1.” . Lam LIBRARY IVIIu. ., ~. e mate University This is to certify that the dissertation entitled BY PARTICIPATING IN THE TGF— fi/SMAD SIGNALING PATHWAY, LRP12 REGULATES THE TUMORIGENICITY OF HUMAN F IBROSARCOMA DERIVED CELL LINE SHAC presented by Jie Zhang has been accepted towards fulfillment of the requirements for the Ph.D. degree in Biochemistry and Molecular Biology \ ~ ~. A .' \_ . f .1 ~ \ \ .. If (I ' ’ " x L \:m L.‘\_ ( ( (“L‘Mr’CA "'7 /" Major Professor’s Signature I) g is”: Q cot- .r /" k...‘(;l.‘. ) .3 // Date MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 KthrojIAccaPres/ClRC/DateDue.indd BY PARTICIPATING IN THE TGF-B/SMAD SIGNALING PATHWAY, LRP12 REGULATES THE TUMORIGENICITY OF HUMAN FIBROSARCOMA-DERIVED CELL LINE SHAC By Jie Zhang A DISSERTATION Submitted to Michigan Sate University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry and Molecular Biology 2006 ABSTRACT BY PARTICIPATINT IN THE TGF-B/SMAD SIGNALING PATHWAY, LRP12 REGULATES THE TUMORIGENICITY OF HUMAN FIBROSARCOMA- DERIVED CELL LINE SHAC By Jie Zhang It is generally accepted that tumorigenesis is a multi-step process that involves a series of genetic and/or epigenetic changes. In an effort to identify the nature of these changes, McCormick and Maher established a cell lineage in which normal human fibroblasts were transformed into malignant fibroblasts by acquiring a series of genetic changes. Using this cell lineage, differential display of mRNA from an infinite life span, non- tumorigenic human fibroblast cell strain and one of its carcinogen-transformed, malignant derivatives led to the identification of a novel gene designated LRP12. LRP12 is expressed in normal fibroblasts, but is absent or only expressed at very low levels in human fibrosarcoma cells, suggesting it is a tumor suppressor gene. What is more, expression of LRP12 in human fibrosarcoma-derived cell line SHAC inhibits tumor formation in athyrnic mice. Structurally, LRP12 belongs to the low-density lipoprotein receptor (LDLR) family. Although members of this family are best known as endocytic receptors, recent studies demonstrate that the cytoplasmic tails of several LDLR family members interact with adaptor and scaffold proteins that are implicated in signal transduction. A yeast two—hybrid assay in this laboratory suggested that the cytoplasmic tail of LRP12 interacts with several signaling proteins, including a truncated form of the SMAD Anchor for Receptor Activation (SARA). The fact that SARA is a protein involved in TGF-B signaling suggests that LRP12 participates in the TOP-[3 signaling pathway. Using Western blotting analysis and luciferase assays, I determined that LRP12 expression increases TGF-B-induced phosphorylation of Smad2 and Smad3. This function of LRP12 depends on the presence of its cyt0plasmic tail, which 1 demonstrated to interact with the full-length SARA and the Smad7-Smurf2 complex. Using Wesem blotting analysis, I showed that expression of LRP12 significantly decreases the ubiquitination of TGF-B receptor type I (TBRI) and activated Smad2. in contrast, LRP12 lacking the majority of the cytoplasmic tail cannot associate with SARA and Smuer, nor does it increase TGF-fi-induced phosphorylation of Smad2. Using an in vitro cell migration assay and invasion assay, I further demonstrated that expression of LRP12 inhibits the ability of tumor cells to migrate and invade by enhancing the TGF-Bl induction of PAH expression. These data support the hypothesis that LRP12 is a tumor suppressor. Furthermore, they indicate that LRP12 regulates tumorigenicity by participating in the TGF-B signaling pathway and by enhancing the TGF-Bl-induced PAI-l expression. DEDICATION I dedicate my dissertation to my parents, And especially to my wife Yun and my daughter Emma. I love you. Acknowledgement I would like to thank Dr. J Justin McCormick for accepting me into the Carcinogenesis Laboratory and providing me that LRP12 project. Especially, I want to thank Dr. McCormick for all the support and advice he provides to both my research and my life. I would also like to thank Dr. Veronica Maher, the co-director of the Carcinogenesis Laboratory, for her support, advice, and serving on my committee. I would also like to thank my committee members Dr. Katheryn Meek, Dr. Kathy Gallo, and Dr. John Wang, for all the support and advice in these years. I want to thank all the members of the Carcinogenesis Laboratory. Sandra, Terry, Igor, Jessica, Rick, Piro, and all other member of the carcinogenesis family, I thank you for helpful scientific discussions and friendships over the years. Special thanks to Jessica Apostol, and Martin Furey from the College of Osteopathic Medicine for their many hours of proofreading my dissertation. I want to thank my parents. They worked very hard and tried their best to support my sisters, my brother, and me so that we all could go to college. They taught me that there is a huge world outside the small village where I grew up and encouraged me to pursue my dreams in it. They always provide me unconditional support and believe that I am the best. Thank you, mom and dad. Without you, I wouldn’t be where I am now. I want to thank a very special person in my life, my wife Yun. Yun and I have been married for seven years. She has always been giving me unconditional support and encouragement so that I could go on even during very difficult times. She believed in me even when I doubted myself. She took the stress off me when I struggled. She shared the happiness when I was happy and shared the sadness when I was upset. Thank you and I love you. Finally, I would like to thank my little angel, Emma. She is really the joy of my life. Her laughter, even her crying shed light in my life. I enjoy every second I spend with her, from a newborn baby to a four-year-old girl. She also brings new thoughts in my mind and new meanings to my life. Thank you for giving me so much joy and making my life happier every day. vi TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ x LIST OF ABBREVIATIONS ............................................................................................ xi INTRODUCTION .............................................................................................................. 1 REFERENCE ...................................................................................................................... 3 Chapter I .............................................................................................................................. 4 LITERATURE REVIEW ................................................................................................... 4 1. TGF-B signaling pathway ................................................................................. 4 a. The TGF-B superfamily growth factors ......................................................... 5 i. TGF-B subgroups .................................................................................... 5 ii. Latent TGF-B complexes ........................................................................ 8 b. Signaling receptors ....................................................................................... 12 i. TGF-B receptor families ........................................................................ 13 ii. Structural features of TGF-B receptors ................................................. 14 1. GS domain ...................................................................................... l7 2. Kinase domain ................................................................................ 17 c. Li gand-receptor interactions ........................................................................ 18 i. The binding of ligands to receptors ...................................................... 18 ii. Accessory receptors: Betaglycan and Endoglin .................................... 20 iii. Activation of the TGF-B receptors ....................................................... 22 1. Proteins regulating the TGF—B receptors ........................................ 22 2. Mechanism of receptor activation .................................................. 24 3. Receptor trafficking ........................................................................ 26 iv. Smad proteins ....................................................................................... 28 1. Identification of Smads as mediators of TGF—B signaling ............. 29 2. Smads subfamilies .......................................................................... 31 3. Structural features of Smads ........................................................... 33 4. Smad nucleocytoplasmic shuttling ................................................. 38 5. Subcellular retention of Smads ....................................................... 40 d. Smad-dependent signaling pathway ............................................................ 42 i. Smad adaptors for receptor activation .................................................. 42 ii. Smads activation ................................................................................... 43 iii. Regulation of Smads by phosphOrylation ............................................ 49 iv. Smad oligomerization .......................................................................... 50 v. DNA-Smad transcriptional complex interaction .................................. 52 vi. Transcriptional activation by Smads .................................................... 58 vii. Transcriptional repression by Smads .................................................. 61 viii. Self-enabling gene response by Smads .............................................. 62 ix. Inhibition by inhibitory Smads ............................................................. 63 x. Regulation of TGF-B/Smads signaling by ubiquitination ..................... 65 e. TGF-B signaling pathway crosstalks with other pathways .......................... 69 i. TGF—B signaling pathway crosstalks with MAPK pathways ................ 69 vii ii. Other TGF-B-induced signaling pathways ............................................ 72 2. TGF-B biological functions and cancer ........................................................... 72 a. TGF-B biological functions .......................................................................... 73 b. Tumor suppression ....................................................................................... 74 i. TGF—B-dependent growth control ......................................................... 74 ii. TGF—B-induced apoptosis ..................................................................... 78 iii. Loss of TGF—B-induced suppression in cancer .................................... 80 iv. TGF-B signaling and replication potential ........................................... 82 c. Tumor progression ....................................................................................... 83 i. Epithelial-mesenchymal transition (EMT) ........................................... 84 ii. Tissue invasion and metastasis ............................................................. 85 iii. Angiogenesis ........................................................................................ 88 iv. Evasion of immune surveillance .......................................................... 89 d. Target TGF—B for cancer therapy ................................................................. 91 3. Conclusion and perspective ............................................................................ 93 REFERENCES ................................................................................................................. 95 Chapter II ........................................................................................................................ 130 BY PARTICIPATING IN THE TGF—B/SMAD SIGNALING PATHWAY, LRP12 REGULATES THE TUMORIGENICITY OF HUMAN FIBROSARCOMA- DERIVED CELL LINE SHAC ................................................................................ 130 ABSTRACT .................................................................................................................... 13 1 INTRODUCTION .......................................................................................................... 133 EXPERIMENTAL PROCEDURES ............................................................................... 137 Materials .................................................................................................................. 137 Cell Culture .............................................................................................................. 137 Immunoblot Analysis of Smad2 Phosphorylation ................................................... 138 Luciferase Reporter Assay ....................................................................................... 138 Co-immunoprecipitation .......................................................................................... 139 Monolayer Wound Healing Assay ........................................................................... 139 Tumor Cell Trans-well Invasion Assay ................................................................... 139 3H-Thymidine incorporation assay .......................................................................... 140 Statistical analysis .................................................................................................... 140 RESULTS ....................................................................................................................... 142 LRP12 increases the TGF-Bl-induced Smad2 phosphorylation .............................. 142 LRP12 expression increases both pSmad2- and pSmad3-dependent transcriptional activity ...................................................................................................................... 143 LRP12 associates with full-length SARA ................................................................ 143 LRP12 associates with the Smurf2-Smad7 complex. .............................................. 145 Ubiquitination of TBRI and phosphorylated Smad2 are inhibited in 02 cells ........ 146 Inhibition of proteosome-mediated degradation increases TGF—Bl induction of Smad2 phosphorylation in D2 cells, but not in 02 cells .......................................... 147 The cytoplasmic tail is required for TGF-Bl-induced Smad2 phosphorylation. ..... 148 LRP12 is required for TGF—Bl induction of PAI-l expression. .............................. 149 LRP12 is required for TGF-Bl induction of tumor cell migration inhibition .......... 150 LRP12 is required for TGF—Bl induction of tumor cell invasion inhibition ............ 151 viii DISCUSSION ................................................................................................................. 152 Chapter III ....................................................................................................................... 180 Identification of Proteins that Interact with the Extracellular Domain of LRP12 .......... 180 ABSTRACT .................................................................................................................... 18 1 INRODUCT ION ............................................................................................................. 183 EXPERIMENTAL PROCEDURES ............................................................................... 188 Construction of Baculovirus Transfer Vectors: ....................................................... 188 DNA Sequencing: .................................................................................................... 188 Generating Recombinant Baculovirus by Co-transfection: ..................................... 189 Plaque Assay: ........................................................................................................... 190 Cell Culture .............................................................................................................. 190 Expression and purification of LRP12 using the Baculovirus Expression Vector System ...................................................................................................................... 191 Immunoblot Analysis of LRP12 expression ............................................................ 191 Ligand pull-down assay ........................................................................................... 191 Silver staining .......................................................................................................... 192 Mass spectrometry analysis ..................................................................................... 192 RESULTS ....................................................................................................................... 193 Construct the recombinant baculovirus. .................................................................. 193 Ligand pull-down assay ........................................................................................... 193 Mass spectrometry analysis ..................................................................................... 194 DISCUSSION ................................................................................................................. 196 Future Work .................................................................................................................... 200 1. Determine the interaction between LRP12 and putative ligands. ........................ 200 2. Determine if LRP12 binds the putative ligands on cell surface ........................... 200 3. Determine if LRP12 mediates the internalization of the putative ligands. .......... 200 REFERENCE .................................................................................................................. 202 Chapter IV ....................................................................................................................... 208 Proposed future study of LRP12 ..................................................................................... 208 Accomplishment of my project: ............................................................................... 208 What are the molecular mechanisms of the LRP12-SARA and the LRP12- Smurf2/Smad7 interactions? .................................................................................... 210 Does down-regulation of LRP12 in non-tumorigenic cells by siRNA increase tumorigenicity? ........................................................................................................ 212 Is LRP12 an endocytic receptor? ............................................................................. 213 Is LRP12 involved in SARA-regulated endosomal trafficking? ............................. 215 LIST OF FIGURES CHAPTER I: LITERATURE REVIEW Figrue 1: formation of the latent TGF-B complex from the precursor molecules ............ 10 Figrue 2: Type I and type H TGF-B receptors ................................................................... 15 Figure 3: Determinants of specificity in TGF-B signal transduction ................................ 36 Figure 4: The TGF-B/Smad signaling pathway ................................................................ 46 CHAPTER II: LITERATURE REVIEW Figure 1: Expression of LRP12 enhances TGF- B l-induced Smad2/3 phosphorylation ................................................................................................ 162 Figure 2: LRP12 expression enhances TGF- B l-induced transcription of the luciferase reporter gene from the pSmad2- (ARE-Luc) and pSmad3-dependent (SBE—Luc) promoters ................................................ 164 Figure 3: LRP12 forms a protein complex with full-length SARA ................................ 166 Figure 4: LRP12 interacts with Smurf2 and inhibits ubiquitination of TBRI and phosphorylated Smad2 ................................................................ 168 Figure 5: Inhibition of proteosome-mediated protein degradation increases TGF-Bl induced Smad2 phosphorylation in D2 cells, but not in 02 cells ............................................................................................ 170 Figure 6: Interaction between LRP12 and SARA is required for TGFBl-induced Smad2 phosphorylation ......................................................... 172 Figure 7: LRP12 expression increases TGF—Bl induced PAI-l expression ................... 174 Figure 8: LRP12 expression inhibits SHAC cell migration through TGF-Bl induction of PAH expression ........................................................... 176 Figure 9: LRP12 expression inhibits SHAC cell invasion through TGF-Bl induction of PAH expression ......................................................................... 178 CHAPTER III Figure 1: Expression and secretion of the LRP12ext-V5/His and LRP12full-V5/His ..................................................................................... 204 Figure 2: Identification of proteins interacting with the extracellular domain of LRP12 ........................................................................ 206 a2-M ActR-II ActR-IIB ALK AMH AMHR-II APC ApoER2 ARE ATP BAMBI BMP BMPR-II BRAMI BRE CamKII CBP cDN A CDK Co-Smad CTGF daf-4 DPP ABBREVIATIONS (_1_2_-_M_acroglobulin porivin type fl receptor a_ctivin type II receptor B pctivin-receptor-Iike l_(inase pnti-Muellerian hormone pnti-Muellerian hormone type fl receptor antigen-presenting gells applipoprotein _E_ receptor 2 activin-responsive plement Adenosine rn'phosphate BMP and a_ctivin membrane pound inhibitor pone morphogenetic proteins pone morphogenetic proteins type _II receptor _B_MP receptor associated molecule 1 _BMP response plement gplcium/leodulin-dependent protein l_§mrr.mmnmmfwfififw' TBRF+~AW* ‘*““ ACtin -+ m" “Wm-«W ' Cell strain D2 02 X2 pSmad2—> ....- 2...... .. . ____,, .. ' M TGF-B1-++-++-++ Cell strain D2 02 X2 Figure 1 163 Figure 2: LRP12 expression enhances TGF- B l-induced transcription of the luciferase reporter gene from the pSmadZ- (ARE-Luc) and pSmad3-dependent (SBE-Luc) promoters. 02, X2, and D2 cell strains were transiently transfected either with a pSmadZ-dependent ARE-Luc reporter construct, a FoxHZ construct (the Smad2 transcriptional cofactor) and Renilla luciferase reporter control construct (A) or a pSmad3-dependent SBE-Luc reporter construct along with Renilla luciferase reporter control construct (B). 24 hours after transfection, cell were serum-starved for 8 hours and then treated with serum-free media or serum-free media containing 2.5ng/ml TGF- B 1 as indicated for 16 hours. Cells were then lysed and analyzed for the luciferase activity using the Dual-Luciferase Reporter Assay System from Promega. Each luciferase measurement is corrected for Renilla luciferase. Each bar represents triplicate measurements from a representative experiment. Each experiment has been repeated at least three times. 164 ARE—LL10 wad: mumuufinum «Haaaom\o=A TGF-131: SBE-Luc ifiap humuufinum madam X2 02 D2 + TGF-B1 Figure 2 165 Figure 3: LRP12 forms a protein complex with full-length SARA. (A). 293T cells were transiently transfected with LRP12-V5 (lane 1-3), or SARA-Flag (lane 4-6), or both of the expression constructs (lane 7-12). Cell lysates (containing 250pg of total protein) were processed for immunoprecipitation with the anti-Flag antibody. The presences of LRP12 and SARA protein were analyzed by Western blotting with anti—V5 antibody and with anti-Flag antibody. Cell lysates containing 25ug of total protein were used as a positive control. The experiment has been repeated at least three times. (B). 293T cells were transiently transfected with empty vector (lane 1-3), or SARA-Flag (lane 5-8). Cell lysates (containing 250ug of total protein) were processed for immunoprecipitation with the anti-Flag antibody. The presences of Ku80 and SARA protein were analyzed by Western blotting with anti-Ku80 antibody and with anti-Flag antibody. Cell lysates containing 25 pg of total protein were used as a positive control. 166 LRP12-V5 LRP12-V5 Plasmids and ' and __LRP12-V5 We SABA=flag SABAJlag TGF- B 1 6‘ <9. _‘__\.)\_ —i-—$~. IP: a-flag _ + s9 _ + \«Q _ + sq - + ~62 IB: a-vsal .- h ---a- . -- ----j 18: a-flag-tf 6 M ‘ i‘bfi - -— - 123.4 5 6 7 8 9161112 B. 293T 293T/SARA-flag C)‘ 8‘ 5‘ . Q lea-flag - + ‘62 - + ‘QQ ‘0 lB: a-Ku80-> . t ' lB: a-flag -> ’ Figure 3 167 Figure 4: LRP12 interacts with Smurfl and inhibits ubiquitination of TBRI and phosphorylated Smad2. (A) 02 cells were cultured in serum-free media with or without 2.5ng/ml TGF-B1 for 1 hour as indicated. Cell lysates (containing 250 pg of total protein) were processed for immunoprecipitation with the anti-V5 antibody. Presence of Smad7, Smurf2 and LRP12-V5 were analyzed by Western blotting with the anti-Smad7, anti- Smuer and anti-V5 antibodies. Cell lysates containing 25 pg of total protein were used as positive control. (B-D) D2 and 02 cells were cultured in serum-free media without (lane 1, 3) or with 2.5ng/ml TGF-B1 (lane 2, 4) for four hours as indicated. Whole cell lysates were collected and cell lysates (containing 250 pg of total protein) were processed for immunoprecipitation with the anti-phospho-SmadZ, anti-TBRI and anti-EGFR antibodies. The ubiquitination of phosphorylated Smad2 (B), TBRI (C) and EGFR (D) were analyzed by Western blotting using the anti-ubiquitin antibody. Each experiment has been repeated at least three times. 168 WB: Smurf2 +1 - [1,“. i ‘ j .: M1EM ml WB: LRP12-V5 “ IP: TGF B 1 anti-V5 Normal lgG anti- Y PAK 1P: p-Smad2 WB: Ub D2 D2 02 02 Cell strain -—++ -+-+ C. Ub TB R1 1P: TR1 D. 1P: EGFR WB: Ub WB; Ub 1111 11:15.], 1 DZ D2 02 02 LRP12 - - + + TGF B 1 - + - + Figure 4 169 Figure 5: Inhibition of proteasome-mediated protein degradation increases TGF-B1 induced Smad2 phosphorylation in D2 cells, but not in 02 cells. D2 cells (lane 1-3) and 02 cells (lane 4-6) were cultured in serum-free media without (lane 1, 4) or with (lane 2, 5) 2.5ng/ml TGF-B1, or with both 2.5ng/ml TGF-Bl and 10 pM proteasome inhibitor MGl32 (lane 3, 6) for 6 hours as indicated. Cells were then collected in NP-40 lysis buffer. Cell lysates containing 50 pg of total protein were denatured and separated by SDS-PAGE. The presence of phosphorylated Smad2 was analyzed with Western blotting analysis using the anti-phospho-SmadZ antibody. Actin was used as protein loading control. The experiment has been repeated at least three times. 170 2 3 4 5 6 P-Smad2—> ACIln+ . m. ' Cell strain D2 LRP12: - TGF B 12 - MG132: - Figure 5 171 Figure 6: Interaction between LRP12 and SARA is required for TGFBl-induced Smad2 phosphorylation. (A) D2, 02 and another SHAC-derived cell strain Trl, which expresses a truncated LRP12 that only contains extracellular domain and the first ten amino acids of the cytoplasmic tail, were cultured in serum-free media with or without 2.5ng/ml TGF-B1 as indicated. Whole cell lysates were collected in NP-40 lysis buffer. Cell lysates containing 50 pg of total protein were denatured and separated by SDS- PAGE. The protein level of phosphorylated Smad2 was analyzed by Western blotting using anti-phospho-SmadZ antibody. Actin was used as loading control. (B) D2, 02 and Trl cells were transiently transfected with SARA-flag expression plasmid. 24 hours after transfection, cells were cultured in serum-free media with or without 2.5ng/ml TGF-B1 as indicated for 1 hour. Cell lysates (containing 250 pg of total protein) were processed for immunoprecipitation with the anti-V5 antibody. The presence of SARA-Flag, Smurf2, LRP12-V5 (lane 3, 4) and LRP12-Trl-V5 (lane 4, 5) proteins was analyzed by Western blotting with the anti-Flag, anti-Smuer and anti-V5 antibodies respectively. Each experiment has been repeated at least three times. 172 P-Smad2—> ~— ' """"". - Cell strain D2 02 Tr1 TGFBl - + - + - + B. 123456789101112 SARA-Flag ' ..'..“.......‘ ‘.'............'.......... ' LRP12-V5 . ---~ ‘- an *m LRP12Tr1-V5 . _ ,. , . Cell strain D2 02 Tr1 D2 02 Tr1 TGFBl -+-+-+-+-+-+ IP: V5 Cell lysate Figure 6 173 Figure 7: LRP12 expression increases TGF-B1 induced PAI-l expression. (A) D2, 02 and X2 cells were cultured in serum-free media with or without 2.5ng/m1 TGF-B1 for different period of time as indicated. Whole cell lysates were collected and PAI-I expression were analyzed by SDS-PAGE and Western Blot using the anti-PAI-I antibody. Actin was used as loading control. (B) D2, 02 and X2 cells were cultured in serum-free media with or without 2.5ng/m] TGF-B1 for different period of time as indicated. Conditioned media were collected and concentrated with Vivaspin 20 concentrators. Normalized amount of media were analyzed by SDS-PAGE and Western blotting using the anti-PAI-I antibody. Each experiment has been repeated at least three times. 174 1 2 3 4 5 6 7 8 9 PAl-1-> m . n W’ I ACUn www.mmwwwwl TGF-B1 - + + - + + - + + Cell strain DZ 02 X2 3- 1 2 3 4 5 6 7 8 PAI-1-f -->~— - 1' S" i ' ”.— TGF-Bl + + ' '1' ‘ "' "' Cell strain D2 02 X2 Figure 7 175 Figure 8: LRP12 expression inhibits SHAC cell migration through TGF-B1 induction of PAI-l expression. D2 and 02 cells were allowed grow to confluence. Then w ounds were generated using sterile pipet tips. Cells were cultured in serum-free media, serum-free media with TGF-B1 (2.5ng/ml), or serum-free media with both TGF-B1 (2 -Sng/ml) and neutralizing anti-PAI-I antibody (100ngng/ml) for 24 hours. Cell migration was observed using phase contrast microscope before and after the treatment. The experiment has been repeated at least three times. 176 A. TGF Anti— B 1 PAl-1 + - + + B. TGF Anti- B 1 PAl-1 + _ + + Figure 8 177 Figure 9: LRP12 expression inhibits SHAC cell invasion through TGF-B1 induction of PAI-l expression. Invasion chambers (Becton Dickinson) were coated with Matrigel (3 ()ug). D2 and 02 cells were plated in the upper chambers with the following treatment: serum-free media, serum-free media with TGF-B1 (2.5ng/ml), or serum-free media with both TGF-B1 (2.5ng/ml) and neutralizing anti-PAI-I antibody (lOOngng/ml). The lower (3 hambers contain culture media supplemented with 10% PBS. The invasion assay was C arried out for 24 hours in tissue culture incubator. Cells were then fixed and stained with crystal violet solution. The cells in the upper chamber were removed with Q-tips. Stained cells at the bottom side of the membrane were collected with acetic acid solution and analyzed with spectrometer. Each bar represents triplicate measurements from a representative experiment. The experiment has been repeated at least three times. 178 e ateive Units r—4 , TGF— B 1: - + + - + + Anti-PAI-l - - + - - + Figure 9 179 Chapter III Identification of Proteins that Interact with the Extracellular Domain of LRP12 180 ABSTRACT Many proteins of the low-density lipoprotein receptor (LDLR) family are known to function as endocytic receptors. The extracellular domains of these proteins bind various extracellular ligands, including lipoproteins, proteinases, proteinases-inhibitor complexes, extracelluar matrix (ECM) components etc. By binding such ligands, LDLR proteins mediate their interanlization for degradation in the lysosome. LRPl is the best—studied receptor from this family. Its huge extracelluar domain (525 kDa) has 31 ligand-binding repeats clustered into four ligand-binding domains. This large extracellular domain enables it to bind more than 30 structurally different ligands and mediate their internalization. LRP12 shares with other members of LDLR family conserved ligand- binding repeats in its extracellular domain and internalization signals in the cytoplasmic tail. Its extracellular domain also has another structural motif involved in protein-protein interaction: two CUB domains. To determine whether LRP12 interacts with other proteins, we expressed and purified the extracellular domain of LRP12 using the Baculovirus Expression Vector System. Purified LRP12 protein was incubated with conditioned medium from cells which has low expression of LRP12 protein. Secreted proteins in the medium bound to the purified LRP12 protein were separated by SDS- PAGE. The result of silver staining showed that several proteins bound to LRP12 specifically compared to the negative control. Mass Spectrometry analysis of these proteins led to the identification of a2-macroglobulin (aZ-M), pregnancy zone protein (PZP), complement component 3 (C3), and Apolipoprotein A-l (Apo A-l). These proteins have been found to be ligands for other LDLR endocytic receptors, including 181 LRPl. Taken together, these data provide evidence that LRP12 binds proteins through its extracellular domain and may functions as an endocytic receptor. 182 INRODUCTION My first project in the Carcinogenesis Laboratory was to identify the ligand(s) of the newly-identified transmembrane protein LRP12. At the time, Dr. Battle was studying the interactions between the cytoplasmic tail of LRP12 and cytoplasmic proteins. Her research led to the discovery of several signaling adaptor/scaffold proteins that associate with the cytoplasmic tail of LRP12, including a truncated form of the Smad Anchor for Receptor Activation (SARA). After Dr. Battle graduated, I went on to study the functional effect of the interaction between LRP12 and SARA, which is involved in the TGF-B signaling pathway. 1 found that expression of LRP12 enhances the TGF-B/Smad signaling pathway. This was a very interesting discovery convincing me to following on this experiment. Because it was very difficult for me to work on two projects at the same time, I spoke with Dr. McCormick and we both agreed that I should concentrate on this signaling project. Therefore, I studied the function of LRP12 in the TGF-B/Smad signaling pathway, which is presented in Chapter H. In Chapter HI, I will discuss the results I obtained in tying to identify the ligands of LRP12. LRP12 was identified in the Carcinogenesis Laboratory at Michigan State University by differential display of mRNA from the non-tumorigenic, infinite life span human fibroblast cell line MSU1.1 and one of its carcinogen-transformed MSU1.1 derived cell line, L210-6A/SB]. Northern and Western blotting showed that LRP12 is expressed in normal human tissues from different organs, but is not expressed or is expressed at a much lower level in malignant cell lines (Qing et al., 1999). Over-expression of LRP12 183 inhibits the tumor formation of human fibrosarcoma-derived cell line SHAC in athyrrric mice (M.A. Battle, unpublished data). These data suggest that LRP12 is a tumor suppressor gene. LRP12 belongs to the low-density lipoprotein receptor (LDLR) family (Battle et al., 2003). Most members of the LDLR family have been shown to function as endocytic receptors (Herz, 2001). They bind the ligands on cell surface and mediate ligands internalization for degradation (Herz, 2001). LRPl is the best-studied receptor from this family. Its huge extracelluar domain (525 kDa) has 31 ligand-binding repeats clustered into four ligand-binding domains. The large extracellular domain enables it to bind more than 30 structurally different ligands. These ligands include lipoproteins, proteinases, proteinases-inhibitor complexes, ECM components, etc (Howell and Herz, 2001). By constructing the mini-receptors containing the different ligand-binding domains of LRP], it was found that the second and the fourth ligand-binding domains are the major ligand- binding domains (Lynn et al., 2000). Most members of the LDLR family are type I transmembrane proteins. They share the following structural similarities: (a) cysteine-rich LDLR class A (LDLRA) repeats, (b) epidermal growth factor (EGF) receptor-like cysteine-rich repeats, (c) spacer repeats containing an YWTD sequence, ((1) a single transmembrane domain and (e) a cytoplasmic domain containing an NPXY motif as an internalization signal. 184 The LDLRA repeats contain about 40 amino acids and have six conserved cysteine residues, which form disulfide bonds in the pattern of one to three, two to five and four to six (Bieri et al., 1995). A cluster of several of these repeats constitutes a ligand-binding site for extracellular ligands. Different receptors of LDLR family contain different numbers of these domains. It might be the differential combination of ligand-binding repeats in a domain that determines the specificity of the ligands for a receptor (Hussain etaL,1999) Compared to the size of extracellular domains, most of the LDLR family receptors have relatively small cytoplasmic tails (Howell and Herz, 2001). Many members have one to three conserved NPXY or NPXY-like motifs (where x is any amino acid) in their cytoplasmic tail, which act as the internalization signal. Not all members of the LDLR family have all of the five structural motifs. LRP12, LRP3 (Ishii et al., 1998) and LRP9 (Sugiyama et al., 2000) do not have the epidermal growth factor (EGF) receptor-like cysteine-rich repeats and the spacer repeats containing YWTD sequence. In the LDLR family, LRP12, LRP3 and LRP9 constitute a unique subgroup characterized by the following unique features: (a) two CUB domains (acronym for _Clr/le, Uegf and BMP-1) in the extracellular domain separated by ligand-binding repeat(s); (b) four or five ligand-binding repeats arranged in two clusters; (e) no EGF precursor-homology repeats; (d) a relatively large cytoplasmic tail with an internalization motif and a proline-rich region (Battle et al., 2003). 185 As an LDLR-related protein, LRP12 protein has five imperfectly conserved ligand- binding repeats. They are arranged in two clusters, which have two and three repeats respectively, separated by the second CUB domain. All of the five ligand-binding repeats have the six conserved cysteine residues that form three disulphide bonds, but only two of them have all the conserved acidic arrrino acid residues in the C-terminus that are involved in ligand binding or coordination of calcium ion (Fass et al., 1997). In LRP12 protein, the first CUB domain has all four conserved cysteine residues, whereas the second domain has only two, and these are involved in one disulphide bond. The presence of both CUB domains and ligand-binding repeats in the extracellular domain of LRP12 protein indicates that LRP12 may bind ligand(s) to its extracellular domain. These differences between the extracellular domain of LRP12 protein and those of other receptors from LDLR family suggest that LRP12 protein has a distinct binding specificity. The cytoplasmic tail of LRP12 is larger than most of the other receptors from the LDRR family. It does not have the conserved NPxY internalization motif, but it has other potential internalization signals, including an YxxL motif and two di-leucine motifs. The structural characteristics strongly suggest that LRP12 is an endocytic receptor like many other LDLR family proteins. In order to understand the function of LRP12 protein, it will be important to identify the protein(s) that interacts with LRP12. The protein(s) can be expected to bind the 186 extracellular domain of LRP12 and be internalized or to bind and activate signaling pathway(s) transducing signals through the cell membrane. We report here that by using the Baculovirus Expression Vector System and Mass Spectrometry, several secreted proteins have been tenatively identified that interact with the extracellular domain of LRP12. These proteins have been reported to be the ligands of other LDLR family members (Hussain et al., 1999). Our data suggest that LRP12, like other LDLR proteins, binds proteins to its extracellular domain and may functions as an endocytic receptor. 187 ‘4 EXPERIMENTAL PROCEDURES Construction of Baculovirus Transfer Vectors: The expression vector encoding LRP12 (pCDNA6-LRP12) was constructed by M. Battle in the Carcinogenesis Laboratory. The sequence of LRP12 or the extracellular domain of LRP12 was obtained by a PCR reaction using the vector pCDNA6-LRP12 as the template and pfu polymerase (Strategen). The primers were designed so that the endonuclease restriction sites were available to introduce the PCR products of LRP12 into the multiple cloning sites (MCS) of transfer vector pAcGP67B directionally. The synthesized oligonucleotides encoding V5/His tag were annealed as followed: the same amount of oligonucleotides were incubated at 95°C for 5 min and then cooled to RT slowly. The annealed V5/I-Iis oligonucleotides were introduced into the 3’ end of LRP12 in the transfer vectors. The structures of transfer vectors pAcGP67B-LRP12ext-V5/His and pAcGP67B-LRP12fuu-V5/His were confirmed by DNA sequencing. The transfer vectors were then amplified in DHSa cells and purified with HiSpeed Plasmid Maxi Kit (Qiagen). DNA Sequencing: The DNA sequencing reaction was done following the protocol of Termo Sequenase Cy5.5 Dye Terminator Cycle Sequencing Kit (Amersham Pharrnacia Biotech). For each template to be sequenced, the following were mixed as the master mix: 2 pg purified DNA (transfer vectors), 3.5 pl reaction buffer, 2 pl primer (1 pmol/pl), 2 p1 ThermoSequenase DNA Polymerase (10 U/ pl) and distilled water to final volume 31.5 188 pl. 7 pl of the master mix was mixed with 1 pl of corresponding Cy5.5 ddNTP termination mix in a labeled tube and overlayed with mineral oil. The cycling program was set up according to the annealing temperature of individual promer. After the cycling program, samples were removed to new tubes and mixed with 2 pl of 7.5 M Ammonium Acetate acid and 30 p] of chilled 100% ethanol by vortexing. The tubes were placed on ice for 20 min to precipitate DNA. The precipitated DNA was collected by centrifugation at 12,000 rpm for 30 min at 4°C. The pellets were washed with 200 pl of chilled 70% ethanol and centrifuged for another 5 min. The pellets were dried for 5 min and resuspended in 6 pl of Formamide loading dye by vortexing. After being incubated at 95°C for 5 min, 2 pl of each sample was loaded in different lanes of a sequencing gel. Sequence data were collected and analyzed in MicroGene Clipper sequencer (Visible Genetics, Inc.) Generating Recombinant Baculovirus by Co-transfection: 2 x106 Sf9 cells were seeded onto each 60 mm tissue culture plate and incubated at 27°C for one hour to let cells attached to the plate. 0.5 pg BaculoGoldTM DNA and 5 pg recombinant Baculovirus Transfer Vector (pAcGP67B-LRP126xt-V5/His or pAcGP67B- LRPlZmu-VSIHis) were mixed in a microcentrifuge tube by flicking the tube. The mixture was incubated for 5 min before adding 1 ml of Transfection Buffer B. The old medium was removed from the cells and replaced with 1 ml of Transfection Buffer A. 1 ml of Transfection Buffer B/DNA solution was added to the plate drop by drop. After every 3—5 drops, the plate was gently rock back and forth to mix the drops with the medium. The plate was then incubated at 27°C for four hours. After the incubation, the 189 medium was removed from the plate. After washing the plate with 3 m] fresh TNM-FH medium, 3 ml of fresh TNM-FH medium was added to the plate and the plate was incubated in a moisturized container at 27°C for 5 days. Plaque Assay: 2 x106 Sf9 cells were seeded on each 60 mm plate and allowed to attach firmly to the plate (1 hour). After the incubation, the medium was replaced with 4 ml fresh TNM-FH. The viral transfection supernatant was added to three plates with 1:1, 1:10 and 1:100 dilution. The plates were incubated at 27 °C for 30 min. A 2% agarose solution was prepared using Agarplaque-PlusTM Agarose (low melting point agarose) in protein-free medium by heat the solution in a microwave until the agarose is dissolved. The solution was cool to 37°C in water bath and mixed with the same volume of pre-warrned TNM- FH medium to get the final agarose solution of 1%. The medium was then removed from plates and cells were overlayed with 4 m] of the prepared Agarplaque-PlusTM Agarose solution by carefully adding agarose to the side of the tilted plate. After the agarose solidified, the plates were incubated in a humid atmosphere at 27°C until visible plaques developed. Cell Culture Cell strains derived from the human fibrosarma cell line, SHAC, were grown in Eagle’s minimal essential medium supplemented with 0.2 mM L-aspartic acid, 0.2 mM L-serine, and 1.0 mM sodium pyruvate, and containing 10% (v/v) supplemented calf serum, 100 units/mL penicillin, 100 ug/ml streptomycin, and 1 ug/ml hydrocortisone. Cells were 190 incubated in a 37 °C humidified incubator with 5% C02 in air. Insect sf-9 cells were cultured in Sf-900H SFM supplemented with Antimyotic (GIBCO) at 27 °C. Expression and purification of LRP12 using the Baculovirus Expression Vector System Sf—9 cells were infected with baculoviruses encoding LRP12-V5. 72 h after infection, culture mediaum were collected and incubated with Ni-NTA agarose beads overnight at 4 °C. Beads were washed with purification washing buffer (50mM NaHZPO4, 300mM NaC], ZOmM Irnidzole, pH 8.0) and purified protein was eluted in elution buffer (50mM NaHZPO4, 300mM NaCl, ZSOmM Imidzole, pH 8.0). Immunoblot Analysis of LRP12 expression 72 h after sf9 cells were infected with recombinant Baculoviruses, cell lysates and medium were separated in 8% SDS-polyacrylamide gel and transferred onto Irnmobilon- P PVDF membrane (Millipore, Massachusetts). LRP12s were detected with an anti-V5 antibody followed by HRP-conjugated secondary antibody and electrochemical luminescence (Pierce). Ligand pull-down assay Purified LRP12 protein on the Ni-NTA agarose beads was incubated with concentrated conditioned medium overnight. After washing, proteins bound to the purified LRP12 protein were eluted with 2M N aCl. 191 Silver staining Proteins eluted from the purified LRP12 protein were separated in 8% SDS- polyacrylamide gel. The gel was stained using the SilverQuest silver staining kit (Invitrogen) following the manufacturer’s basic staining protocol. Mass spectrometry analysis Gel slices of interest were cut out, de-stained, trypsin-digested, and analyzed by mass spectrometry by the Mass Spectrometry Facility at Michigan State University. 192 RESULTS Construct the recombinant baculovirus. To prepare baculoviruses containing the LRP12 genes, 1 subcloned the extracellular domain or full-length LRP12, tagged with V5 and His epitopes, into the transfer vector pAcGB67B. Their structures were confirmed by DNA sequencing. To generate the recombinant viruses, recombinant transfer vectors were co-transfected into insect cell line Sf9 cells, together with the linearized baculovirus genome DNA (BaculoGold DNA, PharMingen). After five days, the culture media containing the recombinant viruses were collected. A plaque assay was used to isolate single recombinant viruses encoding the LRP12ext-V5/His or LRP12full-V5/His protein. Isolated recombinant viruses were amplified to generate a high titer virus stock solution for further research. The recombinant viruses in the collected medium were also used to infect Sf9 cells to confirm the expression of recombinant LRP12 proteins. The infected cells were harvested. Expression of LRP12 proteins in the culture medium and cell lysate were detected by Western blotting analysis using the anti-V5 antibody. The results showed that both of the LRP12ext-V5/His or LRP12full-V5/His proteins were expressed in the infected insect cells. This indicates that the recombinant viruses were successfully generated. The result also showed that LRP12ext-V5/His protein, but not LRP12full-V5/His protein, is secreted into the culture medium (Fig 1, compare lane 1 and 2). Ligand pull-down assay 193 A If Because most ligands of LRP], the well-characterized member of the LDLR family, have been found in the cell culture medium and/or bound to the external cell surface, we presumed that the extracellular ligand(s) of LRP12 would also exist in the cell culture medium or on the cell surface (Howell and Herz, 2001). We predicted that the lignad(s) of LRP12 will be enriched in the conditioned medium of cells with low level of endogenous LRP12, such as SHAC. To identify the ligand(s) of LRP12, the purified LRP12 protein bound to Ni-NTA Agarose beads was incubated with conditioned medium from SHAC cells. The bound proteins were eluted with 2M NaCl. The eluted solution were concentrated and analyzed by SDS-PAGE. Silver staining of the polyacrylamide gel showed that compared to the negative control, several unique protein bands were found when purified LRP12 protein were utilized to bind ligands (Figure 2A). Mass spectrometry analysis Three protein bands, which were specifically pulled down by LRP12-agarose, but not by the negative control, were subjected to trypsin digestion and Mass Spectrometry analysis (with the assistance of Rhonda Hussain from the Mass Spectrometry Facility at Michigan State University). The proteins dentified were compared to the ligands of other LDLR family members, especially those of LRPl. Among the ligands of LRPl, a2- macroglobulin (0.2M), pregnancy zone protein (PZP) and complement component 3 (C3) were identified in an experiment (Howell and Herz, 2001). Apolipoprotein A-l (Apo A-l) was also identified (Figure 1B). Apo A-1 is not a ligand for any LDLR family members, but other apolipoproteins, such as Apo B and Apo B, have been identified as ligands for other LDLR family members (Hussain et al., 1999). This experiment strongly suggests 194 that the extracellular domain of LRP12 interacts with proteins that have been identified as ligands for another well-studied LDLR family member LRP] (Howell and Herz, 2001). 195 DISCUSSION LRP12 was identified in the Carcinogenesis Laboratory as a potential tumor suppressor gene (Qing et al., 1999). LRP12 is homologous to LRP3 and LRP9, two receptors of the LDLR gene family. They constitute a unique subgroup in the LDLR family (Ishii et al., 1998; Sugiyama et al., 2000; Battle et al., 2003). The LDLR family members are involved in the cellular uptake of various extracellular ligands for degradation in the lysosome (Hussain et al., 1999). Some members of this family are also involved in cellular signaling by binding extracellular ligands and cytoplasmic adaptor proteins (Gotthardt et al., 2000; Herz, 2001). LRP12 shares with other members of LDLR family conserved ligand-binding repeats in its extracellular domain. Its extracellular domain also has another structural motif involved in protein- protein interaction: CUB domain. Recent work in the Carcinogenesis Laboratory identified several adaptor proteins that can bind to the cytoplasmic tail of LRP12 (M. Battle, unpublished data). Taken together, this data suggests that LRP12 can bind extracellular ligand(s) and induce endocytosis and/or signaling transduction. The linearized baculovirus genome DNA used in this experiment, the BaculoGold DNA (Pharmaingen), is engineered such that a sequence required for virus survival is deleted. After being co-transfected into insect cells with the recombinant transfer vector, homologous recombination will introduce the target gene as well as this essential sequence into the viral genome. This will make the recombinant virus viable. This 196 _l strategy ensure ~90% viable viruses are recombinant viruses after co-transfection. The expression of the recombinant LRP12 proteins in the insect cells (Figure 2) showed that the recombinant viruses encoding LRP12ext-V5/His and LRP12full-V5/His were successfully constructed. The gplO secretion signals fused to the N-terminus of the recombinant proteins will cause the synthesized proteins to be secreted into the medium. The results showed that about one third of LRP12ext-V5/His protein is secreted into the medium, but almost all LRP12full-V5/His protein exists in the cell lysate. One possible explanation is that LRP12 protein has a transmembrane domain which will localize the newly synthesized proteins in the plasma membrane preventing its secretion into the medium. aZM, PZP and C3 are members of the aZM superfamily. (12M and PZP are strongly homologous. they function as inhibitors of a variety of proteinases and can be cleaved by these proteinases. Once cleaved, a2M and PZP undergo conformational changes allowing them to bind convalently to the proteinases. This process also exposes the receptor recognition sites, which enables the proteinase-inhibitor complexes to bind LRP]. LRP] mediates the rapid internalization and degradation of aZM- and PZP-proteinase complexes, thereby regulating the proteinases activity on the external surface of the cell membrane by receptor-mediated endocytosis (Mikhailenko et al., 1995; Howell and Herz, 2001). C3 is a key molecule of the complement system. It also can be cleaved and undergo comformational changes to become activated. Activated C3 is a ligand of LRP] and it is internalized by receptor-mediated endocytosis (Meilinger et al., 1999). 197 .1? Bovine Apo A-l, which was identified in this experiment, is highly homologous to human Apo A-1 (78% identity and 87% similarity). It is very likely that human Apo A-l also binds to LRP12. Apo A-lis a ligand for Cubulin, which is mainly composed of multiple CUB domains(Kozyraki et al., 1999). CUB domains are also present in the extracellular domain of LRP12 protein. These reports suggest that Apo A-l bind to LRP12, but not other LDLR receptors, because of the presence of CUB domains. Apo A-l is the main protein in high-density lipoprotein (HDL). HDL is an important mediator of reverse cholesterol transport, which regulates plasma cholesterol levels by mediating liver uptake of cholesterol. Both Apo A-1 and HDL have protective effect against atherosclerosis (Shah et al., 2001). It is interesting that the low-density lipoprotein receptor (LDLR) controls the plasma cholesterol levels by mediating the endocytosis of low—density lipoprotein (LDL) particles. This endocytosis is mediated by the interaction between LDLR and Apo B (Hussain, 2001). If it is true that LRP12 is an endocytic receptor of Apo A-1 and mediates the endocytosis of HDL, it could be an additional mechanism for regulating the plasma cholesterol levels and it may play a role in the pathology of atherosclerosis. Identification of a2M, PZP, C3, and Apo A-l strongly suggests that the extracellular domain of LRP12 can associate with these proteins. Combined with the fact that LRP12 has several internalization signals in the cytoplasmic tail, it is very likely that LRP12 can mediate the internalization of these proteins. This is further supported by the reports that these four proteins are ligands for LRP], one of the well-characterized members of the 198 LDLR family. Taken together, our data suggest that LRP12 is a endocytic receptor sharing ligands with other LDLR members. 199 Future Work 1. Determine the interaction between LRP12 and putative ligands. Purified (12M, C3 and Apo A-l are commercially available. These proteins will be iodinated with IODO—BEADS (Pierce) according the manufacturer's protocol. These iodinated proteins will be incubated individually with SHAC cells expressing LRP12- V5/His (02 and X2) in serum-free media at 4°C and then treated with DSS, a cross- linking reagent. The cell lysate will be subjected to immunoprecipitation using anti-V5 antibody. The presence of 125I-ligand-LRPIZ complex will be analyzed by SDS-PAGE and autoradiography. 2. Determine if LRP12 binds the putative ligands on cell surface. These iodinated putative ligands will be incubated individually with SHAC cells expressing LRP12-V5/His (02 and X2) in serum-free media at 4°C, with or without the presence of 100 fold more unlabeled putative ligands. The cells will be washed before collecting the cell lysate. The binding of iodinated putative ligands to LRP12 will be measured by scintillation counting of cell lysate. This assay will be done with appropriate controls, e. g. SHAC cells transfected with empty vector. 3. Determine if LRP12 mediates the internalization of the putative ligands. The same experiment as described above (La. and 1b.) will be carried out, except that this time the cells will be incubated at 37°C after the initial treatment with the iodinated putative ligands at 4°C. Cell lysates and the overlaying culture medium will be collected 200 at different time points over a 1-4 hour period. Proteins in the medium will be precipitated by addition of bovine serum albumin to lOmg/ml and trichloroacetic acid (TCA) to 10%. The TCA-soluble fraction and the cell lysates will be analyzed by scintillation counting. If the internalization and degradation take place, the number of counts in the cell lysates and the TCA-soluble fraction of the medium will be found to increase as the iodinated protein(s) is internalized and degraded in the cells and the degraded peptides are released into the medium. To determine if a lysosomal pathway is used in the ligand degradation, a parallel experiment will be carried out the same way as above except for the addition of IOOmM chloroquine, an inhibitor of the lysosomal proteases. These assays performed with appropriate controls (e.g. SHAC cells transfected with empty vector) will help us determine if LRP12 is the endocytic receptor for (12M, C3 and Apo A-l. 20] If REFERENCE Battle, M. A.,Maher, V. M. and McCormick, J. J. (2003). ST7 is a novel low-density lipoprotein receptor-related protein (LRP) with a cytoplasmic tail that interacts with proteins related to signal transduction pathways. Biochemistry 42(24): 7270-82. Bieri, S.,Djordjevic, J. T.,Jamshidi, N.,Smith, R. and Kroon, P. A. (1995). Expression and disulfide-bond connectivity of the second 1i gand-binding repeat of the human LDL receptor. FEBS Lett 371(3): 341-4. Fass, D.,Blacklow, S.,Kim, P. S. and Berger, J. M. (1997). Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module. Nature 388(6643): 691-3. Gotthardt, M.,Trommsdorff, M.,Nevitt, M. F.,Shelton, J .,Richardson, J. A.,Stockinger, W.,Nimpf, J. and Herz, J. (2000). Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction. J Biol Chem 275(33): 25616-24. Herz, J. (2001). The LDL receptor gene family: (un)expected signal transducers in the brain. Neuron 29(3): 571-81. Howell, B. W. and Herz, J. (2001). The LDL receptor gene family: signaling functions during development. Curr Opin Neurobiol 11(1): 74-81. Hussain, M. M. (2001). Structural, biochemical and signaling properties of the low- density lipoprotein receptor gene family. Front Biosci 6: D4l7-28. Hussain, M. M.,Strickland, D. K. and Bakillah, A. (1999). The mammalian low-density lipoprotein receptor family. Annu Rev Nutr 19: 141-72. Ishii, H.,Kim, D. H.,Fujita, T.,Endo, Y.,Saeki, S. and Yamamoto, T. T. (1998). cDNA cloning of a new low-density lipoprotein receptor-related protein and mapping of its gene (LRP3) to chromosome bands 19q12-q13. 2. Genomics 51(1): 132-5. Kozyraki, R.,Fyfe, J .,Kristiansen, M.,Gerdes, C.,Jacobsen, C.,Cui, S.,Christensen, E. I.,Arrrinoff, M.,de la Chapelle, A.,Krahe, R.,Verroust, P. J. and Moestrup, S. K. (1999). The intrinsic factor-vitamin BlZ receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of hi gh-density lipoprotein. Nat Med 5(6): 656-61. Lynn, E. G.,Siow, Y. L. and O, K. (2000). Very low-density lipoprotein stimulates the expression of monocyte chemoattractant protein-1 in mesangial cells. Kidney Int 57(4): 1472-83. 202 ————v‘—-" Meilinger, M.,Gschwentner, C.,Burger, I.,Haumer, M.,Wahrmann, M.,Szollar, L.,Nimpf, J. and Huettinger, M. (1999). Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)—macroglobulin receptor. J Biol Chem 274(53): 38091-6. Mikhailenko, I.,Kounnas, M. Z. and Strickland, D. K. (1995). Low density lipoprotein receptor-related protein/alpha Z-macroglobulin receptor mediates the cellular internalization and degradation of thrombospondin. A process facilitated by cell-surface proteoglycans. J Biol Chem 270(16): 9543-9. Qing, J .,Wei, D.,Maher, V. M. and McCormick, J. J. (1999). Cloning and characterization of a novel gene encoding a putative transmembrane protein with altered expression in some human transformed and tumor-derived cell lines. Oncogene 18(2): 335-42. Shah, P. K.,Kaul, S.,Nilsson, J. and Cercek, B. (2001). Exploiting the vascular protective effects of hi gh-density lipoprotein and its apolipoproteins: an idea whose time for testing is coming, part I. Circulation 104(19): 2376-83. Sugiyama, T.,Kumagai, H.,Morikawa, Y.,Wada, Y.,Sugiyama, A.,Yasuda, K.,Yokoi, N.,Tamura, S.,Kojima, T.,Nosaka, T.,Senba, E.,Kimura, S.,Kadowaki, T.,Kodama, T. and Kitamura, T. (2000). A novel low-density lipoprotein receptor-related protein mediating cellular uptake of apolipoprotein E-enriched beta-VLDL in vitro. Biochemistry 39(51): 15817-25. 203 Figure Legends Figure 1. Expression and secretion of the LRP12ext-V5/His and LRP12full-V5/I-Iis. The recombinant viruses encoding LRP12ext-V5/His and LRP12full-V5/His were used to infect insect sf9 cells. 72 h after infection, culture medium and cell lysate were separated in 8% polyacrylamide gel and LRP12 proteins were detected by Western blotting using anti-V5 andtibody. The result showed that about one third of LRP12ext- V5/His protein is secreted into the medium, but almost all LRP12full-V5/His protein exists in the cell lysate. One possible reason is that LRP12 protein is a membrane protein. The transmembrane domain will locate the synthesized proteins in the plasma membrane instead of being secreted into the medium. 204 . +LRP12fuu-V5/His (B) ram '7' <— LRP12ext-V5/His (A) medium lysate Figure 1. 205 Figure 2. Identification of proteins interacting with the extracellular domain of LRP12. (A): The purified extracellular domain of LRP12 was incubated with the conditioned medium from SHAC cells expressing low levels of LRP12. Bound proteins were eluted with high-salt solution, seperated by SDS-PAGE and detected by silver staining. Compared to negative control, three unique protein bands were pulled down by LRP12 specifically as indicated. These three gel slices were cut out for trypsin digestion and Mass Spectrometry anaysis was carried out. (B): Proteins identified by Mass Spectrometry. Four proteins were identified from corresponding gel slices as indicated. 206 a. a 2 Macroglobulin ( a 2M) Pregnancy zone protein (PZP) “1.11"... 1"- ? \‘Q’w. g b. .f, Complement component 03 (C3) c. Apolipoprotein A-1 (Apo A-l) .. 41.3.} H ‘_ C LRP12 - + Figure 2. 207 Chapter IV Proposed future study of LRP12 Accomplishment of my project: TGF-B1 initiates the TGF-B/Smad signaling pathway by binding to TGF-B receptors on the external surface of the cell membrane, which results in the phosphorylation and nuclear translocation of Smad2 and Smad3. SARA promotes the TGF-B-induced Smad2/3 phosphorylation by preventing the nuclear translocation of Smad2/3, which ensures the their quick access to the activated TBRI, and by directly recruiting Smad2/3 to the activated TBRI (T sukazaki et al., 1998). A previous study in the Carcinogenesis Laboratory has shown that LRP12 interacted with a truncated form of SARA (Battle et al., 2003), which suggested that LRP12 was involved in the TGF-B signaling pathway. To determine whether LRP12 participates in the TGF-B/Smad signaling pathway, I investigated TGF-Bl-induced phosphorylation of Smad2 and Smad3 in human fibrosarcoma-derived SHAC cells stably transfected with an empty vector or a LRP12 expressing construct. The results of Western blotting analysis and luciferase assays indicate that LRP12 participates in the TGF-B/Smad signaling pathway by increasing the TGF-Bl-induced Smad2 and Smad3 phosphorylation. To determine the mechanisms through which LRP12 modulates the TGF-B/Smad signaling pathway, I determined that the exposure of SHAC cells expressing LRP12 to TGF-B1 induced Smad2/3 phosphorylation which depends on the presence of the LRP12 cytoplasmic tail and its 208 “V —‘|-J' interaction with SARA and/or Smurf2-Smad7 complex (Discussed in Chapter H). A . truncated LRP12 containing only the 10 arrrino acids adjacent to the internal side of the cell membrane, out of the 343 amino acids of the cytoplasmic tail, did not associate with SARA and Smurf2, nor did it increase the TGF-Bl-induced Smad2 phosporylation. What is more, using immunoblotting analysis, I found that expression of LRP12 significantly decreases the ubiquitination of TGF-B receptor type I (TBRI) and activated Smad2. These results indicate that LRP12 participates in the TGF-B/Smad signaling pathway through its interaction with SARA and/or the Smurf2-Smad7 complex. Qing et a1. (1999) found that LRP12 is a putative tumor suppressor gene. The TGF-B signaling pathway has been considered as both a tumor suppressor inhibiting tumor cell proliferation, and as a tumor promoter increasing tumor cell progression and invasion (Derynck et al., 2001). I hypothesized that expression of LRP12 in tumor cells with low level of endogenous LRP12 either restored the tumor-suppressing function by inhibiting cell proliferation, or suppressing the pro-oncogenic effects by increasing tumor cell migration and invasion (Discussed in Chapter I). I demonstrated that LRP12 inhibits the ability of SHAC cells to migrate and invade by enhancing the TGF-Bl induction of PAI-l expression. LRP12 expression had no effect on cell roliferation. Therefore, it is not surprising that LRP12 was discovered because of its greatly reduced expression in a human fibrosarcoma cell line, compared to that of its parental non-tumorigenic cell line (Qing et al., 1999). 209 Taken together, my research is the first to show that LRP12 augments the TGF-B/Smads signal transduction. The interactions I detected between LRP12 and SARA, as well as LRP12 and Smurf2-Smad7, provide detailed evidence demonstrating the involvement of LRP12 in the TGF-B/Smad signaling pathway. The data also indicate that LRP12 inhibits cell migration and invasion by means of enhanced activation of the TGF-B/Smad signaling pathway. As is often the case in science, my studies raise new questions regarding the function of LRP12. If I ware continuing this study, I would carry out experiments to characterize how LRP12 participates in the TGF-B/Smad signaling pathway and functions as an endocytic receptor. I outline these experiments below. What are the molecular mechanisms of the LRP12-SARA and the LRP12- Smurfl/Smad7 interactions? Hypothesis: Interrupting the LRP12-SARA and/or LRP12-Smurf2/Smad7 interactions will abrogate the increased TGF-Bl-induced Smad2 and Smad3 phosphorylation by LRP12. Aim: To determine the amino acids that mediate the interactions between LRP12 and SARA as well as Smurf2/Smad7 using deletion or site-directed mutations and to further determine the effects of these mutations on the TGF-B/Smad signaling pathway. One question to further expand the current project is whether the interaction between LRP12 and SARA or the interaction between LRP12 and Smurf2/Smad7 plays a more 210 important role for the function LRP12 in the TGF-B/Smad signaling pathway. To answer this question, the detailed molecular basis of the LRP12-SARA and the LRP12- Smurf2/Smad7 interactions must be investigated. Battle et al. (2003) determined that the interaction between LRP12 and SARA is mediated by the 62 amino acids most adjacent to the cell membrane of LRPlZ’s cytoplasmic tail (343 amino acids) and C-terminus of SARA (amino acids 731-C- terminus). My project first reports the interaction between LRP12 and the Smurf2/Smad7 complex. However, I failed to determine whether the interaction was mediated by Smurf2, Smad7, or both of the proteins. Deletion and site-directed mutagenesis of amino acids with potential importance for protein-protein interaction could be used to generate mutant proteins. Then the mutations abrogating the LRP12-SARA or LRP12-Smurf2/Smad7 . interaction could be determined by co-transfection in human embryonic kidney 293 cells and co-immunoprecipitation assays. Finally, the cell strains stably expressing protein mutants that interrupt the LRP12-SARA or LRP12-Smurf2/Smad7 interaction could be tested to determine if abrogating these interactions has an effect on TGF-Bl-induced Smad2 and Smad3 phosphorylation. While these experiments would be time-consuming and costly, they are the most accurate way to provide information of the LRP12-SARA and LRP12-Smurf2/Smad7 interactions and to determine the mechanism(s) through which LRP12 participates in the TGF- B/Smad signaling pathway. 21] Does down-regulation of LRP12 in non-tumorigenic cells by siRNA increase tumorigenicity? Hypothesis: Decreasing the protein level of LRP12 by siRNA within cells will interrupt the TGF-B/Smad signaling pathway and increases the tumorigenicity in athynric mice. Aim: To stably transfect cells with siRNA against LRP12 to down-regulate LRP12 protein level and to test TGF-Bl-induced Smad2/3 phosphorylation and tumorigenicty in athymic mice. A confirmatory experiment to determine the role of LRP12 in the TGF—B/Smad signaling pathway and tumori genesis would be to down-regulate the protein level of LRP12 in non- tumorigenic cells, such as MSU1.1 cells. However, lack of working LRP12 antibody makes MSU1.1 an unfeasible choice. SHAC cells stably expressing LRP12-V5 could be used is this study. Dr. Battle demonstrated that SHAC cell strains stably transfected with the LRP12-V5 expressing construct, the same cell strains 1 used in my project, had a much lower frequency of tumor formation in athymic mice compared to vector control cells and parental cells (Unpublished data). The siRNA technique, which has been successfully employed by other researchers in the Carcinogensis Laboratory, could be used to down-regulate the LRP12 protein level. An expression vector encoding siRNA targeting LRP12 would be constructed containing puromycin resistance gene to allow selection. The LRP12-V5 expression construct used to generate the SHAC cell strains contains the Blasticidin-resistance gene. The siRNA construct would be stably transfected into SHAC cells expressing LRP12-V5 and cell 212 clones would be selected with both Blasticidin and puromycin. These clones would be screened for LRP12-V5 expression level by Northern blot and/or Western blot. Clones with a decreased LRP12-V5 expression level would be selected. Several experiments would be performed. First, a Western blotting analysis and/or a luciferase assay would be performed to determine if decreasing the LRP12 protein level abrogates the increased TGF-Bl-induced Smad2/3 phosphorylation observed when LRP12 was over-expressed. If so, the TGF-Bl-induced PAI-l expression, cell migration and invasion ability would be tested to determine if decreasing the LRP12 expression level leads to decreased PAI-l expression, increased cell migration and invasion. If this occurred, it would allow us to definitely conclude that expression of LRP12 was responsible for the increased TGF-B/Smad signaling pathway as well as the decreased cell migration and invasion. The cells with down-regulated LRP12 expression level by siRNA could also be injected subcutanteously into athymic mice to study if lost of LRP12 expression leads to increased tumorigenicity. Is LRP12 an endocytic receptor? Hypothesis: LRP12 is an endocytic receptor that mediates the internalization of its ligands from the cell surface and their degradation in lysosomes. Aim: To incubate SHAC cells expressing or not expressing LRP12 with I25I-labeled coulddidate ligands and determine the internalization and degradation of these proteins. 213 LRP12 is a member of the low-density lipoprotein receptor (LDLR) family. Many proteins of the low-density lipoprotein receptor (LDLR) family are best known as endocytic receptors. The extracellular domains of these proteins bind various extracellular ligands, including lipoproteins, proteinases, proteinases-inhibitor complexes, extracelluar matrix (ECM) components and etc. By binding these ligands, LDLR proteins mediate their interanlization for degradation in the lysosome. In an effort to investigate if LRP12 is an endocytic receptor, I identified aZ-macroglobulin (aZ-M), pregnancy zone protein (PZP), complement component 3 (C3), and Apolipoprotein A-l (Apo A-l) as the potential ligands of LRP12 (Discussed in Chapter H). However, whether LRP12 mediates the internalization and degradation of these potential ligands is not clear. During the assay, SHAC cells expressing or not expressing exogenous LRP12 would be incubated at 4° for one hour in serum-free medium containing 125I-labeled candidate ligand, followed by incubation at 37°C. Cell lysates and the overlaying culture medium would be collected at different time points over a 1-4 hour period. Proteins in the medium would be precipitated by addition of bovine serum albumin to 10mg/ml and trichloroacetic acid (TCA) to 10%. The TCA-soluble fraction and the cell lysates would be counted in a gamma counter. If the internalization and degradation take place in SHAC cells expressing exogenous LRP12, the number of counts in the cell lysates and the TCA-soluble fraction of the medium would be increased as the iodinated protein(s) is internalized and degraded in the cells, and the degraded peptides would be released into the medium. Each of the four potential ligands would be tested in this experiment. It is possible that all of these proteins, or some of them, or perhaps none of them would be 214 internalized by LRP12 protein. To determine if a lysosomal pathway is used in the degradation, 3 parallel experiment would be carried out the same way as above except for the addition of 100mM chloroquine, an inhibitor of the lysosomal proteases (Hahn- Dantona et al., 2001). Is LRP12 involved in SARA-regulated endosomal trafficking? Hypothesis: By interacting with SARA, LRP12 participates in the SARA-regulated endosomal trafficking. Aim: To express the mutant SARA that can not interact with LRP12 to study TGF-Bl- induced Smad2/3 activation and Rab5-dependent endosomal trafficking. SARA has been reported to play an important functional role in Rab5-regualted endosomal trafficking (Hu et al., 2002). Ectopic expression of SARA induces enlargement of endosomes (Hu et al., 2002) and enhances TGF-B-induced Smad activation (Itoh et al., 2002). These data suggest that SARA acts as the functional link between signal transduction and endosomal trafficking. Indeed, the best characterized function of the other FYVE-containing proteins is membrane trafficking (Gillooly et al., 2001). Hepatic growth factor-regulated tyrosine kinase substrate (Hgs) is a FYVE- containing protein that shares little sequence similarity to SARA Mura et al., 2000). However, it has a similar function as SARA. Hgs interacts with Smad2/3 and recruits the proteins to the activated TGF-B receptors. In addition to the function in the TGF-B signaling pathway, Hgs is better known as protein regulating intracellular membrane trafficking by recruiting clathrin to early endosomes (Raiborg et al., 2001). Endofin, 215 another FYVE domain-containing protein, is closely related to SARA (Seet and hong, 2001). Unlike SARA, Endofin does not interact with Smad2/3. Instead, Endofin regulates endosomal trafficking in a similar manner of SARA and recruits clathrin to early endosomes (Seet and Hong, 2005). These reports suggest that SARA may play an important role in regulating endosomal trafficking, and in this way regulating the TGF- B/Smad signaling pathway. Whether the interaction between LRP12 and SARA is required for this function is not clear. In this study, SARA mutant lacking the ability to associate with LRP12 would be transfected in cells. Endosoma] enlargement and TGF-Bl-induced Smad2/3 activation in these cells would be compared with those observed in cells transfected with wild-type SARA to determine whether abrogation of LRP12-SARA interaction leads to the lost of SARA-dependent endosomal enlargement and TGF-Bl-induced Smad2/3 activation. If this occurred, the same study would be carried out with the co-transfection of SARA and LRP12 with mutations of potential internalization signals on the cytoplasmic tail. This study would determine if the internalization of LRP12 is required to regulate SARA- dependent endosomal enlargement and TGF-Bl-induced Smad2/3 activation. If so, it would allow us to conclude that the internalization of LRP12 and its interaction with SARA is required for SARA to regulate endosomal trafficking and through this to regulate the TGF-B/Smad signaling pathway. This would indicate that in addition to SARA, LRP12 also plays a role as the functional link between endocytosis and TGF-B signaling. 216 REFERENCE Battle, M. A.,Maher, V. M. and McCormick, J. J. (2003). ST7 is a novel low-density lipoprotein receptor-related protein (LRP) with a cytoplasmic tail that interacts with proteins related to signal transduction pathways. Biochemistry 42(24): 7270-82. Derynck, R.,Akhurst, R. J. and Balmain, A. (2001). TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 29(2): 117-29. Gillooly, D. J .,Simonsen, A. and Stenmark, H. (2001). Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins. Biochem J 355(Pt 2): 249- 58. Hahn-Dantona, E.,Ruiz, J. F.,Bomstein, P. and Strickland, D. K. (2001). The low density lipoprotein receptor-related protein modulates levels of matrix metalloproteinase 9 (MMP-9) by mediating its cellular catabolism. J Biol Chem 276(18): 15498-503. Hu, Y.,Chuang, J. Z.,Xu, K.,McGraw, T. G. and Sung, C. H. (2002). SARA, a FYVE domain protein, affects Rab5-mediated endocytosis. J Cell Sci 115(Pt 24): 4755-63. Itoh, F. ,Divecha, N .,Brocks, L.,Oomen, L.,Janssen, H.,Calafat, J .,Itoh, S. and Dijke Pt, P. (2002). The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/Smad signalling. Genes Cells 7(3): 321-31. Miura, S.,Takeshita, T.,Asao, H.,Kimura, Y.,Murata, K.,Sasaki, Y.,Hanai, J. I.,Beppu, H.,Tsukazaki, T.,Wrana, J. L.,Miyazono, K. and Sugamura, K. (2000). Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol Cell Biol 20(24): 9346-55. Qing, J .,Wei, D.,Maher, V. M. and McCormick, J. J. (1999). Cloning and characterization of a novel gene encoding a putative transmembrane protein with altered expression in some human transformed and tumor-derived cell lines. Oncogene 18(2): 335-42. Tsukazaki, T.,Chiang, T. A.,Davison, A. F.,Attisano, L. and Wrana, J. L. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell 95(6): 779-91. 217 rslt . s. 551%... rest... 3..