The Cardiotonic Steroid Marinobufagenin (MBG) Induces Epithelial-Mesenchymal Transition in LLC-PK1 Cells
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Health Science Campus FINAL APPROVAL OF THESIS Master of Science in Biomedical Sciences The Cardiotonic Steroid Marinobufagenin (MBG) induces Epithelial-Mesenchymal Transition in LLC-PK1 Cells. Submitted by: Vanamala Raju In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences Examination Committee Major Advisor: Joseph Shapiro, M.D. Academic Advisory Deepak Malhotra, Ph.D. Committee: Zijian Xie, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: August 1, 2007 The cardiotonic steroid Marinobufagenin (MBG) induces Epithelial-Mesenchymal Transition (EMT) in LLC-PK1 cells. Vanamala Raju MD Department of Internal Medicine University of Toledo College of Medicine Toledo, Ohio 1 Acknowledgements I thank Dr.Joseph Shapiro for giving me this wonderful opportunity to pursue my Masters thesis under his expert guidance. He has been instrumental in helping me develop my critical thinking and has made my experience as a graduate student memorable. He has taught me the way to conduct research and how to apply it clinically. I cannot thank Dr.Larisa Fedorova enough for her efforts in training me. She taught me the essence of research with immense patience and understanding of my limitations. The knowledge I have acquired today about conducting an experiment is wholly due to her effort. I have turned to Dr.Sankaridrug Periyasamy on numerous occasions and he has been extremely supportive and helpful in times of need. I also thank Dr.Jiang Liu for his support and his encouragement. My colleagues in the lab – David Kennedy, Sandeep Vetteth, Nasser El-Okdi, Liang Wu, Hai Ping Cai, Jihad El-Kareh, Amjad Shidyak and Shalini Gupta have made my experience in the lab enjoyable. I thank my husband for all the support he has given me and for being there when I have needed him the most. I am most grateful to my family for their support and motivation. I owe everything I am today to them. 2 Index Acknowledgements 2 Introduction 4 Hypothesis and Specific Aims and Objectives 16 Materials and Methods 19 Results 32 Discussion 37 Figures 43 Conclusions 57 Abstract 58 Bibliography 59 3 Introduction Chronic Kidney Disease and End-Stage Renal Disease Chronic kidney disease is a pathophysiological process with multiple etiologies leading to the destruction and loss of nephrons frequently leading to end-stage renal disease (ESRD). This progressive loss takes place over a period of months or years leading to abnormally low kidney function as shown by a low glomerular filtration rate. Diabetes and hypertension are among the leading causes of ESRD accounting for more than 60% of new cases each year[1, 2]. Other causes include infection, inflammation of renal blood vessels and glomeruli, kidney stones and cysts. With the only treatment for ESRD being hemodialysis, ESRD is one of the most expensive diseases to treat. Progression from chronic renal failure to ESRD is predominantly characterized by the development of fibrosis in the glomerular, interstitial and vascular compartments. Renal fibrosis is a complex dynamic process involving several factors – inflammatory agents, cytokines, vasoactive agents and enzymes involved in extracellular matrix assembly, anchoring and degradation. In spite of all these causes for fibrogenesis, there appears to be a final common pathway for fibrosis[3] and massive interstitial myofibroblast activation. Renal fibrosis is a dominant determinant of the clinical outcome of patients and current therapies are, at best, marginally effective [4]. 4 Patients with chronic kidney disease have been noted to have significant elevations in circulating concentrations of digitalis-like substances (DLSs). Also, they have been noted to develop an oxidant stress state due to increased production of reactive oxygen species (ROS) due to DLS stimulation [5]. It has also been known for some time that the Na,K-ATPase (NKA) pump is abnormal in chronic renal failure and that circulating inhibitors can be demonstrated in the serum of uremic patients [6]. Cardiotonic steroids Cardiotonic steroids (CTSs), also known as cardiac glycosides or DLSs, have been used for centuries to treat congestive heart failure. CTSs in the form of digoxin and digitoxin are still an important component of the clinical treatment of cardiac diseases [7-10]. CTSs are found in digitalis plants and in the toad venom [11, 12]. They are the only known molecules that have binding site in the extracellular domain of animal enzyme Na,K-ATPase (NKA). In the early 90s endogenous CTSs in form of cardienolides and bufadienolides were found in body fluids of mammals[13-15]. Previously, elevated level of endogenous CTS like marinobufagenin (MBG) and ouabain (stereoisomer of plant ouabain) have been shown to be associated with different pathological conditions including essential hypertension, preeclampsia, experimental diabetes, uremic cardiomyopathy, chronic cardiac and renal failure [5, 16-27]. There is recent evidence to show the production of endogenous ouabain, marinobufagenin and marinobufotoxin in the adrenal complex and hypothalamus (ouabain only) of mammalian species [28-30]. 5 Structurally, CTS are composed of three major components, a steroid ring system, a five- or six-membered lactone moiety, and on some CTSs, at least one carbohydrate residue [31-34]. CTS belong to two different classes - cardienolides represented by ouabain and digoxin, digitoxin, and bufadienolides represented by bufalin, proscillaridin A, cinobufagin, cinobufotoxin, telobufotoxin, marinobufagenin, and marinobufotoxin. Cardienolides Bufadienolides Bufalin Proscillaridin A Ouabain Digoxin Cinobufagin Marinobufagenin Na+/K+ ATPase Na-K/ATPase (NKA) is a ubiquitous transmembrane protein that utilizes energy gained from ATP hydrolysis to transport Na+ and K+ ions across cell membranes in opposite directions against their chemical gradient and electrical potential. The resulting ion gradients are necessary for a wide range of physiological processes [35, 36]. 6 The NKA is composed of two protein subunits termed α and β. Almost all known functions of NKA pertain to operation and aspects of α subunit. However, separation of α and β subunits results in loss of function of the enzyme [37]. It is believed that the β subunit has an important scaffolding function, controls trafficking and delivery of NKA to the cell membrane [38] and is also involved in cell cooperation in a cell specific manner [39] Multiple isoforms of α and β subunits have been identified. Most tissues express α1 and β1 isoforms and these forms are predominant in organs involved in ion homeostasis, especially kidney. Other isoforms, α2, α3, α4, β2, and β3, are more tissue specific [40-43]. These isoforms exhibit altered affinities for Na+ and K+ ions and also to ouabain and MBG and show different reaction kinetics compared with the α1 isoform [13, 14, 44-47]. The primary sequence of α1 isoform shows tremendous conservation throughout the animal kingdom. It has been shown that the α1 subunit has 10 transmembrane segments with both the N and C terminals exposed to cytoplasm [48, 49]. Transmembrane regions are most likely involved in the transport of ions. Largest intracellular portion of NKA is located between 4th and 5th loop of transmembrane domain and contains phosphorylation and nucleotide binding domains. The N-terminal chain of NKA (A domain) is believed to have regulatory or functional significance [50]. The extracellular face of NKA contains multiple binding and release sites for ions and CTS as well. 7 Unlike other steroid hormones, CTS do not penetrate the plasma membrane and exert their action almost exclusively by binding to NKA in most tissues [51, 52]. The complex inhibitory effect of CTSs on NKA has been extensively studied. It has been assumed that physiological and pharmacological functions of CTSs are secondary to their effect on intracellular ion concentrations. However, the concentration of endogenous CTS even under pathological conditions is not high enough to cause changes in cytosolic ion concentration. During recent years there have been a large number of reports that the non-inhibitory doses of ouabain and other CTSs can modulate cell proliferation, apoptotic threshold, cell-cell contacts and cell migration [53-59]. It has been previously shown that binding of the ouabain to NKA promotes its interaction with Src family of protein kinases and their subsequent activation in different types of cells including cardiac myocytes, smooth muscle cells, renal epithelial cells and in skeletal muscles [60-63]. NKA-Src complex indirectly affects the phosphorylation of downstream proteins that are associated with or are proximal to the receptors. Thus, activated Src trans-activates EGFR, which in turn recruits adapter protein ShC to relay the ouabain signal to Ras. [58, 60, 61, 64, 65]. Ras activation leads to two signal transduction cascades: 1. Communicating with the mitochondria to increase the generation of mitochondrial reactive oxygen species (ROS) resulting in NF-κB activation 2. Consisting of Ras/Raf/MEK/ERK[65]. 8 The latter pathway not only leads to gene activation and proliferation but also provides a link between Ca2+ -dependent protein kinase C (PKC) activation and other cellular signaling pathways induced by NKA/CTS interaction [58, 64, 66, 67]. Thus, in rat 2+ cardiac myocytes, a ouabain-induced increase in [Ca ]I leads to activation of PKC that in turn activates ERK1/2 via the Raf/MEK cascade [65], leading to expression of the transcription