ANGIOTENSIN 1 - 7/ MAS PROMOTES ALVEOLAR EPITHELIAL CELL SURVIVAL THROUGH UPREGULATION OF MAP KINASE PHOSPHATASE - 2 By Indiwari Gopallawa A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology Doctor of Philosophy 2015 ABSTRACT ANGIOTENSIN 1 - 7/ MAS PROMOTES ALVEOLAR EPITHELIAL CELL SURVIVAL THROUGH UPREGULATION OF MAP KINASE PHOSPHATASE - 2 By Indiwari Gopallawa Apoptosis is now known to be an important regulator of maintaining normal organ homeostasis. However, in recent years experimental studies support the concept that excessive alveolar epithelial cell (AEC) apoptosis contributes to pathogenic conditions in lung. Studies show that local activation of angiotensin system (ANG) in the lung plays a major role in AEC apoptosis. Autocrine generation of angiotensin II ( ANGII ) an effector peptide, initiates AEC apoptosis through AT1 rec eptor, phosphorylating c - Jun - N - terminal kinase (pJNK), both of which are required events in AEC apoptosis. Angiotensin converting enzyme - 2 (ACE - 2) is a vital enzyme that converts ANGII to angiotensin 1 - 7 ( ANG1 - 7 ), promoting cell survival by limiting the accumulation of ANGII . Although the downstream signaling mechanisms of ANG1 - 7 / Mas are unclear, experimental studies have shown anti - apoptotic effects of ANG1 - 7 in AECs. In this study, the molecular mechanisms by which ANG1 - 7 and its receptor Mas promote AEC survival are invest igated. Previous studies from the Uhal laboratory indicated that under normal conditions ANG1 - 7 levels are higher than ANGII levels in the AEC culture. Hence, it was theorized that ANG1 - 7 activates a map kinase phosphatase - 2 (MKP - 2) and maintains low pJNK levels, as a cell survival mechanism in AECs. The data show blockade of the Mas receptor diminished the induction of MKP - 2 by ANG1 - 7 which confirmed that Mas acts through MKP - 2. Further, silencing MKP - 2 abolished the ability of ANG1 - 7 to block ANGII - induce d phospho - JNK and apoptosis. Silencing of MKP - 2 significantly prevented the blockade of all apoptotic markers such as caspase - 9, loss of mitochondrial membrane potential (MMP) and DNA fragmentation by ANG1 - 7 . These data support the theory that ANG1 - 7 upreg ulates the phosphatase MKP - 2 through Mas and thereby maintains low phospho - JNK levels to promote AEC survival. In conclusion, this study implies that ANG1 - 7 / Mas activation inhibits JNK phosphorylation and apoptosis by constitutively activating MKP - 2, and further demonstrates the critical role of the ANG1 - 7 receptor Mas in AEC survival. Copyright by INDIWARI GOPALLAWA 2015 v ACKNOWLEDGEMENTS First and foremost I would like to express my sincere gratitude to my mentor Dr. Bruce Uhal who provided me the opportunity to be his graduate student and since early days he has continuously supported me. I want to express my special appreciation for his guidance, time, patience, scientific advice and most importantly, his insightful discussion s about the research. Dr. Uhal has been a great advisor to me and has always directed me towards developing and improving my research skills. Therefore , I would like to take this opportunity to immensely thank him for guiding me countless times during my g raduate school career, providing helpful recommendations and his moral support. I would also like to acknowledge my gui dance committee members, Dr. John LaPres, Dr. Karl Olson, Dr. Erik Martinez - Hackert and Dr. Kevin Walker for their support during my dis sertation research. I am grateful for their time, comments to improve my research, discussions and advice in general. I wish to thank the biochemistry and molecular biology program and the physiology d epartment for continuously supporting me. I also like to thank Dr. Jon K aguni, the graduate program director of biochemistry and molecular b iology for his support at numerous times. I want to express my thanks to Dr. Adele Denison for her helpful comments to improve my teaching skills and allowing me to teach her physiology class. cooperation over the years. I thank to all my friends in the Biochemistry, Physiology and vi Chemistry departments for all their support, encouragement and for the friendships that I needed throughout my graduate work. A very special thanks to my parents and my brother for all their understanding, support and care throughout these years. I am i ncredibly grateful for the encouragement and motivation that my parents provided along the way. Finally, this work is supported by HL - 45136. Many thanks to all those of you who stood by to support me. vii TABLE OF CONTENTS LIST OF FIGURES ................................ ................................ ................................ ........................... viii KEY TO ABBREVIATIONS ................................ ................................ ................................ .................. x CHAPTER 1: GENERAL INTRODUCTION ................................ ................................ .......................... 1 INTRODUCTION ................................ ................................ ................................ ........................... 2 Definition of Apoptosis ................................ ................................ ................................ ............ 2 Characteristics of Apoptosis ................................ ................................ ................................ .... 2 Major Apoptotic Signaling Path ways ................................ ................................ ...................... 3 Extrinsic Apoptotic Signaling Pathway ................................ ................................ ................ 3 Intrinsic Apoptotic Signaling Pathway ................................ ................................ ................. 4 ALVEOLAR EPITHELIAL CELLS ................................ ................................ ................................ ................ 5 Type I and Type II Cells ................................ ................................ ................................ ...................... 5 Alve olar Cell Defense Mechanisms ................................ ................................ ................................ .. 6 Apoptosis in the Lung Epithelium ................................ ................................ ................................ .... 8 Lung Epithelial Repair Mechanisms ................................ ................................ ................................ . 9 Epithelial to Mesenchymal Transition (EMT) ................................ ................................ ............... 11 RENIN ANGIOTENSIN SYSTEM (RAS) ................................ ................................ ................................ .. 12 Local Angiotensin (ANG) System ................................ ................................ ................................ .... 13 Angiotensin Receptors and Signaling ................................ ................................ ........................ 14 Angiotensin Converting Enzymes (ACE and ACE - 2) ................................ ................................ .. 15 ANG SYSTEM AND AEC APOPTOSIS ................................ ................................ ................................ .... 17 Blockade of Apoptosis in AECs ................................ ................................ ................................ ....... 19 ANG System in Heart ................................ ................................ ................................ ....................... 20 ANG System in Kidney ................................ ................................ ................................ ..................... 21 ANG Syst em in Vascular Smooth Muscle Cells (VSMCs) ................................ ............................. 22 ANGIOTENSIN 1 - 7 (ANG1 - 7)/ MAS RECEPTOR ................................ ................................ ................. 23 ANG1 - 7 Synthesis and Catabolism ................................ ................................ ................................ . 26 Inhibitory Actions of the ANG1 - 7/Mas Pathway on Pulmonary Injury ................................ ..... 27 Signaling Mechanisms Underlying ACE - 2/ANG1 - 7/Mas Action in Lung Cells .......................... 37 ACE - 2/ANG1 - 7/Mas in Non - Pulmonary Cells ................................ ................................ ............... 42 Novel Therapeutic Targets of ACE - 2/ANG1 - 7/Mas ................................ ................................ ..... 44 MITOGEN ACTIVATED PROTEIN KINASE (MAPK) SIGNALING ................................ ......................... 45 C - jun - N - terminal kinase (JNK) signaling ................................ ................................ ........................ 45 JNK Pathway and Diseases ................................ ................................ ................................ .............. 47 Map Kinase Phosphatases (MKPs) ................................ ................................ ................................ . 49 CHAPTER 2: ANGIOTENSIN 1 - 7/MAS INHIBITS APOPTOSIS IN ALVEOLAR EPITHELIAL CELLS THROUGH UPREGULATION OF MAP KINASE PHOSPHATAS E - 2 ................................ .................. 52 Abstract ................................ ................................ ................................ ................................ ............. 53 viii Introduction ................................ ................................ ................................ ................................ ...... 54 Materials and Me thods ................................ ................................ ................................ ................... 56 Results ................................ ................................ ................................ ................................ ................ 59 Discussion ................................ ................................ ................................ ................................ .......... 79 CHAPTER 3 : INVESTIGATION OF THE ROLE OF ANGIOTENSIN 1 - 7/ACE - 2 IN ALVEOLAR EPITHELIAL CELLS DURING ENDOPLASMIC RETICULUM STRESS AND HYPEROXIA .................... 8 5 Abstract ................................ ................................ ................................ ................................ ............. 86 Introduction ................................ ................................ ................................ ................................ ...... 87 Materials and Methods ................................ ................................ ................................ ................... 90 Results ................................ ................................ ................................ ................................ ................ 92 Discussion ................................ ................................ ................................ ................................ .......... 97 CHAPTER 4 : SUMMARY AND CONCLUSIONS ................................ ................................ ............ 102 SUMMARY ................................ ................................ ................................ ................................ ........... 103 Upregulation of Map Kinase Phosphatase - 2 in Alveolar Epithelial Cells ................................ 103 Role of Angiotensin 1 - 7/ACE - 2 in ER Stress and Hyperoxia ................................ ..................... 105 CONCLUSIONS ................................ ................................ ................................ ................................ ..... 107 REFERENCES ................................ ................................ ................................ ................................ 109 ix LIST OF FIGURES Figure 1 - 1 : Functions of type I and type II alveolar epithelial cells. 6 Figure 1 - 2 : Local angiotensin system in alveolar epithelial cells. 17 Figure 1 - 3 : Known signaling mechanisms in alveolar epithelial cells (AECs). .. 19 Figure 1 - 4 : Angiotensin 1 - 7 synthesis and catabolism. 27 Figure 1 - 5 : Known actions of ANG1 - 7 in lung injury. 37 Figure 1 - 6 : Downstream signaling pathways of the ANG1 - 7/Mas pathway in non - epithelial lung cell types. 42 Figure 2 - 7 : Mas blocker increases pJNK and decreases MKP - 2 in lung epithelial cells. 65 Figure 2 - 8 : ANG1 - 7 induces MKP - 2 in lung epithelial cells. 67 Figure 2 - 9 : Blockade of MKP - 2 induction by a mas receptor blocker. 68 Figure 2 - 10 : Mas knockdown prevents ANG1 - 7 induction of MKP - 2. 70 Figure 2 - 11 : Verification of MKP - 2 knockdown by small interfering RNA (siRNA) in A549 cells but not by a scrambled siRNA. 71 Figure 2 - 12 : MKP - 2 knockdown prevents inhibition of JNK phosphorylation by ANG1 - 7. .. 72 Figure 2 - 13 : MKP - 2 knockdown increases basal pJNK and caspase - 9 levels. 73 Figure 2 - 14 : Silencing MKP - 2 prevents ANG1 - 7 rescue of mitochondrial membrane potential (MMP). 74 Figure 2 - 15 : Silencing of MKP - 2 prevents inhibition of caspase - 9 activation, DNA and nuclear fragmentation by ANG1 - 7. 75 Figure 2 - 16 : Mas knockdown increases basal pJNK and caspa se - 9 levels. 77 Figure 2 - 17 : Mas knockdown induces caspase - 9 activation and apoptosis in lung epithelial cells. 78 x Figure 3 - 18 : Blockade of ER stress induced JNK phosphorylation by angiotensin 1 7 or by saralasin. 94 Figure 3 - 19 : ADAM17 blocker prevented the induction of soluble ACE - 2. 95 Figure 3 - 20 : Ectodomain shedding of angiotensin - converting enzyme - 2 (ACE - 2) in hyperoxia - induced lung injury. 96 xi KEY TO ABBREVIATIONS ACE Angiotensin Converting Enzyme ACE - 2 Angiotensin Converting Enzyme - 2 ACEi Angiotensin Converting Enzyme Inhibitor AEC Alveolar Epithelial Cell AGT Angiotensinogen AIF Apoptosis Inducing Factor ANG1 - 5 Angiotensin 1 - 5 ANG I Angiotensin I ANG1 - 7 Angiotensin 1 - 7 ANGII Angiotensin II ANG Angiotensin System ARB Angiotensin Receptor Blocker ATI Angiotensin Type I Receptor BH - 3 Bcl - 2 Homology cAMP Cyclic Adenosine Monophosphate DISC Death Induced Signaling Complex DUSP Dual Specific Phosphatase ECM Extra Cellular Matrix EMT Epithelial Mesenchymal Transition ER Endoplasmic Reticulum xii ERK Extra Cellular Regulated Kinase GPCR G - Protein Coupled Receptor HGF Hepatocyte Growth Factor IAP Inhibitor of Apoptosis ILD Interstitial Lung Disease JNK Jun N - terminal Kinase KGF Keratinocyte Growth Factor KIM Kinase Interacting Motif MAP3K MAPK Kinase Kinase MAPK Mitogen Activated Protein Kinase MKP - 2 Map Kinase Phosphatase - 2 MLK Mixed Lineage Kinase MMP Matrix Metalloproteinase MMP Mitochondrial Membrane Potential MOMP Mitochondrial Outer Membrane Potential NEP Neprilysin NF - Nuclear - factor kappa B PDGF Platelet Derived Growth Factor PEP Prolylendopeptidase PKC Protein Kinase C PLC Phospholipase C RAS Renin Angiotensin System xiii ROS Reactive Oxygen Species siRNA Small interfering RNA SP Surfactant Protein TACE TNF - verting Enzyme TGF Tumor Growth Factor TNF - Tumor Necrosis Factor Alpha VSMC Vascular Smooth Muscle Cell 1 CHAPTER 1 : GENERAL INTRODUCTION 2 I NTRODUCTION Definition of Apoptosis In recent years, excessive apoptosis of alveolar epithelial cells (AECs) has shown to contribute to progressive lung diseases such as idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI) and chronic obstructive pulmonary disease (COPD, Li, 2004; Uhal, 2003) . Apoptosis is a form of programmed cell death that has a fundamental role in maintaining homeost asis which is essential for normal organ development. It is an active , hi ghly regulated physiological process that removes unnecessary cells without initiating an immune response. Although apoptosis is beneficial to maintain normal physiological function, excessive cell death has been reported in many disease types. Therefo re, investigati on of the factors that control apoptosis is essential to gain insights in certain disease states. Characteristics of Apoptosis During apoptosis, eukaryotic cells undergo a series of morphological and bi ochemical characteristics that can be d ist inguished from normal cells . C el ls that undergo apoptosis are rapidly phagocytosed by macrophages witho ut dam aging the adjacent cells . In normal living cells phosphatidylserine (PS) resides in the inner leaflet of the lipid bilayer, and during early a poptosis, PS can be seen on the outer leaflet of the membrane (Mariño and Kroemer, 2013) . Apoptotic cells initially retain the integrity of the plasma membrane. However, during the early process of apoptosis, the membrane forms cell blebs which lead to decreased cell size. Extensive blebbing causes decreased cytoplasm, tightly packed organelles and formation of apoptotic bodies (Hotchkiss et al., 2009) . Activation of cysteine proteases known as caspases 3 pla y a major role in apoptosis which cleave other cellular proteins and activate endogenous DNases leading to DNA fragmentation (Saraste and Pulkki, 2000) . During the late apoptotic phase , nuclear fragments are commonly seen and this event is one of the m ost commonly measured hallmarks of apoptosis (Henson and Tuder, 2008) . Major Apoptotic Signaling Pathways Extrinsic Apoptotic Signaling Pathway The methodical events in cell death are controlled by an apoptotic signaling network, which includes an extrinsic and intrinsic pathway. The extrinsic pathway is activated when cytokines such as Fas ligand (FasL) and tumor necrosis factor (TNF - cell - death receptors. These death - receptors belong to the TNF - their cytosolic regions comprise a death domain. Once the death - receptors are bound by t heir ligands they form trimers. When the ligand binds to its receptor , the receptor is activated, recruiting factors s uch as Fas - associated death domain (FADD) and pro - caspases 8/10 that form the death in ducing signaling complex (DISC, Fattman, 2008) . The caspases are major players in initiating a proteolytic cascade. Caspases are generated in the cell as pro - caspases which are inactive precursors that are activated upon cleavage at aspartic acids by other already activated caspases. The initiator caspases , including caspase 8 and 10 are responsible for activating the e xecutioner caspases that can cleave cellular proteins. Autocatalytic activation of initiator caspase - 8/10 activate downstream effector caspase - 3/7 eliciting ultimate breakdown of the cell (Elmore, 2007) . Moreover, c ellular FLICE inhibitory protein (cFLIP) has shown to inhibit apoptosis by interacting with the DISC complex. 4 Intrinsic Apoptotic Signaling Pathway The intrinsic pathway or the mitochondrial pathway of apoptosis is triggered when an apoptotic stimulus disrupts the balance between pro - apoptotic an d anti - apoptotic Bcl - 2 proteins . Bcl - 2 family of proteins contains both pro and anti - apoptotic proteins that regu late cell survival or death. Bcl - 2 itself and Bcl - XL are anti - apoptotic proteins while Bax and Bak are pro - apoptotic proteins that increase per meability in mitochondria. Some pro - apoptotic protein s contain only Bcl - 2 homology 3 (BH - 3) domain which bind s to anti - apoptotic proteins and inhibit cell survival (Hotchkiss et al., 2009) . Upon receiving the death signal , the cytoplasmic protein Bax translocate s to the outer mitochondrial membrane. Oligomerization of Bax and Bak results in mitochondrial outer membrane permeabilization (MOMP), releasing mitochondrial protei ns including cytochrome c (cyt c) and apoptosis inducing factor (AIF, Tait and Green, 2010) . Cyt c combines and stimulates oligomerization of apoptosis pr otease activating factor 1 (Apaf - 1) creating a large scaffold complex called the apoptosome which recruits pro - caspase - 9 . Activation of caspase - 9 results in activating the proteolytic cascade and the activation of caspa se - 3 and 7 leading to apoptosis (Green, 2005) . A cross talk between extrinsic and intri nsic path occurs , when caspase - 8 cleaves and activates the pro - apoptotic BH - 3 protein Bid to a truncated Bid (tBid ) which causes MOMP. Inhibitor of apoptosis (IAP) is a family of proteins that are able to bind to pro - caspases and prevent the activation of caspases . X - chromosome linked inhibitor of apoptosis (XIAP) is a key member of the IAP family that inhibits caspase 3, 7 and 9 and prevents cell death. This inhibition of apoptosis by IAP s , is blocked by certain factors including Smac and Omi , which are re leased from mitochondria that activate the ca spases thus promoting apoptosis (Green, 2005) . 5 ALVEOLAR EPITHELIAL CELLS Type I and Type II Cells The lung epithelium is c omposed of type I and II epithelial cells. These cells are present in similar numbers but are functionally distinct. Type I cell has an extended cytoplasm that covers most of the surface area in the lung and is also terminally differentiated. Therefore, type I cell is quite susceptible to injury a nd cannot initiate self - repair. The thin cytoplasm primarily facilitates resp irat ory gas exchange and minimizes the diffusion distance between al veoli and pulmonary capillaries (Uhal, 1997) . Type II cell is cuboidal and is the stem cell of the lung. Type II cells are located in the corners of the alveoli. In contrast to type I cell s , type II cell s are resistant to injury (Selman and Pardo, 2006) . T he multi - functional type II cell is capable of proliferating and differentiati ng into both type I and II cell s (Uhal, 2008) . Type II cell synthesizes , stores and secretes the pulmonary surfactant, a heterogeneous mixture of lipids and proteins to reduce surface tensio n in the lung (Uhal, 2003) . Type II cell s are capable of synthes izing immune - modulatory proteins that are important for host defense and turnover of extracellular matrix molecules (F igure 1 - 1 ) . The equilibrium between type I and type II cell s depend on the proliferation, differentiation and cell death of type II cells. Due to the high proliferative capacity of type II cells, during an injury type II cells repair the damaged epithelium and maintain barrier functions. Failure to initiate re - epit helialization leads to destruction of the healthy alveolar epithelium (Uhal and Nguyen, 2013) . Hence, loss of type II cel ls is particularly significant. 6 Figure 1 - 1: Funct ions of type I and type II alveolar epithelial cells. Type I cells are the squamous cells that facilitate majority of the gas exchange. Type II cells produce the pulmonary surfactant that reduces pulmonary surface tension and is the stem cell of the lung. They also produce factors like prostaglandin (PGE - 2) and plasminogen activators that inhibits fibroblast proliferation and degrade fibrin respectively. Type II cells produce matrix - metalloproteinase (MMP) that degrade the extra cellular matrix (ECM). 7 Alveolar Cell Defense Mechanisms The lung epithelium is equipped with several defense mechanisms to maintain barrier functions. The pulmonary surfactant produced by type II cells, especially surfactant protein s (SP s ) A and D bind to pathogens and enable their destruction via alveolar macrophages ( Fehrenbach, 2001) . Both SP - A and D have been shown to bind to bacteria and viruses and increase phagocytosis. Alveolar macrophages are large mononuclear phagocytes that play a vital role by engulfing various inhaled particles and destroying them . Several studies suggest that alveolar macrophages can release factors that enhance the recruitment of fibroblast s a nd promote collagen deposition (Uhal et al., 2007) . Further, macrophages are known to secrete many enzymes including MMPs , cytokines and growth fac tors that modulate other cells. These phagocytes interact with a variety of pathogens through cell surface receptors that bind to specific ligands . A recent study demonstrated that angio tensin II ( ANGII ) upr egulates MMP - 9 and MMP - 2 through partial activation of the ATI receptor in macrophages (Uhal et al., 2007) . Type II cells also produce growth inhibitors like prostaglandin E2 (PGE - 2) and matrix metalloproteinases (MMPs) which inhibit fibroblast proliferation and degrade collagen, respectively (Uhal, 2008). Similarly, plasmino gen activators convert plasminogen to plasmin that degrade s interstitial f ibrin and restrict s cytokines from reaching the underlying tissue. Toxic agents including industrial dusts, c igarette smoke, air pollutants , and certain disease conditions may weaken or inhibit pulmonary defense mechanisms. Therefore, c hanges in macrophage function may be central in determining the effectiveness of defense mechanisms . 8 Apoptosis in the L ung Epithelium Apoptosis in the lung is essential to maint ain normal tissue homeostasis. It is vital during development and restoration of the normal lung after injury. The intact lung epithelium protects the underlying tissue by providing a physical barrier. The normal lung cells are generally quiescent; h owever, when epithelial cells are damage d , it is essential to repair the site of damage in order t o maintain the physical barrier (Camelo et al., 2014) . I ncreased alveolar epithelial cell (AEC) apoptosis may lead to a coll apse in the barrier and halt its pro tective functions (Uhal, 2008) . A ctions that stimulate repeated insults to lung epithelial cells and contribute to dysregulated repai r mechanisms are not completely understood (Selman and Pardo, 2006) . Further, loss of AECs contributes to a reduction of growth inhibitors and a r eduction of matrix metalloproteinase, thus crea ting a damaged microenvironment (Uhal, 2008) . Unlike terminally differentiated type I cells, type II cells are capable of repairing the damaged alveolar epith elium ( Li, 2009) . Upon injury, damaged type I cells are removed and type II cells begin to prolifer ate rapidly forming a so called hyper plastic epithelium and then differentiate into type I cells to maintain the integrity of the epithelium (Uhal and Nguyen, 2013) . The exact process es of type II cell differentiation into type I cell in vivo are not completely understood (Selman and Pardo, 2006) . It was shown that type II cell cultures loose lamellar bodies and alter the gene expression ex hibiting type I characteristics (Guo et al., 2001b) . Type II cells also produce factors that facilitate the migration, differentiation and adhesion of fi broblasts. Thus, injury to type II cells or delayed epithelial repair can result in loss of the intact epithelium allowing the fibroblasts to reach the alveolar air sp ace contributing to the obliteration of the lung architecture. 9 Alveolar e pithelial cell apoptosis is a persistent finding in a number of interstitial lung diseases (ILD). I t was demonstrated that upregulation of DNA fragmentation and apoptosis in alveolar epithelia l cells, contributed to ILD (Kuwano et al., 1996) . Increased expression of pro - apoptotic proteins and decreased expression of anti - apoptotic prot eins have been reported in AECs (Uhal, 2008) . An earlier study showed incre ased labeling of fragmented DNA that was used as a marker of apoptosis in lung tissue s isolated from experimental animal models treated with bleomycin (Hagimoto et al., 1997) . The Fas receptor is expressed in type II cells and administration of Fas activating antibodi es stimulated apoptosis in AECs (Fine et al., 2000) . It was also shown that bleomycin, TNF - all physiologically relevant molecules that can induce apoptosis in AECs (Uhal, 2003) . The AECs can be exposed to a variety of reactive nitrogen species and under certain conditions NO 2 may interfere wi th epithelial repair and induce apoptosis selectively (Fine et al., 2000) . Blockade of AEC apoptosis has shown to reduce lung injury and further, AECs lacking the pro - apoptotic protein Bid, were resistant to tumor growth factor - - cell death (Budinger et al., 2006). Lung Epithelial Repair Mechanisms T he exact signaling mechanisms through which AECs induce a repair process remain unclear, but the current knowledge on the pathway is described in brief. The epithelial repair process consists of epithelial cell - cell communication and epithelial cell - extra cellular matrix (ECM) communication. I nhibition of interleukin - - 1 in vitro suggesting epithelial repair occurs throu gh an IL - (Geiser, 2003) . 10 These data were further strengthened by demonstrating the induction of alveolar epithelial re pair by a recombinant form of IL - Evidence show s the involvement of the epidermal growth factor (EGF ) and its receptor (EGFR) in alveolar epithelial repair both in vivo and in vitro . Neutralizing antibodies against tumor growth factor (TGF - , reduced IL - epithelial repair. Blockade of the EGFR or intracellular signaling pathways inhibited IL - induced repair showing through an in vitro model that IL - may induce epithelial repair through the EGF/TGF - (Geiser, 2003) . Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are members of the fibr oblast growth factor family . These growth factors were shown to protect the epithelium against injury. Several studies done in animal models, suggest a major role of HGF and KGF in epithelial repair a fter injury. In rats , administration of bleomycin increased the levels of KGF and c orrelate d with AEC proli feration (Barazzone et al., 1999) . HGF may also act as a potent mitogen for AECs after injury. Several studies also suggest that effects of KGF on the alveolar epithelium are mediated in part by the EGFR pathway. Treatment with HGF significantly stimulated DNA synthesis an d proliferation (O hmichi et al., 1996) . In addition, several studies have shown the anti - apoptotic effects of HGF on alveolar epithelial cells (Uhal, 2008) . Further, HGF stimulated proliferati on of bronchial epithelial cells were dependent on cyclooxygenase - 2 (COX - 2) mediated MAPK/Akt pathway (Crosby and Waters, 2010) . 11 Epithelial to Mesenchymal Transition (EMT) EMT is the phenotypic transition of a differentiated epithelial cell to a fully differentiated mesenchymal cell. It is known that, during development or injury to the lung can induce epithelial cells to differentiate to a mesenchymal pheno type to restore the normal architecture (Crosby and Waters, 2010; Willi s et al., 2006) . But in aberrant would healing models, it is suggested that EMT can contribute to interstitial lung diseases. In the p resence of TGF - alveolar epithelial cells undergo a loss of epithelial markers and express a variety of mesenchymal markers - - SMA), vimentin and type 1 collagen that leads to motility, and cytoskeletal rearrangements . Type II AECs are the main source of surfactant protein - C (SP - C) production and exposure to TGF - has shown to downregula te SP - C and gain fibroblast like morphology (Willis et al., 2006) . E - cadherin is expressed in type II AECs and EMT causes loss of E - cadherin whi ch leads to a reduction in cell cell communication an d contributes to cell migration (Crosby and Waters, 2010) . An earlier study demonstrated exposure of alveolar epithelial cells to TGF - , expressed morphological changes an d fibroblast phenotypic markers (Kasai et al., 20 05) . Further, siRNA mediated gene silencing of Smad2 inhibi ted TGF - The findings from this study indicate d that TGF - signal ing pathway (Kasai et al., 2005) . Similarly, when primary cultures of rat AECs were exposed to TGF - , the cells increased expression of mesenchymal markers and decreased ex pression of epithelial markers (Willis et al., 2005) . These data were strongly supported by another study - galactosidase - gal) that allows to determine whether EMT occurs in vivo . Overexpression of TGF - - gal positive 12 cells expressing mesenchymal marker s (Kim et al., 2006) . Nevertheless, c omplete understanding of the signaling process in EMT and its dysregulation is needed. RENIN ANGIOTENSIN SYSTEM (R AS) The renin - angiotensin system (RA S) plays a central role in regulating blood pressure. Renin is produced i n the juxtaglomerular cells and a decrease in blood pressure or increased activity in the sympathetic nervous system can release this hormone to the circulatory system. The circulating r enin can cleave liver produced angiotensinogen (AGT) to generate angiotensin I (ANG I) . Angiotensin converting enzyme (ACE) hydrolyzes ANG I to angiotensin II ( ANGII ). B in ding of ANGII to ATI exerts harmful actions , if not properly counterbalanced. ANGII n arrows blood vessels (vasoconstriction ) increasing blood pressure. ANGII releases vasopressin and aldosterone which act on the distal tubule, media ting water and sodium retention (Fyhrquist and Saijonmaa, 2008; Simões E Silva and Flynn, 2012) . The effects of the ATII receptors have not been completely unde rstood but generally are known to counteract ANGII induc ed signaling (Robles et al., 2014) . In the last decade, several angiotensin peptides have been discovered including angiotensin III ( ANGII I) , angiotensin IV (ANG IV) , and angiotensin I - 7 ( ANG1 - 7 , Robles et al., 2014) . Recently discovered, prorenin /renin receptor increases t he conversion of AGT to ANG I and there by contributes to accumulation of ANGII when the receptor is bound by renin. Also this interaction stimulates intracellular signaling and enhances synthesis of tumor growth factor (TGF - ignaling, collagen and fibronectin in the kidney (Nguyen, 2011; Robles et al., 2014) . Recent discoveries show t hat classical RAS is expressed locally within tissues, independent or 13 dependent of the circulatory RAS. In the lung a local RAS is expressed independent of the circulatory RAS and is known as the ANG system. Numerous experimental studies with both animal models and human tissues ex vivo now support the concept that activatio n of a local angiotensin (ANG) system plays a major role in lung injury . A subset of these studies demonstrated the existence of an intrinsic (i.e., all components expressed by the target cell itself) angiotensin system in lung alveolar epithelial cells. Local Angiotensin (ANG) System Apoptotic causing agents induce apopt osis in AECs by upregulation of AGT synthesis (F igure 1 - 2 ) . AGT is a 58 kDa protein which is the only known pre cursor for angiotensin peptides (Uhal, 2003) . In response to Fas and TNF - , AGT synthesis was increased in human alveolar epithelial - derived cell line A549 and moreover , antisense oligonucleotides against AGT completely abrogated a poptosis (Wang et al., 1999, 2000a) . Those data clearly show the autocrine production of AG T locally in AECs. Cleavage of the NH2 - termi nal end of AGT by cathepsin D (c at - D) or other aspartyl proteases generates ANG I which is the substrate of angi otensin converting enzyme (ACE, Li et al., 2004) The biologicall y active peptide angiotensin II ( ANGII ) is generated from cleavage of two amino acids from the COOH end of ANG I. ACE is the primary enzyme that could generate ANGII . However, chy m as e or other peptidases can form ANGII , an octa - peptide that has shown detri mental effects in many cell types including AECs (Uhal et al., 2012) . In lung epithelial cells, ANGII induced apoptosis could be blocked by ATI receptor antagonist losartan but not by an ATII antagonist conforming that the physiological rec eptor for ANGII in AECs is ATI (Papp et al., 2002) . Angiotensin converting enzyme - 2 (ACE - 2) 14 is an vital enzyme that removes a single amino acid from the COOH end of ANGII , to gen erate angiotensin 1 - 7 ( ANG1 - 7 , Uhal et al., 2011) . The hepta - peptide ANG1 - 7 has shown to inhibit many injurious actions of ANGII through its receptor Mas wh ich belongs to the G - protein coupled receptor (GPCR) family (Uhal et al., 2012) . Inhibition of ACE - 2 by a competitive inhibitor DX - 6 00 or siRNA meditated knockdown, caused a significant increase in ANGII levels (Uhal et al., 2011) . Therefore ACE - 2 demonstrates a protective role against AEC apoptosis by limiting the accumulation of ANGII . In AECs , a balance between ANGII and ANG1 - 7 is a critical determinant of cell death and survival. Angiotensin Receptors and Signaling ANGII is a vasoconstrictor and has demonstrated pro - apopto tic, proliferative, hypertrophic , fibrotic, mitogenic and tissue remodeling actions. There are two types of ANGII receptors; ATI and ATII, both of which are members of the seven - transmembr ane domain, GPCR su perfamily but are functionally distinct with a sequence homology of 30% (Goodfriend et al., 1996) . Two types of ATI receptors have been identified; ATI A and ATI B which are prese nt in rodents but not in h umans (Guo et al ., 2001a) . The ANGII /ATI receptor signaling pathway is mediated by heterotrimeric G q/11 - proteins that activate phospholipase C (PLC, Goodfriend et al., 1996) . Cleavage of Phosphatidylinositol 4, 5 - bisphosphate (PIP2) by PLC yields inositol 1, 4, 5 - triphosphate (IP3) and diacylglycerol (DAG). DAG activates Protein k inase C (PKC) which can add a phospho group to serine/threonine in cellular proteins and stimulate proliferation. In AECs, both ATI a nd ATII mRNA are present but were demonstrated that ATI is the main receptor responsible for JNK phosphorylation and AEC apoptosis. Signal transduction of ATII receptor is 15 still unclear although, in several cell types ATII receptor has shown to activate a tyrosine phosphatase and counteract ANGII indu ced effect through ATI receptor (Papp et al., 2002) . Angiotensin Converting Enzymes (ACE and ACE - 2) The balance between ACE and ACE - 2 plays a major role in many diseases and ACE - 2 is known to counteract actions of ACE. Both of these enzymes are important regulators of the ANG system. ACE is a type 1 transmembrane protein and two f orms of ACE has been reported; somatic ACE and germinal A CE (Danilczyk et al., 2003) . Somatic ACE is composed of two active - site domains; N - domain and C - domain both of which differ in substrate specificity whereas germinal ACE has a single active site (Danilczyk et al., 2003) . ACE converts the decapeptide ANG I to ANGII and also degrades the vasodilator bradykinin. Somatic ACE can be cleaved by a secretase from the cell surface although the biological importance of s oluble ACE is poorly understood (Tipnis et al., 2000) . In AECs , blockade of ACE by ACE inhibitors such as l isinopril has shown to prevent apoptos is (Filippatos and Uhal, 2003) . ACE - 2, a 805 amino acid, type 1 transmemb rane glycoprotein, was discovered recently and is a close homologue of ACE. Unlike ACE, ACE - 2 contains a single active - site domain which has a high substrate specificity of ANGII . ACE - 2 also hydrolyze s ANG I to generate ANG 1 - 9, but relatively poorly. The large extracellular active site of the ACE - 2 shows 40% overall sequence identity with t he N - domain and C - domain of ACE (Zisman, 2005) . ACE - 2 can be released from the cell membrane by ADAM 17 which belongs to the adamalysin protein family. El evated levels of sheded ACE - 2 has been rep orted in myocardial dysfunction (Clarke and Turner, 2011) . Knockdown or competitive inhibition of ACE - 2 has shown to alte r the levels of ANGII and ANG1 - 7 (Uhal et al., 2011) . Further, o ver - 16 expression of ACE - 2 has demonstrated to treat several diseases in experimental models and has a protective role against AEC apoptosis (Imai et al., 2010) . 17 Figure 1 - 2: Local angiotensin system in alveolar epithelial cells. Apoptotic inducers activate angiotensinogen which is cleaved by cathepsin D (cat D) to generate angiotensin I (ANG I). Angiotensin converting enzyme (ACE) generates angiotensin II by cleaving two amino acids from ANG I. Angiotensin converting enzyme - 2 (ACE - 2) cleaves a single amino acid from ANGII to generate angiotensin 1 - 7, the ligand for Mas receptor. 18 ANG SYSTEM AND AEC APOPTOSIS Recently , experimental studies have demonstrated the involvement of ANG system in AEC apoptosis. In response to apoptotic inducers such as Fas ligand, TNF - is produced in cultured AECs which after enzy matic cleavage generates ANGII (Li et al., 2003a; Wang et al., 1999, 2000a) . Antisense oligonucleotides against AGT mRNA, neutralizing antibodies against ANGII , ACE inhibitors ( ACEi) and ATI receptor antagonists have all blocked apoptosis in AECs (Uhal et al., 2012) . Collectively, these studies demonstrate d the local production of ANGII and activation of ATI receptor. In primary cultures of rat alveolar epithelial cells , ANGII induc ed apoptosis measured by nuclear fragmentation was blocked by chelerythr ine, an inhibitor of PKC which suggests the activation of PKC downstream of ANGII /ATI (Papp et al., 2002) . Phosphorylation of jun - N - terminal kinase (pJNK) is a required event in AEC apoptosis (F igure 1 - 3 ) . ANGII induced pJNK was blocked by ATI receptor antagonist but not by an ATII receptor antagonist, showing that ATI receptor mediates the induction of pJNK (Uhal et al., 2011) . Clearly, these data suggest ATI is the functional receptor that mediates JNK phosphorylation and ap optosis in AECs. Activation of caspase - 9 by ANGII shows that AEC apoptosis occurs through the mitochondrial pathway. Further , this activation of caspase - 9 was prevented by a JNK inhibitor. Blockade of ACE - 2, one of the beneficial enzymes that generate anti - apoptotic peptide ANG1 - 7 , by DX - 600, a competitive inhi bitor induces caspase - 9 in AECs (Uhal et al., 2011) . Bo th ANGII and bleomycin induced pJNK was blocked by pre - incubating the cells with ANG1 - 7 . This blockade was prevented by A779, the receptor antagonist for Mas , showing the importance and presence of the Mas receptor in AECs. Similar results were obtained wi th propidium iodide (PI) assay that measures nuclear fragmentation. 19 Figure 1 - 3: Known signaling mechanisms in alveolar epithelial cells (AECs). The octapeptide ANGII induces JNK phosphorylation and apoptosis through the AT1 receptor (Uhal et al., 2011 ). ACE - 2 degrades the pro - apoptotic ANGII to the anti - apoptotic ANG1 - 7, which inhibits both JNK phosphorylation and apoptosis through the Mas receptor. These inhibitory effects of ANG1 - 7 are blocked by A779, a specific antagonist of Mas. Current studies su ggest that ANG1 - 7/Mas activation prevents JNK phosphorylation by constitutively activating the JNK - selective map kinase phosphatase - 2 (MKP - 2) and further, demonstrate the involvement of the Mas receptor in MKP - 2 activation. The mechanism(s) by which Mas ac tivation induces MKP - 2 (??) are currently unclear (PM ¬ plasma membrane). 20 Blockade of Apoptosis in AECs Since apoptosis requires activation of many signaling proteins, in the recent years studies have shown blockade of apoptosis holds a great pharmacological potential to prevent lung diseases that involve apoptosis. In a rat model, it was demonstrated excessive collagen deposition after administration of bleomycin was blocked by captopril , an angi otensin converting enzyme inhibitor (ACEi) or b y a broad spectrum caspase inhibitor (ZVADfmk) that in hibits the activity of caspases (Filippatos and Uhal, 2003) . Deletion of genes that are necessary for apoptos is have proven to be beneficial (Uhal et al., 2012) . Th is was shown by mice lacking Bid , one of the pro - apoptotic proteins demonstrated resistance to transforming growth factor (TGF - th in alveolar epithelial cells (Budinger et al., 2006) . Induction of AEC apoptosis by fas lig and, TNF - saralasin (non - selective ATI antagonist). Apoptosis was also blocked by antisense oligonucleotides against AGT or by antibodies that neutralize ANGII . Collectively, these studies demonstrated the autocrine production of ANGII rega rdless of the apoptotic stimuli (Filippatos and Uhal, 2003; Li et al., 2003a; Wang et al., 1999, 2000a) . It was demonstrated that ANGII induced apoptosis in A549 cells or in primary rat cells were inhibited by ATI selective blocker losartan but not by ATII selective blocker PD - 126055 (Papp et al., 2002) . ANG System in He art The contribution of ANGII as an apoptotic regulator was first hypothesized by cardiologists. The local presence and synthesis of the ANG peptides in the heart suggest the modulation of cardiac structu re and function by ANG peptides (Filippatos et al., 2001) . Clinical 21 and experimental studies have documented the vital role of ANGII in pathological conditions of the heart. ANGII has been shown to upregulate AGT mRNA synthesis, activation of p53 and JAK/S TAT pa thway in cardiac myocytes (Do stal and Baker, 1999) . ANGII induced apoptosis in neonatal cardiac myocytes were blocked by ATI selective antagoni sts (Filippatos and Uhal, 2003) . Further ANGII induced cardiac damage in Sprague - Dawley rats were blocked by ACE - 2 gene transfe ction using lenti - viral vectors (Santos et al., 2013) . The hepta - peptide ANG1 - 7 has shown to attenuate cardiac remodeling. This was further investigated by Tallant et al . in cardiac fibroblasts (McCollum et al., 2012a) . ANGII stimulated phosphorylation of extracellular signal regulated kinase (ERK1/ERK2) was blocked by ANG1 - 7 through upregulation of dual specific phosphatase - 1 and the modulatory effects of ANG1 - 7 were blocked by Mas receptor antagonist. These data show the involvement of the ANG system in heart and beneficial e ffects of the ANG1 - 7 / Mas pathway. ANG System in Kidney Proliferation of resident renal cells and deposition of extra - cellular matrix components are well - studied features of progressive renal diseases. A significant amount of studies have demonstrated the involvement of a tissue specific ANG system in pathological conditio ns in the kidney. ACE inhibitors and ATI selective antagonists have demonstrated beneficial actions against ANGII induced apoptosis and cell proliferation in kidney disease (Filippatos and Uhal, 2003) . Experimental studies have demonstrated a prote ctive role of ACE - 2 in different models of renal damage by genetic deletion of ACE - 2 that aggrava tes the pathological conditions (Santos et al., 2013) . Along s imilar line s, studies have reported a downregulation of ACE - 2 22 associated with kidney disease and moreover, ANGII induced oxidative stress was attenuated by a recombinant fo rm of ACE - 2 (Simões E Silva and Flynn, 2012) . Although , the exact ANG1 - 7 signaling is unclear and ANG1 - 7 effects can be quite complex in the kidney , studies have reported the inhibition of ATI receptor mediated signaling by ANG1 - 7 in nephron injury (Brewster and Pera zella, 2004) . It is also documented that ANG1 - 7 effects could be controversial in the kidney and this might be due to cell type specificity and diff erences in models that are used (Santos et al., 2013) . ANG System in Vascular Smooth Muscle Cells (VSMCs) Angiotensin peptides play a key role in regulating vascular reactivity and experimental studies have suggested a vital role of ANGII in VSMCs. ANGII has been found to exert hypertrophy and in some cases hyperplasia . S timulation of ANGII /ATI pathway activates PLC and intracellular Ca 2 + , in addition to the activation of NADH oxidase in VSMCs (Griendling et al., 1997) . Administration of ANGII significantly stimulated proliferation in VSMCs isolated from aorta of Sprague - Dawley rats measured by DNA synthesis (Freeman et al., 1996; Tallant et al., 1999) . The proliferation of cells was attenuated after incubation with ANG1 - 7 showing the regulation of vascular growth by the two major peptides in the ANG system . Further, ANGII induced activity of ERK1/ERK2 was significantly prevented by ANG1 - 7 (Tallant and Clark, 2003) . T he signaling mechanisms of ANG1 - 7 in VSMCs have been investigated and experimental data illustrate that prostacyclin , nitric oxide (NO) and prostaglandin have shown to inhibit VSMC growth. Further, this inhibition occurs through a cyclic adenosine monophosphate ( cAMP ) 23 mediated pathway which was demonstrated through pharmacological agents that induce cAMP levels can contribute to i nhibition of VSMC proliferation (Tallant et al., 1999) . ANGIOTENSIN 1 - 7 ( ANG1 - 7 )/ MAS RECEPTOR The local ANG system has recently been extended with new key players that oppose actions of AC E/ ANGII /AT I. The components of the new axis consist of ACE - 2/ ANG1 - 7 / Mas that have shown to exert many beneficial actions in many cell types includ ing in AECs. The b iologically active peptide ANG1 - 7 is one of the key regulators in the locally expressed ANG system in AECs. ACE - 2 degrades the pro - apoptotic, proliferative and vasoconstrictive octa - peptide ANGII to generate anti - apoptotic, anti - proliferative and vasodilatory hepta - peptide ANG1 - 7 . Recent data demo nstrated the importance of ANG1 - 7 which counteracts ANGII induced deleterious effects in many tissues including in the lung, heart, vascular smooth muscle cells and k idney. The hepta - peptide was first discovered in the brainstem as a product of ANG I and little over 20 yea rs since its discovery, a large body of evidence has shown the import ance of ANG1 - 7 , especially after the discovery of its receptor Mas . It was found that the inhibitory effects of ANG1 - 7 was blocked by the receptor antagonist A779 (Asp - Arg - Val - Tyr - Ile - His - D - Ala) which was discovered by Santos et al (Santos et al., 1994) . This synthetic analogue of ANG1 - 7 showed to inhibit ant i - diuresis in water loaded rats and was unable to block any of ANGII induced agonist effects. Moreover, A779 (D - Ala - ANG1 - 7 ) did not displace iodine labelled ANGII in tissues that are rich of ATI receptor subtype. Further, changes in blood pressure by ANG1 - 7 were blocked by A779 but not by an A TI or ATII 24 antagonist. Collectively, the data demonstrate that A779 is a s elective antagonist for ANG1 - 7 (Santos et al., 1994) . Before the discovery of Mas , R ent et al . showed vasodilation in rabbit afferent arteriole by ANG1 - 7 was not blocked by ATI or ATII selective a ntagonists but was blocked by A 779 compound (Ren et al., 2002) . Similarly, vasodilation of microvessels in the mesenteric circulation wa s not blocked by losartan but by A779 (Oliveira et al., 1999) . These data were strengthened by similar studies done in other cell types. VSMC growth by A NGII was opposed by the effects of ANG1 - 7 . The bl ockade was not attenuated by ATI or AT II selective antagonists but by A 779 (Fr eeman et al., 1996) . These data provid ed the evidence of a novel ANG1 - 7 specific receptor. However, a t higher concentrations of ANG1 - 7 , studies have shown that it can produce ANGII like effects through binding to ATI (Santos et al., 2000) . The Mas proto - oncogene was discovered by its tumorigenic properties that encodes a seve n transmembrane receptor which w as considered as an orphan GPCR (Santos et al., 2003) . The tumorigenic properties of Mas appears to be really low and it was initially thought to be the functional receptor for ANGII (Jackson et al., 1988) . Santos et al . demonstrated through radio - ligand binding studies in mouse kidney , that the physiological receptor for ANG1 - 7 is the G - protein coupled receptor Mas (Santos et al., 2003) . In this study the authors demonstrated the binding of I 125 labelled ANG1 - 7 with high affini ty to Mas transfected CHO cells. Moreover, this binding was d isplaced by both unlabeled ANG1 - 7 and A779 with high affinity. To examin e the functional effect of ANG1 - 7 , the release of arachidonic acid (AA) was measured in CHO cell tr ansfected with the Mas receptor. The induction of AA release was blocked by A779 but not ATI or ATII selective antagonists , demonstrati ng a specific receptor for ANG1 - 7 distinct from ATI 25 or ATII (Santos et al., 2003) . Additionally, relaxation of the aortic rings in rats was investigated with ANG1 - 7 treatment. Mas deficient aortas lost their ability to induce relaxation effect in response to ANG1 - 7 compared to the wild type demonstrating Mas as a functional recep tor for ANG1 - 7 (Santos et al., 2003) . It is no w evident that GPCRs may exist as homo or hetero dimers. Recently , it was discovered that ANG1 - 7 / Mas mediated inhibition occurs through interaction with ATI receptor. In transfected mammalian cells ANGII /ATI induced generation of inositol phosphates and in tracellular Ca 2+ were reduced by half after the expression of Mas receptor. The authors also demonstrated the formation of a hetero - oligomeric complex between ATI and Mas receptor that does not respond to agonists or an tagonists of the both receptors (Kostenis et al., 2005) . Along similar lines, a different group demonstrated that ATII receptor is an antagonist of ATI receptor that prevents ANGII signaling in fetal fibroblasts. ATI receptor induced inositol phosphate levels were augmented in fibroblasts after a reduction of ATI/ATII hetero - dimerization when antisense construct against ATII was used. Further, ANGII induced signaling was blocked by ATI selective antagonist but not by ATII antagonist. But the reduction in ATI/ATII hetero - dimer complex caused an induction in ATI signaling demonstrating that ATI signaling is prevented (at least partially) by the ATII receptor, forming a hetero - dimeriz at ion between the two receptors (AbdAlla et al., 2001) . Castro et al . sh owed, ANG1 - 7 mediated vasodilation was significantly influenced in the presence of losartan demonstrating a comp lex interaction between ATI/ Mas and possibly with ATII receptors (Castro et al., 2005) . However, more studies are required to elucid ate the functional interactions among these receptors . 26 ANG1 - 7 Synthesis and Catabolism The physiological and th e most accepted pathway of ANG1 - 7 generation is hydrolysis of ANGII by ACE - 2 (F igure 1 - 4). The enzyme ACE - 2 can also generate ANG I - 9 by ANG I. However, It is known that ACE - 2 has 400 fold higher affinity for ANGII than ANG I (Xu et al., 2011) . ACE or Neprilysin (NEP) cleaves two amino acids from ANG 1 - 9 to generate ANG1 - 7 . NEP, thimet oligopeptidase and prolylendopeptidase (PEP) can remove three amino acids directly from ANG I to generate ANG1 - 7 . The hydrolysis of ANG1 - 7 has been investigated in the rat lung. The authors demonstrated the breakdown of radio - labeled ANG1 - 7 primarily to ANG1 - 5 . ACE has been shown to bind to ANG1 - 7 with high affinity and inhibition of ACE by lisinopril a brogated the generation of ANG1 - 5 . Further, d egradation of AN G1 - 5 yielded ANG3 - 5 , independent of ACE and might due to other enzymes like diamino - peptidases (Allred et al., 2000) . 27 Figure 1 - 4: Angiotensin 1 - 7 synthesis and catabolism. ANG1 - 7 can be synthesized by several different enzymatic mechanisms (Allred et al., 2000). ACE - 2 degrades the octa - peptide ANGII to the hepta - peptide ANG1 - 7. ANG I can be metabolize d by neprilysin (NEP), prolylendopeptidase (PEP) into ANG1 - 7. ACE - 2 also can cleave ANG I to generate angiotensin 1 - 9 (ANG 1 - 9) which then is degraded by ACE resulting in ANG1 - 7. ACE is also involved in degrading ANG1 - 7 to angiotensin 1 - 5. 28 Inhibitory Actions of the ANG1 - 7/Mas Pathway on Pulmonary Injury Exactly how the ANG1 - 7 / Mas axis affects injurious signaling pathways is curr ently a topic of intense focus. The protective actions of the ANG1 - 7/Mas axis on lung cells in non - neoplastic lung injury are briefly discussed here. The local ANG system is activated after tissue injury in a variety of organs to promote repair, but abnormalities in the process promote lung injury. Many experimental studies have elucidated the contribution of AEC apoptosis to th e pathogenesis of lung fibrosis (Fattman, 2008; Hagimoto et al., 1997; Li et al., 2006) . Many years ago and more recently, seminal research works reported data to support the concept that the death o f AECs, by itself, could create a profibrotic microenvironment without the involvement of an inflammatory response (Uhal and Nguyen, 2013) . Consistent with this concept, bloc kade of apoptosis of AECs during lung injury by angiotensin receptor blocker s (ARBs), ACEi or by a broad - spectrum caspase inhibitor decreased the fibrotic response in animal models (Filippatos and Uhal, 2003) . However, issues that might limit the applicability of this approach to human subjects have been discussed, such as potential sid e effects, gender differences and in the case of ARBs, the potential for systemic hypotension in some patients (Ferreira et al., 2012) . Althou gh ACEi have shown to reduce lung fibrogenesis in some animal models, clinical trials of ACE inhibitors in humans have failed to detect beneficial effects on lung fibrosis. This might be explained by the presence of other enzymes independent of ACE that co uld generate ANGII. Thus, it is crucial to understand the underlying mechanisms of the counter - regulatory axis ACE - 2/ ANG1 - 7 / Mas , which may hold potential for future therapeutics for lung diseases. In AECs, constitutively expressed ACE - 2 converts the pro - a poptotic octapeptide ANGII to the anti - apoptotic heptapeptide ANG1 - 7 , and thereby limits the accumulation of ANGII to 29 promote cell survival (Uhal et al., 2012) . Evidence for a beneficial role of ACE - 2/ ANG1 - 7 is strengthened by in vivo studies of experimental animals that used genetic manipulation of ACE - 2 or specific inhibitors of ACE - 2 to establish a protective role of the enzyme (Soler et al., 2008) . Previous work in this laboratory showed that ACE - 2 is protective against experimen tal fibrosis, but is down - regulated in both human lung fibrosis and experimental lung fibrosis in animal models (Li et al., 2008) . Uhal et al . demonstrated that ACE - 2 mRNA, protein and enzymatic activity were severely decreased in lung b iopsy specimens isolated from IPF patients and also in the lung tissue of experimental animals made fibrotic by administration of bleomycin (Li et al., 2008) . In these studies, intratracheal administration of either ACE - 2 - specific siRNAs or DX600, a competitive inhibitor of ACE - 2, enhanced bleomycin - induced lung collagen accumulation. Moreover, in the lungs of animals in which ACE - 2 was manipulated in these ways, ANGII levels were increased and the resulting increase in lung collagen was blocked by an ANG receptor blocker. Together, these studies showed that ACE - 2 is protective against lung fibrogenesis by controlling local ANGII generation. In the lungs of patients with IPF, many alveolar epithelial cells are proliferating in the so - calle d "hyperplastic epithelium" described by pathologists, whereas AECs in the normal lung are primarily quiescent (Li, 2004) . On this basis, it was hypothesized that cell cycle regulation plays an important role in ACE - 2 expression by AECs. This was verified in a recent study that showed significant differences in ACE - 2 mRNA, protein and enzymatic activity in sub - confluent (proliferating) vs. post - confluent (quiescent) human lung cells in culture, and within normal or fibrotic human lung specimens (Uhal et al., 2013a) . The data clearly showed a down - regulation of ACE - 2 mRNA, protein and enzymatic activity in proliferating cells and an up - regulation in 30 quiescent cells. Additionally, the up - regulation of ACE - 2 that occurs in cells approaching density - dependent quiescence in vitro is blocked by the transcription blocker actinomycin D or by an inhibitor of JNK phosphorylation. Taken together, these results illustrated the cell cycle - dependent and JNK - mediated reg ulation of ACE - 2 expression in AECs. Recent work in our laboratory showed that both ANGII generation and JNK phosphorylation are required events in AEC apoptosis and subsequent lung injury (Uhal et al., 2011) . It was speculated that ACE - 2, as well as its product ANG1 - 7 , might regulate AEC apoptosis. This theory was confirmed by the findings that ANG1 - 7 could block JNK phosphorylation, caspase activation and nuclear fragmentation in a cultured mouse lung epithelial cell line (MLE - 12 cells) or in primary cultures of rat lung alveolar type II epithelial cells (Uhal et al., 2011) . Furthermor e, pretreatment with A779 , a specific antagonist of the Mas receptor, prevented th e inhibitory actions of ANG1 - 7 and thus implicated the involvement of Mas receptor (Uhal et al., 2011) . A subsequent study of the human lung epithelial cell line A549 and primary cultures of human lung AECs evaluated apoptosis of these cells, induced by either MG132 (a proteasome inhibitor and inducer of ER stress) or by the surfactant protein C (SPC) BRICHOS domain mutation G100S (an inducer of the Unfolded Protein Response and ER stress), one of several recently discovered SPC mutations that cause interstitial lung disease. In response to either of these inducers, the apoptosis was completely abrogated by ANG1 - 7 (Uhal et al., 2013b) . Specifically, ANG1 - 7 prevented the induction of caspases, loss of mitochondrial membrane potential, cytochrome c release, JNK p hosphorylation and nuclear fragmentation in the cultured human AECs. Further, the Mas antagonist A779 blocked the inhibition of apoptosis by ANG1 - 7 and demonstrated the involvement of Mas . This study also demonstrated a 31 reduction of ACE - 2 expression when t he cultured AECs were challenged with the proteasome inhibitor MG132 or the SPC mutant G100S. This reduction was prevented by an inhibitor of the ACE - 2 ectodomain shedding enzyme ADAM17/TACE (TAPI - 2). Together, these data demonstrate that AEC apoptosis is mediated by the autocrine ANGII/ ANG1 - 7 system expressed by these cells, and suggest that the hepta - peptide ANG1 - 7 may hold therapeutic potential for lung diseases in which the UPR and/or ER stress play a role in pathogenesis. The exact mechanisms of the ac tivation of the ANG system in response to ER stress are currently unclear but are under investigation. Consistent with these observations, Shenoy et al. demonstrated that intratracheal administration of a lentiviral - packaged ANG1 - 7 expression construct or ACE - 2 cDNA into Sprague Dawley (SD) rats significantly inhibited bleomycin - induced collagen deposition, expression of TGF - mRNA and AT1 receptor protein levels in the rat lungs (Shenoy et al., 2010) . Additionally, protective effects against lung fibrosis were also obtained by overexpression of ACE - 2. This study is consistent wi th a previously published study that demonstrated that exposure of cultured rat or human AECs to bleomycin in vitro caused a robust expression of AGT mRNA and the processed peptide ANGII, both of which are required for the apoptotic response of these cells (Li et al., 2003a) . Attempts to determine which profibrotic genes might be activated in response to ANGI I have shown the induction of TGF - - collagen - 1 mRNA levels in vitro in human fetal lung (HFL - 1) cells exposed to the octapeptide (Meng et al., 2013a) . Pre - or co - incubation with ANG1 - 7 prior to the application of ANGII inhibited the induction of the profibrotic genes. However in this study, pre - incubation with A779 did not preven t the inhibitory actions of ANG1 - 7 . The cause for this incongruity is not 32 clear, but may depend on the cell type specificity (fibroblast vs. epithelial cell) or the experimental conditions that were used (Filippatos and Uhal, 2003) . Acute respiratory distress syndrome (ARDS) is one of the most devastating forms of acute lung inju ry (ALI). Each year in the United States around 200,000 patients suffer from ARDS (Wösten - van Asperen et al., 2011) . Apoptosis of AECs has been discovered in the lungs of ARDS patients and was ass ociated with increased Fas/FasL expression (Albertine et al., 2002) . It is currently believed that anomalies of the ANG system contribute to the pathogenesis of ARDS. Abo ut 60% of ARDS patients are shown to develop pulmonary fibrosis with increased mortality rates (Phua et al., 2009) . A considerable number of in vivo studies demonstrate the beneficial actions of the ACE - 2/ ANG1 - 7 axis in acute lung injury in several animal models. For example, intratracheal administration of lipopolysaccharide (LPS) induced acute lung injury in C57BL/6 mice, which resulted in substantial induction of collagen accumulation, pulmonary edema and infl ammation (Chen et al., 2013b) . However, subcutaneous infusion of ANG1 - 7 significantly reduced hydroxyproline levels (a marker of total collagen) as well as TGF - /3 protein levels. Treatment with A779 prevented the protective effect of ANG1 - 7 on collagen deposition and lung remodeling, observations that provide in vivo evidence that Mas mediates the protective role of ANG1 - 7 on lung injury and fibrogenesis (F igure 1 - 5). Similarly, overexpression of a recombinant form of ACE - 2 prevented ALI induced by acid aspiration or sepsis in mice (Imai et al., 2005) . The same authors also demonstrated that mice deficient in ACE had markedly decreased ALI. On the other hand, the importance of ACE - 2 is strengthened by the use of ACE - 2 knockout mice (Hamming et al., 2007) . Experimental ARDS induced in mice by acid aspiratio n was more severe in ACE - 2 knockouts compared to wildtype 33 controls that express functional ACE - 2; the loss of ACE - 2 in the knockout mice increased neutrophil accumulation and worsened pulmonary edema. These studies demonstrated the protective role of ACE - 2 in vivo in models of ALI and showed that part of this defensive role is due to limitatio n of the accumulation of ANGII. Recently, many studies have accumulated to support the view that an imbalance between the enzymatic activity of ACE and ACE - 2 determine s the local tissue levels of ANGII a nd ANG1 - 7 . For example, a study conducted to determine pulmonary ACE and ACE - 2 activity in patients with ARDS, demonstrated increased ACE activity and decreased ACE - 2 activity compared to the control group (Wösten - van Asperen et al., 2013) . In an animal model of ARDS, the reduction of ACE - 2 activity was also present, but could be reestablished by in vivo treatment with ANG1 - 7 . These findings are promising but since the clinical study was limited to fourteen ARDS patients, larger clinical st udies are needed for confirmation. Along the same line of thinking, Imai et al . demonstrated that pharmacological inhibition of AT1 receptor or ACE - knockout mice showed improved ALI symptoms in the absence of functional AT1 receptor or ACE (Imai et al., 2008) . On t he basis of this work and that summarized in preceding paragraphs, it has been theorized that ACE/ANGII/AT1 can promote ALI, but the counter - regulatory axis ACE - 2/ ANG1 - 7 is protective against ALI. Related experimental studies showed that ALI in mice follo wing hindlimb ischemia - reperfusion (LIR) is also due to the dysregulation of the ANG system (Chen et al., 2013a) . Changes in the ACE/ACE - 2 mRNA level and protein levels were measured a fter 2 hour of hind limb ischemia in mice. In addition, ANGII and ANG1 - 7 levels in the blood se rum and in lung tissues were measured by enzyme - linked immunosorbent assay. In the beginning of the 34 reperfusion period, the authors found higher levels of ANG1 - 7 than ANGII, but in later stages of reperfusion ANGII levels were higher than ANG1 - 7 levels. Th is change agreed with varying levels of ACE/ACE - 2 expression (Chen et al., 2013a) . Consistent with other works mentioned above, genetic deletion of ACE - 2 showed increased disease progression in this model. Collectively, the above studies all demonstrate the protective role of ACE - 2 in lung injury. On the other hand, the efficacy of ANG1 - 7 administration in vivo has been less well documented to date, in part due to the many proteases that degrade the heptapeptide very rapidly. This problem was bypassed by the addition of a thi oether ring to the peptide to form cyclic ANG1 - 7 (c ANG1 - 7 ), which has been shown to increase resistance to proteolytic degradation in vivo in Sprague Dawley rats (Kluskens et al., 2009) . Experimental studies have found enhanced stability of the c ANG1 - 7 and also evidence that it binds to the ANG1 - 7 receptor Mas with high affinity. Efficacy of the cyclic analog of ANG1 - 7 was confirmed by the abrogation of LPS - induced acute lung injury by treatment with c ANG1 - 7 in vivo (Wösten - van Asperen et al., 2011) . These authors further found that c ANG1 - 7 acted very quickly (< 4 hrs ) to improve lung function and increase oxygenation. Thus, administration of the modified heptapeptide has a protective role against LPS - induced experimental ARDS. Nevertheless, the ability of c ANG1 - 7 to inhibit acid aspiration - induced and sepsis - induced ARDS also needs to be validated in animal models. It was also demonstrated that agonists of the Mas receptor or angiotensin type II receptor may hold therapeutic benefits against chronic lung disease (CLD) by counterbalancing ANGII induced pulmonary inflam mation. Wagenaar et al . demonstrated cardiopulmonary effects by examination of lung and heart histopathology in neonatal rats challenged with 35 constant exposure to 100% oxygen for 10 days in the presence of c ANG1 - 7 or an AT2 agonist. Additionally, mRNA leve ls of crucial genes that are involved in the ANG system and alveolar development were evaluated. Treatment with the agonists reduced the influx of macrophages and neutrophils into the lungs. However, treatment with the agonists did not affect alveolar deve lopment in neonatal rats with CLD (Wagenaar et al. , 2013) . A non - peptide compound AVE 0991 (AVE) has also shown to mimic the beneficial effects of ANG1 - 7 in a murine model of ovalbumin (OVA) - induced chronic allergic lung inflammation. Mice were challenged with OVA in the presence or absence of AVE (Rodrigues - Machado et al., 2013) . While OVA increased airway and pulmonary vascular wall thickness, OVA + AVE - treated mice displayed reduced airway wall and pulmonary vasculature thickness. Further, cytokine levels and airway contractile response were also reduced in mice treated with AVE compound. Together, these studies suggested the potential of analogues of ANG1 - 7 in the treatment of chronic pulmo nary remodeling associated with asthma. The protective effects of ACE - 2 and ANG1 - 7 have also been demonstrated in models of pulmonary hypertension (PH). In animal models of PH, ANGII contributes to pulmonary remodeling and binding of ANGII to AT1 receptor is increased in rats with experimental pulmonary hypertension (Kuba et al., 2006) . Moreover, ACE expression in vivo is also increased in these animals (Shrikrishna et al., 2012) . However, preliminary clinical trials did not have major success in demonstrating beneficial effects of ACE inhibit ors or ARBs on COPD - related PH (Morrell et al., 2005) . Monocrotaline - induced animal models of PH have revealed that experimental overexpression of ACE - 2 can inhibit and reverse the induction in r ight ventricular pressure, suggesting ACE - 2 as a potential therapy (Shenoy et al., 2011; Yamazato et al., 2009) . 36 development of a plant - based oral delivery system, consisting of purified ACE - 2 and ANG1 - 7 bioencapsulated in plant cells, has displayed protec tion against experimental PH (Shenoy et al., 2014) . The bioencapsulation defends against gastric enzymatic degradation and improved systemic absorption from the intestine . Sprague - Dawley rats with monocrotaline - induced experimental PH were treated with bioencapsulated ACE - 2 and ANG1 - 7 , which significantly halted the disease progression in these animals. This novel approach of delivery system using transplastomic technology may be beneficial for future treatments of other types of lung injuries, but needs to be investigated further. As discussed, ultimately these experimental studies may provide ideas to develop novel therapeutic strategies to control lung diseases and conce ivably other diseases that involve the ANG system. 37 Figure 1 - 5: Known actions of ANG1 - 7 in lung injury. In vitro studies of lung fibroblasts have demonstrated the down - regulation of profibrotic genes. Further, ANG II - induced apoptosis of AECs and JNK phosphorylation were reversed by treating the epithelial cells with ANG1 - 7. In vivo studies have shown the reduction of collagen levels, TGF - - 7. Moreover, ANG1 - 7 enhanced lung function in experimental mice after lung injury. 38 Signaling Mechanisms Underlying ACE - 2/ANG1 - 7/Mas Action in Lung Cells The recent advances discussed above have enhanced our under standing of the tissue specific ANG system and mainly the counter - regulatory role of the ACE - 2/ ANG1 - 7 / Mas axis which opposes the many deleterious actions of the ACE/ANGII/AT1 axis. However, the exact intracellular signaling mechanisms of the ANG1 - 7 / Mas pathway are currently unclear in lung cells. A num ber of cell signaling mechanisms are thought t o be involved downstream of ANG1 - 7 binding to Mas , but only a few studies have explored this subject in lung injur y. In this section the experimental studies that demonstrate the downstream signaling pathways t hat are involved in ANG1 - 7 / Mas signaling in lung cells are briefly summarized . A recent study from the Uhal laboratory demonstrated that ANGII - induced JNK phosphorylation and apoptosis in AECs were potently blocked by ANG1 - 7 (Uhal et al., 2011). At baseli ne (without added inducers and in serum - free media), ANG1 - 7 levels in the medium of cultured AECs are ~10 - fold higher than ANGII levels, and thereby function to maintain cell survival (Uhal et al., 2011). The same study demonstrated that ANG1 - 7 could poten tly reduce the JNK phosphorylation induced by ANGII, and JNK phosphorylation is required for AEC apoptosis. In accord with these observations, it was hypothesized that ANG1 - 7 binding to Mas activates a JNK - selective phosphatase which reduces the accumulation of phospho - JNK as a cell survival mechanism. This idea is currently being investigated through the use of gene knockdown strategies. In an earlier study of primary cultures of AECs, it w as observed that ANGII binding to AT1 activates PKC and is required for apoptosis (Uhal et al., 2012). Other recent studies showed that blockade of protein kinase A (PKA) by a specific inhibitor led to a rapid increase in p JNK, which suggests a possible ro le for PKA in AEC apoptosis (unpublished 39 data). Therefore, it will be of high interest to investigate the possible involvement of the cAMP/PKA pathway in the inhibitory ac tions of ANG1 - 7 on lung cells . Other signaling pathways of ANG1 - 7 / Mas have been repo rted by various research groups studying lung injury and fibrosis (Figure 1 - 6) . Meng and colleagues showed that the ACE - 2/ ANG1 - 7 / Mas axis protects against pulmonary fibrosis by inhibiting the MAPK/NF - in homogenates of whole lung tissue, thereby - collagen - I and synthesis of TGF - human fetal lung (HFL) - 1 cells, that the resistance of fibroblasts to bleomycin - or ANGII - induced apoptosis was prevented by ANG1 - 7 through inhibition of the MAPK/ NF - Moreover, they found the activation of caspase - dependent mitochondrial apoptotic pathway and BAX protein in response to ANG1 - 7 in lung fibroblasts. The inhibitory effects of ANG1 - 7 could be blocked by A779, the Mas receptor blocker, thus showing the involvement of Mas . However, these authors also demonstrated that administration of ANG1 - 7 alone activated ERK1/2 and moreover, blunted JNK phosphorylation in the HFL - 1 cells. The dual effects of ANG1 - 7 that were shown in this study were explained by the state of activation of the ACE/ANGII/AT1 axis and a possible mechanism of ANG1 - 7 acting through AT1 receptor. However, no data were provided to support the proposed mechanism of ANG1 - 7 action thro ugh AT1 rather than Mas , and thus this concept should be evaluated carefully, since cell type specificity and the ratio of AT1 vs. Mas receptors are likely to play roles in the activation of this pathway. A later study from the same research group revealed that the ANG1 - 7 / Mas axis protects against fibroblast migration by inhibiting the NADPH oxidase - 4 (NOX - 4) - derived ROS - mediated RhoA/Rho kinase pathway (Meng et al., 2015). 40 Recent reports have established the contribution of oxidative stress to the pathogen esis of pulmonary fibrosis (Carnesecchi et al., 2011; Kliment and Oury, 2010). NOX - 4 is an important source for the generation of reactive oxygen species (ROS) believed to be involved in initiating lung fibrosis. Consistent with this notion, the induction - collagen - I synthesis and fibroblast migration by ANGII was abrogated by an inhibitor of RhoA/Rock pathway (Y - 27632) or by siRNA - mediated silencing of NOX - 4. A direct inhibitory effect of ANG1 - 7 was investigated by the ability of ANG1 - 7 to block ANGII - induced RhoA and Rock - 2 mRNA induction. Additionally, the authors showed that lentiviral - mediated expression of ACE - 2 suppressed ANGII - induced fibroblast migration and collagen synthesis by blockade of the RhoA/Rho kinase pathway. In agreement with this st udy, another group showed that ANGII - induced human airway smooth muscle cell (HASMC) contraction was reversed by ANG1 - 7 through the RhoA/Rho kinase signaling pathway (Li et al., 2012). In this study, HASMCs that were isolated from main bronchus biopsies ob tained from lung resection donors were incubated with a RhoA/Rho kinase inhibitor Y - 27632, which blocked ANGII induced HASMC contraction. These studies clearly demonstrate a down - regulation of RhoA/Rho kinase signaling pathways by ANG1 - 7 acting thro ugh its receptor Mas . However, further studies are required in other cell types in the lung and to identify the factor(s) that may influence this pathway. Hashim et al. studied bronchoalveolar lavage fluid (BALF) in allergen - challenged mice and demonstrated that ovalbumin increased total cell numbers of neutrophils, eosinophils and lymphocytes, but this was attenuated by ANG1 - 7 through suppression of ERK1/ERK2 (El - Hashim et al., 2012). Although the exact mechanism of the suppression is unknown, ANG1 - 7 attenuated the ovalbumin - - , all of which was prevented by 41 pre - treatment with the Mas receptor antagonist A779 (El - Hashim et al., 2012). Transcriptional regulation of the ANG1 - 7 / Mas pathway is poorly understood in any organ system. To da te, only one study has reported transcriptional regulation (or at least partial regulation) of the ANG1 - 7 / Mas pathway in A549 cells (Verano - Braga et al., 2012). Forkhead bo x protein O 1 (FOXO - 1) is a transcription factor that regulates cell growth and apoptosis; when treated with ANG1 - 7 , FOXO - 1 transcriptional factor in A549 cells was phosphorylated and translocated to the nucleus upon stimulation (Verano - Braga et al., 2012) . However, further studies are required to understand the regulation of this system in vivo. Several groups have shown the involvement of vascular endothelial growth factor (VEGF), cyclooxygenase (COX - 2) and PI3K/AKT pathway in A549s and demonstrated the a bility of ANG1 - 7 to prevent tumor angiogenesis (Menon et al., 2007; Ni et al., 2012; Soto - Pantoja et al., 2009). Nevertheless, further study is needed to determine the role of these pathways in non - neoplastic lung injury. 42 Figure 1 - 6: Downstream sig naling pathways of the ANG1 - 7/Mas pathway in non - epithelial lung cell types. In lung fibroblasts (Left), ANG1 - 7/Mas protects against bleomycin - induced lung fibrosis by inhibiting the mitogen - activated protein kinase (MAPK)/NF - tion (Middle) was prevented by inhibition of the RhoA/Rho kinase pathway. Ovalbumin - induced infiltration of lung tissues by eosinophils, lymphocytes and neutrophils (Right) were prevented by ANG1 - 7 through suppression of ERK1/2. PM plasma membrane. 43 ACE - 2/ANG1 - 7/Mas in Non - Pulmonary Cells In the heart, many actions of ACE - 2 and ANG1 - 7 / Mas are described in cardiac myocytes. Mas or ACE - 2 deficient mice have exhibited a decrease in cardiac contractile which was re scued by administration of ANG1 - 7 (Bader, 2013) . A lthough , the exact mechanisms by which ANG1 - 7 / Mas abolishes ANGII /ATI are currently unclear , a role of ANG1 - 7 mediated release of nitri c oxide production was reported. Significant indu ction in cardiac output by ANG1 - 7 in Wistar rats were prevented by the Mas receptor blocker, A 779 demonstrating the importance of Mas in heart (Ferreira and Santos, 2005) . Recently , many studies indicate a cardio - protective role of ANG1 - 7 in the heart through anti - remodeling ef fect in different models of cardiomyopathy (Santos, 2014) . Further , Ferreira et al . demonstrated a significant reduction in cardiac arrhythmias in isolate d rat heart in response to ANG1 - 7 . Additionally, i n cardiac fibroblasts ANGII induced hypertrophic effects were prevented by ANG1 - 7 / Mas activation (Stewart et al., 2008) . It was shown that ANGII induced kidney damage was reversed by a recombinant form of ACE - 2. Further , ANG1 - 7 infusion or a Mas agonist has shown to ameliorate renal damage in rats. The mechanisms involving t hese beneficial effects of ANG1 - 7 / Mas , appear to be due to a reduction in oxidative stress (Bader, 2013) . Either of ACE - 2 gene therapy or delivery of recombinant ACE - 2 seem to be protective in diabetic nephropathy. W hether this protective effect is due to an increase in degradation of ANGII or a production of ANG1 - 7 remains unclear in the kidney (Zimmerman and Burns, 2012) . The brain is one of the organs with highest Mas expression. Depending on the brain area th at is under investigation, ANG1 - 7 / Mas have shown to regulate blood pressure. For example, 44 overexpression of ACE - 2 in the medulla decreased blood pressure . ANGII induced reduction of ACE - 2 mRNA and protein was prevented by an ATI receptor antagonist in cerebellar or medullary astrocytes in neonatal rat (Xu et al., 2011) . In vitro e xperiments have shown ANG1 - 7 induces vasopressin release and prostaglandin - releasing activity which promotes neuron al activity in the hypothalamus (Xu et al., 2011) . A protective role by ACE - 2/ ANG1 - 7 / Mas has also been noted in the endothelium. Overexpr ession of ACE - 2, generates ANG1 - 7 and improves endothelial function in hypertensive rats (Rentzsch et al., 2008) . Further, ANG1 - 7 treatment increased release of nitric oxide and im proved endothelial function in Mas transfected Ch inese hamster ovary (CHO) cells (Ferreira et al., 2012) . Novel Thera peutic Targets of ACE - 2/ ANG1 - 7 / Mas In a recent human study, administration of a recombinant form of ACE - 2 intravenously showed a decrease in ANGII in the plasma. Results from this study demonstrated the high possibility of cardiovascular protecti ve effects of recombinant ACE - 2 (Jiang et al., 2014) . In support of this theory, a recombinant form of ACE - 2 is currently in clinical tr ials to tre at acute lung injury (Imai et al., 2005) . Similarly, an ACE - 2 activator, named as XNT was identified and has revealed to decrease blood pressure in hypertensive rats with improved cardiac function (Ferreira et al., 2012) . Activation of ACE - 2 by XNT increased ANG1 - 7 levels and co - administra tion of A779 abolished the ANG1 - 7 effects. Th e therapeutic potential of ANG1 - 7 is limited due to its short half - life. To circumvent this problem a cyclic form of ANG1 - 7 has been introduced to produce a bio - stable peptide 45 ana logue . It has demonstrated improved resistance to ACE, increased half - life and most importantly, it exerted Mas dependent vasodilato r effects in isolated rat aorta (Jiang et al., 2014; Kluskens et al., 2009) . AVE 0991 , a non - peptide , is the first small molecule agonist that w as discovered and has shown to bind to the Mas receptor with high affinity. This compound has shown to mimic ANG1 - 7 i n the heart, kidney and vessels (Ferreira et al., 2012) . AVE 0991 has recently reported to protect against cardiac dysfunction ind uced by isoproterenol (Iusuf et al., 2008) . Another method to deliver ANG1 - 7 is through liposomal delivery, encapsulated in lipid vesicles to protect against degradation. Inject ion of liposome containing ANG1 - 7 into rats exhibited prolonged hypotens ion compared to control animals (Ferreira et al., 2012; Iusuf et al., 2008) . In addition, development of hydroxypro - ANG1 - 7 ) has shown significant attenuation of impaired cardiac functions and cardiac remodel ing ( Ferreira et al., 2012) . MITOGEN ACTIVATED PROTEIN KINASE (MAPK) SIGNALING C - jun - N - terminal kinase (JNK) signaling Cells respond to various stimuli including temperature changes, pH, growth factors, cytokines, hormones, stress and other chemical stimulations (Cui et al., 2007) . The m itogen activated protein kinase (MAPK) family is one of the major signaling system s that consist of protein kinases that regulate signals from outside the cell to activate many intracellular signaling cascades. After the activation of MAPKs, they phosphorylate many proteins that affect the function of the cell. Activation of MAPKs lead to physiologic al processes including cell 46 proliferation, differentiation, apoptosis, stress response s and development (Dickinson and Keyse, 2006) . MAPKs ar e activated by dual phosphorylation on threonine (thr) or tyrosine (ty r) residues by upstream MAP2Ks (MAPK kinase) , which are activated after phosphorylation by MAP3Ks (MAPK kinase kinase) . There are three major classes of MAPKs; e xtra cellular regulated signaling kinase (ERK) 1 and 2 were the first MAPKs identified. In the following years JNK and p38 MAPKs were discovered. JNK is also recognized as stress activated protein kinases that play a key role in apoptosis. There are three JNK genes in mammals; JNK1, JNK2 and JNK3. JNK1 and JNK2 are ubiquitously expressed where as JNK3 is limited to the central nervous system (Cui et al., 2007) . JNK is phosphorylated in the thr or tyr residues of the TXY motif by upstream protein kinases MKK4 /MKK7 that are activated by mixed lineage kinases (MLK) including ASK , TAK and MEKK . MKK7 has been shown to activate JNK with high affinity whereas MKK4 can activate both JNK and P38 MAPKs (Dhanasekaran and Reddy, 2008) . Deletion of MKK4 in mice, prevented the activation of JNK and reduced cardiac hypertrophy. Studies performed in vitro have shown MKK4 phosphorylates JNK on the tyr residue and may be required for opt imal activation of JNK (Haeusgen et al., 2011) . MKK7 phosphorylates JNK on the thr residue which is vital to trigger JNK activity. cFLIP, an anti - apoptotic protein has shown to bind to MKK7 and inhibit JNK activation. Phosphorylated JNK can activate downstream targets including c - jun , c - fos, ATF by phosphorylating ser/thr residue or by modulating pro - apoptotic proteins by phosphorylation . Upon activation, JNK can translocate in to mitochondria and mediate the release of cyt c to form the apoptosome (Chauhan et al., 2003; Dhanasekaran and Reddy, 2008) . Although the exact mechanisms of cyt c release by JNK is unclear, studies have shown a role of pro - apoptotic 47 protein Bid in the pro cess where it can activate Bax. Also the activation of Bid leads to release of apoptotic proteins from the mitochondria that c ould induce apoptotic signaling (Dhanasekaran and Reddy, 2008) . In line with this, JNK also interacts with other pro - apoptotic proteins such as Bim and Bmf (Lei and Davis, 2003) . During UV stimulated apoptosis, the p hosphorylation of Bim and Bmf by JNK, release them from hold and activate Bax and Bak in HEK293 cells. A ctivated JNK also has shown to specifically phosphorylate a serine residue on Bad, one of the pro - apoptotic proteins . Typically pro - survival kinases such as Akt and PKA inhibi t Bad by phosphorylating at a ser residue. Activation of JNK, phosphorylate s ser at a different position than pro - s urvival factors do and promote apoptosis in rat neuronal cells (Dhanasekaran and Reddy, 2008) . In A ECs, JNK phosphorylation is a required event in apoptosis. It was shown ANGII induced pJNK w as blocked by ATI selective antagonist. Inhibition of pJNK by a selective JNK inhibitor prevented th e caspase - 9 activation by bleomycin (Uhal et al., 2011) . Similarly, Chandel et al . demonstrated in mouse lung epithelial cells that bleomycin - induced activa tion of Bax was abrogated by the expression of a dominant negative JNK. Bleomycin exposed both MLE - 12 cells and in primary rat alveolar type II cells, prevented cell death by the dominant negative JNK (Lee et al., 2005) . Collectively, t hese data indicate that JNK dependent activation of cell death in lung epithelial cells. JNK Pathway and Diseases Unusual a ctivation of JNK pathway has shown to contribute to pathological conditions. Acute respiratory distress syndrome is a severe form of acute lung injury (ALI) and endothelial 48 cell injury has shown to contribute to ALI. Apoptosis signal - regulating kinase (ASK) - 1 is a ubiquitously expressed MAP3K that activates JNK. Knock down of ASK - 1 has show n to reverse caspase activation, JNK phosphorylation and apoptosis in endothelial cells (Li, 2004) . Oxidative stress has shown to con tribute to lung injury as well , through signaling mechanisms that activate JNK and phosphorylation of its downstream targets including c - jun and activat ing transcription factor (ATF - 1, Li, 2004) . Endothelin ( EL - 1 ) is mainly generated in endothelial cell but also is pro duced in mesenchymal cell types (A braham, 2008) . The receptors for EL - 1 are widely expressed in tissues and abnormally high levels are reported in lung diseases. Elevated levels of EL - 1 in pulmonary blood vessels and macrophages have been detected in lung injury. Experimental studies ha ve shown TGF - - 1 in fibroblasts through a JNK dependent mechanism and additionally, JNK activation is reduced when th e EL - 1 receptors were inhibited (Abraham, 2008) . Some studies have implicated a role of JNK in several cancer types. Recently, experimental data ha ve suggested a different role in function of JNK in normal and tumor cells. Antisense oligonucleotides m ediated knockdown of JNK showed to inhibit stress induced apoptosis whereas knockdown of JNK prevented the tumor growth (Davis, 2000) . A lthough the exact role of JNK in tumor development is controversial, JNK was shown to induce hepato - carcinogenesis and may relate on its ability to stimulate cell proliferation. Mutation of JNK phosphorylation sites preve nted the tumor size and number (Weston and Davis, 2002) . Whether the induction of JNK leads to apoptosis or survival mechanisms seem to depend on the interactions of JNK with signaling pathways (Davis, 2000) . 49 Liver samples from patients with both acute liver injury and fatty liver disease, had high levels of JNK activity. Mice exposed to carbon tetrachloride (CCL4) exhibited elevated levels of JNK activity and mice deficient in J NK prevented liver injury. Further, in vitro studies demonstrated JNK inhibitors prevented platel et - derived growth factor (PDGF) and transforming grow th factor (TGF - epatic stellate cells (HSC) . Further, the authors demonstrate d, angiotensin II - induced HSC activation was prevented by JNK inhibitors by reducing TGF - and PDGF levels (Kluwe et al., 2010) . Map Kinase Phosphatase s (MKPs) MAPKs play a vital role in determining duration and magnitude of physiological signaling. The activity of MAPK signaling is regula ted by the activation of MKP that remove the phospho group and limit the actions of MAPK signaling. Thus, MKPs form a signaling complex to negatively regulate MAPK signaling (Owens and Keyse, 2007) . MKPs are a group of proteins that belong to the dual specific phosphatase (DUSP) family which consists of ten catalytically active proteins. The MKPs are capable of dephos p horylating both threonine/serine and tyrosine residues of MAPKs. MKPs can recognize and bind to different MAPKs with high specificity but the interaction depends on cell type, cellular localization and re sponse to extracellular stimuli (Lawan et al., 2012) . MKPs contain a N - terminal non catalytic domain and C - terminal catalytic domain. The N - terminal domain c onsists of two short regions that are homologous to c dc25 cell cycle regulatory phosphatase catalytic site. There are three amino acids (asp artate , arg inine and cysteine ) in the catalytic site that are absolutely essential for the catalytic activity. Some MKPs 50 do contain a PEST sequence which is abundant with proline, serine, glutamat e and threonine residues. Removal of the PEST seque nce, stabilizes the phosphatase (Theodosiou and Ashworth, 2002) . The kinase interacting motif (KIM) consists of positively charged amino acids that bind to negative ly charged amino acids in M APKs (Theodosiou and Ashworth, 2002) . Map kinase phosphatase - 2 (MKP - 2) , a 43 kDa protein is one of the earliest identified MKPs (Cadalbert et al., 2005) . It is encoded by the DUSP4 gene and is expressed in a variety of tissues. It belongs to the above mentioned DUSP family and is known to negatively regulate MAPK s by dephosphorylating them . MKP - 2 has been shown to dephosphorylate JNK selectively in vitro and interaction with JNK has shown to increase the catalytic activity. Mouse embryonic fibroblasts derived from a DUSP4 knockout mouse exhibited increased apoptosis and decreased cell proliferation (Lawan et al., 2012) . O verexpression of MKP - 2 has shown to negatively reg ulate JNK signaling and prevent JNK dependent apoptosis (Al - Mutairi et al., 2010a) . In this study, human endothelial cell s were infected with adenoviral MKP - 2 , which selectively eliminated TNF - - mediated JNK activation . Further , cellular damage, apoptosis and caspase - 3 act ivation were all rever s ed by overexpression of MKP - 2 in endothelial cells. UV light has shown to induce apoptosis through mi tochondrial dependent pathway and o verexpression of MKP - 2 rescued UV induced apoptosis in embryonic kidney cells 293. Although , more studies in vivo are needed to clarify the role of MKP - 2 , the authors showed that MKP - 2 specifically dephosphorylates JNK and a high specificity of MKP - 2 for JNK in vivo (Cadalbert et al., 2005) . A recent study done by a different group found , H 2 O 2 induce d JNK phosphorylation was abolishe d by WT - MKP - 2 expressing clones in endothelial cells compared to cells expressing a catalytically inactive form of MKP - 2. Additionally, activation of ERK1/2 was not prevented by 51 overexpression of the MKP - 2 (Robinson et al., 2001) . These data express the importance of phosphatas es in regulation of MAPKs which control many signaling pathways. 52 CHAPTER 2 : ANGIOTENSIN 1 - 7/ MA S INHIBITS APOPTOSIS IN ALVEOLAR EPITHELIAL CELLS THROUGH UPREGULATION OF MAP KINASE PHOSPHATASE - 2 53 Abstract Earlier work from this laboratory show ed that autocrine generation of ANGII and pJNK are both required events in AEC apoptosis. Although earlier data showed that ANG1 - 7 protects against AEC a poptosis, the pathways by which ANG1 - 7 / Mas activation prevent JNK phosphorylat ion and apoptosis are poorly understood. Therefore , in the current study, it was theorized that ANG1 - 7 activates a map kinase phosphatase (MKP - 2) and thereby reduces JNK phosphorylation to inhibit apopt osis and promote cell survival. This hypothesis was ev aluated in both the human and mouse alveolar epithelial cell lines A549 and MLE12, respectively. Cells were transfected with small interfering RNAs, antisense oligonucleotides or inhibitors specific for MKP - 2 or Mas , and were then assayed for phospho - JNK, caspase - 9, loss of mitochondrial membrane potential (MMP) and nuclear fragmentation. Silencing of MKP - 2 significantly prevented the blockade of all apoptotic markers by ANG1 - 7 . Knockdown or blockade of Mas receptor by antisense oligonucleotides or by the r eceptor antagonist A779, respectively, caused significant decreases in MKP - 2, and simultaneously increased the apoptotic markers of caspase - 9 activation and nuclear fragmentation. These data show that the ANG1 - 7 / Mas activation prevents JNK phosphorylation and apoptosis by constitutively activating the JNK - selective phosphatase MKP - 2, and further demonstrate the critical role of the ANG1 - 7 receptor Mas in AEC survival. 54 Introduction It is well established that AEC apoptosis contributes to the pathoge nesis of lung injury (Li, 2004) . Understanding the underlying signaling mechanisms of AEC apoptosis is cri tical to determine the pathogenesis interstitial lung diseases (ILD). Blockade of apoptosis by broad spectrum caspase inhibitors or genetic deletion of ap optotic genes prevented lung injury in animal models (Tallant et al ., 2005) . In recent years, activation of a local ANG in the lung has shown to play a major role in AEC apoptosis a nd subsequent lung fibrosis (Uhal, 2008) . Previous work from this laboratory demonstrated that inducers of apoptosis generate AGT , the 58 kDa protein which, after enzymatic cleavage generates the effector peptide ANGII ( Li et al., 2003a; Wang et al., 1999, 2000a) . Moreover, it was shown that autocrine generation of ANGII is required in AEC apoptosis, through experiments that blocked apoptosis by either antisense oligonucleotides agains t AGT mRNA, AT1 receptor antagonists, or by neutralizing antib odies against ANGII itself (Uhal, 2002) . Subsequent in vitro studies showed that binding of ANGII to its receptor AT1 causes phosphorylation of JNK, a member of the MAPK family, which is required for AEC apop tosis (Uhal et al., 2012) . Recent work showed that ACE - 2 is protective against experimental lung fibrosis. Lung tissues from idiopathic pulmonary fibrosis (IPF) patients showed significantly reduced levels of ACE - 2 mRNA, protein and enzymatic activity, sugge sting that loss of ACE - 2 contributes to accumulation of ANGII causing AEC apoptosis and lung injury (Li et al., 2008) . Accordingly, in both pulmonary and non - pulmonary systems ANG1 - 7 has shown to counteract detrimental effects of ANGII t hrough the Mas receptor (Jiang et al., 2014) . Radio - ligand binding studies have 55 provided evidence that ANG1 - 7 binds to its receptor Mas , which is distinct from the AT1 an d AT2 receptor subtypes (Santos et al., 2003) . Experimental studies in this laboratory demonstrated that ANG1 - 7 inhibits ANGII - or bleomycin (bleo) - induced JNK phosphorylation in AECs (Uhal et al., 2011) . Further, ANG1 - 7 also inhibited caspase activation an d apoptosis which were blocked by the Mas receptor antagonist, A779, which has very low affinity for the AT1 or AT2 receptors. Although the exact downstream signaling mechanisms of the ANG1 - 7 / Mas pathway are currently unclear, several groups have shown the activation of a phosphatase in different cell types (Burgun et al., 2000; Gallagher et al., 2008) . Map kinase phosphatases are important negative regulators of MAPKs through dephosphorylating the Thr - X - Tyr (T - X - Y) motif of MAP Ks (Dickinson and Keyse, 2006) . A recent publication by Uhal et al. showed that at baseline (without stimulation) ANG1 - 7 is more abundant than ANGII in the cell culture media bathing primary AECs and that ANG1 - 7 dephosphorylates pJNK a s a cell survival mechanism (Uhal et al., 2011) . Therefore, it was theorized that the ANG1 - 7 / Mas pathway activates a JNK - selective MKP - 2 to reduce pJNK levels, thus promoting cell survival. The data herein report the fi ndings that silencing MKP - 2 prevents the blockade of JNK phosphorylation and apoptosis ( ANGII signaling) by ANG1 - 7 in AECs. Further, we also report that silencing Mas decreases MKP - 2 and promotes apoptosis. 56 Materials and Methods Reagents and materials: ANGII and ANG1 - 7 were purchased from Sigma - Aldrich (St. Louis, MO). Mas receptor antagonist A779 (D - Ala7 - ANG1 - 7 ) was purchased from GenScript (Piscataway, NJ). Antibodies for the detection of MKP - 2, Mas receptor as well as MKP - 2 specific small interfering RNAs (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for th e detection of pJNK , active forms of caspase - - actin were all obtained from Cell Signaling Technology (Boston, MA). Antisense oligonucleotides against the Mas receptor and the control a ntisense were obtained from Genemed Synthesis (San Antonio, TX). 3 , 3 - dihexyloxacarbocyanine iodide (DiOC6) was obtained from Life Technologies (Carlsbad, CA). All the other materials were of reagent grade and were purchased from Sigma Aldrich. Cell culture : The human type II epithelial c ell - derived cell line (A549) was obtained from American Type Culture Collection (ATCC, Manassas, VA) and was grown in F - 12 medium containing 10% serum. The mouse lung epithelial cell line (MLE - 12), a generous gift from the laboratory of Dr. J. Whitsett, Un iversity of Cincinnati, was grown in complete HITES media. All the cells were grown in 6, 12 or 24 well culture plates and were analyzed at sub - confluent densities. All subsequent incubations with ANG 1 7 and/or A779 ( Mas receptor antagonist) were performe d in serum - free medium. In all studies cells were exposed to ANG1 - 7 (10 - 7 M) for 40 min and/or A779 (10 - 7 M) for 30 min before exposure to ANGII (10 - 7 M) for 5 min to 20 h as indicated. Exposure to siRNAs and control siRNAs were done prior to treatment wit h ANG1 - 7 and ANGII . 57 Gene knockdown: Antisense oligonucleotides against human Mas were designed using Antisense design tool from Integrated DNA Technologies (IDT, Coralville, IA) and were synthesized as phosphorothioated 20 - mers. A549 cells were transfected with antisense oligonucleotides or control antisense (final conc. 0.1 µM) by using Lipofectamine2000 reagent (Life Technologies, Grand Island, NY) at 2 µl/ml as the vehicle dissolved in F - 12 media without any serum or antibiotics. After transfections, the cells were incubated at 37°C with 5% CO2 for six hours followed by addition of normal growth medium with 3 times the normal serum and antibiotic concentration (3x normal growth medium). At 24 h, the transfection reagen ts were removed and was replaced by complete F - 12 media for an additional 24 h. Afterwards, the cells were serum starved overnight and immediately thereafter ANG1 - 7 (10 - 7 M) w as added for 40 min to 12 h as indicated. The siRNAs against human MKP - 2 were co mmercially synthesized and purchased from Santa Cruz Biotechnologies. The siRNA - to - lipofectine ratios were optimized to yield effective knockdown which was confirmed by Western blotting. A549s were transfected (final conc. 0.1 µM) similarly as described ab ove and were treated with ANG1 - 7 (10 - 7 M) followed by ANGII (10 - 7 M). A scrambled siRNA of the same sequence was used as a negative control. The MKP - 2 - GAAGGACACUAUCAGUACAtt - and antisense - UGUACUGAUAGUGUCCUUCtt - - GGACUCCGAAUACAUAAUAtt - and antisense - UAUUAUGUAUUCGGAGUCCtt - - CACAGAUCCUAGCAAAUGUtt - and antisense - ACAUUUGCUAGGAUCUGUGtt - 58 Detection of apoptosis: Apoptotic cells were detected by nuclea r fragmentation assay using propidium iodide (PI) as described earlier after enzymatic digestion of ethanol - fixed cells with DNase - free RNa se in PBS containing 5 µg/ml PI (Wang et al., 1999, 2000a) . During fixation with 70% ethanol, detached cells were retained by centrifugation of the 24 - well culture plates. Cells with discrete nuclear fragments with condensed chromatin were counted as apoptotic using epi - fluorescence microscopy. Apoptotic cells were scored over a minimum of four separate microscopic fields from each of at least three culture vessels per treatment group. As in earlier publications from this laboratory, the induction of apoptosis is verified by in situ end la beling (ISEL) of fragmente d DNA (Uhal et al., 1998) . Briefly, the cells were washed with distilled water for 10 min followed by incubation with 0.23% periodic acid for 10 min. After five washes with 0.1 5 M PBS, cells were incubated with saline - sodium citrate solution at 80°C for 20 min followed by four washes with 0.5 M PBS and 3x with buffer A (50 mM Tris.HCl, 5 mM - mercaptoethanol and 0.005% BSA in water). Next, cells were incubated with an ISEL solution (0.001 mM biotin - dUTP, 0.01 mM of each dATP, dCTP, dGTP, 20 U/ml DNA polymerase I in buffer A) for 2 h at 20°C. Afterwards, cells were washed with 3x in buffer A and with 0.5 M PBS for 5x times. Then the cells were incubated with a Vectast ain ELITE solution that contains avidin and biotin - peroxidase solution dissolved in buffer B (1% BSA and 0.5% Tween 20 in 0.5 M PBS). After 30 min, cells were washed with PBS followed by incubation with 0.25 mg/ml diaminobenzidine (DAB) solution in 0.05 M Tris - HCl containing 0.01% H 2 0 2 to detect end labeling. The active forms of caspase - 9 were detected by Western blotting using antibodies specific to the cleaved forms. 59 Estimation of mitochondrial mem brane potential: The MMP) in A549 cells transfected with MKP - 2 siRNA as described above, were assessed with the lipophilic probe DiOC6. After treatment with ANGII (10 - 7 M) for 8 h in the presence or absence of ANG1 - 7 (10 - 7 M), the cells we re incubated with PBS containing 50 nM DiOC6 for 15 min at 37°C followed by an assay in a fluorescence plate reader (BioTek, Winooski, VT) at 360 nm excitation and 420 nm emission. To determine the total DNA, cells were fixed with 70% ethanol for 30 min fo llowed by an incubation with 10 µm Hoechst 33342 dye dissolved in PBS for 10 min. Then the cells were reanalyzed at the same wavelengths for quantitation of total cellular DNA. Data were then normalized. Western blotting: Cells were lysed either with a mod ified lysis buffer for phospho - proteins, containing 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X - 100, 1M EGTA, 1.5 mM MgCl2, 100 µM sodium orthovanadate, and the protease inhibitor cocktail (Complete Mini, Roche, Nutley, NJ) or with a Nonidet P - 40 - b ased lysis buffer containing protease inhibitors (for MKP - 2, Mas and caspase - 9). After harvesting, proteins were run on polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Next, the bands were visualized by chemiluminescent s ubstrate West Femto detection systems (Pierce, Rockford, IL). 60 Results In a previous study , it was found that steady state levels of ANG1 - 7 levels are much higher in the cell culture media than ANGII levels (Uhal et al., 2011) . Thus, it was theorized that ANG1 - 7 maintains cell survival by dephosphorylating p JNK , by upregul ating a map kinase phosphatase (MKP - 2) . To test this hypothesis, lung epithelial cells were treated with A779 (10 - 7 M) for 30 min after an overnight serum starvation to block the endogenous accumulation of ANG1 - 7 and cell s were harvested to detect pJNK. Figure 2 - 7A shows that blocking the action s of the endogenous ANG1 - 7 , using the specific Mas blocker A779 added in the f inal 30 min of a 12 h serum starvation, induces pJNK levels compared to control cells without added A779. To determine if the blockade of endogenous ANG1 - 7 also reduces MKP - 2, AECs were incubated with A779 (10 - 7 M) in similar protocol to the previous experiment. Thereafter , the cells were harvested and subjected for Western blotting. T reating the cells with A779 lead to a reduction in MKP - 2 levels in both mouse (Fig. 2 - 7B) and in human (Fig. 2 - 7C) alveolar epithelial cells. Further, treatment with A779 (10 - 7 M) reduced MKP - 2 levels in primary cultures of human alveolar epithelial ce lls (Fig. 2 - 7D) confirming the results from mouse and human cell lines. Further , these data also demonstrate that M as receptor is functional in primary cultures of human AECs . Next, to determine whether addition of exogenous ANG1 - 7 would upregulate MKP - 2, the endogenous ANG1 - 7 was removed after an overnight incubation followed by addition of exogenous ANG1 - 7 (10 - 7 M) for 40 min . As shown in F igure 2 - 8 , MKP - 2 levels were increased in both mouse (2 - 8A) and human (2 - 8B) lung epithelial cells compared to the control cells that did not receive exogenous ANG1 - 7 . In F igure 2 - 9A&B, AECs that received exogenous ANG1 - 7 61 showed increased MKP - 2 protein compared to the control cells . F urther, the induction of MKP - 2 by ANG1 - 7 was blocked by pre - incubating the cells with A779 (10 - 7 M), the Mas receptor antagonist, which demonstrates the involvement of Mas on the induction of MKP - 2 by ANG1 - 7 (in both MLE - 12 and A549 cells). Additionally, to show that map kinase phosphatase - 1 (MKP - 1) and map kinase phosphatase - 5 (MKP - 5) protein levels did not change with treatment of ANG1 - 7 /A779, cells were treated and harvested similar ly as in Fig. 2 - 9A&B . As shown in F igure 2 - 9C, the protei n levels for both MKP - 5 and MKP - 1 did not change in response to ANG1 - 7 /A779. As shown previously in Figure 2 - 9, treatment with A779 reduced the induction in MKP - 2 protein compared to the control cells. To confirm this result with antisense oligonucleotides against the Mas receptor, A549 cells were cultured and were treated with antisense in the presence of ANG1 - 7 (10 - 7 M) for 40 min followed by cell harvesting to detect MKP - 2. Silencing the Mas receptor significantly reduced the levels of MKP - 2 compared to the cells with ANG1 - 7 alone (Fig. 2 - 10) confirming that MKP - 2 upregulation occurs through the Mas receptor. Data from the Figures 2 - 7, 2 - 8 & 2 - 9 clearly demonstrated the upregulation of MKP - 2 in alveola r epithelial cells. To further investigate the functiona l roles of MKP - 2 in AECs, it was important to determine whether MKP - 2 protein can be silenced effectively in A549 cells. After the cells were cultured, they were treated with specific siRNA (final conc. 0.1 µM) against MKP - 2. As shown in Fig. 2 - 11 A , treatment with the siRNA silenced the MKP - 2 protein compared to the negative control with a scrambled sequence (also compared to transfecting reagents only). To test whether MKP - 2 siRNA are specific only for MKP - 2 protein and not any of other JNK selective phos phatases, the same samples above were subjected for Western blotting for other known MKPs . F igure 2 - 11B shows, map kinase phosphatase - 3 (MKP - 3) and map kinase 62 phosphat ase - 7 (MKP - 7) protein levels did not change with MKP - 2 siRNA . This illustrated that MKP - 2 silencing is specific and does not interfere with the expression of the two other known JNK - selective phosphatases MKP - 3 and MKP - 7, which were unaffected. Earlier wo rk showed that JNK phosphorylation is a required event in AEC apoptosis (Uhal et al., 2011) ; to determine whether silencing MKP - 2 induces JNK phosphorylation, A549 cells were treated with MKP - 2 siRNAs (final conc. 0.1 µM) in the presence or ab sence of ANG1 - 7 (10 - 7 M) and /or ANGII (10 - 7 M) for 40 min and 5 min respectively. Figure 2 - 12 shows that silencing MKP - 2 prevented ANG1 - 7 blockade of JNK phosphorylation. Previously, it was shown that at baseline ANG1 - 7 levels are protective against AEC apoptosis. Therefore, it was imp ortant to access the effects of silencing MKP - 2 in the absence of exogenous ANG1 - 7 and ANGII . To test whether knockdown of MKP - 2 increases basal levels of pJNK and caspase - 9, cells were tre ated with MKP - 2 siRNA and were harvested after an additional 12 h to det ect MKP - 2 protein. Figure 2 - 13A show s the increase in basal pJNK levels when MKP - 2 is silenced. Further, silencing MKP - 2 also increased the active form of caspase - 9 compared to the control cells that did not receive MKP - 2 specific siRNA (Fig. 2 - 13B) . Prev iously , it was demonstrated that the blockade of mitochondrial membrane potential (MMP) loss by ANG1 - 7 (Uhal et al., 2013b) . To determine the effects of sil encing MKP - 2 could reverse the effects of ANG1 - 7 on MMP ( one of the apoptotic markers ) , A549 cells were cultured and treated with MKP - 2 - specific siRNA followed by treatment with the peptides ANG1 - 7 (10 - 7 M) and /or ANGII (10 - 7 M) for 40 mi n and 8 h , respectively. A549 cells were then incubated with the lipophilic dye DiOC6 for 30 min and fluorescence was measured. As shown, Fig. 2 - 14 demonstrates that the loss of MMP induced by 63 ANGII , was prevented by ANG1 - 7 . Further, the blockade by ANG1 - 7 was inhibited by siRNA - mediated silencing of MKP - 2 . To determine the effects of silencing MKP - 2 on inhibition of caspase - 9 activation by ANG1 - 7 , A549 cells were cultured and were transfected similarly to the above experim ent in Fig. 2 - 14 . Next, cells were treated with ANG1 - 7 (10 - 7 M) and /or ANGII (10 - 7 M). As shown, in Figure 2 - 15A , silencing MKP - 2 significantly prevented the ability of ANG1 - 7 to inhibit generation of the cleaved form of caspase - 9. Given that, knockdown of MKP - 2 increased p JNK, MMP an d active form of caspase - 9 ( Figures 2 - 12, 14&15A) , it was of high interest to determine whether silencing MKP - 2 would also increase apo ptosis in AECs . To measure nuclear fragmentation (apoptotic body formation) by propidium iodide (PI) assay , A549 cells were cultured and were transfected with MKP - 2 specific siRNA followed by incubation with ANG1 - 7 (10 - 7 M) and /or ANGII (10 - 7 M) . Figure 2 - 15C shows that a fter 20 h exposure to ANGII , knockdown of MKP - 2 significantly prevented the ANG1 - 7 blockade of nuclear fragmentation induced by ANGII . The results of the nuclear fragmentation assay were also confirmed by in situ end labeling (ISEL) to - diaminobenzidine (DAB) method. Again, knockdown of MKP - 2 signific antly prevented blockade by ANG1 - 7 (Figure 2 - 15B). To illustrate that silencing MKP - 2 also reverses ANG1 - 7 signaling in well - differentiated lung epithelial cells, human primary cultures of AECs were cultured and were treated similarly as in Fig 2 - 15C. As s hown in Fig 2 - 15D , silencing MKP - 2 significantly reversed ANG1 - 7 blockade of ANGII induced nuclear fragmentation , confirming the results wit h A549 cells. To investigate the downstream signaling of the Mas receptor in AECs, antisense olig onucleotides (final conc. 0.1 µM) were used to knock down the Mas receptor, which 64 significantly silenced the receptor compared to the controls incubated wi th a scrambled sequence (Figure 2 - 16 A ). Next, to determine the importance of Mas receptor in AEC survival, A549 ce lls were incubated with Mas antisense oligonucleotides, but in the absence of exogenous ANG1 - 7. The cells were then harvested to detect basal levels of pJNK and caspase. S ilencing the Mas receptor significantly induced basal levels of pJNK compared to the control cells (Fig. 2 - 16B) and similarly , induced the active form of caspase - 9 (Fig. 2 - 16C) . These data demonstrated the importance of Mas receptor in AEC survival. To determine if silencing the Mas receptor induces the active form of casp ase - 9 in the presence of ANG1 - 7 , A 549 cells were treated with Mas antisense oligonucleotides and were treated with ANG1 - 7 (10 - 7 M) for an additional 8 h . F igure 2 - 17A shows the increase in active form of caspase - 9 when Mas receptor is silenced . Similarly, the cells were cultured and were treated with ANG1 - 7 (10 - 7 M) for an additional 12 h. N uclear fragments assessed with PI assay was significantly increased after the Mas re ceptor was silenced (Figure 2 - 17B ). 65 Figure 2 - 7 : Mas blo cker increases pJNK and decre a ses MKP - 2 in lung epithelial cells . A) Mouse l ung epithelial (MLE) cells were incubated with the Mas receptor antagonist A779 (10 - 7 M) for 30 min after a 12 h serum starvation, without removing the endogenous ANG1 - 7 . Bars are mean + SE of n=4 over two experiments. * P < 0 .05 vs. CTL by unpaired t test . B) MLE cells 66 Fig ure 2 - 7 ( ) were cultured and serum starved for 12 h before challenging with A779 (10 - 7 M) for 30 min followed by harvesting for Western blotting. Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL by unpaired t test. C) Similarly, A549 cells were treat ed as described above in Fig. 2 - 7B . Bars are means + SE of n=4 over two ex periments. * P < 0.05 vs. CTL by unpaired t test. D) Bars are means + SE of n=3 in primary human alveolar epithelial cells . * P = 0.05 vs. CTL by unpaired t test . 67 Figure 2 - 8: ANG1 - 7 induces MKP - 2 in lung epithelial cells. A) MLE - 12 cells were serum starved for 12 h, after which the media was removed and were challenged with freshly prepared ANG1 - 7 (10 - 7 M) for 40 min before harvesting for Western blotting. The control group did not receive ANG1 - 7. Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL by unpaired t test. B) A549 cells were treated similarly as in above Fig. 2 - 8A . Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL by unpaired t test. 68 Figure 2 - 9 : Blockade of MKP - 2 induction by a mas receptor blocker. A) A549 cel ls were pre - incubated with ANG1 - 7 (10 - 7 M) for 40 min in the presence or absence of A779 (10 - 7 M), the mas receptor antagonist followed by cell harvesting for Western blotting. B ) MLE - 12 cells were treated and h arvested simil arly as in Fig 2 - 9A . Bars are means + SE of at least 6 69 Fig ure 2 - 9 ( ) separate cell cultures over two or three experiments.* P < 0.05 vs. CTL and * * P <0.05 vs. ANG 1 - 7 by ANOVA and Student - New man - Keuls post hoc analysis. C) Same s amples as in above were subjected for detection of map kinase phosphatase - 5 and 1 as shown. 7 0 Figure 2 - 10: Mas knockdown prevents ANG1 - 7 induction of MKP - 2. A) A5 49 cells were treated with antisense oligonucleotides (final conc. 0.1 µM) in the presence of ANG1 - 7 (10 - 7 M) for 30 min followed by harvesting. Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL and ** P < 0.05 vs. ANG 1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis . 71 Figure 2 - 11: Verification of MKP - 2 knockdown by small interfering RNA (siRNA) in A549 cells but not by a scrambled siRNA. A) A549 cells were cultured and were transfected with either MKP - 2 siRNA or a scrambled sequence using Lipofectamine2000 reagent. After 48 h cells were harvested and subject ed t o Western blotting. B) The s ame samples in Fig. 2 - 11A were used to determine any knockdown effects on other phosphatases (map kinase phosphatase - 7 and 3) by Western blotting. 72 Figure 2 - 12: MKP - 2 knockdown prevents inhibition of JNK phosphorylation by ANG1 - 7. A549 cells were transfected with MKP - 2 siRNA for 48 h, followed by an incubation with ANG1 - 7 (10 - 7 M) and ANGII (10 - 7 M) for an additional 40 min and 5 min respectively. Next, the cells were * P < 0.05 vs. siCTL, ** P < 0.05 vs. ANGII and *** P < 0.05 vs. ANGII/ANG1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis. 73 Figure 2 - 13: MKP - 2 knockdown increases basal pJNK and caspase - 9 levels. A) A549 cells were treated with MKP - 2 siRNA or a scrambled sequence as described in Fig 2 - 11A . After 48 h , cells were serum starved and were incubated for an additional 12 h. Next, cells were harvested for Western blotting to detect basal levels of pJNK. Bars are means + SE o over two experiments. * P < 0.05 vs. siCTL by ANOVA and Student - New m an - Keuls po st hoc analysis. B) The s ame samples above in Fig. 2 - 13 A were subjected for active form of caspase - 9 over two experiments. * P < 0.05 vs. siCTL by unpaired t test. 74 Figure 2 - 14 : Silencing MKP - 2 prevents ANG 1 - 7 r escue of mitochondrial membrane potential (MMP). A) A549 cells were cultured in 24 well plates and were treated as in Fig. 2 - 12 with MKP - 2 siRNA and ANG1 - 7 (10 - 7 M) followed by ANGII (10 - 7 M) for an additional 8 h. Next, cells were - dihexyloxacarbocyanine iodide (DiOC6) for 15 min at 37°C for the estimation of mitochondrial membrane potential (MMP). Bars are means + SE of n in two or more cell cul tures. * P < 0.05 vs. CTL and ** P < 0.05 vs. ANGII by ANOVA and Student - Newman - Keuls post hoc analysis. 75 Figure 2 - 15 : Silencing of MK P - 2 prevents inhibition of caspase - 9 activation, DNA and nuclear fragmentation by ANG 1 - 7. A) After transfection of A549 cells, ANG1 - 7 (10 - 7 M) was added for 40 min followed by ANGII (10 - 7 M) for an additional 8 h. Next, the cells were harvested for Western blotting and the densitometry bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL, ** P < 0.05 vs. ANGII and *** P < 0.05 vs. ANGII/ANG1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis. B) A549 cells were treated with MKP - 2 siRNA followed by incubation with ANG1 - 7 76 Fig ure 2 - 15 ( ) (10 - 7 M) for an additional 40 min. Thereafter, the cells were exposed to ANGII (10 - 7 M) for 20 h followed by fixation of cells and were subjected to in situ end la beling (ISEL) procedure for the - diaminobenzidine (DAB) detection method. Bars are means + SE of n=3; * P < 0.05 vs. CTL and ** P < 0.05 vs. ANG II/ ANG1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis. C ) A549 cells were treated as in Fig. 2 - 15B and were treated with propidium iodide (PI), followed by microscopic quantitation of nuclear fragmentation. Bars are mean s + SE of n=3; * P < 0.05 vs. CTL, * * P < 0.05 vs. ANG II and *** P < 0.05 vs. ANG II/ ANG 1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis. D) Primary human AECs were cultured and were t reate d with MKP - 2 siRNA as in Fig. 2 - 15C. Bars are means + SE of n=3; * P < 0.05 vs. CTL, * P < 0.05 vs. ANGII/ANG 1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis . 77 Figure 2 - 16: Mas knockdown increases basal pJNK and caspase - 9 levels. A) A549 cells were treated with antisense oligonucleotides (0.1 µM final concentration) against mas receptor to verify mas knock down. At 48 h, cells were harvested for Western blotting. B) A549 cells were treated with antisense oligonu cleotides as described in Fig. 2 - 16A . At 18 h (post transfection), cells were harvested for Western blotting to detect basal levels of pJNK. Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL - As by Student - Newman - Keuls post hoc analysis. C) Same samples above in Fig. 2 - 16B were subjected for caspase - 9 detection. Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL - As by unpaired t test . 78 Figure 2 - 17 : Mas knockdown induces caspas e - 9 activation and apoptosis in lung epithelial cells. A) A549 cel ls were transfected as in Fig. 2 - 16 A in the presence of ANG 1 - 7 (10 - 7 M) followed by cell harvesting for Western blotting. Bars are means + SE of n=4 over two experiments. * P < 0.05 vs. CTL and ** P < 0.05 vs. ANG 1 - 7 by ANOVA and Student - Newman - Keuls post hoc a nalysis. B) A5 49 cells were treated with anti sense oligonucleotides for 48 h as described above and were subjected to PI assa y after 12 h treatment with ANG 1 - 7 (10 - 7 M). Bars are means + SE periments. * P < 0.05 vs. CTL and ANG 1 - 7 by ANOVA and Student - Newman - Keuls post hoc analysis. 79 Discussion The exact downstream signaling mechanisms of the ANG1 - 7 / Mas pathway are currently of high interest to investigators seeking to understand the regulation of AEC apoptosis and its contribution to lung disease. Past studies from this laboratory have shown the involvement of the ANG system in AEC apoptosis and subseq uent lung injury (Fine et al., 2000) . It is well known that in many organ systems, detrimental effects of the ANGII /AT1 pathway are counteracted by the opposing axis of the ANG1 - 7 / Mas signaling pathway (Iwai and Ho riuchi, 2009) . Recently, it was demonstrated that JNK phosphorylation is a required event in AEC apoptosis in response to bindin g of ANGII to the AT1 receptor (Uhal et al., 2011) . These authors also showed that bleomycin - or ANGII - induced JNK phosphorylation and apoptosis were blocked by ANG1 - 7 through its binding to Mas receptor . The enzyme ACE - 2 functions as a mono - carboxypeptidase and is one of t he enzymes that could degrade the pro - apoptotic ANGII to form the anti - apoptotic ANG1 - 7 (Hamming et al., 2007) . Lentiviral overexpression of ACE - 2 has been shown to protect against experimental acute lung injury and cardiac fibrosis in response to bleomycin and ANGII , respectively (Huentelman et al., 2005; Shenoy et al., 2010) . Sim ilarly, infusion of ANG1 - 7 subcutaneously in C57BL /6 mice attenuated lung injury and moreover, treatment with a specific Mas blocker A779 aggravated collagen depositio n and lung tissue remodeling (Chen et al., 2013b) . Several studies have shown that imbalance in the levels of extracellular ANGII and ANG1 - 7 contribute to the pathogenesis of lung injury and defects in other organs (Wösten - van Asperen et al., 2013) . A recent s tudy of AECs by Uhal et al. showed that extracell ular ANG1 - 7 levels in serum - free cell culture media are much higher than extracellular ANGII levels under 80 unstimulated conditions, which was interpreted as a mechanis m to maintain cell survival . In the same study it was found that JNK phosphorylation is a required event in AEC apoptosis. Consistent with those findings, in the present study blocking the action of the endogenous ANG1 - 7 with the Mas antagonist A779 significantly increas ed JNK phosphorylation (Fig. 2 - 7 A ). Other research groups have shown that in non - pulmonary cells, ANG1 - 7 inhibits ANGII - induced signaling through activation of a phosphatase. Given that phospho - JNK was increased in response to the Mas block er (Fig. 2 - 7A ), it was high of interest to determine whether blockade of Mas could reduce MKP - 2 prot ein; this result was observed (F igur e 2 - 7A&B ) in AECs . Furthermore, removing the endogenous ANG1 - 7 and adding freshly prepared exogenous ANG1 - 7 significantly induced MK P - 2 (Fig. 2 - 8 ), demonstrating that ANG1 - 7 regulates AEC survival by upregulating a MAPK - selective phosphatase and dephosphorylating JNK. The further finding that the induction of MKP - 2 by ANG1 - 7 was prevented by the Mas antagonist A779 (F ig. 2 - 9 ) showed that ANG1 - 7 induces MKP - 2 through its rece ptor Mas . These results are consistent with earlier work from this laboratory which demonstrated Mas - mediated blockade of ANGII - or bleomycin - i nduced signaling by ANG1 - 7 (Uhal et al., 2011) . Together, those da ta and the results from Fig. 2 - 9 strongly suggested that MKP - 2 mediates its action through the Mas receptor in AECs. The data showing that antisense oligonucleotides against the Mas receptor significantly prevented the ANG1 - 7 - induced MKP - 2 protein levels (Fig. 2 - 10) provide further support for this concept. Together, these data are consistent with experimental results that several other research groups have obtained by studying non - pulmonary cell types. In proximal tubular cells, ANG1 - 7 activated a tyrosine phosphatase and thereby prevented high glucose - stimulated 81 phosphorylation of p38 (Gava et al., 2009) . In studies of cardiac myocytes, it was found that ANGII stimulated the phosphorylation of ERK1/ERK2, but this was reduced by cotreatment with ANG1 - 7 (McCollum et al., 2012b) . Moreover, the same authors found that ANG1 - 7 induces map kinase phosphatase - 1 (MKP - 1) and further, at tenuates cardiac remodeling (McCollum et al., 2012b) . Consistent with these findings, transgenic mice with constitutive overexpression of MKP - 1, did not activate JNK , p38 or ERK1/ERK2 in the heart and further, catecholamine induced hypertrophy was prevented by overexpression of MKP - 1 . Those data showed that dual specific phosphatases (DUSPs), primarily MKP - 1, are important in counter - regul ating MAPKs in cardiac cells (Bueno et al., 2001) . Activation of MKP - 1 was also demonstrated in vascular smooth muscle cells (VSMCs), and this activation antagonized ANGII /A T1 - mediated vascular injury (Takeda - Matsubara et al., 2002 ) . However, the data presented herein strongly demonstrate the upregulation of MKP - 2 in alveolar epithelial cells as a cell survival mechanism and treatment with ANG 1 - 7 and/or A779 did not change MKP - 1 or MKP - 5 as shown in Fig. 2 - 9C. These differences could be due to cell type specificity and various signaling mechanisms involved. In light of the data implicating MKP - 2 in AEC survival, it was of high interest to determine the functional effects of MKP - 2 silenci ng in AECs with siRNAs (Fig. 2 - 11 ). As illustrated here, knockdown of MKP - 2 induced basal levels of pJNK and caspase - 9 demonstrating its importance in AEC survival (Fig. 2 - 13). The siRNA - mediated knockdown of MKP - 2 caus ed a blockade of the ability of ANG1 - 7 to inhibit ANGI I - induced JNK phosp h orylation (Fig. 2 - 12 ), caspase - 9 activation, DNA and nuclear fragmentation (Fig. 2 - 15 ). These data are consistent w ith findings by Gava et al. who showed that blockade of a tyrosine phosphatase by the inhibitor phenylarsine oxide reve rsed the effects of A NG1 - 7 . Moreover, o verexpression of 82 MKP - 2 in human endothelial cells prevented tumor necrosis factor (TNF - - induced apoptosis by preventing JNK phosphorylation, and the induction of apoptotic markers by these cells was also reve rsed by overexpressing MKP - 2 (Al - Mutairi et al., 2010b) . A recent publication from this laboratory demonstrated ER - stress induced mitochondrial dysfunction in AECs was blocked by ANG1 - 7 (Uhal et al., 2013b) . In the present study ANG1 - 7 significantly prevented ANGII - induced reduction of the mitochondrial me mbrane potential (MMP, Fig. 2 - 14 ) and moreover, the prevention by ANG1 - 7 was blocked in the absence of MKP - 2. Cadalbert et al. showed that MKP - 2 protects against stress - induced apoptosis in human embryonic kidney cells 293 and moreover, the authors showed specificity of MKP - 2 to dephosphorylate JNK in vivo (Cadalbert et al., 2005) . The data from these groups and our current data suggest that ANG1 - 7 activates different phosphatases and cau ses multiple different biological effects in different cell types. In non - pulmonary cell types, ANG1 - 7 has shown physiological responses that are opposite to those of ANGII. The hepta - peptide ANG1 - 7 has been shown to inhibit ANGII - induced MAPK signaling i n cardiac myocytes (Tallant et al., 2005), endothelial cells (Sampaio et al., 2007), smooth muscle cells (Freeman et al., 1996) and renal proximal tubular cells (Su et al., 2006). Furthermore, cellular responses to ANG1 - 7 were blocked by pretreatment with the Mas selective blocker A779. It was shown that Mas knockout mice exhibit impaired cardiac function in vivo and in vitro, which demonstrated the physiologic significance of Mas receptor (Santos et al., 2006). Likewise, the data shown here demonstrate a s imilar role for the ANG1 - 7/Mas pathway in AECs; knockdown of the mas recepto r, in the absence of ANG II/ANG1 - 7 showed an induction of basal levels of pJNK and caspase - 9 (Fig. 2 - 16) demonstrating a critical role of mas in 83 AECs. Moreover, antisense oligonucleotide - mediated Mas knockdown induced the activated form of caspase - 9 and nuclear fragmentation as shown in Fig. 2 - 17. These data confirm the involvement of Mas in AEC survival, and moreover are consistent with previously published data showing th at blockade of the Mas receptor with A779 in mouse lun g epithelial cells prevented the inhibition of apoptosis by ANG1 - 7 (Uhal et al., 2011). To date, only a few studies have investigated the downstream signaling of the ANG1 - 7 / Mas pathway. It was shown tha t in rat neurons, ANG1 - 7 induces phosphatase and tensin homolog (PTEN), which dephosphorylates membrane phosphorylated lipids to prevent recruitment of Akt (Modgil et al., 2012) . By contrast in isolated adult myocytes, ANG1 - 7 increased nitric oxide (NO) production associated with induction in endothelial NO synthase and Akt signaling, which were all blocked by treatment with A7 79 (Dias - Peixoto et al., 2008) . These different mechanisms of ANG1 - 7 action could be due to cell type s pecifici ty. Tallant et al. (Tallant and Clark, 2003) demonstrated that ANG1 - 7 stimulates prostacyclin (PGI2) and stimulated cAMP production in rat VSMCs. However, the molecular mechanisms by which t he ANG1 - 7 / Mas pathway stimulates MKP - 2 are currently unknown. In vascular smooth muscle cells, blockade of the NO/cGMP pathway preve nted the induction of MKP - 1 (Jacob et al., 2002) . Along similar lin es, it was demonstrated that activation of Na + - ATPase in response to ANGII was blocked by ANG1 - 7 through th e cAMP/PKA - mediated pathway (Lara et al., 2010) . Therefore, it will be of high interest to determine whether cAMP/PKA or cGMP pathways a re involved in the induction of MKP - 2 in AECs. It is also a possibility that phospho - JNK might be a substrate for other phosphatases, but the results of the siRNA knockdowns shown here suggest that MKP - 2 is uniquely responsible for maintaining AEC survival . Although studies have demonstrated the 84 activation of phosphatases by ANG1 - 7 in different organs, transcriptional regulation and mRNA stability of the various phosphatases are poorly understood and need to be investigated. Collectively, the experimental studies herein showed that the ability of ANG1 - 7 to block ANGII - induced phospho - JNK, caspase - 9, MMP, DNA fragmentation and apoptosis is abolished if MKP - 2 is silenced. These data support the concept that ANG1 - 7 upregulates the phosphatase MKP - 2 through Mas and thereby maintains low phospho - JNK levels to promote AEC survival. Blockade or knockdown of the Mas receptor by the antagonist A779 or antisense oligonucleotides attenuated the induction of MKP - 2 by ANG1 - 7 and confirmed that Mas acts through MKP - 2. The se signaling mechanisms suggest the potential for pharmacologica l manipulation of AEC apoptosis through Mas and MKP - 2. 85 CHAPTER 3 : I NVESTIGATION OF THE ROLE OF ANGIOTENSIN 1 - 7/ACE - 2 IN ALVEOLAR EPITHELIA L CELLS DURING ENDOPLASMIC RETICULUM STRESS AND HYPEROXIA 86 A bstract Previous e xperimental studies showed that apoptosi s of AECs in response to apoptotic inducers is regul ated by a n ANG system. A utocrine generation of ANGII and its counter - regulatory peptide ANG 1 7 have shown to regulate the ANG system in AECs . Endoplasmic reticulum (ER) stress in AECs is a prominent finding in interstitial lung diseases . It was theorized that i nduction of ER stress causes apoptosis and may also be regulated by the ANG system in AEC s. To test this hypothesis, ER stress was induced in MLE - 12 cells by the proteasome inhibitor MG132. ER stress induced a poptosis was measured by assays of pJNK , caspase activation, mitochondrial function and nuclear fragmentation. Induction of pJNK by MG132 was significantly inhibited by the non - selective ANG receptor blocker saralas in and was completely blocked by ANG1 - 7 . Hyperoxia is known to contribute to lung injury and ANGII is also involved in experimental hyperoxia induced lung diseases. Both ER stress and hyperoxia decre ase d the ANGII degrading immunoreactive protein angiotensin converting enzyme - 2 (ACE - 2). A n inhibitor of ADAM17/TACE, significantly reduced ER stress induced reduction of ACE - 2 by MG 132. Similarly, exposure of human fetal lung fibroblasts (IMR90) to hyperoxic (95% O 2 /5%CO 2 ) gas reduced ACE - 2 immunoreactive protein and enzyme activity. Moreover, soluble ACE - 2 protein was increased in the cell culture media, suggesting a role of ectodomain sh edding in hyperoxia. These data illustrate that ER stress - induced JNK phosphorylation and apoptosis is regulated by the ANGII and ANG1 - 7 in AECs . Moreover , ACE - 2 is significantly decreased by ER stress/ hyperoxic gas through a shedding mechanism mediated by ADAM17/TA CE. Further, these data demonstrate ACE - 2 and its product ANG1 - 7 may hold therapeutic strategies for ER stress or hyperoxia induced pathological conditions in the lung . 87 Introduction A poptosis of AECs is a critical event that contri butes to several lung diseases. Earlier experimental studies demonstrated that apoptotic inducers activate the autocrine synthesis of AGT and its effector peptide ANGII (Li et al., 2003a) . Further work illustrated the autocrine production of ANGII and JNK phosphorylation through the ATI receptor are r equired events in AEC apoptosis (Uhal et al., 2011) . Mor e recent studies showed that actions of ACE / ANGII /ATI are opposed by the counter - regulatory axis ACE - 2/ANG 1 7/ Mas which limits the accumulation of ANGII and prevents AEC apoptosis through the ANG1 - 7 / Mas receptor pathway . In AECs, ER stress can be induced by many deleterious agents that cause apoptos is and subsequent lung diseases (Uhal et al., 2013b; Weiche rt et al., 2011) . Surfactant protein (SP) - C is a type II alveolar epithelial cell specific protein that is synthesized as proSP - C . After subsequent posttranslational processing , the mature SP - C protein is stored in lamellar bodies (Maguire et al., 2012) . Mutations in the SP - C protein cause accumula tion o f misfolded proteins which activate unfolded protein response (UPR). The UPR activates signaling pathways that inhibit protein translation , enhance s metabolism, increase s protein degradation enzymes and induces the production of chaperon e proteins (Lawson et al., 2008) . However, prolonged activation of UPR can activ ate apoptotic signaling pathways and ER stress has shown to induce apoptosis in AECs. The present study is aimed to investigate the hypothesis that ER stress - induced apoptosis of AECs may also be regulated by the ANG system. To evaluate the hypothesis, AE C apoptosis was induced in MLE - 12 or A549 cell line by a synthetic proteasome inhibitor, MG132 . The findings herein indicate pro - apoptotic modifications in the ANG system by ER stress and 88 can be preven ted by either blockade of the ANG receptor or by the anti - apoptotic peptide ANG 1 7. In the recent years, experimental studies have also demonstrated that hyperoxia can directly cause lung injury. Damage to the alveolar microenvironment has been reported during hyperoxia and is well characterized in rodents (Pagano and Barazzone - Argiroffo, 2003) . It has been reported that exposure of lung tissue to hyperoxia induces reactive oxygen species (ROS) originating in the mitochondria and significantly decreases alveolarization in mice (Ratner et al., 2009) . Several reports indicate that hyperoxia - induced lung injury can be prevented by beneficial effects of ACE - 2 in neonatal rats (Wagenaar et al., 2013) . Further , in vivo data suggest that recombinant expression of ACE - 2 can prevent lung injury in mice (Imai et al., 2005) . Therefore, ACE - 2 may play a major role, since the components of the local ANG system is expressed and is functional in human lung myofibroblasts, the primary source of collagen deposition in the lung (Abdul - Hafez et al., 2009; Oarhe et al., 2015) . A NGII , the effector peptide in the ANG system , induces collagen synthesis through the ATI receptor in the lung fibroblast s . Both angiotensin receptor blockers and ACE inhibitors have demonstrated to protect against lung injury (Marshall et al., 2004; Wang et al., 2000b) . In vivo experimental studies performed in fetal lung fibroblasts exposed to oxygen showed the upregulation of the ANG system (Lang et al., 2010) . Prior wor k from this laboratory, found that ACE - 2 is downregulated in response to bleomycin and additionally, a purified recombinant human ACE - 2 diminished, bleomycin - induced lung collagen accumulation (Li et al., 2008) . Further, it was revealed hyperoxia significantly increased total collagen content in neonatal Sprague - Dawley rats. Moreover, ANG system components including ATI receptor and ACE were 89 induced in hyperoxia (Jiang et al., 2012) . Additionally, hyperoxia induced collagen deposition was blocked by ATI antagonist losartan demonstrating the upregulation of the ANG system in hyperoxia (Chou et al., 2012) . The role of TNF - converting enzyme (TACE), also known as ADAM17, was evaluated by several groups and demonstrated its ability to cleave the ectodomain of ACE - 2 (H aga et al., 2008, 2010) - 2 ectodomain was blocked by the TACE antagonist, TAPI - 2. Given the known involvement of the ANG system components in lung fibroblast function it was hypothesized that ACE - 2 might be downregulated b y hyperoxia in lung fibroblasts and further, downregulation of ACE - 2 could be prevented by inhibiting TACE. 90 Materials and Methods Reagents and m aterials : The proteasome inhibitor MG132 (carboxybenzoxy - Leu - Leu - leucinal) was obtained from GenScript , Piscataway, NJ. ANG1 7 and t he non - selective ANG receptor saralasin was obtained from Sigma Chemical, St. Louis, MO. Antibodies fo r t he detection of phospho - JNK, total JNK - actin and secondary antibodies were purchased from Cell Signaling, Dan vers, MA. Antibodies for the detection of ACE - 2 were obtained from Abcam Biotechnology, Cambridge, MA . Protease inhibitor cocktail and broad spectrum phosphatase inhibitors were obtained from Roche, Nutley, NJ. TAPI - 2, an inhibitor of ADAM17 was purchased from Calbiochem, San Diego CA. For sample dialysis, Spectra/por biotech cellulose ester dialysis membranes with a MWCO 10kDa were obtained from Spectrum Laboratories, Rancho Dominguez, CA. All other materials were of reagent grade and were obtained from Sigma Aldrich, St. Louis, MO. Cell c ulture : The mouse lung epit helial cell line MLE - 12, a gift from Dr. Jeffrey Whitsett, University of Cincinnati, OH, was cultured in complete HITES medium as described previously . All cells were grown in 6 - well chambers a nd were analyze d at sub - confluent densities of 6 0 80% except where indicated. All sub sequent incubations with ANG1 - 7 (10 - 7 M) or saralasin (50 µg/ml) or other test agents were performed in serum - free medium. In all studies, cells were exposed to antagonists 30 min before exposure to MG132 (10 µM) for 5 min. The fetal lung fibroblast cell line ( IMR90 ) was purchased from ATCC (Manassas, VA) and was cultured in complete media. At confluence fibroblasts were exposed to hyperoxic (95% oxygen with 5% CO 2 ) or normoxic (21% oxygen with 5% CO 2 ) gas for 72 h, in 5% fetal bovine serum. Some cells were also treated with TAPI - 91 cells were aspirated and rinsed once with serum - free media. Cells were then allowed to recover in serum - free media for 24 h in room air. At the end of the recovery period, cells were harvested and assayed for ACE - 2 protein by Western blotting. Cell Culture Media Handling : Media on cell culture (a total volume of 1 ml) were ad ded to a 15 - free cocktail. After centrifugation to remove cell debris, samples were subjected for dialysis. Next, membranes containing the samples were kept with gentle stirring in a 2L beaker with deionized water at 4 °C for two days till samples were clear. Samples were then transferred to a new 15 ml c anonical °C and were lyophilized using the Labconco Freeze Dryer/Freezone 4.5 deionized water and ACE - 2 protein levels were analyzed by Western blotting. Western blotting : Following treatment, c ells were lysed with 200 µl of modified lysis buffer containing 50 mM HEPES, 150 mM NaCl , 10% glycerol, 1% Triton X - MgCl 2 , 1 tor cocktail tablet and phosphatase inhibitor for detection of phospho - proteins . After harvesting, p roteins were run on 10% polyacrylamide gels in tris/glycine/SDS buffer and w ere transferred t o PVDF membranes. After transferring the proteins, the membrane was blocked in 5% nonfat dry milk in 0.1% tween 20 in tris - buffered saline (TBS). Western blot analysis of pJNK was performed with stress activated protein kinase (SAPK)/JNK a ntibody (1:000 dilution) with overnight incubation at 4 °C followed by HRP - conjugated secondary anti - mouse antibody (1:2000) incubation the next day . For the detection of total JNK, the membranes were stripped and re - probed with total JNK antibody. 92 Immunoreactive bands were visualized by W est F em to substrate systems ( Thermo Scientific, Rockford, IL). 93 Results Previous work from this laboratory showed , blockade of bleomycin induced JNK phosphorylation and apoptosis by ANG1 - 7 (Uhal et al., 2011) . Additionally, it was found that JNK phosphorylation is mediated by the ATI receptor through experiments that prevent JNK phosphorylation by ATI receptor antagonists . Therefore, the effect of the anti - apoptotic peptide ANG 1 - 7 or the non - selective receptor blocker saralasin on JNK phosphorylation was determined in AECs treated with the proteasome inhibitor MG132 . Mouse lung epithelial cells were cultured and were treated with saralasin - 7 (10 - 7 M) for 30 min followed by treatment with MG132 for an additional 5 min. Next, the cells were harvested. Figure 3 - 18 shows partial blockade of MG132 - induced JNK phosphorylation by saralasin and complete blockade by ANG 1 - 7. To investigate, whether exposure of the fetal fibroblasts to hypero xic gas with recovery, increase the cleavage of membrane bound ACE - 2 by ADAM17/TACE, cell culture media was collected after exposure of IMR90 cells to hyperoxia (95% O 2 ) in the presence or absence of TAPI - 2 and were analyzed for soluble ACE - 2 protein. Figure 3 - 19 shows expos ure of IMR90 cells to hyperoxia significantly induced the soluble form of ACE - 2 in the culture media compared to the control cells demonstrating the cleavage by ADAM17/TACE. Further, addition of TAPI - 2 prevented the induc tion of soluble ACE - 2 by ADAM17. 94 Figure 3 - 18 : Blockade of ER stress induced JNK phosphorylation by angiotensin 1 7 or by saralasin . MLE - 12 cells were grown in 6 well culture plates and were serum starved overnight at sub - confluent densities. Thereafter, saralasin angiotensin 1 7 (10 - 7 M) were applied to cells for 30 min immediately prior to challenge with MG132 ditional 5 min. Next , cells were harvested for Western blot analysis of phosphorylated JNK . Results are indicative of those obtained in at least 3 separate experiments. 95 Figure 3 - 19: ADAM 17 blocker prevented the induction of soluble ACE - 2. IMR90 cells were cultured and exposed to hyperoxic or normoxic gas as described in method section, in the presence or absence of the ADAM17 inhibitor TAPI - - free culture media were then collected separately from the cell monolayer. Lyophilized samples were subjected to western blotting for ACE - 2. Ba rs are means + SE * P < 0.05 vs. CTL and ** P < 0.05 vs. 95% O2 by ANOVA and Student - Newman - Keuls multiple comparisons test. 96 Figure 3 - 20 : Ectodomain shedding of angiotensin - converting enzyme - 2 (ACE - 2) in hyperoxia - induced lung injury. Hyperoxia ind uces ADAM17/TACE which in turn mediates the release of the ectodomain of ACE - 2 from the cell. Loss of ACE - 2 promotes accumulation of ANGII and reduces the production of ANG1 - 7 , contributing to lung injury. 97 Discussion The notion that induction of apoptosis in AECs is sufficient to induce a pathological microenvironment in the lung is supported by experimental studies that began almost 40 years ago (Uhal et al., 2013) . Recent experiments that demonstrated blockade of apoptosis with broa d spectrum caspase inhibitors or by deletion of genes that are essential in apoptosis prevented lung injury (Budinger et al., 2006; Kuwano et al., 2001; Wang et al., 2000b) . Recently ER stre ss has shown to induce apoptosis in AECs and contribute to interstitial lung diseases. Accumulation of unfolded proteins in the ER activates UPR which attem pts to enhance protein folding. However, overwhelming ER stress may trigger AEC apoptosis (Korfei et al., 2008) . Previ ous e xperimental studies from this laboratory showed activation of the ANG system in response to bleomycin, f as ligand and TNF - which induces the production of AGT and ANGII in the AEC itself (Li et al., 2003a; Wang et al., 1999, 2000a) . Further, ANGII or bleomycin induced JNK phosphorylation and apoptosis was blocked by selective ATI antagonists showing AEC apoptosis i s mediated through ATI receptor (Uhal et al., 2011) . Moreover, addition of AGT to cultured AECs in serum free media increased ANGII receptor - dependent apoptosis demonstrating the constitutive expression of required enzymes in AECs (Li et al., 2004) . Recent studies have revealed the regulation of AEC apoptosis by the counter - regulatory axis of the ANG system, ACE - 2/ANG 1 7/ Mas . Constitutive expression of ACE - 2 in AECs, limits the accumulation of ANGII by degrading the octa - peptide to ANG1 - 7 which inhibits AEC apoptosis by decreasing JNK phosphory lation through the receptor Mas (Uhal et al., 2011) . SP - C is s olely produced and secreted by the t ype II AECs. A ccumulation of mutant SP - C or protein complexes in the ER has shown to cause injury to AECs (Korfei et al., 2008; Whitsett, 98 2002) . Overexpression of Bcl - 2, an anti - a poptotic protein demonstrated blockade of ER stress induced loss of m itochondrial membrane potential (Schröder and Kaufman, 2005) . Additionally, prolonged activation of the UPR showed induction of pJNK and its downstream targets (Maguire et al., 2011) . Activation of JNK has shown to phosphorylate BH - 3 family related p roteins and also anti - apoptotic proteins including Bcl - 2 in ER stress conditions (Szegezdi et al., 2006) . Deletion of a poptosis - signal - regulating kinase ( ASK1), a MAP3K that activates JNK, ex hibited resistance to ER stress (Nishitoh et al., 2002) . Together, these data presented by other research groups may suggest that phosphorylation of JNK by ER stress targets Bcl - 2 family proteins and causes dimerization of B ax and Bak leading to apoptosis (Hetz et al., 2006; Tanjore et al., 2013) . The data presented herein , show the regulation of ER stress induced AEC apoptosis by the ANG system . Figure 3 - 18 shows, MG132 induced JNK phosphorylation was blocked by ANG1 - 7 and was only partially blocked by saralasin . This result show s a more potent role of ANG1 - 7 in ER stress induced apoptosis in AECs compared to ANG receptor blockers . Consistent with this finding , a clinical trial demonstrated positive results of ANG receptor blockers but was limited to a smaller number of patients (Couluris et al., 2012; Woo et al., 2003) . A purified recombinant ACE - 2 or ANG 1 - 7 has alread y demonstrated to protect against lung injury in animal models (Li et al., 2008; Shenoy et al., 2014) . Recently, it was foun d that hyperoxia also upregulates the ANG system including ATI receptor expression. Oxidative injury is a well - documented mechanism that is associated with abnormalities in alveolar development (Ratner et al., 2009) . Experimental studies have shown proliferation of fibroblasts and differentiatio n to myofibroblasts, a major source of the 99 extracellula r matrix production in hyperoxia - induced lung injury (Rehan and Torday, 2003) . It w as shown that siRNA mediated knockdown of the ATI receptor attenuated hyperoxia induced type I collagen expression, suggesting a role of ANGII /ATI in lung fibr oblasts (Lang et al., 2010) . Earlier work from this laboratory and other studies conducted by different groups, have demonstrated the protective role of ACE - 2 in lung injury and a recombinant form of human ACE - 2 (rhACE - 2) is in clinical trials t o treat acute lung injury. However, the role of ACE - 2 has not been investigated in the hyperoxic - induced injury in lung fibroblasts. ACE - 2 has been found to play a vital role in virus - induced lung diseases. ACE - 2 is the cellular receptor for severe acute respiratory syndrome (SARS - Coronavirus) which is essential for coronaviral entry (Dimitrov, 2003) . SARS - Coronavirus spike protein has shown to induce s hedding of ACE - 2 in lung tissue (Haga et al., 2010) . Additionally, ACE - 2 has also shown to downregulate in influenza A (H7N9) virus - induced acute lung injury (ALI) and deficiency of ACE - 2 aggravated ALI in mice (Yang et al., 2014) . Similarly, a downregulation of ACE - 2 expression in the lung in response to H5N1 virus resulted in an induction in serum ANGII l evels (Zou et al., 2014) . Genetic deletion of ACE - 2 in H5N1 challenged mice caused severe lung injury and recombinant ACE - 2 improved H5N1 induced lung injury confirming a protective role of ACE - 2. ACE - 2 is a type I transmembrane protein with a N - terminal ectodomain containing the active site. Ectodomain shedding has been observed in many transmembrane proteins and seems to be mediated by ADAM (a disintegrin and metalloproteinase) protein family. ADAM17 or TNF - - other membrane proteins (Garton et al., 2003; Wang et al., 2002) . Inhibition of AD AM17 by siRNA mediated knockdown decreased ACE - 2 shedding and overexpression caused increased 100 shedding, suggesting the role of ADAM17/TACE in ACE - 2 shedding (Lambert et al., 2005) . In cardiovascular diseases circulating levels of ACE - 2 has been reported although the physiological role of shedding remains unclear (Shaltout et al., 2009) . In line with this, another group demonstrated ADAM17/TACE mediated ACE - 2 ectodomain shedding in the brain which de creased membrane bound ACE - 2, thus stimulating the development of neurogenic hypertension (Xia et al., 2013) . AECs are the primary site of ACE - 2 expression in mice and has been studied extensively demonstrating its protective role against lung injury (Uhal et al., 2011; Wiener et al., 2007) . Exposure of Primary cultures of rat AECs to hyperoxic gas caused apoptosis of alveolar epithelial cells (Wang et al., 2014) . However, exposure of lung fibroblasts to hyperoxia did not upregulate markers of apoptosis and this might be due to activation of different signaling mechanisms in lung cell types. In the present study it is shown that TAPI - 2, significantly prevented the induction of soluble ACE - 2 in response to hyperoxia in the culture media of IMR - 90 cells (Fig. 3 - 19 ; adapted from Oarhe et al., 2015 ). It is prominent that the reduction in ACE - 2 protein in IMR90 cells occurred only if the hyperoxic exposure was followed by normoxic period of recovery. This is a notable concept that was also seen in ischemia reperfusion injury, in which many vital signali ng cascades are activated during the return to normoxia but not during the ischemic period (Kulkarni et al., 2007) . In summary, t he exact signaling mechanisms by which ER stress induce the ANG system to initiate AEC apopt osis are currently unclear but possibly involve both axis of the ANG system (Uhal et al., 2013b) . The ability of ANG1 7 to completely abolish JNK phosphorylation suggests that the ANG1 7/Mas pathway is a more effective regulator of ER stress - induced apoptosis. It 101 was also shown that exposure of human lung fibroblasts to hyperoxic gas seems to involve the upregulation of ADAM17/TACE protein which facilitates the ectodomain shedding of ACE - 2 protein (Fig. 3 - 20). The exact signaling mechanism(s) by which hyperoxia upr egulates TACE still remain unclear and need to be elucidated. Together , the data reported here show that both ER stress and hyperoxia upregulate the ANG system and that ACE - 2 or its product ANG1 - 7 may hold the therapeutic potential to treat lung diseases t hat involve ER stress and h y peroxia . 102 CHAPTER 4 : SUMMARY AND CONCLUSION S 103 SUMMARY U pregulation of Map Kinase Phosphatase - 2 in Alveolar Epithelial Cells Previous work found that apoptosis in AECs requires the local production of angiotensinogen and its effector peptide ANGII (Xiaopeng Li, 2003) . Recent studies showed that ACE - 2 is protective but severely downregu lated in human and experimental lung tissue in response to bleomycin (Li et al., 2008) . Earlier experimental studies from this laboratory demonstrated that ACE - 2 balances the steady - state levels of both ANGII and ANG1 7 in alveolar epithelial cells (Uhal et al., 2011) . Blockade of ACE - 2 by a competitive inhibitor DX - 600 or siRNA mediated knockdown showed activati on of apoptotic markers in AECs (Uhal et al., 2011) . ACE - 2 limits the accumulation of ANGII and produces ANG1 7 which inhib its JNK phosphorylation and apoptosis induced by either ANGII or by bleomyci n. This protective role of ACE - 2 was further strengthened by in vivo exper iments conducted by I mai et al. showing that inhibition or knockout of ACE - 2 in mice worsens acid aspiration induced lung injury. Additionally, a recombinant form of ACE - 2 protects ag ainst acute lung injury in mice (Imai et al., 200 5) . Moreover, the specific ANG1 - 7 inhibitory effects were blocked by A779, a receptor antagonist for the Mas receptor which has shown to mediate the anti - apoptotic effects of ANG1 - 7 . Experimental studies to date noticeably demonstrate the protective actions of ANG1 - 7 / Mas receptor pathway on lung injury through activation of signaling mechanisms (Li et al., 2008; Uhal et al., 2011) . Beneficial actions of ANG1 - 7/Mas include inhibition of tissue remodeling and/or collagen deposition, anti - proliferative and have shown to prevent apoptosis i n epithelial cells . These cell type specific si gnaling pathways downstream of the Mas receptor 104 demonstrate the importance of ANG1 - 7 / Mas signaling in individual cell types of the lu ng. Experimental data from our group and others, suggest the potential of ANG1 - 7 , or activators of the Mas receptor , in regulation of apoptosis in AECs . Although, a significant number of experimental studies elucidated the inhibitor y actions of the ANG1 - 7 / Mas pathway in pulmonary cell types and in other cell types, the exact mechanism(s) by which ANG1 - 7 / Mas inhibits JNK phosphorylation and apoptosis in AECs needed to be evaluated. Therefore, it was hypothesized that ANG1 - 7 constitutively activates a map kinase phosphatase - 2 and maintains cell survival. Th e experimental data herein showed the ability of ANG1 - 7 to block ANGII - induced apoptotic markers including phospho - JNK, caspase - 9, MMP, DNA fragmentation and apoptosis in alveolar epithelial cells . Further, i nhibition of ANG1 - 7 actions by the siRNA mediated knockdown of MKP - 2 s how the upregulation of MKP - 2 by the ANG1 - 7 / Mas pathway. These data support the concept that ANG1 - 7 upregulates the phosphatase MKP - 2 through Mas receptor and thereby maintains lo w phospho - JNK levels to pr omote survival in alveolar epithelial cells . Together , these studies showed the up regulation of MKP - 2 by the ANG1 - 7 / Mas pathway that constitutively dephosphorylates JNK and maintain s cell survival . Since the benef icial actions of ACE - 2 and ANG1 - 7 / Mas are already known, the demonstration of MKP - 2 downstream of the ANG1 - 7 / Mas pathway suggest s the potential for pharmacological manipulation of AEC apoptosis and related pathogenic conditions in the lung through Mas and MKP - 2. 105 Role of Angiotensin 1 - 7/ACE - 2 in ER Stress and Hyperoxia E R stress induced by physiological conditions or by pharmacological agent s cause accumulation of unfolded proteins which in turn activates UPR ( Pluquet et al., 2005) . Many recent experimental studies have demonstrated that ER stress in AECs can be induced by many harmful agents that lead to AEC apoptosis and lung injury. Th erefore, it was theorized t hat ER stress may induce apoptosis through the autocrine generation of ANGII / ANG1 - 7 in AECs. To test this hypothesis, mouse lung epithelial cells were challenged with MG132 , a synthetic proteasome inhibitor to induce ER stress . T he results demonstrate, JNK phosphorylation induced by MG132 was significantly attenuated by the non - selective ANG receptor blocker saralasin and was completely abrogated by ANG1 - 7 . Further, the result suggests a more important role of the ANG1 - 7 in regulating ER stress - induced a poptosis in lung epithelial cells , relative to the ANGII /ATI receptor interaction . These data show that in human AECs, ER stress - induced JNK phosphorylation is mediated by the peptides ANGII / ANG1 - 7 and demonstrate d effective blockade of pJNK by ANG1 - 7. These data also may suggest ACE - 2/ ANG1 - 7 as therapeutic strategies for ER stress - in duced pathogenic lung diseases and might be prevented by manipulation of the ANG system. Activation of the ANG system was also investigated in lung fibroblasts exposed to hy peroxia. AECs are the main source of ACE - 2 in the adult lung . Regulation of cell survival by ACE - 2 was investigated by Uha l et al. in AEC s demonstrating the knockdown of ACE - 2 disrupts the balance between ANGII and ANG1 - 7 levels in AEC culture media (Uhal et al., 2011) . Similarly inhibition of ACE - 2, significantly increased ANGII levels in the mouse lung (Li et al., 2008) . Additionally, inhibition of A CE - 2 by a competitive inhibitor increased caspase - 9, an apoptotic 106 marker in AECs . It was also found that ACE - 2 mRNA, immunoreactive protein and enzymatic activity were all high in quiescent mouse and human lung epithelial cells, but were severely downregulated in actively proliferating cells (Uhal et al., 2013a) . In support of t his theory, downregulation of ACE - 2 was observed in lung tissues of experimental mice exposed to bleomycin . This downregulation or loss of ACE - 2 observed in the lungs might be due to the high fraction of AECs that are proliferating. I nhibition of ACE - 2 , induced lung collagen accumulation and i n addition, a purified recombinant human ACE - 2, attenuated bleomycin - induced lung collagen accumulation in mice (Li et al., 2008) . In the recent study conducted, the data show ACE - 2 is expressed no t only by AECs , but also by fetal human lung fibroblasts. Exposure of human lung fibroblasts to hyperoxia decreased ACE - 2 protein levels, but a soluble form of ACE - 2 was increased in IMR90 cell culture media. TAPI - 2, an inhibitor of ADAM 17/TACE significantly reduced the shedding of ACE - 2 from the cell surface which shows that hyperoxia with normoxic recovery induces soluble ACE - 2 by an ADAM17/TACE sensitive mechanism . Collectively , data here show the upregulation of the ANG system in ER stress a nd hyperoxia in lung cells. Further , ACE - 2 / ANG1 - 7 might be useful in the managem ent and prevention of hyperoxia or ER stress induced lung injury and may hold the potential as therapeutic strategies for treating pathological conditions in the lung. 107 CONCLUSION S It is well documented that in many organ systems, signaling mechanisms of the ANGII /ATI pathway are counteracted by the opposing axis ANG1 - 7 / Mas pathway. O nly a few studies have explored the downstream signaling factors of the ANG1 - 7 / Mas pathway. However, the different signaling mechanisms of ANG1 - 7 that were reported could be due to different experimental conditions that were being used in various cell types . The data presented here showed upregulation of MKP - 2 by the ANG1 - 7 / Mas pathway and constitutively dephosphorylates p JNK as a cell survival mechanism in lung epithelial cells. However, the molecular mechanism(s) by which ANG1 - 7 / Mas pathway upregulates MKP - 2 are currently unknown. It was demonstrated that in vascular smooth muscle cell s ANG1 - 7 releases prostaglandin I 2 (PGI 2 ) and increases cAMP levels to reverse ANGII induced phosphorylation of ERK1/ERK2. Further, inhibition of cAMP dependent protein kinase activity prevented the anti - proliferative actions of ANG1 - 7 (McCollum et al., 2012b) . It was also shown that cAMP elevating agents increase MKP - 1 in pheochromocytoma (PC - 12) cells ( Burgun et al., 2000) . Similarly, La ra et al . found that activation of a Na + - ATPase in the proximal tubule in response to ANGII was prevented by ANG1 - 7 through the cAMP/PKA - mediated mechanism and further , ANG1 - 7 effects were blocked by the Mas receptor antagonist . Moreover, it was found that ANG1 7 prevented ANGII - induced activation of N a + - ATPase through a PKA - mediated mechanism that reversed elevated levels of PKC activity (Lara et al., 2010) . In an earlier publication, it was shown that ANGII activates PKC and further , blockade of PKC prevented AEC apoptosis (Uhal et al., 2011) . Thus , it will be of hig h interest to determine whether cAMP/PKA pathway is involved in upregulation of MKP - 2 in AECs and whether s iRNA mediated silencing of cAMP - dependent 108 protein kinase could reverse the ability of ANG1 - 7 to block ANGII induced effects. Likewise, it would also be interesting to investigate whether c AM P analogues could prevent ANGII induced JNK ph o sphorylat ion and AEC apoptosis . Further, to investigate other signali ng pathways by which ANG1 - 7/Mas inhibits AEC apoptosis, signaling arrays can be used to test the activation of genes in control vs. treated groups. Although , multiple signaling pathways might be involved in downstream of ANG1 - 7/Mas pathway, the data presente d here demonstrate a key role of MAPK/MKP signaling and suggest s the potential for pharmacological manipulation of pat hogenic conditions in the lung through M as and MKP - 2. The data here also demonstrated that manipulation of the ANG system can rescue lung cells in response to ER stress or hyperoxia. The exact mechanism ( s ) by which ER st ress activates the ANG system are not understood clearly. But it is highly likely that both axes of the autocrine ANG system are involved in regulating ER stress induced apoptosis of AECs. More over, previous work showed a significant loss or downregulation of ACE - 2 in r esponse to apop totic inducers th rough mechanisms that are yet to be elucidated. Further, identifying the key signaling mechanisms that upregulate ADAM17/TACE in hyperoxia/ER stress induced lung injury will be an in teresting topic to investigate. 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