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 factors activator protein 1 (AP-1).
Epithelial-mesenchymal transition (EMT)
Epithelial-mesenchymal transition (EMT) is a major cellular mechanism of embryonic tissue remodeling [68].It was first discovered in the primitive streak of chick embryo, where it was defined as a process during which, epithelial cells acquire new features of mesenchyme characterized by enhanced motility and invasive phenotype [69].
Growing number of studies have shown that in an aging animal, EMT causes several pathological conditions such as organ fibrosis and tumor metastasis [70-72]. It is now believed that renal tubular epithelial cells are one of the major sources of activated fibroblasts responsible for the development of interstitial fibrosis in kidney [4, 73, 74].
Tubular EMT is described as a process in which renal tubular cells lose their epithelial phenotype and attain new characteristic mesenchymal features. Interestingly, the majority of renal tubules in adult kidney excluding collecting ducts are developmentally originated from the metanephrine mesenchyme through mesenchymal – epithelial transdifferentiation. Tubular epithelial cells under normal conditions are tightly attached to each other forming an integrated epithelial sheet. Theses cells are rich in
9 adherens junction proteins – E-cadherin in complexes with β-catenin and α-catenin linked to the cytoskeleton. EMT of renal epithelial cells can be triggered by different growth factors among which transforming growth factor β1 (TGFβ1) is considered the most important [4, 74-76]. TGFβ1 promotes complete EMT of renal epithelial cells by coordinated activation of multiple signaling pathways. EMT is composed of two steps:
1. The loss of epithelial phenotype characterized by loss of polarity, disruption of adherent junctions in epithelial cells that liberates them from the epithelial sheets and anchoring tubular basement membrane.
2. Acquisition of mesenchymal phenotype is characterized by alteration in cellular morphology (elongated spindle-like cell shape and front end-back end polarity) and the gain of motility enabling them to invade into interstitium [68, 72, 74, 75, 77-79].
The typical mesenchymal cell has no E-cadherin but can form transitory gap junctions with other mesenchymal cells. It is polarized in vivo for cell locomotion with a trailing pseudopodium, and filopodia that interact with ECM in a three-dimensional (3D) configuration. When removed from the body and cultured on flat substrates lacking exogenous ECM, mesenchymal cells flatten out and lose their elongated, polarized form. The healthy mesenchymal cell is a secretory cell; it mainly produces collagen and fibronectin that are secreted via Golgi complex at the front end just behind the filopodia [80].
10 Biochemically, EMT is assessed by analyzing expression level of epithelial and
mesenchymal markers proteins (summarized below):
Epithelial marker proteins Mesenchymal marker proteins In vitro functional markers e-cadherin vimentin elongation of cell shape occludin fibronectin increased scattering claudin-1 Snail Increased invasion LEF-1 collagen I
Loss of the epithelial phenotype
Epithelial cells are characterized by cobblestone morphology and strong adherent
junctions. E-Cadherin is a major structural protein of adherent junction in epithelial
cells. and is a distinct adhesion receptor which plays a principal role in preserving the
structural integrity of renal epithelia and its polarization. Down regulation of this protein
is considered as a first and pivotal step of EMT in normal and cancerous cells [68, 77,
81-83]. E-Cadherin at protein level is regulated by its tyrosine phosphorylation [84].
An E3 ubiquitin-ligase, Hakai, binds to e-cadherin when e-cadherin is tyrosine
phosphorylated, ubiquinating it and enchancing endocytosis [85]. The tyrosine on e-
cadherin, which is critical for Hakai interaction, is a substrate for the soluble tyrosine
kinase c-Src. Cell surface e-cadherin normally exists in equilibrium with a pool of
endocytosed e-cadherin [86]. The down regulation of e-cadherin function by
endocytosis is considered as the major immediate event leading to loss of cell-cell
adhesion and EMT [87].
11 Transcriptional down regulation of E-cadherin by transcription factors Snail, Slug,
Twist was detected in many types of cancerous cells [88, 89]. In normal renal epithelial cells TGFβ1 activated Ras/Raf/MEK/ERK/AP-1 signaling pathway promotes up regulation of transcription factor Snail [81, 88]. Transcription factor Snail can be up- regulated by increased production of ROS as well [90]. Besides e-cadherin, Snail also suppresses other epithelial genes such as tight junction proteins claudin and occludin
[68].
Acquisition of mesenchymal phenotype
Mesenchymal cell is characterized by expression of mesenchymal proteins and distinguished morphological features such as spindle-like, elongated shape, front-end back-end polarity and increased motility [68]. It is widely believed that transcription factor LEF-1 is the master gene which is responsible for the signaling of almost all significant EMT genes [75, 91]. LEF-1 is a member of LEF/TCF transcription factors which are responsive to TGF-β and Wnt signaling pathways [92]. LEF/TCF bind to
DNA weakly and with moderate specificity. Such features demand LEF/TCF cooperation with other factors to bind more tightly and specifically to the correct set of target genes. In case of LEF-1, such factors are members of the TGF-β signal transduction pathway, Smad2/4 and β-catenin [91-93]. Interestingly, expression of
LEF-1 gene is also up regulated by Smads during embryonic development and TGF-
β1 induced EMT. LEF-1 transcription factor is stimulated by the same Smad2/4 complex to activate genes involved in EMT [76, 77, 91, 94]. Smad2/3-Smad4
12 transcription complex translocates to the nucleus and induces expression of LEF-1 in renal epithelial cells[81, 95-99].
In the canonical Wnt pathway, up regulation of expression of the mesenchymal genes is triggered by co-activation of LEF-1 by activated (non-phosphorylated) β- catenin[100]. In epithelial cell β-catenin is associated with e-cadherin. When β-catenin is released from adherent junction, it is either degraded in the cytoplasm by GSK-3β
(by phosphorylation) or translocated to the nucleus to activate LEF-1.
EMT in Renal Fibrosis
ESRD is characterized by severe interstitial fibrosis. Irrespective of the primary renal injury or disease process, fibrogenesis underlies progressive kidney disease leading to ESRF. Tubular epithelial-to-mesenchymal transition(Tubular EMT) has been currently proposed as an essential event in renal tubulointerstitial fibrosis [101].
Recent studies have implicated EMT in the pathogenesis of experimental and native kidney diseases in both animals and humans .Interstitial fibroblasts are the important effector cells in organ fibrosis. Previous studies have shown that the source for these fibroblasts are from the tubular epithelial cells via EMT during renal fibrogenesis in injured kidneys [78].
Renal tissue fibrosis is an irreversible process which occurs as a consequence of excessive accumulation of extra cellular matrix (ECM) components which
13 subsequently leads to the development of chronic kidney disease (CKD) and eventually to end- stage renal disease[70].
The underlying cellular events causing the characteristic histological presentation seen in renal fibrosis are complicated and is justified by the participation and interaction of many regional and infiltrated cell[102]. Fibroblast activation, in isolation is not capable of initiating and maintaining the entire process of renal fibrosis.
Regardless of the initial causes, interstitial fibrosis is a monotonous process characterized by de novo activation of α-smooth muscle actin (αSMA) – positive myofibroblasts, the fundamental effector cells that are responsible for the excess deposition of interstitial ECM under certain pathologic conditions [101].
The presence of EMT in renal fibrosis was first demonstrated a decade ago by using the fibroblast-specific protein-1 (FSP-1) as a marker. The tubular epithelial cells could express FSP-1, which is normally expressed by fibroblasts but not epithelia[78]. Large amount of cells co-expressing both αSMA and tubular marker was detected in unilateral ureteral obstruction – induced obstructive nephropathy, denoting that these cells stand at a transitional stage between epithelia and mesenchyme [101].
Furthermore, these epithelial cells lost E-Cadherin, acquired mesenchymal features, and produced extra cellular matrix components such as fibronectin and type I collagen
[103].All these evidences strongly suggests EMT as a pivotal process in renal fibrosis.
14 Analysis of numerous studies of different mechanisms underlying EMT as well as mechanisms by which NKA transduces signals from its extracellular ligands to its intracellular targets has led us to hypothesize that CTSs may present a new class of
EMT inducers. Marinobufagenin (MBG) has been identified as a digitalis-like substance (cardiotonic steroid) in mammals. MBG has been found to be elevated in fibrotic diseases such as hypertension, diabetes, chronic heart and renal failure which indicates its profibrotic potential. MBG is also elevated in the serum and urine of experimental animals and patients who have extracellular fluid volume expansion [18]
Like oubain, MBG binds to NKA and it is well known that NKA is co-localized with e- cadherin in the baso-lateral membrane of renal epithelial cells. Oubain and MBG have also been found to induce endocytosis of the plasmalemmal Na/K-ATPase in LLC-
PK1 cells [104]. Recent findings have also shown that MBG causes cardiac [5, 105] and renal fibrosis in rats. All these data suggest that MBG could serve as one of the endogenous initiators of EMT in kidney by regulation of cell contact integrity and de novo expression of proteins specific to the mesenchymal phenotype. Therefore, cumulative facts point to a potential involvement of MBG in the induction of renal fibrosis. In this context, it can be proposed that MBG could be one of the unidentified endogenous factors that could trigger EMT.
15 Hypothesis
The cardiotonic steroid Marinobufagenin (MBG) induces epithelial- mesenchymal transition in renal tubular epithelial cells.
Specific Aims and Objectives
Specific Aim 1: Test whether MBG has distinct potency to induce EMT in
Porcine renal proximal epithelial cell lines (LLC-PK1).
Specific Aim 2: Study of molecular mechanisms of MBG-induced EMT in
Porcine renal proximal epithelial cell lines (LLC-PK1).
Specific Aim 1:
1. Test for loss of the epithelial phenotype and down regulation of epithelial markers in
LLC-PK1 Cells treated with MBG in a time and dose dependent manner. a. LLC-PK1 cells are grown to 90-100% confluence and treated with MBG in doses ranging from 0.1nM to 100nM for 24-96 hours and cell lysates are tested for Western blotting for down-regulation of the epithelial markers — E-Cadherin, occludin and claudin-I.
16 b. LLC-PK1 cells grown on chamber slides are treated with MBG and immuno-stained for specific epithelial markers (E-Cadherin, occludin and claudin) and also examined for loss of the epithelial phenotype and down-regulation of the epithelial markers.
2. Test for the acquisition of the mesenchymal phenotype and mesenchymal markers in MBG treated renal epithelial cells. a. LLC-PK1 cells are treated with MBG and the cell lysates are tested by Western blotting for up-regulation of the mesenchymal markers – fibronectin and vimentin.
b. LLC-PK1 cells grown on chamber slides are treated with MBG and immuno-stained for mesenchymal markers, fibronectin and vimentin and examined for acquisition of the mesenchymal phenotype.
c. LLC-PK1 cells grown on collagen gels are treated with one dose of MBG (100nM) for 72-96 hours and are tested for the gain of invasive motility which is a feature of mesenchymal cells. Cells on collagen gels are stained with Coomassie blue and invasion into the gel after MBG treatment is examined under the light microscope and imaging done using DIC.
Specific Aim 2:
1. To investigate the involvement of Wnt/ß-catenin signaling pathway in MBG induced
EMT.
17 a.MBG-treated LLC-PK1 cell lysates and nuclear extracts are probed for changes in the levels of ß-catenin by Western Blotting and cells treated with MBG are immunostained for ß-catenin and its translocation into the nucleus is looked for.
2. To test for expression of transcription factors LEF-1 and SNAIL in MBG treated
LLC-PK1 cells. a. Cell lysates and nuclear extracts from MBG-treated cells are tested by Western blotting for transcription factors LEF-1 and SNAIL which have consistently been shown to be involved in EMT.
b. LLC-PK1 cells grown on chamber slides are treated with MBG and immuno-stained for the expression of these transcription factors and their translocation into the nucleus.
3. To study the role of Src tyrosine kinase and Reactive Oxygen Species (ROS) in the molecular mechanism of MBG-induced EMT. a. LLC-PK1 Cells are grown in the presence of MBG, Src inhibitor PP2 and ROS quencher NAC. Cell lysates are probed for epithelial and mesenchymal proteins by western blotting and any differences in their levels are looked for. b. MBG treated cells are also immunostained for epithelial and mesenchymal proteins and any difference in the morphology is noted.
18 Materials and Methods
Cell culture
The cells used for this study, the porcine kidney proximal tubule cell line (LLC-PK1) were obtained from the American Tissue Culture Collection (ATCC, Manassas,
VA,USA.).LLC-PK1 cells were grown in sterile six well plates for extracting whole cell lysates and 15 cm plates for obtaining nuclear extracts(Both the 6 well plates and the
15 cm plates were from Corning Incorporated).Cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM) from Sigma containing 10% fetal bovine serum(FBS- from Hyclone) and 1% Penicillin Streptomycin for 1-2 days till they reached 90-100% confluence. Prior to treatment;cells were serum starved with DMEM medium containing no serum(0% DMEM) for a duration of 12-18 hours.Cells were then treated with different concentrations of MBG – ranging from 0.1nM to 100nM for a duration starting from 24 hrs upto 96 hours.In some of the experiments cells were also treated with 5 to 10 ng/ml of TGF-ß1(Recombinanat from Biovision).One well with cells was left without any treatment which served as a control. The serum free medium was changed every 24 hrs and fresh MBG and TGF-β added daily for the entire duration of treatment. Cells were treated in this manner for a duration of 24-96 hours.
In studies done to determine implication of Src and ROS production on MBG-induced
EMT, cells were grown on 6 well plates as described above and treated with 100nM
MBG in the presence and absence of Src inhibitor PP2 and ROS quencher N-Acetyl
Cysteine (NAC). Src inhibitor PP2 was used in the concentration of 1µM.MBG was
19 added 30 minutes after adding PP2.Cells were treated in this fashion for 24 to 96 hours. NAC was used in a concentration of 10nM for treatment .Fresh solution of NAC was prepared everyday prior to treatment, pH adjusted to 7.3-7.5, filtered sterile under the hood and used for treatment. Cells were pretreated with NAC for 2 hours before adding 100 nm of MBG. Cells were treated in this fashion for 24 to 96 hours and whole cells lysates extracted.
Sample Preparation for Western Blotting
Western Blot is based on the principle of subjecting protein samples to electrophoresis on a polyacrylamide gel and probing the proteins with specific antibodies. When same amount of proteins are loaded from each sample, the quantities of protein specifically binding the antibodies can be estimated. Western blots were used to detect levels of epithelial and mesenchymal proteins in the whole cell lysates and levels of transcription factors in the nuclear extracts from MBG treated and control LLC-PK1 cells.
Extraction of whole cell lysates:
Whole cell lysates were obtained from the control and the treated cells. Cells were washed twice with ice-cold phosphate buffer saline (PBS) at a pH of 7.4.Cells were scraped from the plates using a cell scraper and proteins were immediately extracted into Radio-immune Precipitation Assay buffer (RIPA buffer containing-50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Nonidet® P-40 Substitute, 5% sodiumdeoxycholate, 0.1%
SDS) containing protease inhibitor cocktail (Sigma p8340, 2μl of 100x in 1 ml RIPA)
20 and 5mM EDTA. When whole cell lysates were used for detecting phosphorylated proteins, phosphatase inhibitor cocktail containing 10mM β Gylcerophosphate, 2mM sodium pyrophosphate, 1mM Sodium orthovanadate, 1mM Sodium Fluoride and
10nm Okadaic acid was added into the RIPA extraction buffer. Cell extracts were then spun down at 14,000 g at 4ºC for 15 minutes and supernatant was collected. The supernatant containing proteins were stored at -80ºC till further use.
Preparation of nuclear extracts:
Nuclear extracts were prepared from the treated and the control cells grown on 15cm plates. Cells were washed with ice cold PBS pH 7.4 twice. Cells were then scraped using a cell scraper and extracted into 2ml of ice cold PBS, centrifuged at 1500 rpm for 10 minutes at 4ºC.Supernatant was discarded and the pellet was washed again in
1ml of cold PBS, and centrifuged at 3000 rpm for 10 minutes at 4ºC.Supernatant was discarded and the pellet containing cells were lysed in 500μl of Lysis buffer (Lysis buffer contains-10mM Tris Hcl pH 7.4,3mM MgCl2,10mM NaCl and 0.5% Nonidet® P-
40 Substitute) containing protease and phosphatase inhibitor cocktail in concentrations mentioned above and allowed to sit on ice for 15 minutes with intermittent vortexing. The cell suspension was then centrifuged at 500 rpm for 15 mins at 4ºC.The supernatant was discarded and to the pellet 500μl of Buffer A (Buffer
A contains-10mM HEPES pH 7.9,1.5 mM MgCl2,10mM KCl and 0.5mM DTT) containing protease and phosphatase inhibitor cocktail in concentrations mentioned above and allowed to sit on ice for 15 minutes with intermittent vortexing. The cell suspension was then centrifuged at 5000 rpm for 15 minutes. The supernatant
21 containing cytosolic proteins was discarded. The pellet was then dissolved in 200μl of
Buffer B(Buffer B contains 20mM HEPES pH 7.9,1.5mM MgCl2,0.2mM EDTA , 0.42M
NaCl, 25%Glycerol, 0.5mM DTT and 0.5mM PMSF) containing protease and phosphatase inhibitor cocktail in concentrations mentioned above and left in the cold room overnight on a rotatory shaker. The suspension was spun down the next day at
14000 rpm for 45 minutes at 4ºC.The supernatant containing nuclear proteins was collected and stored at -80ºC till further use.
Protein Estimation:
The amounts of proteins in the samples were estimated using the microplate method
(Bio-Rad protein assay). 1:10 dilutions of the protein samples were prepared and used for the protein estimation. Protein standards were prepared using Bovine Serum
Albumin (BSA). A standard stock BSA solution of 4µg/µl was prepared by dissolving
20mg of BSA in 5ml of dH2O. 1ml of this standard stock was dissolved in 7ml of dH2O to make the working solution W1 (1:8 dilution of the stock). 1ml of W1 was diluted in
4ml of dH2O to prepare the working solution W2 (1:5 dilution of W1). Diluted protein samples and the protein standard solutions were loaded in triplicate in a 96 well micro plate. Control wells were loaded in the 1st column of 3 wells in triplicate and 10µl of dH2O was used as a blank control. The protein standard solutions were loaded onto
nd rd th the plate as follows: 2 column of 3 wells- 5µl W2, 3 column -10µl W2, 4 column-
th th 2.5µl W1, 5 column -5µl W1 and 6 column- 10µl W1 and made up to 10µl with dH2O.The concentrations of protein standards ranged from 0.05µg/µl, 0.10µg/µl,
0.125µg/µl, 1.25µg/µl and 0.5µg/µl.10µl of each diluted protein samples were loaded
22 in triplicates in the remaining columns on the plate. The protein assay reagent dye was prepared by diluting 3ml of BioRad Protein Assay Reagent in 12ml of dH2O (in ratio of 1:4) and 200µl of this assay dye was added to each well. The plate was incubated at room temperature for 5 min and the plate was read on the spectrophotometer (VersaMax Microplate Reader) at room temperature. Sample dilutions were adjusted to ensure that the readings are in the range of the protein standards. Final protein concentrations in the samples in µg/µl were calculated by multiplying the readings with the appropriate dilution factor.
SDS-PAGE, Western Blotting and Autoradiography:
Based on the amount of proteins in each samples, samples were prepared such that depending on the protein probed for, each sample had protein amount ranging from
10µg to 100µg and the amount of sample loaded into each well ranged from 20µl to
80µl. Each sample was prepared by solubilising protein samples in a third of the volume of 3X loading buffer (10ml of 3x sample buffer contains-2.4mL 1M Tris-Cl pH
6.8, 3mL 20% SDS, 3mL Glycerol, 1.6mL β Mercaptoethanol, 6mg bromophenol blue
) and the remaining volume made up with Tris Hcl pH 6.8. The proteins were then loaded onto self made SDS-Polyacrylamide gels, concentrations ranging from 7.5% to
15% depending on the protein of interest. SDS-polyacrylamide gels were made using the Bio-Rad Mini PROTEAN® 3 system gel making apparatus. To make one 10% mini gel, 10 mL of the resolving gel solution was made. Each 10% of resolving gel solution had 3.3ml of 30% Bis/Acrylamide, 2.5ml of resolving gel buffer (1.5M Tris-Hcl, pH 8.8)
0.1ml 10% SDS and 4.1ml dH2O.Depending on the concentration of the gel, the
23 amount of 30% Bis/Acrylamide varied. After mixing the contents well, the solution was degassed under vacuum for 15 minutes. The mini gel casting plates were assembled using one thick spacer plate with 1.0mm spacers and one short plate (from BIO RAD) and clamped together using casting frame and set on casting stand. To the degassed resolving gel solution 50μl of APS and 5μl of TEMED was added and gently mixed and poured into the set glass plates. Top of the gel was layered with a thin layer of water saturated isopropanol and it was replaced with water once the gels polymerized.
After letting the gels to polymerize for 2 hours, stacking gel was made. Stacking gel was always 4% gel.10 ml of stacking gel solution had 1.3ml of 30% Bis/Acrylamide,
2.5 ml of stacking gel buffer(0.5M Tris-Hcl, pH 6.8) 0.1ml 10%SDS and 6.1 ml dH2O.The solution was mixed well and degassed under vacuum for 15 minutes. To this solution, 50μl of APS and 10μl of TEMED was added, gently mixed and poured on the resolving gel. Depending on the number of samples used to run the gel, either 9 well comb or 10 well comb was inserted into the stacking gel solution and allowed to polymerize. After 30 minutes, the gels were set up in the tank ready to be loaded with samples. When nuclear proteins were probed larger gels were made using the same procedure. The prepared samples were loaded into each well. The first well was loaded with protein standard (Bio Rad Precision plus Kaleidoscope).Gels were run in
1x running buffer (1L 10x Running buffer was made by dissolving 30g Tris Base, 144 g Glycine and 10 g SDS in 1L water) at 150 volts for 15 minutes and later at 200 volts for 50 minutes. After the proteins were well separated, gels were transferred using
® Lauriere semi-dry method using trans-Blot SD Semi Dry Transfer Cell(Bio Rad).This method is based on the use of an original set of buffers which creates a stable pH
24 boundary between the two faces of the blotted membrane. The gel to be transferred is placed on the basic side (pH 8.4) of this boundary, which also contains SDS. Briefly the procedure is as follows. After the gels were run, they were removed from the glass plates and were equilibrated for 15 minutes in buffer II (Buffer II contains 0.1% SDS
,15mM Lactic Acid, 25 mM Tris pH 8.4.in the meantime Whatman No. 3 papers were cut to the size of the gel. Two thick blotting papers were also cut to the size of the gel.
Immobilon-P (PVDF Millipore) membranes cut to the size of the gel was first rinsed with methanol and later soaked in water. The gel and the membrane with the papers were assembled as follows. The first layer was formed by the thick blotting paper wetted in buffer IV pH 10.4(Buffer IV contains 20% Methanol, 100 mM Tris, pH 10.4).
The second layer was formed by three Whatman No. 3 papers wetted in buffer III pH
3.8(Buffer III has 20% methanol, 60mM Lactic Acid, 20mM Tris, pH 3.8). PVDF membrane was kept as layer three. Gel was placed on the membrane. On the top of the gel three more Whatman No. 3 papers wetted in buffer II pH 8.4 were placed. The layers were rolled with a roller to remove any trapped air bubble in each step. The top layer was formed by thick blotting paper wetted in buffer I pH 8.4(Buffer I has 0.4%
SDS, 60mM Lactic acid, 100mM Tris, pH 8.4) .After the gel sandwich was set, gels were transferred onto PVDF membrane at 10-15 volts and current of 3.5mA/cm2 for
50-55 minutes depending on the proteins. Multiple gels can be transferred in parallel.
After transfer was done, membrane was blocked in 5% non-fat dry milk in TBS-T (1L
10x TBS was made using 24.2g Tris Base, 80g NaCl, and pH 7.5) and 0.1% Tween-
20(1ml Tween 20 in 1L 1x TBS) for one hour room temperature on a rotatory shaker.
When probed for LEF-1, membranes were blocked in 2% blocker (Milk from ECL plus
25 Kit-Amersham) in TBS containing 0.05% Tween-20 and 1% PVP-40(TBS-PT) overnight in cold room on a shaker. After blocking, membranes were probed with primary antibodies in 5% non-fat dry milk in TBS-T (0.1% Tween) for one hour room temperature on a shaker. The following antibodies were used in specified dilutions.
Mouse Anti-E-Cadherin (BD Biosciences clone 36)1:1500, Rabbit Anti- Occludin and
Claudin I (Zymed Labs) 1:1000, Rabbit Anti-β-Catenin (Sigma) 1:20,000 Mouse Anti-
Active-β-Catenin, clone 8E7(Upstate) 1:1000, Goat-Anti LEF-1 (N-17 Santa Cruz)
1:750, Rabbit-Anti SNAIL (abcam) 1:1000, Rat-Anti SNAIL(SN9H2 Cell Signaling)
1:1000,Mouse Anti-Vimentin (Serotec) 1:500, Rabbit Anti-Fibronectin 1:1000(AB 1954,
CHEMICON). For Protein loading control, Mouse Anti-α Tubulin (TU-02 Santa Cruz)
1:1000, Mouse Anti-Actin (Sigma clone AC-40) 1:1000, mouse Anti-GAPDH (ab 9484)
1:1000 and mouse Anti-Cox IV (ab 14744) 1:1000 were used.
When probed for LEF-1 and β-Catenin (Sigma), membranes were probed with primary antibodies prepared in TBS-PT and SNAIL was probed with primary antibodies prepared in 5%BSA in TBS-T and incubated at room temperature for one hour and kept in the cold room overnight.
After probing with primary antibodies, membranes were washed in TBS-T every 5 minutes for a total of 6 times. Membranes were then probed with secondary antibody in the following dilutions:
For E-cadherin-1:3000 goat anti-mouse HRP,
Occludin and Claudin-I and Fibronectin -1:2000 goat anti-rabbit HRP,
26 β- Catenin -1:40000 goat anti-rabbit HRP in TBS-PT,
Active β- Catenin-1:2000 goat anti- mouse HRP,
LEF-1-1:1500 donkey anti-goat HRP in TBS-PT,
SNAIL (abcam)-1:5000 goat anti-rabbit HRP,
SNAIL (cell signaling)-1:2000 goat anti-rat HRP,
Vimentin-1:1500 goat anti-mouse HRP,
α –Tubulin, Actin, GAPDH and Cox IV -1:2000 goat anti-mouse HRP.
All the secondary antibodies were made in 5% milk in TBS-T, unless specified and incubated at room temperature for one hour. Membranes were washed in TBS-T every 5 minutes for a total of 6 times. Electrochemiluminescence liquid (ECL® Western
Blotting Analysis System by GE Amersham) was added to the membrane ensuring that the membrane was completely covered by the ECL liquid, wrapped in Saran wrap, excess ECL liquid blotted away after a minute and radiographic films are exposed in the dark room and films developed after 30sec and upto 5 minutes exposure.
Immunocytochemistry
For immunocytochemistry, cells were grown on 8-well Lab-Tek II Chamber® SlidesTM under the same conditions described above and were treated with MBG in concentrations varying from 0.1nM to 100nM for a duration of 24 to 96 hrs .In the later experiments MBG concentration of 100nM and for the duration of treatment for 72 and
96 hours was followed. Cells were also treated with Src inhibitor PP2 and ROS quencher NAC as described above. After treating the cells for specified time, cells
27 were washed two times in phosphate buffered saline pH 7.4 and fixed in ice cold methanol for 10 minutes at 4ºC (or) 4% formaldehyde for 10 minutes at room temperature. After 10 minutes, cells were rinsed with cold PBS and were stored at 4ºC in PBS till further use. When staining was done, cells were incubated in permeabilization buffer (PBS-DOC 4mM sodium deoxycholate in PBS) for 10 minutes at room temperature. Cells were then washed in TBS containing 0.025% Triton for 5 minutes and placed in blocking buffer (1.5% horse serum in PBS-Serum PBS) for 30 to 60 minutes depending on the antibody used for staining. Cells were then incubated at room temperature for 1 hour with primary antibodies of appropriate dilutions made in serum PBS, washed for 5 minute in PBS and incubated for an additional 30 minutes with respective secondary antibodies. The following Primary antibodies were used
Mouse Anti-E-Cadherin (1:125, BD Biosciences 610404) or Pre-diluted Mouse Anti-E-
Cadherin antibodies (Cell Marque ECH-6), Goat Anti-LEF-1 (N-17) 1:50 [ Santa Cruz
Biotechnology SC-8591], SNAIL- 1:200 Rabbit PolyclonalAbcam-ab17732),Mouse
Anti-β-catenin(14)[Pre-diluted–Cell Marque] RabbitAnti-Claudin-1 and Occludin 1:50
(Zymed Laboratories), Rabbit Anti-Fibronectin 1:330 [AB 1954, CHEMICON], Mouse
Anti-Vimentin –1:10 Serotec MCA862.
For Fluorescent staining, following FITC and Rhodamine tagged secondary antibodies were used in concentrations of 1:200 in serum PBS.
1. Goat polyclonal to Rabbit IgG (FITC) ab 6717 [ABCAM]
2. Donkey polyclonal to Mouse IgG (Texas Red) ab 6818 [ABCAM]
3. Goat polyclonal to Rabbit IgG (Rhodamine) ab 6718 [ABCAM]
4. Alexa Fluor 555 donkey anti-mouse IgG [Invitrogen]
28 5. Alexa Fluor 488 donkey anti-goat IgG [Invitrogen]
6. Oregon Green 488 Goat anti-rabbit IgG [Invitrogen]
7. Fluorescein goat anti-mouse IgG [Invitrogen].
After incubating the cells in fluorescent tagged secondary antibodies for 30 minutes in dark, cells were washed in PBS and mounted with Vectamount with P.I from Vector
Labs (when nuclear counter stain was desired) and mounted with Gold antifade (from
Vector) when nuclear stain was not required. Confocal images were captured using a
Leica TCS SP5 broadband confocal microscope (Leica, Mannheim, Germany) equipped with Argon-488 and diode pumped solid state-561 laser sources and 63.0x
1.40 N.A. oil immersion objective.
When immuno-peroxidase staining was done, protocol from the ABC Kit was followed
-- after permeabilization step, cells were washed in PBS for 5 minutes and quenched in 1.6% Hydrogen Peroxide(H2O2)(6.4ml 30% H2O2 393.6mL TBS) for 10 minutes.
Cells were then washed in TBS containing 0.025% Triton for 5 minutes and placed in blocking buffer (1.5% horse serum in PBS-Serum PBS) for 30 to 60 minutes depending on the antibody used for staining. Cells were then incubated at room temperature for 1 hour with primary antibodies of appropriate dilutions made in serum
PBS, washed for 5 minute in PBS and incubated for an additional 30 minutes with respective AB (Avidin-Biotin) conjugated secondary antibody made in Serum PBS.
Cells were washed for 5 minutes in PBS and incubated in ABC reagent for 30 minutes. Secondary antibodies and ABC reagent are from the Vecstatin® Elite ABC
Kit.cells were washed again in PBS for 5 minutes and developed using Peroxidase
29 substrate solution prepared from the Vector Nova Red Substrate Kit for peroxidase
(SK-4800).Nuclear counterstaining was done using Sigma Hematoxylin. Slides were then dehydrated after passing through xylene and ethanol and mounted using
Permount (Fisher). Images were made using normal microscope.
Cell Invasion Assays/Collagen Motility Assay
To asses for the invasive property of the transformed cells, LLCP-K1 cells were grown on collagen gels. Collagen gels were made in a concentration of 2.5mg/ml and were
5mm thick. Rat tail Collagen Type I in 0.02N Acetic Acid (BD Biosciences) having a collagen concentration of 3.10mg/ml was used. Using the protocol provided by the company, amount of collagen solution sufficient to make gels in two six well plates was calculated-for making 26 ml of collagen solution with a final collagen concentration of 2.5mg/ml , 2.6ml of Sterile 10x PBS ,16.9ml of Rat tail Collagen solution and 400µl of Sterile 1N NaOH were added and the final volume made up with sterile distilled water(6.1ml).The entire procedure was done on ice under sterile conditions in a Hood. All the contents were mixed well and 2ml of this collagen solution was added into each well of a six well plate and was allowed to gel at room temperature in the hood for 2 hours. After the gel was polymerized, the surface was washed once with sterile PBS and once with 10% DMEM medium and cell suspension was added. Cells were grown up to 90-100% confluence before they were serum starved and treated. Cells were treated with 100nM MBG for 72 and 96 hours. After treatment, the collagen gels were separated from the wells and the cells on the
30 collagen gels were washed twice with cold PBS and fixed in 4% formaldehyde for 30 minutes at room temperature.
They were then stained with 0.1% Coomassie (R 250, Bio-Rad) in 10% methanol and
10% acetic acid until sufficient staining was achieved. Gels were then destained with
50% methanol and 10% acetic acid with frequent changes. Destaining was continued overnight until gels were destained sufficiently. Gels were later mounted on glass slides using aqueous or gelatin mount. (Crystal Mount Biomeda). Gels were later visualized for cellular motility by invasion using confocal microscope and DIC images were made.
Statistical analysis: Data are presented as the mean±standard error of the mean. All data presented in this report were felt to be normally distributed based on the Shapiro-
Wilks W test. One way ANOVA was used to compare experimental groups with post hoc analysis employing Bonferroni’s correction for multiple comparisons as appropriate.
Statistical significance was reported at the p<0.05 and p<0.01 levels.
31 Results
1. MBG induces down regulation of epithelial marker E-Cadherin in LLC-PK1 cells.
To test our hypothesis whether MBG could induce EMT in LLC-PK1 cells, we treated the cells with MBG in a time and dose dependent manner. After 24 to 96 hours post treatment, morphological and biochemical changes were assessed. Firstly, changes in the levels of epithelial markers E-Cadherin, Claudin I and Occludin were tested. We noticed that treatment of LLC-PK1 cells with increasing concentrations of MBG from
0.1 nM to 100 nM for 24 to 96 hours down regulated epithelial marker protein E- cadherin. But significant down regulation was noticed with 10-100nM of MBG after 96 hours treatment. Western Blot analysis of the cell lysates with antibody against the protein E-cadherin showed that the expression was decreased by 50% after 96 hours of treatment with 100nm MBG (Fig 1). The cell lysates were also probed for the other tight junction proteins, occludin and Claudin-1 by western blotting. The expression of these proteins was down regulated by around 30% after 72 and 96 hours treatment with 100nm MBG (Fig 2a and 2b).
2. MBG induces morphological changes in LLC-PK1 Cells.
To check whether MBG treatment changes the morphology of LLCP-K1 cells, cells were grown on chamber slides and treated with MBG as above. Cells were immunostained for protein E-cadherin. We noticed that control cells formed typical polarized epithelial sheets with individual cells tightly attached to each other-
32 cobblestone appearance, whereas 10-100 nM of MBG induced significant changes in the cellular morphology. 10nM of MBG caused cell detachment at 96 hours post treatment. But more significant changes in morphology were noticed with 100nM of
MBG after 96 hours with around 60-70% of cells showing transformation. Cells acquired fibroblast-like phenotype-characterized by spindle like elongated shape with a front end-back end polarity. They also showed increased scattering, detachment from the adjoining cells, and flattening. These morphological changes were also associated with down regulation of E-cadherin, as assessed by immunostaining for E- cadherin (Fig 1).
3. MBG induces up-regulation of mesenchymal proteins in LLC-PK1 Cells and the transformed cells acquire invasive property.
Since MBG treatment caused down regulation of E-cadherin and changed cellular morphology, we tested the treated cell lysates for any changes in the expression of mesenchymal proteins Fibronectin and Vimentin by western blot. Expression of
Fibronectin was increased two fold after 72 and 96 hours of treatment with 10 and
100nM MBG (Fig 3). But remarkable increase (two fold) in expression was seen at 96 hours with 100nM MBG. The other important mesenchymal marker we tested was
Vimentin.100nM of MBG for 72 and 96 hours considerably increased the levels of expression of Vimentin as determined by western blot against anti-vimentin antibody.
There was a two fold increase in the expression of vimentin after 72 hours of treatment (Fig 4). The effect on vimentin was evident even after 48 hours of treatment.
MBG treated cells immunostained for fibronectin and vimentin showed increased
33 staining for these proteins in cells with elongated spindle like morphology with fibronectin and vimentin concentrated more in the filopodia (spindles), which is characteristic of fibroblast like cells. On the other hand, the control cells showed very minimal staining for these proteins.
Another functional feature of mesenchymal cells is their invasive property. Since MBG treatment transformed the epithelial cells to mesenchymal cells, we tested if these transformed cells acquired invasive motility. Treatment with 100nM MBG for 72 and 96 hours of cells grown on collagen gels showed more scattering and faster transformation. Cells at 72 hours were approximately 50% transformed, but still remained on the surface of the gel whereas at 96 hours transformed cells acquired invasive motility and invaded the collagen gel as determined by DIC images taken from different planes. Cells were found at around 20 microns distance from the surface (Fig 5a and 5b).
4. MBG treatment causes up-regulation and subsequent nuclear translocation of transcription factors LEF-1 and SNAIL.
The strongest evidence for EMT is the expression of transcription factors LEF-1 and
SNAIL. To test whether MBG treatment induces expression of these factors in LLCP-
K1 cells, nuclear extracts from treated cells were probed for antibody against LEF-1 using western blotting and cells were also immunostained with anti-LEF 1 antibody.
Treatment with 10 to 100 nM of MBG induced de novo expression of LEF-1. At 72 hours post treatment, significant amount of LEF-1 was detected in nuclear extracts
34 and immunostaining also showed nuclear accumulation of this protein. Cells that showed nuclear accumulation of LEF-1 were characterized by elongated spindle-like morphology-characteristic of fibroblasts (Fig 6).
Wnt signaling pathway involving ß-catenin/LEF-1 is one of the major signaling pathways in EMT. To test whether ß-catenin is involved in EMT induced by MBG treatment, we looked for changes in the expression and nuclear translocation of ß- catenin in MBG treated cell lysates and nuclear extracts by western blot as well as immunostaining of treated cells with anti-ß-catenin antibody. Incidentally, no significant changes in the levels of ß-catenin were seen at 72 and 96 hours after treatment and also ß-catenin did not translocate to the nucleus. This was true even with the antibody against activated ß-catenin (non-phosphorylated) (Fig 7).
The other transcription factor implicated in EMT is SNAIL. To test for expression of
SNAIL in MBG treated cells, we probed both the cell lysates and nuclear extracts for anti-Snail antibody by western blotting. Control cells showed some basal levels of
SNAIL, whereas treated cell lysates showed increased expression of SNAIL. Probing of nuclear extracts revealed increased accumulation of SNAIL in the nucleus after treatment. This effect was seen as early as 48 hours post treatment and was maximum at 72 hours. Immunostaining of cells with Snail confirmed these results of control cells showing basal staining for Snail and treated cells showing increased accumulation of Snail in the nucleus as early as 48 hours (Fig 8).
35 5. ROS and Src tyrosine kinase inhibitors reverse the effects of MBG in LLC-PK1
Cells.
MBG and other CTSs signal through the Na/K-ATPase (sodium pump) which further activates Src tyrosine kinase. MBG binding to pump is also known to result in increased ROS production. Therefore we decided to test if the molecular mechanism of MBG induced EMT involves activation of Src and ROS production. When we treated the cells in the presence of ROS quencher, NAC and MBG, NAC completely inhibited the morphological changes induced by MBG at 72 and 96 hours (as assessed by immunostaining) (Fig 9). Cells treated with NAC alone and with NAC and
MBG together did not show elongated, spindle like morphology. They looked very similar to control cells. Expression of mesenchymal marker Fibronectin, as determined by western blotting decreased by 40% in cells treated with NAC and MBG (Fig 10).
When Src tyrosine kinase inhibitor, PP2 was used to treat the cells along with MBG, there was almost complete reversal in the morphological changes induced by MBG at
72 hours and even more at 96 hours. Around 10-15% of cells showed transformation in MBG and PP2 treated cells (as assessed by immunostaining) (Fig 11) at 72 and 96 hours. Fibronectin expression was decreased 30-40% in these cells (Fig 12).
36 Discussion
End stage renal disease is characterized by severe interstitial fibrosis. Tubular epithelial-to-mesenchymal transition (Tubular EMT) has been currently proposed as an essential event in renal tubulointerstitial fibrosis [101] and has been implicated in the pathogenesis of experimental and native kidney diseases in both animals and humans.TGF-ß1 is considered to be the principal fibrogenic growth factor promoting
EMT in renal epithelial cells [4, 74-76].
Patients with CRF show increases in the circulating concentrations of cardiotonic steroids. Cardiotonic steroids are inhibitors of Na+/K+ ATPase. MBG, a cardiotonic steroid, has been found in body fluids of patients with pre-eclampsia, CRF and heart failure and also in experimental diabetes. It has also been associated with hypertension in experimental and clinical conditions. In animal models of uremic cardiomyopathy characterized by hypertension and cardiac fibrosis, increased levels of circulating MBG were found. It has been shown that administration of pathophysiologically high doses of MBG for four weeks triggers fibrotic alterations in renal and cardiac tissues resulting in considerable collagen accumulation in rat heart tissues. In the kidneys MBG administration stimulated modest accumulation of interstitial collagen. Also, administration of MBG-specific antibody resulted in dramatic attenuation of fibrosis in heart but surprisingly, animals were still hypertensive[5, 105].
These findings led us to hypothesize that increased level of MBG could trigger EMT in tubular part of nephron, and thus induce interstitial fibrosis. Since CTS, upon binding
37 to NKA, activates multiple signaling cascades eventually leading to changes in gene expression and alteration in cellular functions, we hypothesized that MBG could induce EMT by signaling through NKA. Thorough analysis of signaling pathways promoting TGFβ1-induced EMT, one of the major mechanisms of renal fibrosis, shows significant similarity with NKA signaling.
Numerous cell lines and primary cultures of normal (MDCK-distal renal tubular cells) and malignant epithelial cells have been shown to undergo EMT upon treatment with growth factors, primarily with TGF-β1. Specific experimental conditions at which these cells were treated with the growth factors have been shown to dramatically influence cell transformation. For our study, we used LLC-PK1 cells which are the porcine renal proximal tubule cells. LLC-PK1 cell line is resistant to TGF-β1 induced EMT if the cells are confluent and with intact adherent junctions [106, 107]. In all our experiments,
LLC-PK1 cells were treated with MBG when the cells reached 90-100% confluence.
Confluent epithelial cells are characterized by a specific actin cytoskeleton structure.
In order to maintain the columnar shape characteristic for epithelial cells, actin fibrils form an actin ring. This ring is in turn connected to e-cadherin through a complex of different catenins and to Na,K-ATPase. When epithelial cells undergo transformation the actin ring is reorganized into a pattern similar to that of fibroblasts.
LLC-PK1 cells express less E-cadherin and NKA than distal tubular cells [108, 109]. It has been shown earlier that in LLC-PK1 cells, binding of cardiotonic steroids to the cells leads to internalization of NKA [62].
38 Firstly, to test the ability of MBG to induce EMT in LLC-PK1 cells, we tested the levels of epithelial markers E-cadherin, occludin and claudin-1. Treatment with 10-100nM concentrations of MBG caused a 50% down regulation of E-cadherin at 72 and 96 hrs treatments. This down regulation was also observed with other epithelial markers occludin and claudin-1. E-cadherin endocytosis is regulated by its tyrosine phosphorylation. This tyrosine on E-cadherin is a substrate for c-Src [84, 85]. Down regulation of e-cadherin by endocytosis is considered as a major event leading to loss of cell-cell adhesion and EMT. Loss of epithelial markers was also associated with a change in the cellular phenotype. MBG treated cells lost the polarity and the cellular adhesion seen among epithelial cells. These cells acquired fibroblast-like phenotype- characterized by spindle like elongated shape with a front end-back end polarity. They also showed increased scattering, detachment from the adjoining cells, and flattening.
EMT is characterized by a loss of the epithelial phenotype and gain of the mesenchymal phenotype. Since our cells showed a loss of the epithelial phenotype and a gain of the mesenchymal phenotype, we tested whether these cells showed an up regulation of the mesenchymal markers. Predictably, cells treated with similar concentrations of MBG showed an up regulation of the mesenchymal markers fibronectin at 72 and 96 hours and vimentin at 48 and 72 hours.
Another characteristic of mesenchymal cells is their invasive ability and motility. Cells treated with MBG for 96 hours were found to invade collagen gels (on which they were grown). This was further evidence for the gain of mesenchymal phenotype the property of motility and invasion.
39 LEF-1 is considered widely as the master transcription factor responsible for the induction of EMT genes [75, 91]. LEF-1 is a member of LEF/TCF transcription factors which are involved in TGF-β and Wnt signaling pathways [92]. In our system of MBG induced EMT, we tested for involvement of LEF-1. We found that upon treating the cells with 10-100nM MBG for 72 hours, there was de novo expression of LEF-1. The up regulated LEF-1 was also found to translocate to the nucleus. In the canonical Wnt signaling pathway, up regulation of mesenchymal gene expression is triggered by
LEF-1 co-activation which in turn is activated by nuclear β-catenin [100]. Incidentally, in our system, β-catenin was not found to translocate to the nucleus in the MBG treated cells.
Another transcription factor known to be involved in EMT is SNAIL. SNAIL causes transcriptional down regulation of e-cadherin in many cancerous cells [88, 89]. In normal renal epithelial cells, TGF-β1 activated signaling pathways promotes up regulation of SNAIL [81, 89]. SNAIL can also be up regulated by increased production of ROS [90]. Patients with chronic renal failure develop oxidant stress. It is known that
CTS binding to NKA activates Src-tyrosine kinases. This Src-activation stimulates production of ROS [22]. When tested for SNAIL involvement in our system, we found that treatment with 100 nM MBG caused increased SNAIL expression as early as 48 hours and up to 72 hours. The up regulated SNAIL was found to translocate to the nucleus.
Since Src-tyrosine kinases and ROS are involved in NKA signaling, we wanted to test the effect of blocking Src-tyrosine kinases and ROS by using PP-2 and N-acetyl
40 cysteine respectively. When ROS quencher NAC was used, it led to a complete blockade of MBG-induced morphological changes. It also blocked MBG induced up regulation of fibronectin expression by 40%. PP-2 also caused a significant reduction in the MBG induced transformation. PP-2 also blocked MBG induced up regulation of fibronectin expression by 35-40%.
Cardiotonic steroids (CTSs) are commonly used for treatment of congestive cardiac failure. Elevated levels of CTS (MBG) have been found in patients with essential hypertension, renal and cardiac failure and in pre-eclampsia. Na+, K+-ATPase (NKA) is the only one known natural target of CTS. For years, it was assumed that pharmacological and physiological actions of CTS are secondary to their effect on intracellular ion concentration. However, CTS circulates in human body in concentrations lower than that necessary for Na+, K+-ATPase inhibition. These findings together with multiple observation that CTS if applied in low doses trigger specific cell responses as hypertrophic growth, proliferation and apoptosis have led to a discovery of a new function of CTS-as an endogenous steroid hormone and NKA as a specific signal transducer. EMT is one of the most important mechanisms in embryonic development, but in ageing, EMT causes several pathological conditions as organ fibrosis and tumor metastasis. This study indicates that MBG triggers EMT in cultured renal epithelial cells. As a result of treatment with MBG epithelial cells lose their morphological and biochemical features and acquires mesenchymal features.
This transformation is accompanied by de novo expression of transcription factor LEF-
1 and by increased SNAIL expression. Complete EMT requires coordinated activation
41 of multiple pathways. Our study provides a new insight into the pathophysiology of
EMT and this knowledge obtained will improve our understanding of physiological functions of CTSs and facilitate new research in developing drugs and treatment for fibrosis in chronic kidney disease.
42 Fig 1
Figure 1. Treatment of LLC-PK1 cells with MBG for 96 hrs induces down regulation of E-cadherin. Top panels represent immunostaining with anti E-cadherin antibody. Bottom panel is the representative Western Blot of cell lysates against anti E-cadherin antibody. ** P<0.05.
43 Fig 2a
Figure 2a. Treatment of LLC-PK1 cells with MBG for 72 and 96 hrs induces down regulation tight junction proteins occludin. Immunostaining and representative Western Blot of cell lysates against occludin antibody.
44 Fig 2b
Figure 2b. Treatment of LLC-PK1 cells with MBG for 72 and 96 hrs induces down regulation tight junction protein-Claudin I. Immunostaining and representative Western Blot of cell lysates against anti Claudin antibody.
45
Fig 3
Figure 3. Treatment of LLC-PK1 cells with MBG up regulates expression of Fibronectin after 72 and 96 hours treatment. Immonofluorescent staining-upper panels (Green- Fibronectin and Red-nucleus) and western blotting-bottom panels with anti-Fibronectin antibody. **p≤0.001
46
Fig 4
Figure 4. Treatment of LLC-PK1 cells with MBG up regulates expression of Vimentin 48 and 72 hrs treatment. Immonofluorescent staining-upper panels (Green-Vimentin) and western blotting-bottom panels with anti-Vimentin antibody. * p≤0.05
47
Fig 5a
Figure 5a. LLC-PK1 cells grown on collagen gel change their morphology from epithelial to fibroblast-like. Cells were treated for 72 and 96 hours and stained with Coomassie.
48 Fig 5b
Figure 5b. Treatment with 100 nM of MBG increases invasive motility of epithelial cells. DIC images of LLC-PK1 cells grown on collagen gel.In control-epithelial cells completely cover surface of collagen gel; after 72 hours of treatment with 100 nM of MBG approximately 50% of cells acquire mesenchymal phenotype but these cells are still located on the surface of collagen gel; after 96 hours of treatment part of transformed cells invade collagen as it is shown by DIC images taken on surface and inside the gel. (20 microns distance). Images were taken on IX71 Inverted research microscope (Olympus).
49 Fig 6
Figure 6. MBG induces up-regulation of transcription factor LEF1 at 72 hours as shown by immunostaining of LLC-PK1 cells and western blotting of nuclear extracts from these cells with anti-LEF1 antibody. (Green-LEF-1).
50 Fig 7
Figure 7. LLC-PK1 cells treated with 100 nM MBG immunostained with antibody against β-catenin (Green) and Western Blot with Cell lysates from the treated cells- show no difference in the levels and no nuclear translocation of β-catenin.
51 Fig 8
Figure 8. MBG induces up-regulation of transcription factor Snail at 48 and 72 hours as shown by immunostaining of LLC-PK1 cells and western blotting of nuclear extracts from these cells with anti-SNAIL antibody.
52 Fig 9
Figure 9 LLC-PK Cells treated with MBG and NAC for 72-96 Hours and immunostained for E-Cadherin. NAC completely blocks the transformation induced by MBG
53 Fig 10
Figure 10 LLC-PK Cells treated with MBG and NAC for 72-96 Hours and cell lysates were probed for Fibronectin by western blot. NAC completely blocks the fibronectin up regulation induced by MBG by 40%. * p≤0.05
54 Fig 11
Figure 11. LLC-PK Cells treated with MBG and PP2 for 72-96 Hours and immunostained for E-Cadherin. PP2 almost completely blocks the transformation induced by MBG.
55
Fig 12
Figure 12 LLC-PK Cells treated with MBG and PP2 for 72-96 Hours and cell lysates were probed for Fibronectin by western blot. PP2 blocks the fibronectin up regulation induced by MBG by around 30-40%. * p≤0.05
56
Conclusions
MBG, at a concentration that is non-toxic and that does not measurably inhibit the pumping activity of Na+, K+-ATPase (NKA), executes an important physiological effect of EMT by activating the signaling pool of NKA leading to pathological fibrosis.
1. MBG treated LLC-PK1 cells show a down regulation of epithelial marker e-by mesenchymal markers fibronectin and vimentin.
2. MBG treated LLC-PK1 cells lose the morphological features of epithelial cells and a gain of features consistent with mesenchymal cells. Also, MBG treated cells gained an invasive property.
3. Transcription factors LEF-1 and SNAIL were up regulated by MBG in LLC-
PK1 cells. LEF-1 and SNAIL subsequently translocated into the nucleus.
4. Canonical Wnt signaling pathway involving ß-catenin is not involved in MBG- induced EMT.
5. MBG induces EMT in a Src-tyrosine kinase and ROS dependent manner.
57 Abstract
Cardiotonic steroids such as MBG have been shown to induce cardiac fibrosis. MBG
contributes to renal fibrosis as well by inducing epithelial to mesenchymal transition
(EMT). The porcine kidney cell line, LLC-PK1, acquires mesenchymal features such as
a fibroblast-like phenotype, scattering and invasive properties and increased expression
of mesenchymal proteins fibronectin and vimentin in a time and concentration
dependent fashion. To examine the mechanisms which are operant, Western blotting
and immunostaining were used. LLC-PK1 cells were grown to complete confluence and
treated with 100 nM of MBG for 72-96 hours. At this concentration, MBG induces
profound EMT (more than 50% of cells show fibroblast-like morphology; scattered cells
occupy more than 80% of the cell- grown surface). It decreases the expression of epithelial proteins-E-cadherin, occludin and claudin-1 and increases the expression of
mesenchymal proteins-vimentin and fibronectin by two fold. These alterations of LLC-
PK1 cells are accompanied by 1) translocation of transcription factor Snail into nuclei
after 72-96 hours post treatment and 2) de novo expression of transcription factor LEF1
at 72 hours post treatment, and its accumulation in the nuclei at 96 hours. Interestingly,
distribution of the LEF1 co-activator, beta-catenin, does not change with MBG
treatment. ROS scavenger NAC completely prevents morphological alteration of LLC-
PK1 cells and inhibits the up-regulation of fibronectin caused by MBG. The Src inhibitor,
PP2, shows only partial attenuation of the morphological alterations of LLC-PK1 cells
induced by MBG but it completely abolishes MBG-induced up regulation of fibronectin.
This study indicates that MBG induces EMT in LLC-PK1 cells and the two transcription
regulators of the EMT transcriptome, Snail and LEF1 in a process requiring reactive
oxygen species.
58
Bibliogaphy
1. Incidence and prevalence of ESRD. United States Renal Data System. Am J Kidney Dis, 1998. 32(2 Suppl 1): p. S38-49.
2. Valderrabano, F., et al., Report on management of renal failure in Europe, XXV, 1994 end stage renal disease and dialysis report. The EDTA-ERA Registry. European Dialysis and Transplant Association-European Renal Association. Nephrol Dial Transplant, 1996. 11 Suppl 1: p. 2-21.
3. Chatziantoniou, C., et al., Progression and regression in renal vascular and glomerular fibrosis. Int J Exp Pathol, 2004. 85(1): p. 1-11.
4. Okada, H. and R. Kalluri, Cellular and molecular pathways that lead to progression and regression of renal fibrogenesis. Curr Mol Med, 2005. 5(5): p. 467-74.
5. Kennedy, D.J., et al., Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy. Hypertension, 2006. 47(3): p. 488-95.
6. Stokes, G.S., et al., Effect of dialysis on circulating Na,K ATPase inhibitor in uremic patients. Nephron, 1990. 54(2): p. 127-33.
7. Bauman, J.L., R.J. Didomenico, and W.L. Galanter, Mechanisms, manifestations, and management of digoxin toxicity in the modern era. Am J Cardiovasc Drugs, 2006. 6(2): p. 77-86.
8. Gheorghiade, M., D.J. van Veldhuisen, and W.S. Colucci, Contemporary use of digoxin in the management of cardiovascular disorders. Circulation, 2006. 113(21): p. 2556-64.
59 9. Hoppe, U.C. and E. Erdmann, Digitalis in heart failure! Still applicable? Z Kardiol, 2005. 94(5): p. 307-11.
10. Pervaiz, M.H., M.G. Dickinson, and M. Yamani, Is digoxin a drug of the past? Cleve Clin J Med, 2006. 73(9): p. 821-4, 826, 829-32 passim.
11. Garraffo, H.M. and E.G. Gros, Biosynthesis of bufadienolides in toads. VI. Experiments with [1,2-3H]cholesterol, [21-14C]coprostanol, and 5 beta-[21-14 C]pregnanolone in the toad Bufo arenarum. Steroids, 1986. 48(3-4): p. 251-7.
12. Shimada, K. and T. Nambara, Isolation and characterization of cardiotonic steroid conjugates from the skin of Bufo marinus (L.) Schneider. Chem Pharm Bull (Tokyo), 1979. 27(8): p. 1881-6.
13. Bagrov, A.Y., et al., Plasma marinobufagenin-like and ouabain-like immunoreactivity during saline volume expansion in anesthetized dogs. Cardiovasc Res, 1996. 31(2): p. 296-305.
14. Fedorova, O.V., D.E. Anderson, and A.Y. Bagrov, Plasma marinobufagenin-like and ouabain-like immunoreactivity in adrenocorticotropin-treated rats. Am J Hypertens, 1998. 11(7): p. 796-802.
15. Hamlyn, J.M., et al., Observations on the nature, biosynthesis, secretion and significance of endogenous ouabain. Clin Exp Hypertens, 1998. 20(5-6): p. 523-33.
16. Bagrov, Y.Y., et al., Endogenous digitalis-like ligands and Na/K-ATPase inhibition in experimental diabetes mellitus. Front Biosci, 2005. 10: p. 2257-62.
17. Doris, P.A. and A.Y. Bagrov, Endogenous sodium pump inhibitors and blood pressure regulation: an update on recent progress. Proc Soc Exp Biol Med, 1998. 218(3): p. 156-67.
60 18. Fedorova, O.V., et al., Marinobufagenin, an endogenous alpha-1 sodium pump ligand, in hypertensive Dahl salt-sensitive rats. Hypertension, 2001. 37(2 Part 2): p. 462-6.
19. Fedorova, O.V., et al., Endogenous ligand of alpha(1) sodium pump, marinobufagenin, is a novel mediator of sodium chloride--dependent hypertension. Circulation, 2002. 105(9): p. 1122-7.
20. Fedorova, O.V., et al., Coordinated shifts in Na/K-ATPase isoforms and their endogenous ligands during cardiac hypertrophy and failure in NaCl-sensitive hypertension. J Hypertens, 2004. 22(2): p. 389-97.
21. Fridman, A.I., et al., Marinobufagenin, an endogenous ligand of alpha-1 sodium pump, is a marker of congestive heart failure severity. J Hypertens, 2002. 20(6): p. 1189-94.
22. Lopatin, D.A., et al., Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia. J Hypertens, 1999. 17(8): p. 1179-87.
23. Manunta, P., B.P. Hamilton, and J.M. Hamlyn, Structure-activity relationships for the hypertensinogenic activity of ouabain: role of the sugar and lactone ring. Hypertension, 2001. 37(2 Part 2): p. 472-7.
24. Manunta, P., B.P. Hamilton, and J.M. Hamlyn, Salt intake and depletion increase circulating levels of endogenous ouabain in normal men. Am J Physiol Regul Integr Comp Physiol, 2006. 290(3): p. R553-9.
25. Manunta, P., et al., High circulating levels of endogenous ouabain in the offspring of hypertensive and normotensive individuals. J Hypertens, 2005. 23(9): p. 1677- 81.
61 26. Pitzalis, M.V., et al., Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy. Eur J Heart Fail, 2006. 8(2): p. 179- 86.
27. Puschett, J.B., The role of excessive volume expansion in the pathogenesis of preeclampsia. Med Hypotheses, 2006. 67(5): p. 1125-32.
28. Dmitrieva, R.I., et al., Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that Is independent of cholesterol side-chain cleavage. Hypertension, 2000. 36(3): p. 442-8.
29. Lichtstein, D., et al., Bufodienolides as endogenous Na+, K+-ATPase inhibitors: biosynthesis in bovine and rat adrenals. Clin Exp Hypertens, 1998. 20(5-6): p. 573- 9.
30. Yoshika, M., et al., Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats. Hypertension, 2007. 49(1): p. 209-14.
31. Keenan, S.M., et al., Elucidation of the Na+, K+-ATPase digitalis binding site. J Mol Graph Model, 2005. 23(6): p. 465-75.
32. Lingrel, J.B., et al., Cation and cardiac glycoside binding sites of the Na,K-ATPase. Ann N Y Acad Sci, 1997. 834: p. 194-206.
33. Lingrel, J.B., et al., Ligand binding sites of Na,K-ATPase. Acta Physiol Scand Suppl, 1998. 643: p. 69-77.
34. Scheiner-Bobis, G., The sodium pump. Its molecular properties and mechanics of ion transport. Eur J Biochem, 2002. 269(10): p. 2424-33.
35. Aperia, A.C., Regulation of sodium transport. Curr Opin Nephrol Hypertens, 1995. 4(5): p. 416-20.
62 36. Therien, A.G. and R. Blostein, Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol, 2000. 279(3): p. C541-66.
37. Martin, D.W., Structure-function relationships in the NA+,K+-pump. Semin Nephrol, 2005. 25(5): p. 282-91.
38. Geering, K., The functional role of beta subunits in oligomeric P-type ATPases. J Bioenerg Biomembr, 2001. 33(5): p. 425-38.
39. Rajasekaran, S.A., S.P. Barwe, and A.K. Rajasekaran, Multiple functions of Na,K- ATPase in epithelial cells. Semin Nephrol, 2005. 25(5): p. 328-34.
40. Farman, N., et al., Localization of alpha-isoforms of Na(+)-K(+)-ATPase in rat kidney by in situ hybridization. Am J Physiol, 1991. 260(3 Pt 1): p. C468-74.
41. Herrera, V.L., et al., Developmental cell-specific regulation of Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and alpha 3-isoform gene expression. Am J Physiol, 1994. 266(5 Pt 1): p. C1301-12.
42. Martin, D.W. and J.R. Sachs, Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites. Biochemistry, 1999. 38(23): p. 7485-97.
43. Reeves, A.S., J.H. Collins, and A. Schwartz, Isolation and characterization of (Na,K)-ATPase proteolipid. Biochem Biophys Res Commun, 1980. 95(4): p. 1591- 8.
44. Blanco, G. and R.W. Mercer, Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol, 1998. 275(5 Pt 2): p. F633-50.
45. Munzer, J.S., et al., Tissue- and isoform-specific kinetic behavior of the Na,K- ATPase. J Biol Chem, 1994. 269(24): p. 16668-76.
63 46. Segall, L., S.E. Daly, and R. Blostein, Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase. J Biol Chem, 2001. 276(34): p. 31535-41.
47. Segall, L., et al., Structural basis for alpha1 versus alpha2 isoform-distinct behavior of the Na,K-ATPase. J Biol Chem, 2003. 278(11): p. 9027-34.
48. Hu, Y.K. and J.H. Kaplan, Site-directed chemical labeling of extracellular loops in a membrane protein. The topology of the Na,K-ATPase alpha-subunit. J Biol Chem, 2000. 275(25): p. 19185-91.
49. Karlish, S.J., R. Goldshleger, and P.L. Jorgensen, Location of Asn831 of the alpha chain of Na/K-ATPase at the cytoplasmic surface. Implication for topological models. J Biol Chem, 1993. 268(5): p. 3471-8.
50. Segall, L., L.K. Lane, and R. Blostein, New insights into the role of the N terminus in conformational transitions of the Na,K-ATPase. J Biol Chem, 2002. 277(38): p. 35202-9.
51. Dostanic-Larson, I., et al., Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site. Am J Physiol Regul Integr Comp Physiol, 2006. 290(3): p. R524-8.
52. Dostanic-Larson, I., et al., The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation. Proc Natl Acad Sci U S A, 2005. 102(44): p. 15845-50.
53. Dmitrieva, R.I. and P.A. Doris, Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells. J Biol Chem, 2003. 278(30): p. 28160-6.
64 54. Kurosawa, M., et al., Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells. Am J Physiol Cell Physiol, 2001. 280(3): p. C459-64.
55. Lichtstein, D., et al., Digitalis and digitalislike compounds down-regulate gene expression of the intracellular signaling protein 14-3-3 in rat lens. Hypertens Res, 2000. 23 Suppl: p. S51-3.
56. Liu, L., et al., Role of caveolae in ouabain-induced proliferation of cultured vascular smooth muscle cells of the synthetic phenotype. Am J Physiol Heart Circ Physiol, 2004. 287(5): p. H2173-82.
57. Nasu, K., et al., Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis. Mol Hum Reprod, 2005. 11(11): p. 817-23.
58. Xie, Z., et al., Intracellular reactive oxygen species mediate the linkage of Na+/K+- ATPase to hypertrophy and its marker genes in cardiac myocytes. J Biol Chem, 1999. 274(27): p. 19323-8.
59. Yeh, J.Y., et al., Effects of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells. Prostate, 2003. 54(2): p. 112- 24.
60. Haas, M., et al., Src-mediated inter-receptor cross-talk between the Na+/K+- ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases. J Biol Chem, 2002. 277(21): p. 18694-702.
61. Kotova, O., et al., Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism. J Biol Chem, 2006. 281(29): p. 20085-94.
65 62. Liu, J., et al., Effects of cardiac glycosides on sodium pump expression and function in LLC-PK1 and MDCK cells. Kidney Int, 2002. 62(6): p. 2118-25.
63. Liu, J., et al., Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations. J Biol Chem, 2000. 275(36): p. 27838-44.
64. Mohammadi, K., et al., Role of protein kinase C in the signal pathways that link Na+/K+-ATPase to ERK1/2. J Biol Chem, 2001. 276(45): p. 42050-6.
65. Xie, Z., Molecular mechanisms of Na/K-ATPase-mediated signal transduction. Ann N Y Acad Sci, 2003. 986: p. 497-503.
66. Wang, H., et al., Ouabain assembles signaling cascades through the caveolar Na+/K+-ATPase. J Biol Chem, 2004. 279(17): p. 17250-9.
67. Yuan, Z., et al., Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex. Mol Biol Cell, 2005. 16(9): p. 4034-45.
68. Hay, E.D., The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev Dyn, 2005. 233(3): p. 706-20.
69. Trelstad, R.L., E.D. Hay, and J.D. Revel, Cell contact during early morphogenesis in the chick embryo. Dev Biol, 1967. 16(1): p. 78-106.
70. Kalluri, R. and E.G. Neilson, Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest, 2003. 112(12): p. 1776-84.
71. Lee, J.M., et al., The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol, 2006. 172(7): p. 973-81.
72. Thiery, J.P. and J.P. Sleeman, Complex networks orchestrate epithelial- mesenchymal transitions. Nat Rev Mol Cell Biol, 2006. 7(2): p. 131-42.
66 73. Iwano, M. and E.G. Neilson, Mechanisms of tubulointerstitial fibrosis. Curr Opin Nephrol Hypertens, 2004. 13(3): p. 279-84.
74. Zeisberg, M. and R. Kalluri, The role of epithelial-to-mesenchymal transition in renal fibrosis. J Mol Med, 2004. 82(3): p. 175-81.
75. Nawshad, A. and E.D. Hay, TGFbeta3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development. J Cell Biol, 2003. 163(6): p. 1291-301.
76. Nawshad, A., et al., Transforming growth factor-beta signaling during epithelial- mesenchymal transformation: implications for embryogenesis and tumor metastasis. Cells Tissues Organs, 2005. 179(1-2): p. 11-23.
77. Medici, D., E.D. Hay, and D.A. Goodenough, Cooperation between snail and LEF- 1 transcription factors is essential for TGF-beta1-induced epithelial-mesenchymal transition. Mol Biol Cell, 2006. 17(4): p. 1871-9.
78. Strutz, F., et al., Identification and characterization of a fibroblast marker: FSP1. J Cell Biol, 1995. 130(2): p. 393-405.
79. Zavadil, J. and E.P. Bottinger, TGF-beta and epithelial-to-mesenchymal transitions. Oncogene, 2005. 24(37): p. 5764-74.
80. Grunert, S., M. Jechlinger, and H. Beug, Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol, 2003. 4(8): p. 657-65.
81. Ayalon, O. and B. Geiger, Cyclic changes in the organization of cell adhesions and the associated cytoskeleton, induced by stimulation of tyrosine phosphorylation in bovine aortic endothelial cells. J Cell Sci, 1997. 110 ( Pt 5): p. 547-56.
67 82. Roura, S., et al., Regulation of E-cadherin/Catenin association by tyrosine phosphorylation. J Biol Chem, 1999. 274(51): p. 36734-40.
83. Strutz, F., et al., Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int, 2002. 61(5): p. 1714-28.
84. Fujita, Y., et al., Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol, 2002. 4(3): p. 222-31.
85. Le, T.L., A.S. Yap, and J.L. Stow, Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J Cell Biol, 1999. 146(1): p. 219-32.
86. Khew-Goodall, Y. and C. Wadham, A perspective on regulation of cell-cell adhesion and epithelial-mesenchymal transition: known and novel. Cells Tissues Organs, 2005. 179(1-2): p. 81-6.
87. Peinado, H., M. Quintanilla, and A. Cano, Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Biol Chem, 2003. 278(23): p. 21113-23.
88. Nieto, M.A., The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol, 2002. 3(3): p. 155-66.
89. Radisky, D.C., et al., Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature, 2005. 436(7047): p. 123-7.
90. Zhou, B.P., et al., Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol, 2004. 6(10): p. 931- 40.
91. Arce, L., N.N. Yokoyama, and M.L. Waterman, Diversity of LEF/TCF action in development and disease. Oncogene, 2006. 25(57): p. 7492-504.
68 92. Eastman, Q. and R. Grosschedl, Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol, 1999. 11(2): p. 233-40.
93. Labbe, E., A. Letamendia, and L. Attisano, Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-beta and wnt pathways. Proc Natl Acad Sci U S A, 2000. 97(15): p. 8358-63.
94. Tsukazaki, T., et al., SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor. Cell, 1998. 95(6): p. 779-91.
95. Miura, S., et al., Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol Cell Biol, 2000. 20(24): p. 9346-55.
96. Rusten, T.E. and H. Stenmark, Analyzing phosphoinositides and their interacting proteins. Nat Methods, 2006. 3(4): p. 251-8.
97. Simonsen, A., et al., The role of phosphoinositides in membrane transport. Curr Opin Cell Biol, 2001. 13(4): p. 485-92.
98. Stenmark, H. and R. Aasland, FYVE-finger proteins--effectors of an inositol lipid. J Cell Sci, 1999. 112 ( Pt 23): p. 4175-83.
99. Stenmark, H., et al., Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger. J Biol Chem, 1996. 271(39): p. 24048-54.
100. Clevers, H., Wnt/beta-catenin signaling in development and disease. Cell, 2006. 127(3): p. 469-80.
101. Liu, Y., Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol, 2004. 15(1): p. 1-12.
69 102. Eddy, A.A., Molecular basis of renal fibrosis. Pediatr Nephrol, 2000. 15(3-4): p. 290-301.
103. Yang, J. and Y. Liu, Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol, 2001. 159(4): p. 1465-75.
104. Liu, J., et al., Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism. Kidney Int, 2004. 66(1): p. 227-41.
105. Elkareh, J., et al., Marinobufagenin stimulates fibroblast collagen production and causes fibrosis in experimental uremic cardiomyopathy. Hypertension, 2007. 49(1): p. 215-24.
106. Masszi, A., et al., Central role for Rho in TGF-beta1-induced alpha-smooth muscle actin expression during epithelial-mesenchymal transition. Am J Physiol Renal Physiol, 2003. 284(5): p. F911-24.
107. Masszi, A., et al., Integrity of cell-cell contacts is a critical regulator of TGF- beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin. Am J Pathol, 2004. 165(6): p. 1955-67.
108. Prozialeck, W.C., P.C. Lamar, and D.M. Appelt, Differential expression of E- cadherin, N-cadherin and beta-catenin in proximal and distal segments of the rat nephron. BMC Physiol, 2004. 4: p. 10.
109. Wetzel, R.K. and K.J. Sweadner, Immunocytochemical localization of Na-K- ATPase alpha- and gamma-subunits in rat kidney. Am J Physiol Renal Physiol, 2001. 281(3): p. F531-45.
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