESULTS Truncated forms of CIEBPB that lack activation domains retain the abilig to su ort LPS inflction of lL-6 and MCP-1. We previously found that CIEBPa, B and 6 were all effective in supporting the LPS-induced transcription of IL-6 and MOP-1 (Hu et al., 1998). In order to test whether this redundancy was based on the bZIP domain which is highly conserved among these CIEBP family members (Williams et al., 1991), we examined the expression of a tmncated form of CIEBPB (amino acids 192-276; CIEBPB192-276) that lacks all conventional activation and regulatory domains and is simply the bZIP domain of CIEBPB (VVilliams et al., 1995) (Figure 1). It was expected that the activity of CIEBPBm- 273 would be similar to a truncated form of CIEBPB that initiates at Met 132 and lacks activation domains (Descombes and Schibler, 1991) (Figure 1). This Protein, referred to as LIP, reportedly cannot activate transcription and, in fact, inhibits CIEBPB-mediated transcriptional activation. We performed stable transfections of P388 cells with murine retroviral vectors expressing either CIEBPB192-275 or LIP, and compared those transfectants to P388-CB cells that had been transfected with a vector expressing CIEBPB as well as a control population transfected with the same vector lacking a expressed insert (P388-Neo) (Bretz et al., 1994; Hu et al., 1998). P388 is a murine B lymphoblastic cell line (Bauer et al. 1986) that lacks CIEBPa, CIEBPB, CIEBP6, and CIEBPe expression (Bretz et al., 1994; Hu et al., 1998). Populations of cells transfected for C/EBPB192-276 expression were designated P388-CB192-276, and 84 DNA Activation and Regulatory Binding Domain Domain Leucine Zipper C/Eapg 1 [ . ... -...i 276 LIP ,. a 276 C/EBPBmm .. ”mi 276 ClEBPamm ] 353 C/EBP8,,,,,, 11“ -; I 272 11"“??? CIEBPfizcu W Figure 1. Structures of the various altered CIEBP isoforms used in the studies described in this paper. 85 populations transfected for LIP expression were designated P388-LIP. Surprisingly, P388-CB192-276 cells behaved similarly to P388-CB cells in their ability to induce lL-6 and MCP-1 transcription in response to LPS (Figure 2). P388-LIP cells also showed activity in this assay. Their more modest activity can likely be attributed to the negative regulatory domains that are retained in LIP (Williams et al., 1995). Electrophoretic mobility shift assays (EMSA) of nuclear extracts from the transfected populations, as well as western blot analyses were performed in order to verify proper expression of the stably transfected CIEBPB genes. In Comparison to nuclear extracts from P388-Neo, nuclear extracts from P388-CB, P388'CB192-276, and P388-LIP yielded supershifted protein-DNA complexes upon incubation with an antibody specific for CIEBPB (Figure 3). The EMSA species that gave rise to the supershifts were also evident in the samples incubated with normal IgG. As expected the CIEBPB-specific EMSA species from P388-CB192-276 and P388-LIP cells were of higher mobility than those of P388-CB cells reflecting the truncated structure of their CIEBPB proteins. Additionally, the CIEBPB192-276 and LIP EMSA species could only be supershifted with antibody specific to the carboxyl-terminus of CIEBPB, while EMSA species of intact CIEBPB could be supershifted by both amino— and carboxyl-terminus specific antibodies. To further ensure the specificity of the EMSA, competitions were performed with the unlabeled CIEBP binding site and an unlabeled CTFINF-1 binding site (Figure 4). All of the supershifted protein-DNA complexes observed upon incubation with CIEBPB-specific antibody were effectively competed by a 30-fold excess of the 86 nae-cam l————j 024324 "P15? - nut. -'. . a ‘31. q .5: ru' Tm). W'Q' IW‘J MCP-l Figure 2. Transfectants of P388 that stably express CIEBPB (P388-CB), LIP (p388-LIP), and C/EBPB192-273 (P388-CB 192-276) are stimulated by LPS to PFOduce lL-6 and MCP-1 mRNAs, while cells that express CIEBPBzGLz (P388- 0 Bea) are not induced to produce these mRNAs. Northern analyses of IL-6 and NICP-l expression in P388 transfectants. RNA was isolated over time courses of LPS treatment as indicated. Twenty micrograms of RNA analyzed on northem blots that were hybridized in parallel to probes for IL-6, MCP-l, and GAPDH. 87 P388-Nee nee-cg P388-LIP P388-CM“ Mlle” N 19an N am pr N pm B—(‘ N B—NB-C N en in F igure 3. EMSA of CIEBP DNA binding activities in P388 cells stably transfected With CIEBPB, LIP, C/EBPB192-276, and C/EBPBzGu expression vectors. Cell line nomenclature is explained in Results. Binding reactions included normal rabbit 'QG (N), amino-terminal-specific CIEBPB antibody (B-N), or carboxyl-tenninal- Specific CIEBPB antibody (B-C). The positions of CIEBP-specific antibody supershift species are indicated by arrowheads on the right. 88 mas-c8 P388-LIP P388-C3,”. l I I (‘JIIBI’ CTFINF1 CIEBP CTFINF1 CIEBP CIT/NH AA AA A N (H‘Jomo 30 IOON “3010030100 N (14‘3010030100 Figure 4. EMSA of CIEBP binding activities in P388 transfectants in the presence of unlabeled competing oligonucleotide binding sites. Binding reactions included normal rabbit IgG (N), amino-terminal-specific CIEBPB antibody (B-N), or carboxyl-terminal-specific CIEBPB antibody (B-C). Some binding reactions, in addition to specific antibody, included 30-fold and 100-fold excess quantities of unlabeled CIEBP or CTFINF1 binding oligonucleotides. The positions of CIEBP- specific antibody supershift species are indicated by arrowheads on the right The bar to the left indicates the position of the likely lglEBP (CIEBPy) EMSA Species. 89 CIEBP binding site, while a 100-fold excess of the CTFINF-l binding site had little effect. The prominent protein-DNA complex that was not supershifted by specific antibody, but was effectively competed by unlabeled CIEBP binding site is most likely lglEBP (CIEBPy) (Hu et al., 1998), which is highly expressed in P388 cells and other immature B cells (Roman at al., 1990). A western analysis of nuclear extracts from the transfected populations was performed in order to examine the actual levels of protein expression of the various forms of CIEBPB (Figure 5). The levels of expression of CIEBPB192-276 and LIP in transfected cells were much lower than that of CIEBPB making their ability to support the LPS-induction of lL-6 and MCP-1 all the more remarkable. Interestingly, although the levels of protein expression for the truncated forms of CIEBPB are much lower than that of intact CIEBPB, their binding activity in the EMSA is quite high. CIEBPB192-275 has been reported to have enhanced affinity for its binding site (Williams et al., 1995). A chimeric form of CIEBPB with a heterologous leucine zipper has no activig in stable transfectants. Since CIEBPB192-276 retains only the DNA binding and leucine zipper domains of CIEBPb yet is still active on the lL-6 promoter, we decided to examine whether the leucine zipper domain contains determinants for activation other than those necessary for dimerization with other CIEBP family me"'ibers. To that end, a chimeric CIEBPB where the CIEBPB leucine zipper was replaced with that of yeast GCN4 (CIEBPBzGLz) was stably expressed in P388 cells (P388-CBGLZ) (Figure 1). CIEBPBzGtz retains wild type amino terminal aetivation and regulatory domains as well as the DNA binding domain , and has 90 Figure 5. Western analysis of LIP, CIEBPB192.273 (B192.m) and CIEBPB:GLz(GLZ) expression compared to that of CIEBPB (B) in the P388 transfectants. Control P388-Nee (Neo) cells are included as a control. Arrowheads mark the position of the CIEBP proteins. The proteins on the left panel were detected with a carboxyl- t(“-‘l’rninaI-specific CIEBPB antibody, while the proteins on the right panel were detected with an amino-terminaI-specific CIEBPB antibody. 91 previously been shown to activate transcription from an albumin DE-l site-driven reporter (\Nilliams et al., 1995). P388-CBGLZ cells did not induce lL—6 or MCP-1 mRNA in response to LPS (Figure 2). In order to verify that CIEBPBzGLz was properly expressed in P388-CBGL2 cells, both EMSA and western blot analyses were performed. EMSA revealed a protein-DNA complex that could be supershifted with amino-terminal specific but not carboxyl-terminus specific antibodies (Figure 3). This result was consistent with the replacement of the leucine zipper at the carboxyl-terminus of the CIEBPB protein. This data also demonstrates that CIEBPBzGLz retains the capacity to dimerize and is capable of binding to the CIEBP optimal binding site. As in the EMSA for the other stable transfectants, competitions were performed with the unlabeled CIEBP binding site and an unlabeled CTFINF—1 binding site (Figure 6). Again, the supershifted protein-DNA complexes observed upon incubation with amino terminal CIEBPB-specific antibody were effectively competed by a 30-fold excess of the CIEBP binding site, while a 100-fold excess 0f the CTFlNF-1 binding site had little effect. Western analyses of nuclear extracts from P388-CBGLz cells and other transfectant populations show that, while not expressed at as high a level as CIEBPB, the CIEBPBzGiz protein was SXpressed at a level comparable to CIEBPB192.276 and LIP (Figure 5). All of the variant proteins have similar low levels of expression in comparison to CIEBPB. The data suggest that some determinant for CIEBPB activity on the lL-6 promoter apart from dimerization is located in the leucine zipper. 92 P388-CB P388-CBC” I CIEBP CIT/Nir: CIEBP (TIT/NF] AA Nfl-NJOIWMIWN fl-N 3010030loo - 'v - - - O o u- ‘ Fig ure 6. EMSA of CIEBP binding activities in P388 transfectants in the presence of unlabeled competing oligonucleotide binding sites. Binding reactions included normal rabbit lgG (N) or amino-terminal-specific CIEBPB antibody (B-N). Some binding reactions. in addition to specific antibody, included 30-fold and 100-fold excess quantities of unlabeled CIEBP or CTFINF1 binding oligonucleotides. The positions of CIEBP-specific antibody supershift species are indicated by the arrowhead on the right. The bar to the left indicates the position of the likely lglEBP (C/EBPy) EMSA species. 93 CIEBPfi1gg-376 and UP are cagble of supporting the LPS-induced activation of the IL-6 promoter in transient transfections. We decided to further examine the abilities of CIEBP6192-276, LIP, and CIEBPfizGLz to activate the IL-6 promoter in transient transfections with an lL-6 promoter-driven luciferase reporter. We wanted to confirm the surprising results obtained in stable transfections and to facilitate comparisons of CIEBPB with its stmctural variants and analogous truncations of other CIEBP family members. P388 cells were cotransfected with an lL-6 promoter-luciferase reporter and expression vectors for CIEBPB, CIEBPB192-275, LIP, or CIEBPfizGLz. These transfections were carried out with and without LPS treatment. In addition to CIEBPB, both CIEBPB192-275 and LIP were capable of supporting LPS-induction of the lL-6 promoter (Figure 7). While CIEBPB transfectants with LPS treatment induced luciferase expression by a mean value of 12-fold over an untreated, “reporter-only" control , CIEBPB192-275 and LIP transfectants treated with LPS had levels of luciferase expression 8.3- fold and 62-fold of the control value, respectively. On the other hand, replacement of the leucine zipper in the chimeric CIEBPfizGLz showed a more dramatic reduction in the level of luciferase expression than loss of the activation domains, 4.8-fold of the control value. LPS stimulation by itself without transfection of a CIEBP expression vector produced a mean value of luciferase eXpression only 2.6-fold of the control value. The greater activity of CIEBPB:GIZ Observed in transient transfections in comparison to stable transfectants may be due to a higher level of expression in individual cells or differences in the extent 0f the promoter in the two assays. EMSA and western blot analysis did not 94 Control +LP8 Cm CIBPIHLPS fin LIP LIP+LP8 (3192-276 3192-279LP8 GL2 GLZ+LP8 o i A E; 515151;”; blame Luciferase Expression Figure 7. CIEBPB192-276 (6192-276) and LIP, although lacking activation domains, can support the LPS induced activation of the lL-6 promoter in transient transfections of P388 cells. CIEBPfizGLz (GL2), possessing a heterologous leucine zipper, has reduced activity in comparison to CIEBPB. Transient transfections were carried in out duplicate. Luminometer values were normalized for expression from a cotransfected SV40 early promoter-B-galactosidase reDorter. These values were then normalized to a relative value of 1 for the cells not receiving a CIEBP expression vector and untreated with LPS. The data presented are the mean of 3 experiments with their standard deviation. 95 detect CIEBPfizGLz species in the transient assays, so its level of expression relative to the other forms of CIEBPB could not be assessed as it was for the stable transfections (data not shown). Nonetheless, in either assay system, alteration of the leucine zipper domain has a greater impact on activity than complete loss of the conventional activation and regulatory domains(i.e. CIEBPB192-276)- Other investigators may not have observed significant activity of CIEBPB192-275 and UP because they were tested on promoter-reporter constructs that are based on the DE-l site of the albumin promoter and were thus solely CIEBP- dependent (Descombes and Schibler, 1991; Williams et al., 1995). In order to test if C/EBPB192-276 and LIP are, in fact, inactive on the albumin DE-l promoter in P388 cells, we performed transient transfections of CIEBPB and its truncated forms with an albumin DE-l promoter-reporter (Figure 8). These transient assays show that LIP and CIEBPB192-276 are, indeed, inactive on the simpler DE-l promoter, both in the presence and absence of LPS stimulation; presumably their activity on the lL-6 promoter is dependent on the interactions with other transcription factors that are available on the more complex promoter. .A truncated form of CIEBP§ analogpus to CIEBPEIQZ-Z7§ is capable of supporting the LPS-induced activation of the IL-6 promoter. We have previously shown that CIEBPa, B, and 6 have virtually redundant activities in regards to the lL-6 Promoter (Hu et al., 1998). These earlier results suggested that whatever Structural feature that allows activity of CIEBPBmms might be a general feature 0‘ ClEBPs. In order to test this hypothesis, we performed transient transfections 96 Control +LP8 cream cm +LP8 LIP ”EL” [3 192-270 B 192-270+LP8 0 50 100 150 lilatlvo Luciferase Exprosslon Figure 8. CIEBPB192-276 (6192-276) and UP fail to activate an albumin DEI site- reporter in transient transfections of P388 cells with and without LPS stimulation. Transient transfections were carried in out duplicate. Luminometer values were normalized for expression from a cotransfected SV40 early promoter- B—galactosidase reporter. These values were then normalized to a relative value of 1 for the cells not receiving a CIEBP expression vector and untreated with LPS. The data presented for transfections not treated with LPS are the mean of 3 experiments with their standard deviation. The data for transfections treated with LPS are the mean of one experiment with the standard deviation of the duplicate values. 97 "'3' :K-l‘ m 31"” Im‘?. l ‘v '- w! VE (D v In with truncated forms of CIEBPa and CIEBP5 (CIEBPa273.358 and CIEBP5181.272) in comparison to intact CIEBPB in P388 cells (Figure 9). Again, the transfections were performed with and without LPS stimulation, and the expression vectors were cotransfected with an lL-6 promoter-luciferase reporter. CIEBP6181-272 was almost as active as intact CIEBPB. In this series of transfections, CIEBPB transfectants with LPS treatment expressed luciferase at a mean value of 24- fold over an untreated, “reporter-only” control, while CIEBP6131.272 transfectants treated with LPS had levels of luciferase expression of 20-fold over the control value. CIEBPCX273.353 transfectants treated with LPS had levels of luciferase expression 7-fold over the control value and LPS treatment by itself without transfection of a CIEBP expression vector produced a mean value of luciferase expression only 3.8-fold of the control value. EMSA and western blot analysis did not detect either CIEBP6181-272 or C/EBPa273.353 species, so their level of expression relative to CIEBPB could not be assessed (data not shown). The modest level of activation with C/EBPa273.358 is consistent with the more modest activity of intact CIEBPa observed in LPS inductions of the endogenous lL-6 Promoter in stable transfections (Hu et al., 1998). 98 Control +LP8 cm cmaps (1273.353 a273-358+LP8 6181-272 6 181-272+LP8 l 1 fi Y Y T 0 5 1O 15 20 25 30 BMW. Lucltorau Btpruslon Figure 9. CIEBP5181-272 (5181-272), although lacking an activation domain, can support the LPS induced activation of the IL-6 promoter in transient transfections of P388 cells. CIEBPa273-358 (a273-358) has modest activity in comparison to CIEBPB. Transient transfections were carried out in duplicate. Luminometer values were normalized for expression from a cotransfected SV40 early promoter-B-galactosidase reporter. These values were then normalized to a relative value of 1 for the cells not receiving a CIEBP expression vector and untreated with LPS. The data presented are the mean of 3 experiments with their standard deviation. 99 dom: unpl ollL DISCUSSION The data presented in this paper demonstrate that the conventional activation domains of CIEBPB (VVIIIiams et al., 1995) and CIEBP6 (P. F. Johnson, unpublished results) are dispensable for their roles in the LPS-induced activation of IL-6 and MOP-1 expression. Both CIEBPBmma and LIP, truncated forms of CIEBPB lacking their first 192 and 132 amino acids respectively, are capable of supporting the LPS-induced activation of IL-6 and MCP-1 transcription both in stable and transient transfections of P388 lymphoblasts. Transient transfections showed that CIEBP5131-272, a similarly truncated form of CIEBP6 was also effective in activating the lL-6 promoter with LPS stimulation. A truncated form of CIEBPa, ClEBPaznasa, also showed modest activity. The activity of the bZIP domains of CIEBP isoforms and of LIP is quite surprising. LIP, particularly, has been found to have very little or no transcriptional activity (Descombes and Schibler, 1991; Cooper et al., 1994; Cooper et al., 1995; Williams et al., I995). Previous investigators may not have observed this activity because they used different forms of CIEBPB, different reporters and/or different cell types in their transfection systems. Matsuaka et al. (1993), using an embryonic carcinoma cell line, failed to observe activity of an internally deleted form of CIEBPB on the lL-6 promoter. This mutant, however, would have retained one of three activation domains, as well as sequences that inhibit transactivation potential and mediate cell specificity (Williams et al., I995). Others have assayed UP and CIEBPB192-276 on promoter-reporter constructs 100 ....J based on the DE-I site of the albumin promoter (Descombes and Schibler, 1991; Williams et al., 1995) or other tandem arrangements of C/EBP binding sites (Cooper et al., 1994; Cooper et al., 1995), all of which are solely CIEBP- dependent. We also find that these forms of CIEBPB are inactive on a DE-I albumin-based promoter in P388 lymphoblasts (Figure 8). Presumably, the activity that we have observed is dependent on the interactions with other transcription factors that are available on the more complex lL-6 and MOP-1 promoters. There is good evidence that CIEBPB and NF-icB synergistically activate the lL-6 promoter (Matsuaka et al., 1993). CIEBPa, (3, and 6 have been shown to synergize with NF-KB in activating the lL-8 promoter (Stein and Baldwin, 1993; Kunsch et al. 1994). This synergy may not only involve binding of the factors to their cognate binding sites, but direct physical association through their respective basic region-leucine zipper (bZIP) and Rel homology domains (Stein et al, 1993; Stein and Baldwin, 1993). The basis for the activity of truncated ClEBPs on the IL-6 promoter may rest in the capacity of the bZIP domain by itself to effect synergy with NF-KB in the absence of CIEBP activation domains. We have only observed robust activation of the IL-6 promoter by CIEBPs under conditions of LPS stimulation (Bretz et al., 1994; Hu et al., 1998; this paper) and LPS stimulation does indeed induce NF-icB binding activity in P388 cells (Hu et al. 1998). Future experiments will need to examine the ability of truncated forms of CIEBPB, as well as point mutants within the bZIP domain, to synergize with NF-icB in activating the lL-6 promoter. 101 prom: intra induc Another possible mechanism by which a CIEBP bZIP domain could of itself support the LPS induction of IL-6 and MOP-1 expression is as a structural component in “enhanceosome” assembly (Thanos and Maniatis, 1995; Merika et al., 1998). Perhaps even in the absence of any inherent activation potential, the bZIP domain through its occupation of the C/EBP binding site on the lL—6 promoter allows enhanceosome assembly. Arguing against this is the fact that untransfected P388 cells, which cannot effectively express lL-6 upon LPS induction, exhibit CIEBPy (lglEBP) DNA binding activity (Hu et al., 1998). CIEBPy is a transdominant negative regulator of transcription that binds to the CIEBP consensus binding site (Roman at al., 1990; Cooper et al. 1995). Presumably CIEBPy could perform the role of simply occupying the CIEBP binding site with a bZIP domain. Whatever the mechanism of bZIP activity in the LPS induction of lL-6 and MCP-1 expression, our findings suggest that the basis for CIEBP redundancy in the activation of these genes (Hu et al., 1998) resides in this well-conserved region that is shared by all CIEBP isoforms. Our experiments replacing the leucine zipper of CIEBPB with that of GCN4 suggest that the critical structural feature for activity of bZIP domains may be further localized to the leucine zipper itself. The replacement of the leucine zipper that produces the chimeric CIEBPflzGLz protein shows a greater decrement in activity in comparison to intact CIEBPB than removal of the activation domains. There is some uncertainty in the interpretation of the result because the levels of CIEBPfizGLz expression are below that for intact CIEBPB. On the other hand, the level of CIEBPfizGLz 102 expression is similar to that of ClEBPBmma expression (Figure 5), which is a far more potent transcriptional activator (Figures 2 and 7). It is likely that the leucine zipper possesses critical determinants for the activity of CIEBPs on the IL-6 promoter other than mediating dimerization to known CIEBP family members. For example, the leucine zipper might mediate dimerization to an as yet unknown dimerization partner with inherent activation activity or, as proposed above, it might mediate the synergistic activation of NF-icB activity. The leucine zippers of CIEBP proteins have previously been implicated in functions beyond dimerization. The leucine zipper of ClEBPa has been shown to mediate cell type specificity of albumin promoter activation (Nerlov and Ziff, 1994). This effect can be mediated by a mutant in the nonhydrophobic face of the zipper. Another instance of a non-dimerization function residing in the leucine zipper is that of serine 276 of human CIEBPB (Wegner et al., 1992). Phosphorylation of this serine confers calcium-regulated transcriptional stimulation to a promoter that contains binding sites for CIEBPB. In future experiments, it will be useful to examine single amino acid substitutions in the leucine zipper not only because they may more sharply delineate critical structural features, but because these altered forms of CIEBPB may provide levels of expression more comparable to that of intact CIEBPB and thus allow more direct comparisons of activity. Finally, our results surprisingly show a significant capacity for LIP to support the LPS induction of IL-6 and MCP-1 expression. LIP has previously been proposed to be a transdominant negative inhibitor of transcription (Descombes and Schibler, 1991). It is proposed that high levels of LIP observed in fetal liver 103 constitute a mechanism for inhibiting the activity of other CIEBP isoforms in hepatocytes that are not yet terminally differentiated. The regulation of LIP expression has also been proposed to play a role in the regulation of lactation- associated genes such as B—casein (Raught et al., 1995). More recently, LIP expression has been correlated neoplastic transformation of mammary tissue and has been proposed as a prognostic indicator for breast cancer because of its overexpression in breast tumors that were negative for the estrogen and progesterone receptors (Raught et al., 1996; Zhanow et al., 1997). The central theme of these models is that the expression of LIP in immature proliferating cells suppresses the activity of CIEBPB and other isoforms in activating the expression of gene products associated with terminal differentiation. It is clear from the findings reported here that conditions favoring LIP expression would not universally down-regulate CIEBPB-regulated genes, but would be permissive for the expression of lL-6, MCP-1, and genes with a similar promoter structure. lL-6 and MOP-1 are certainly genes whose expression has been associated with the function of mature terminally differentiated cell types. The findings reported here of LIP activity in the expression of these genes, as well as a recent report that LIP can be the product of proteolysis associated with certain isolation procedures (Baer et al. 1998), call for a reexamination of the role of LIP. It will be a high Priority in future investigations to examine the activity of UP on the promoters of other genes encoding proinflammatory cytokines, as well as several milk protein genes that are apparently regulated by CIEBPB (Robinson et al., 1998). The 104 correlation of LIP activity to promoter structure may provide clues to the mechanism of bZIP activity in the absence of conventional activation domains. 105 r; ..‘b mac) '.1. 05‘ ”it ‘ “j REFERENCES Akira, S., H. lsshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakamima, T. Hirano, and T. Kishimoto. 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a CIEBP family. EMBO J. 921897-1906. Ayoubi, T. A, J. W. Creemers, A. J. Roebroek, and W. J. Van de Van. 1994. Expression of the dibasic proprotein processing enzyme furin is directed by multiple promoters. J. Biol. Chem. 269: 9298-9303. Baer, M., S. C. Williams, A. Dillner, R. C. Schwartz, and P. F. Johnson. 1998. Autocrine signals control CIEBPB expression, localization, and activity in macrophages. Blood (in press). Bauer, 8. R., K L. Holmes, H. C. Morse Ill, and M. Potter. 1986. 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Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc. Natl. Acad. Sci. USA 85: 3738-3742. Roman, C., J. S. Platero, J. Shuman, and K. Calame. 1990. lglEBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to CIEBP and heterodimerizes with CIEBP. Genes & Dev. 4: 1404-1415. 108 Rorth, P. 1994. Specification of CIEBP function during Drosophila development by the bZIP basic region. Science 266: 1878-1881. Shirakawa, F., K Saito, C. A. Bonagura, D. L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron. 1993. The human prointerleukin 18 gene requires DNA sequences both proximal and distal to the transcription start site for tissue- specific induction. Mol. Cell. Biol. 13: 1332-1344. Stein, 8., P. C. Cogswell, and A. S. Baldwin. 1993. Functional and physical associations between NF-xB and CIEBP family members: a rel domain-bZIP interaction. Mol. Cell. Biol. 13: 3964-3974. Stein, 8., and A. S. Baldwin.1993. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between CIEBP and NF- KB. Mol. Cell. Biol. 13: 7191-7198. Tanabe, O., S. Akira, T. Kamiya, G. G. Wong, T. Hirano, and T. Kishimoto. 1988. Genomic structure of the murine lL-6 gene. J. Immunol. 141: 3875-3881. Thanos, D. and T. Maniatis. 1995. \firus induction of human IFNB gene expression requires the assembly of an enhanceosome. Cell 83: 1091-1100. Wegner, M., Z. Cao, and M. G. Rosenfeld. 1992. Calcium-regulated phosphorylation within the leucine zipper of CIEBPB. Science 256: 370-373. VVilIiams, S. C., C. A. Cantwell, and P. F. Johnson. 1991. A family of CIEBP- related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes & Dev. 5: 1553-1567. Williams, S. C., M. Baer, A. J. Dillner, and P. F. Johnson. 1995. CRP2 (CIEBPB) Contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J. 14: 3170-3183. Zhang, Y. and W. N. Rom. 1993. Regulation of the interleukin-1B (IL-1(3) gene by myoobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs. Mol. Cell. Biol. 13: 3831-3837. Zhanow, C. A., P. Younes, R. Laucirica, and J. M. Rosen. 1997. Overexpression 0f CIEBPB-LIP, a naturally occurring, dominant-negative transcription factor, in human breast cancer. J. Natl. Cancer Inst. 89: 1887-1891. 109 Chapter 4 Regulation of the Expression of Primary Granule Proteins by the CIEBP Transcription Factor Family Hsien-Ming Hu and Richard C. Schwartz 110 ABSTRACT CIEBP transcription factors have been implicated in the tissue-specific and temporal regulation of a number of genes encoding primary granule proteins in granulopoiesis. In order to evaluate the abilities of various CIEBP isoforms to regulate the transcription of primary granule genes, we have ectopically expressed CIEBPa, —B, —6, and —e individually in the granulocytic progenitor cell line, 32D cl3. 32D cl3 cells transfected for CIEBPB expression (32D-CB) have the greatest enhancement of mRNA levels for the genes encoding myeloperoxidase (MPO), cathepsin G (Cat G), and lysozyme (LZ). They display levels of expression similar to the G-CSF-induced 32D cl3 parental cells. Transfectants for expression of other CIEBP isoforms also show a modest increase in the mRNA levels of the aforementioned genes. EMSA and Western blot analyses suggest that a ClEBPa-CIEBPB heterodimer is the predominant form of CIEBP present in the nucleus of 32D-CB cells. The predominance of the CIEBPa-CIEBPB heterodimer and increased binding of this form of CIEBP in 32D-CB cells suggest that the ClEBPa—CIEBPB heteodimer is an important regulator of the primary granule-associated genes. 111 INTRODUCTION Neutrophilic granulocytes (also known as polymorphonuclear cells, or PMNs) are specialized phagocytic cells characterized by their distinct mutilobed nuclei and granulated cytoplasms. Neutrophils carry two types of granules (reviewed in Borregaard et al. 1993). Primary or azurophilic granules which contain peroxidase, lysozyme and various hydrolytic enzymes are formed first at the promyelocyte stage of granulopoiesis. Secondary granules which contain collagenase, lactoferrin and lysozyme develop at the later myelocyte stage. The expression of granule-associated genes appears to be regulated primarily at the level of transcription (Grisolano et al. 1996). Most of the primary granule genes are transcriptionally activated at the beginning of the promyelocyte stage and are transcriptionally repressed at the transition to the myelocyte stage. The mechanism that controls this special temporal expression is not well understood. The identification of common cis-acting DNA elements and transcription factors in the regulation of many primary granule genes has shed some light on the mechanism of their stage- and tissue-specific expression (Friedman, 1996). Among the important transcription factors that have been found to be involved in the regulation of many primary granule-associated genes is the CIEBP family. CIEBP proteins belong to a family of basic region-leucine zipper transcription factors with highly homologous DNA binding and dimerization domains (reviewed in JOhnson et. al. 1994). Homo- and heterodimers are readily formed within the family and bind to a similar DNA target site. Within the hematopoietic system, 112 high level expression of CIEBPs is restricted to the myelomonocytic lineages. Indeed, multiple CIEBP family members are expressed in differentiating myelomonocytic cells and show a distinct temporal expression pattern (Scott et al. 1 992). Several studies have shown that the promoters of the genes encoding myeloperoxidase (MPO) (Zhu et al. 1994), neutrophil elastase (NE) (Oelgeschlager et al. 1996), azurocidin (Friedman 1996), and myeloblastin (Zimmer et al. 1992), all components of the primary granule, contain known or predicted binding sites for CIEBP. C/EBPa is shown to bind to the CIEBP site in the NE promoter and transactivate the NE promoter alone or cooperatively with other transcription factors including c-Myb, PU.1 and AML-I (Oelgeschlager et al. 1 996). ClEBPa is proposed to be the major form of CIEBP that regulates the NE gene because it is the most active among CIEBP family members in transient cotransfection experiments. CIEBPB and -6 are also active, although to a lesser eXtent. A distal enhancer, which contains multiple CIEBP binding sites, has been shOwn to be responsible in part for the tissue- and stage-specific expression of the MPO gene (Zhu et al. 1994). It is suggested that CIEBPB and CIEBP6 are the two major CIEBP isoforms that bind to these CIEBP binding sites and activate the transcription of the MPO gene; CIEBPa is mainly present in earlier precursor cans that do not express MPO (Ford et al. 1996). Thus, it remains uncertain as to Which CIEBP family member is the major regulator of these primary granule- asSOciated genes. The answer may be more complicated than is suggested by prior studies, as multiple C/EBPs are expressed by the differentiating Qrahulocytic cells in an overlapping manner. So far, four CIEBP family members, 113 including CIEBPoi, -l3, -6, and -e have been found to be expressed during granulopoiesis. The variety of possible homo— and heterodimers may present a very complex regulatory scheme for the roles CIEBPs in the expression of primary granule-associated genes. We have tried to address this issue by individually over-expressing C/EBPa, -B, -6, and —e in the murine 32D cl3 cell line (Valtieri et al. 1987), which is an immature myeloid precursor that has not yet expressed granule proteins. Stable transfection of a CIEBPB expression vector appears to induce the greatest enhancement in the levels of primary granule protein mRNAs. Furthermore, we have found that the CIEBPa—C/EBPB heterodimer is the predominant form of CIEBP in these transfectants. Increased levels of CIEBPa-CIEBPB binding detected in nuclear extracts of cells transfected for CIEBPB expression suggest that the CIEBPa-C/EBPB heterodimer is an important regulator of the genes encoding primary granule proteins. MATERIALS AND METHODS W. The murine 32o cl3 cells (Valtieri et al. 1987) were maintained at 37°C in a 5% COZ environment in lscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS and 10% WEHl3-conditioned rmedium as a source of interleukin-3 (IL-3). For induction of granulocytic 114 differentiation, cells were washed twice with phosphate-buffered saline (PBS) and plated in IMDM supplemented with 15% heat-inactivated FCS and 15% WEHI274.1-conditioned medium as a source of G-CSF. Stable transfection of 32D cl3 cells was accomplished by using a liposome-mediated transfection protocol. Briefly, 107 cells were incubated with 4 ug of DNA and 15 pl of DMRIE- C reagent in 1.2 ml of Opti-MEM I medium for 12 hr followed by the addition of 2 ml of IMDM supplemented with 15% FCS and 15% WEHl3-conditioned medium. After 36 hr, the medium was replaced by the standard growth medium supplemented with G418 at 1.0 mglml. Stably transfected cells were maintained in the presence of G418 at 0.25 mglml. Expression Vectors. pSV(X)Neo is pZIP-NEO SV(x)1 (Cepko et al. 1984) (Fig. 1) and expresses inserted sequences from the promoter of Moloney murine leukemia virus. This vector expresses the gene for neomycin (neo) resistance through alternative splicing of a transcript from the same promoter. PSV(x)CIEBPa was constructed by insertion of the BamHl/Kpnl fragment e“coding rat C/EBPa from pMEXC/EBP (Williams et al. 1991) into the BamHI Site of pSV(X)Neo with BamHI linkers. pSV(x)C/EBPB was constructed by insertion of the BamHI fragment encoding rat CIEBPB from pMEXCRP2 (Williams et al. 1991) into the BamHI site of pSV(X)Neo. To construct an expression vector for CIEBP6, the sequences encoding murine CIEBP6 (Vlfilliams et al. 1991) were first inserted into the Sphl and Hindlll sites of pMEX (Williams at at . 1991) by a three-part ligation: one inserted fragment extended from a PCR- 115 LTR CIEBP IICO splice donor Figure 1. Structure of the retroviral vector pSV(X)Neo containing the CIEBP cDNA inserted at a BamH1 site. LTR, LTRs are derived from the murine Moloney virus. The transcription start site is indicated by the arrowhead. The donor and splice receptor receptor sites for alternative splicing are also indicated. 116 LTR introduced Sphl site 40 bp upstream of the CIEBP6 initiation codon to an Apal site approximately 100 bp into the coding sequence, and the other fragment extended from the Apal site to a PCR-introduced Hindlll site just downstream of termination codon. The Sphl/Hindlll fragment was then inserted with BamHI linkers into the BamHI site of pSV(X)Neo to produce pSV(x)C/EBP6. To construct an expression vector for CIEBPe, the sequences encoding rat CIEBPe were excised from pMEXC/EBPe (Williams et al. 1998) with Hindlll, and the 5’ overhangs were made blunt using Klenow polymerase, and a BamHI linker was added. The resulting fragment was inserted into the BamHI site of pSV(X)Neo to generate pSV(x)C/EBPs. Nucleic acid isolation and analysis. Total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer's directions. RNAs were electrophoresed through 1% agarosel‘formaldehyde gels. Transfers to membranes were hybridized and washed to a stringency of 0.1% SSPE at 65°C. Hybridization probes were prepared with a random priming kit (Life Technologies) with the incorporation of 5’-[a-32P]dATP (3000 Cilmmol; DuPont- New England Nuclear, Newton, CT). The MP0 probe was a 0.8-kb murine cDNA (Friedman et al. 1991). The lysozyme probe is a 1.0-kb human cDNA. The ciathepsin G probe was a 0.5-kb murine cDNA cloned by differential display PCR Of Control and CIEBPB transfectants of 32D cl3 (Tian and Schwartz, Unpublished). The GAPDH probe was a 1.3-kb rat cDNA (Fort et al. 1985). The C’EBPa probe was a 1.0-kb murine genomic DNA fragment. 117 I s .' ' W- J.uim.‘ Ffii‘ ‘ Western analysis. Nuclear extracts were prepared as described below. The extracts (50 ug) were adjusted to 1x Laemmli sample buffer (Laemmli 1970) and processed on a 12% PAGE gel. The gel was transferred to a Protran membrane (Schleicher and Schuell, Keene, NH), and Ag-Ab complexes were visualized with the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared as follows. Cells were washed in phosphate-buffered saline and lysed in 15 mM KCI, 10 mM HEPES [pH 7.6], 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% [vol/vol] NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 pglml leupeptin, 5 pglml antipain, and 5 pglml aprotinin for 10 min on ice. Nuclei were pelleted by centrifugation at 14,000 x g for 20 sec at 4°C. Proteins were extracted from nuclei by incubation at 4°C with vigorous vortexing in buffer C (420 mM NaCl, 20 mM HEPES [pH 7.9], 0.2 mM EDTA, 25% [vol/vol] glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 jig/IT" leupeptin, 5 (1ng antipain, and 5 rig/ml aprotinin). Nuclear debris was pelleted by centrifugation at 14,000 x g for 1 5 min at 4°C and the supernatant extract was collected and stored at -70°C. The EMSA probe was a double-stranded oligonucleotide containing an optimal CIEBP binding site (5’-GATCCTAGATATCCCTGA'ITGCGCAATAGGC- TCAAAGCTG-a' annealed with 5’-AATTCAGCTITGAGCCTATTGCGCAATC- AGGGATATCTAG-3’) labeled with the incorporation of 5’-[a-32P]dATP (3000 Ci’f'hrnol; DuPont-New England Nuclear) and Klenow DNA polymerase. 118 Underlined sequences correspond to the binding motifs of the specified transcription factors. DNA binding reactions were performed at room temperature in a 25 pl reaction mixture containing 6 pl of nuclear extract (1mg/ml in buffer C)] and 5 pl of 5 x binding buffer (20% [wt/vol] Ficoll, 50 mM HEPES [pH 7.9], 5mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained 1 pg poly(dl- dC), 1.25 ng of probe, bromophenol blue to a final concentration of 0.06% [wt/vol], and water to volume. For supershifts, nuclear extracts were preincubated with antibodies for 30 min at 4°C prior to the binding reaction. Samples were electrophoresed through 5.5% polyacrylamide gels in 1x TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) at 160 V. Antibodies: Rabbit antibodies against CIEBPa (14AA), CIEBPB (C-19), CIEBP6 (C-22) and CIEBPe (C-22) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RESULTS Wrespion of CIEBP family members in 32D cl3 cells. 32D cl3 is an 'nterleukin 3 (lL-3)-dependent hematopoietic precursor cell which can be 'ndUced to differentiate into neutrophilic granulocyte by G-CSF (Valtieri et al. 119 ~ 1987). This cell line has provided a useful system to study the process of neutrophil differentiation (Laneuville et al. 1991, Kreider et al. 1992, Patel et al. 1 993). In order to compare the abilities of the CIEBP family members to induce granule-associated genes, a murine retroviral expression vector pSV(X)Neo was used to ectopically express CIEBP isoforms in 32D cl3 cells. Pools of G418- resistant cells were isolated. Cells transfected with pSV(X)ClEBPa, pSV(X)C/EBPB, pSV(X)C/EBP6 or pSV(X)C/EBPe were designated 32D-Ca, 3ZD-CB, 32D-C6 and 32D-Ce, respectively. Control populations transfected with empty vectors were also generated and designated 32D-Neo. For each construct, two independent G418-resistant populations were examined in order to ensure reproducibility of our findings. To verify that the introduced vectors were properly transcribed, total RNA was isolated from these transfectants and analyzed by Northern blotting. As shown in Fig. 2, the CIEBP transfectants PrOduced two transcripts specific to the gene for neo resistance. As predicted, one transcript is the full length of the retroviral genome from the 5’ LTR to the 3’ LTR. and the other is a shorter transcript resulting from alternative splicing. EleVated e ression of rima ranule otein mRNAs in 32D cl3 transfectants. T-<31Zal RNA was isolated from cultures of 32D-Ca, -C8, -C6, and -Cs as well as the 32D-N90 control cells. As a positive control, total RNA was also isolated from 320 cl3 cells induced by G-CSF for 3 and 7 days. Northern analyses were p e"‘l’ormed to detect the transcripts encoding several granule-associated genes the. uding myeloperoxidase (MPO). cathepsin G (Citt G): lysozyme (L2): and 120 TN—N—N—N Scdééufaééé ZZEUZH’VZ é a doc NNNNNNNNN nnnmnnnnm O N “We ““Mesm “e'l ””nn"n Figure 2. Northern blot analysis of 32D transfectants. Twenty microgram of total RNA was analyzed on a northern blot and was hybridized to a probe for neomycin (neo). The positions of the transcripts containing various CIEBPs as well as neo are indicated by the arrowheads. 121 lactoferrin (LF). Transcripts encoding GAPDH were also examined as a normalization control. As shown in Fig. 3, a strong induction of MPO transcription is observed in 32D cl3 cells transfected for CIEBPB expression. Transfectants for C/EBPa, CIEBP6 and CIEBPe expression showed a more modest elevation in the level of MPO expression in comparison to the 32D-Neo control cells. The level of MPO expression in 32D-CB cells is similar to that of 32D cl3 cells treated with G-CSF for 3 or 7 days. To quantify the level of expression, Northern blots were analyzed using an Ambis radioanalytic scanner. All values for MP0 expression were normalized to those of GAPDH as a control for loading (Table 1 ). A 5.4 fold induction of MPO mRNA expression is observed for 32D-CB cells compared to control cells. The fold of induction for 32D-Ca, -C6, and -Ce is 2.5, 2. 7, and 3.3, respectively (Table 1). Cathepsin G, a serine protease expressed in the primary granule, has been found to be coexpressed with MP0 during granulopoiesis (Hanson et al. 1990). A Northern analysis in Fig. 3 shows that Cat G transcription is also induced in the 320 cl3 transfectants in a manner very similar to that of MPO. One difference, however, is that 32D-Neo control cells express very little, if any, Cat G mRNA, making the enhanced expression in the transfectants even more robust. Quantitation shows that the induction of Cat G mRNA in 32D-CB is 10.2 fold, While 2.6, 3.1 and 3.2 fold inductions are seen for 32D-Ca, -C6 and -C8, respectively (Table 1). L.VSCJZyme is a component of both primary and secondary granules (Borregaard et al - 1 993). The presence of a CIEBP binding site in the promoter of the L2 122 :N—Ff—N—N—N uéddtficfiaboodoibnh “clout-30922000 eeeeeee Add easier-12:22:29. .... sen J” MPO ... H B E“..'.“ ."u .1!!! W6 Eggoiiu.i ” “*“EEIIEV'ES Figure 3. Induction of granule proteins myeloperoxidase (MPO), cathepsin G (Cat G), and lysozyme (L2) in 32D cl3 transfectants. Total RNA was isolated from transfectants of 32D cl3 that express ClEBPa (32D-Ca), CIEBPB (32D-CB), CIEBP6 (32D-C6) and C/EBPe (32D-Ce). RNA was also isolated from the vector controls (32D-Neo) and 320 cl3 parental cells induced by G-CSF for 3 days (32 D-G3) and 7 days (32D-G7). Twenty micrograms of RNA was analyzed on northern blots and hybridized in parallel to probes for MP0, Cat G, [2, and GAPDH. For each construct, two independent transfectants were analyzed. 123 Table 1. Induction of MPO, Cat G and LZ transcriptions in 32D cl3 transfectants‘ Fold of activation Transfectants or treatment MPO Cat G LZ 32D-N60 1.0 1.0 1.0 32D-Ca 2.5 2.6 1.5 32D-C8 5.4 10.2 3.2 32D-C6 2.7 3.1 2.0 32D-Cs 3.3 3.2 1.5 32D-G3 6.2 12.2 2.9 32D-G7 6.9 13.2 1.9 ‘The blots presented in Figure 3 were analyzed by Ambis scanner, and raw data for MP0, Cat G and L2 were normalized to value for GAPDH. The expression levels for 32D-Neo control were set as 1.0. The data shown are average of two experiments. 124 gene has been demonstrated (Goethe et al. 1994). It has also been shown that the avian CIEBPB homolog, NF-M can transactivate the L2 promoter in concert with c-Myb (Ness et al. 1993). Fig. 3 shows that 32D cl3 cells transfected for CIEBPa, B, 6 and 8 expression have a rather modest elevation in their expression of lysozyme mRNA compared to the control populations. Quantitatively, an increase of 3.2 fold is seen in 32D-CB cells, and 2.0 fold in 32D-C6 cells, while 1.5 fold increase in expression are observed for 32D-Ca and —Cs transfectants. The expression of lactoferrin (LF), a protein of the secondary granule (Rado et al. 1987), was also examined in 320 transfectants. LF RNA was not detectable in any of the transfectants (data not shown), a result consistent with the findings that LF is induced only at the mature stage of granulopoiesis and that LF is probably not regulated by CIEBPs (Friedman et al. 1991). Our results clearly show that 320 cl3 populations transfected for CIEBPB expression have elevated levels of transcripts encoding MPO, Cat G and L2. 320 cl3 transfectants for CIEBPa, 6 or 8 expression show more modest elevations in the levels of these transcripts. EMSA of 32D cl3 transfectants. The expression of multiple CIEBP isoforms in 320 cl3 cells (Scott et al. 1992) coupled with the ectopic expression of individual isoforms from introduced expression vectors could yield a diverse repertoire of homo- and heterodimeric CIEBP transcription factors. To determine whether there is any correlation between the levels of primary granule mRNA and the 125 pattern of CIEBP-binding activities, EMSAs were performed on nuclear extracts isolated from the transfectants. Specific antibodies against individual CIEBP family members were used to identify the corresponding CIEBP protein-DNA complexes. Endogenous expression of CIEBPa and CIEBPB, as well as a minor amount of CIEBP6 is observed in 32D-Neo control cells with specific antibodies that yield supershifted protein-DNA complexes. The total amount of CIEBPB-DNA complex appears to be somewhat more abundant than that for CIEBPa. In 320-Ca cells, C/EBPa binding activity is increased compared to control cells, presumably, due to expression from the transfected vector, while the level of CIEBPB binding activity remains similar to that of control cells. An inspection of the CIEBPB binding activity in 32D-CB cells surprisingly does not reveal any increase in CIEBPB-DNA complexes. On the other hand, the CIEBPa binding activity in 32D-CB cells is strongly increased, probably to a level even higher than that of 32D-Cor. This suggests that the ectopic expression of CIEBPB may induce expression of the endogenous CIEBPa gene. EMSA for 32D-C6 cells clearly demonstrates the expected increase in CIEBP6 binding activity. Although the 32D-C6 EMSA may be somewhat underloaded, there is a relatively low abundance of CIEBPa binding activity compared to other transfectants. Lastly, an increase of CIEBPe binding activity is observed in 32D-Ce cells, while the levels of CIEBPa and CIEBPB binding activities are similar to those in 32D-N60 cells. A comparison of supershifts with antibodies to CIEBPa and CIEBPB 126 reveals protein-DNA species (marked by bar number 1 in Fig. 4) that are reactive with either antibody as opposed to species reactive only to anti-CIEBPB (marked bar number 2 in Fig. 4) This suggests that the majority of CIEBPa is present as a heterodimer with CIEBPB. While our EMSAs clearly demonstrate the expected increase in C/EBPa, CIEBP6 and CIEBPe binding activities in their corresponding transfectants, a more accurate assessment of CIEBPB expression is required. Although 32D-CB cells express RNA from the transfected CIEBPB expression vector, no increase in CIEBPB binding activity is detected. Western blot analysis was performed to more accurately examine the abundance of CIEBPB and other CIEBP family members in these transfectants. Western analysis of 32D cl3 transfectants. Western analyses of nuclear extracts from the transfected populations were performed in order to assess the actual levels of CIEBP protein (Fig.5). Antibody to CIEBPa detect two immunoreactive species. The slower-migrating band corresponds to the 42 kDa full-length CIEBPa protein, while the faster-migrating band corresponds to the 30 kDa polypeptide resulting from alternative translation of the CIEBP mRNA. As predicted, 32D-Ca shows an increase in C/EBPa protein expression. Unexpectedly, the expression levels of C/EBPa are also strongly enhanced in 32D-CB and -C6 transfectants. The CIEBPa protein level of 32D-CB is even higher than that of 32D-Ca. This result suggests that ectopic expression of 127 3ZD-Neo SZD-Ca-l 3ZD-CB-l 32D-C84 3ZD-Ce-l I II II II I I l NaflSaNaBficNaBScNaBSa Figure 4. EMSA of CIEBP DNA binding activities in 32D cl3 cells stably transfected with C/EBPa, CIEBPB, CIEBP6 and C/EBPc expression vectors. Reactions included normal rabbit serum (N), ClEBPa antibody (at), CIEBPB antibody ([3), CIEBP6 antibody (6), or C/EBPs antibody (8). The bar to the right indicates the positions of supershifted EMSA species. The bars to the left indicate the positions of protein-DNA complexes reactive with both C/EBPa and CIEBPB antobodies (1), and protein-DNA complexes only reactive with CIEBPB antibody (2). 128 32m: azb-Ca-z 320-034 320-034 szn-C5-1 32D-C54 azb-Ca-i azo-cs-z ’ azb-Neo size-Ca I 83137 tannin 5 smog—2 " JZD-Cb-I JZD-Ca I r JZD-Ce-Z szo-Cp—z 32005-1 320-034 32D-Cs l JZD-Ca-Z SID-V00 32D-Ca-l I. Jzo-Ca-z ' szn-qi-l I It i l i .24 S E If .i i i i 1 32mm I 32D-Ca 1 Cl!) 3P5 .c , 4— K.- —.---..-’" n a ..__ .——- a..- Figure 5. Western blot analyses of CIEBP proteins derived from nuclear extracts of the transfectants. The nomenclature for each cell line is explained in Results. Specific antibodies were used to detect A. CIEBPa, B. CIEBPB, C. CIEBP6, and D. CIEBPa. The positions of CIEBP proteins are indicated by the arrowheads. 129 Another surprising result is obtained in western blot analyses for CIEBPB. The expression level of CIEBPB protein is not increased in either of the CIEBPB transfectants. In fact, all of the transfectants have a similar level of CIEBPB expression. Since the Northern blot demonstrates expression of the CIEBPB ' expression vector at the RNA level (Fig. 2), one possible reason for our inability to detect any increase in either CIEBPB protein or DNA binding activity in the nuclear extract of 32D-CB is that the protein is retained in the cytoplasm. In support of this view, it has been shown in immature myeloid cells that CIEBPB is largely retained in the cytoplasm in an unphosphorylated form. It is possible that it requires an extracellular signal (i. e. the stimulation by G-CSF) and subsequent phosphorylation for translocation into the nucleus (Ford et al. 1996). Western blots using CIEBP6 and C/EBPa antibodies show increased protein expression in the corresponding 32D transfectants, as expected. Induction of endpgenogs CIEBPamsion in CIEBPB and CIEBP6 transfectants. It was unclear from EMSA and western analyses as to what mechanism mediates the strong induction of CIEBPa protein in 32D-CB and -C6 transfectants. In order to determine whether level of endogenous CIEBPa mRNA is increased, a northem analysis was performed. As shown in Fig. 6, a radiolabeled probe for CIEBPa detects two transcripts of different sizes. The longer species represents the transcript from the transfected vector, while the shorter one represents the endogenous CIEBPa mRNA. Clearly, the endogenous CIEBPa mRNA shows increased expression in both 32D-CB and 130 szo-Neo-i 3ZD-Cci-l 3ZD-Cci-2 JZD-CB—l 320.cp-2 3ZD-C6—I _ 32D-C6-2 ' 32D-Cad 32D-Ce—2 C/EBI’a Figure 6. Northern blot analysis of C/EBPa expression in 32D transfectants. Twenty micrograms of RNA was analyzed on a northern blot and hybridized to a probe for CIEBPa. The position of C/EBPa transcripts are indicated by the arrowheads. The upper one indicates the transcript derived from the transfected expression vector while the lower one indicates the endogenous CIEBPa. 131 —C6 cells relative to 32D-Neo control cells. This result shows that CIEBPa mRNA levels are upregulated by either CIEBPB or CIEBP6 expression and suggests a transcriptional mechanism for this increase. 132 DISCUSSION The tissue and stage-specific expression of the granule-associated genes in granulopoiesis has been shown to be regulated mainly at the level of transcription (Friedman et al. 1991, Yoshimura et al. 1992, Sturrock et al. 1996). The CIEBP transcription factor family has been implicated in the regulation of primary granule-associated genes (Ford et al. 1996, Oelgeschlager et al. 1996, Sturrock et al. 1996). We have directly compared the abilities of CIEBPa, -B, -6 and —e to induce the transcription of the genes encoding primary granule- associated proteins including MP0, Cat G, and L2 in the murine myeloid precursor cells, 32D cl3. The data presented in this report show that ectopic expression of CIEBPB in 32D cl3 results in a dramatic increase in the levels of MPO and Cat G mRNA, levels similar to those of G-CSF-induced 320 cl3 cells. A more modest increase in L2 mRNA is also observed. In addition, transfectants for CIEBPa, -6 and -e expression all show a small but significant increase in the transcripts encoding MPO, Cat G and LZ compared with the control cells. The elevated transcription is most likely a direct effect of the elevated expression of CIEBP proteins resulting from their ectopic expression, as binding sites for CIEBP have been identified in the promoter of the L2 gene, as well as the distal enhancer of the MPO gene. Interestingly, no CIEBP binding site has been found in the immediate 5’-region of the Cat G promoter. Our results strongly suggest the involvement of C/EBPs in the regulation of Cat G transcription. It is very likely that a functional CIEBP binding site is present further upstream or downstream of 133 the previously characterized Cat G promoter. On the other hand, the possibility cannot be ruled out that the ectopically expressed CIEBPs may have induced other transcription factors which work in concert with CIEBP proteins in the promoters of the primary granule-associated genes. For example, it is suggested that the transcription of the murine NE gene requires the cooperation of PU.1, c- Myb and CIEBPs for maximal expression. Our data have shown that 32D cl3 cells transfected for CIEBPB expression have the highest level of transcription for the MPO, Cat G and L2 genes among other transfectants. EMSA and Western analyses, however, did not reveal an increase in the level of CIEBPB protein in nuclear extracts of 32D-CB cells. Instead, a large increase in the level of CIEBPa is observed. It is clear that the total level of CIEBP proteins and, particularly, the level of CIEBPa-C/EBPB heterodimer DNA binding activity in the nucleus are increased compared with the 32D cl3 control cells. In fact, in control cells, 32D-CB cells and all transfectants with the exception of 32D-C6, CIEBPa-CIEBPB heterodimers appear to be the predominant form of CIEBP detected by EMSA. This suggests that the increase in CIEBPor-C/EBPB heterodimers caused the up-regulation of the transcripts encoding MPO, Cat G and L2. The observation that 32D-Neo cells have a considerable level of endogenous ClEBPa and CIEBPB but do not express high levels of transcripts for MP0, Cat G and L2 may suggest a threshold for the concentration of C/EBPa-C/EBPB in the nucleus to induce a high level of transcription of these genes. The fact that the levels of these transcripts are not as high in 32D-Ca as they are in 32D-CB may be explained by CIEBPa-CIEBPB 134 being the most active form of CIEBP in regulating these genes. The increased amount of CIEBPa by itself would not be expected to increase the abundance of CIEBPa-CIEBPB. Rather, both isoforms would need to be elevated as in the case of 32D-CB. The more modest induction of transcription for the MPO, Cat G and LZ in 32D-C6 and -Ce may reflect the weaker activities of CIEBP6- or CIEBPe-containing CIEBP dimers. The relative lack of CIEBPa DNA binding activity in 32D-C6 may be a reflection of CIEBPa-C/EBP6 heterodimers having weaker DNA binding activity than CIEBPa-C/EBPB heterodimers. This could be tested by in vitro binding assays using recombinant C/EBPa, B and 6 and heterodimers thereof. It is intriguing that although Western analyses did not detect an increase of CIEBPB protein in nuclear extracts of 32D-CB cells, the cells have the strongest elevation in the mRNA levels of several primary granule genes. This may reflect post-transcriptional regulatory mechanisms of CIEBPB activity. Ford et al. (1996) have shown that CIEBPB is unphosphorylated and localized in the cytoplasm of early progenitor cells, but exists as a phosphorylated form capable of binding to its cognate DNA site in the nuclei of granulocyte-committed cells. Furthermore, G-CSF-induced granulocytic differentiation of multipotential progenitor cells results in the functional recruitment of CIEBPB to the nucleus. It is thus possible that CIEBPB is indeed elevated in the 32D-CB cells but is mainly confined to the cytoplasms. The only change in CIEBPB binding that we can observe is an increase in the amount of ClEBPa-CIEBPB detected. Any change in the nuclear 135 concentration of CIEBPB and/or DNA-binding activity of the CIEBPB homodimer may have been too subtle to be detected by our EMSA and Western blot analyses. If CIEBPB is grossly overexpressed in the cytoplasm of 320 cl3 cells, then cytoplasmic contamination of the nuclear compartment might obscure changes in CIEBPB that would otherwise be detectable. Future analyses should determine CIEBPB levels in the cytoplasm and the procedure for preparation of nuclear extracts should be modified to eliminate cytoplasmic contamination. Since two independent 32D-CB transfectant populations behaved very similarly, it is very unlikely that this is an artifact of clonal variation. We have shown that the level of CIEBPoi protein is highly elevated in the nucleus of 32D-CB cells and, to a lesser extent, is also elevated in the 32D-C6 cells. Northern blot analyses suggest that this induction occurs at the level of transcription. This suggests that CIEBPB and CIEBP6, either as homodimers or as heterodimers with C/EBPa itself, are capable of inducing the transcription of the endogenous CIEBPa gene. In support of this view, a functional CIEBP binding site has been identified in the promoter of the murine C/EBPa gene. This CIEBP site was found to be responsible for the autoregulation of CIEBPa in the liver cells. In addition to CIEBPa, CIEBPB and CIEBP6 can also bind to this site. All three CIEBP isoforms can transactivate a reporter construct driven by the CIEBPa promoter. It is very likely that the CIEBP binding activities expressed during myelopoiesis are not only expressed in a sequential manner (Scott et al. 1992), but regulate their own expression as well. . Finally, the observation that the CIEBPa-B heterodimer is the only form of 136 CIEBP that is detectably increased in P388-CB cells suggests that this form is responsible for the increased expression of MPO, Cat G, and L2 observed in these cells. This conclusion is also consistent with the notion that the upregulation of CIEBPa and —B coincide with the activation of primary-granule associated genes, when 32D cl3 is induced to differentiate along the granulocytic pathway by G-CSF. It would be informative to compare the EMSA pattern of G- CSF induced cells to that of 32D-CB. Additionally, transient transfections of cells with various CIEBP expression vectors, either individually or in combination, and a MP0 or Cat-G promoter-reporter would be informative as to the relative efficacy of the various forms of CIEBP in supporting transcription of these genes. 137 REFERENCES Borregaard, N., K. Lollike, L. Kjeldsen, H. Sengelov, L. Bastholm, M. H. Nielsen, and D. F. Bainton. 1993. Human neutrophil granules and secretory vesicles. Eur. J. Haematol. 51: 187 Cepko, C. L., B. E. Roberts, and R. C. Mulligan. 1984. Construction and applications of a highly transmissible marine retrovirus shuttle vector. Cell 37: 1053. Ford, A. M., C. A. 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Expression of the chronic myelogenous leukemia-associated p210bcrlabl oncoprotein in a murine lL-3 dependent myeloid cell line. Oncogene 6: 275 Ness, S. A., E. Kowenz-Leutz, T. Casini, T. Graf and A. Leutz. 1993. Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types. Genes Dev. 7: 749 Oelgeschlager, M., I. Nuchprayoon, B. Luscher and A. D. Friedman. 1996. CIEBP, c-Myb and PU.1 cooperate to regulate the neutrophil elastase promoter. Mol. Cell. Biol. 16: 4717 Patel, G., B. Kreider, G. Rovera, and E. P. Reddy. 1993. v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation. Mol. Cell. Biol. 13: 2269 Rado, T. A. , X. Wei, and E. J. Benz. 1987. Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis. Blood 70: 989 Scott, L. M., C. l. Civin, P. Rorthand A. D. Friedman. 1992. 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Transcriptional and posttranscriptional modulation of human neutrophil elastase gene expression. Blood 79: 2733 Zimmer, M., R. L. Medcalf, T. M. Fink, C. Mattmann, P. Lichter, and D. E. Jenne. 1992. Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter. Proc. Natl. Acad. Sci. USA 89: 8215 Zhu, J., C. A. Bennett, A. D. MacGregor, M. F. Greaves, G. H. Goodwin and A. M. Ford. 1994. Title. Leukemia 8: 717 140 CONCLUSIONS The results presented in Chapter 2 show that ClEBPa and —6, as well as CIEBPB are expressed by bone marrow-derived macrophages and are all potentially available to support the expression of proinflammatory cytokines In macrophages. It is demonstrated that ClEBPor, -B and —6 are all capable of supporting the LPS-inducible transcription of IL-6 and MCP-1 in P388 lymphoblasts, which normally lack CIEBP factors and do not display LPS induction of proinflammatory cytokines. These results suggest that a largely normal cytokine response to LPS in the macrophages of CIEBPB-deficient mice can be explained by the availability of CIEBPa and/or CIEBP6. The induction of CIEBP6 by LPS in bone marrow-derived macrophages makes it a particularly attractive candidate for replacing CIEBPB activity. Although ClEBPa, -B and —6 are largely redundant in the LPS-inducible expression of proinflammatory cytokines, specific roles for these CIEBP isoforms in the regulation of other inflammation-associated genes are certainly possible. Especially, inflammatory stimuli other than LPS, such as IFN-y, IL-1, lL-6 and TNFa might provide a more complete cytokine response by altering the level of activity and/or specificity of the same CIEBP isoforms that are available during LPS stimulation through post-translational modification (e.g. phosphorylation). Alternatively, these stimuli may activate other transcription factors that are essential for inducing a broad spectrum of proinflammatory cytokines. It will be of interest in future experiments to examine the abilities of these inflammatory 141 stimuli to induce other proinflammatory cytokines in the P388 transfectants generated in this study. The data presented in Chapter 3 show that the bZIP regions of CIEBPB and CIEBP6 are of themselves capable of supporting LPS induction of IL-6 and MCP- 1. The bZIP region of ClEBPa also shows modest activity. Furthermore, the naturally occurring transdominant negative inhibitor LIP is capable of supporting the LPS induction of lL-6 and MCP-1. Replacement of the leucine zipper of CIEBPB with that of yeast GCN4 yields a chimeric protein that can dimerize and specifically bind to a CIEBP consensus sequence, but shows a markedly reduced ability to activate lL-6 and MCP-1. These results implicate the leucine zipper region in some function other than dimerization with known CIEBP family members in the activation of IL-6 and MCP-1 transcription, and suggest that CIEBP redundancy in regulating cytokine expression may result from their highly related bZIP domains. NF-icB has been implicated as an important partner of CIEBP proteins in regulating the genes of proinflammatory cytokines. There is good evidence that CIEBPa , -B and -6 and NF-KB synergistically activate the IL-6 and IL-8 promoters. This synergism may not only involve binding of the transcription factors to their cognate binding sites in the promoters, but direct physical association of the factors through their respective bZIP and Rel homology domains. We have shown that the activation domains of CIEBPs are not necessary for their activities in LPS induction of lL-6 and MCP-1. It is very likely that the bZIP domain that is highly conserved in the CIEBP isoforms may serve 142 to enhance the activity of NF-KB on these promoters. It is also possible that the physical interaction of the bZIP domain with nuclear factors in addition to or other than NF-KB may be critical to its activity. It will be informative in future experiments to examine what structural components of the truncated forms of CIEBPs are required for their physical interactions with NF-KB p50 and p65. In addition, the abilities of truncated forms of CIEBPs to cooperate with other nuclear proteins may also be worthy of investigation. The techniques of co- immunoprecipitation and immunoaffinity chromatography could be applied to these questions. The results in Chapter 4 show that 32D cl3 cells transfected for CIEBPB expression (32D-CB) have the greatest enhancement of mRNA levels for the genes encoding myeloperoxidase (MPO), cathepsin G (Cat G), and lysozyme (LZ) compared to transfectants for other CIEBP isoforms. A level of expression similar to the G-CSF-induced 32D cl3 parental cells is observed in these primary granule protein encoding genes. Other transfectants also show a modest increase in the level of these mRNA. EMSA suggests that the C/EBPa- CIEBPB heterodimer is the predominant form of CIEBP present in the nucleus of 32D-CB cells and that this form of CIEBP DNA binding activity is increased. The CIEBPIa—C/EBPB heteodimer is likely an important regulator of the primary granule-associated genes. An experiment for the immediate future that will strengthen our results is to examine the CIEBPB expression level in the cytoplasmic compartment of 32D-CB cells compared with that of control cells. Also necessary is an improvement of the 143 current protocol for the isolation of nuclear proteins to avoid cytoplasmic contamination. A more accurate assessment of the concentration of CIEBPB and CIEBPB DNA binding activity can then be achieved by Western analyses and EMSA. The elucidation of the mechanism by which the CIEBPB and —6 containing CIEBP isoforms induce the expression of endogenous ClEBPa mRNA may also contribute to the understanding of the special temporal expression pattern of CIEBP proteins observed during granulopoiesis. 144 HI RN STATE UN "Illlljilllllllilillllllllllllllillji” 7