Dissection of Key Events in Tubular Epithelial to Myofibroblast Transition and Its Implications in Renal Interstitial Fibrosis
2001; Elsevier BV; Volume: 159; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62533-3
ISSN1525-2191
Autores Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoMyofibroblast activation is a key event playing a critical role in the progression of chronic renal disease. Emerging evidence suggests that myofibroblasts can derive from tubular epithelial cells by an epithelial to mesenchymal transition (EMT); however, the details regarding the conversion between these two cell types are poorly understood. Here we dissect the key events during the process of EMT induced by transforming growth factor-β1. Incubation of human tubular epithelial cells with transforming growth factor-β1 induced de novo expression of α-smooth muscle actin, loss of epithelial marker E-cadherin, transformation of myofibroblastic morphology, and production of interstitial matrix. Time-course studies revealed that loss of E-cadherin was an early event that preceded other alterations during EMT. The transformed cells secreted a large amount of matrix metalloproteinase-2 that specifically degraded tubular basement membrane. They also exhibited an enhanced motility and invasive capacity. These alterations in epithelial phenotypes in vitro were essentially recapitulated in a mouse model of renal fibrosis induced by unilateral ureteral obstruction. Hence, these results indicate that tubular epithelial to myofibroblast transition is an orchestrated, highly regulated process involving four key steps including: 1) loss of epithelial cell adhesion, 2) de novo α-smooth muscle actin expression and actin reorganization, 3) disruption of tubular basement membrane, and 4) enhanced cell migration and invasion. Myofibroblast activation is a key event playing a critical role in the progression of chronic renal disease. Emerging evidence suggests that myofibroblasts can derive from tubular epithelial cells by an epithelial to mesenchymal transition (EMT); however, the details regarding the conversion between these two cell types are poorly understood. Here we dissect the key events during the process of EMT induced by transforming growth factor-β1. Incubation of human tubular epithelial cells with transforming growth factor-β1 induced de novo expression of α-smooth muscle actin, loss of epithelial marker E-cadherin, transformation of myofibroblastic morphology, and production of interstitial matrix. Time-course studies revealed that loss of E-cadherin was an early event that preceded other alterations during EMT. The transformed cells secreted a large amount of matrix metalloproteinase-2 that specifically degraded tubular basement membrane. They also exhibited an enhanced motility and invasive capacity. These alterations in epithelial phenotypes in vitro were essentially recapitulated in a mouse model of renal fibrosis induced by unilateral ureteral obstruction. Hence, these results indicate that tubular epithelial to myofibroblast transition is an orchestrated, highly regulated process involving four key steps including: 1) loss of epithelial cell adhesion, 2) de novo α-smooth muscle actin expression and actin reorganization, 3) disruption of tubular basement membrane, and 4) enhanced cell migration and invasion. Progression of chronic renal disease is considered to be an irreversible process that eventually leads to end-stage renal failure characterized by wide-spread tissue fibrosis.1Port FK Fenton SSA Mazzuchi N ESRD throughout the world: morbidity, mortality and quality of life.Kidney Int. 2000; 57: S1-S2Crossref Scopus (22) Google Scholar, 2Eddy AA Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 3Remuzzi G Bertani T Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1146) Google Scholar Regardless of the initial causes, renal fibrosis is characterized by massive interstitial myofibroblast activation that is believed to play a central role in the pathogenesis of renal interstitial fibrosis.4Roberts IS Burrows C Shanks JH Venning M McWilliam LJ Interstitial myofibroblasts: predictors of progression in membranous nephropathy.J Clin Pathol. 1997; 50: 123-127Crossref PubMed Scopus (121) Google Scholar, 5Essawy M Soylemezoglu O Muchaneta-Kubara EC Shortland J Brown CB el Nahas AM Myofibroblasts and the progression of diabetic nephropathy.Nephrol Dial Transplant. 1997; 12: 43-50Crossref PubMed Scopus (176) Google Scholar, 6Tang WW Ulich TR Lacey DL Hill DC Qi M Kaufman SA Van GY Tarpley JE Yee JS Platelet-derived growth factor-BB induces renal interstitial myofibroblast formation and tubulointerstitial fibrosis.Am J Pathol. 1996; 148: 1169-1180PubMed Google Scholar Although the exact origins of these α-smooth muscle actin (α-SMA)-positive myofibroblasts remain uncertain,7Ong AC Fine LG Loss of glomerular function and tubulointerstitial fibrosis: cause or effect?.Kidney Int. 1994; 45: 345-351Crossref PubMed Scopus (127) Google Scholar, 8Bohle A Strutz F Muller GA On the pathogenesis of chronic renal failure in primary glomerulopathies: a view from the interstitium.Exp Nephrol. 1994; 2: 205-210PubMed Google Scholar, 9Kliem V Johnson RJ Alpers CE Yoshimura A Couser WG Koch KM Floege J Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats.Kidney Int. 1996; 49: 666-678Crossref PubMed Scopus (258) Google Scholar emerging evidence suggests that they may derive from tubular epithelial cellsby an epithelial to mesenchymal transition (EMT) process under pathological conditions.10Liu Y Rajur K Tolbert E Dworkin LD Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways.Kidney Int. 2000; 58: 2028-2043Crossref PubMed Google Scholar, 11Strutz F Okada H Lo CW Danoff T Carone RL Tomaszewski JE Neilson EG Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (877) Google Scholar, 12Ng YY Huang TP Yang WC Chen ZP Yang AH Mu W Nikolic-Paterson DJ Atkins RC Lan HY Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.Kidney Int. 1998; 54: 864-876Crossref PubMed Scopus (344) Google Scholar However, the details regarding the conversion between these two distinct types of cells are poorly defined.Because tubular epithelial cells and interstitial myofibroblasts dramatically differ in their morphology and phenotypes, and are located in separated tissue compartments within the kidneys, one can envision that there have to be remarkable alterations in the expression of many sets of genes to make this phenotypic conversion possible. Indeed, previous studies have identified altered expression patterns of several genes such as α-SMA and fibroblast-specific protein-1 (Fsp 1) during tubular epithelial to myofibroblast transition (EMT).13Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar, 14Fan JM Ng YY Hill PA Nikolic-Paterson DJ Mu W Atkins RC Lan YH Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro.Kidney Int. 1999; 56: 1455-1467Crossref PubMed Scopus (474) Google Scholar However, the cause-effect relationship of these changes in EMT as well as the key events during the entire EMT course at cellular level remain to be fascinating unanswered questions.Given the fact that several key obstacles have to be overcome to make epithelial to myofibroblast transition possible, we propose that tubular epithelial to myofibroblast transition at the cellular level is likely a step-wise process involving several crucial events that eventually lead to the completion of the entire course. It is conceivable that these tubular epithelial cells lose the key epithelial cell markers that make them epithelium in the first place, while acquiring de novo expression of myofibroblastic markers that define their newly adapted morphology and phenotypes. Likewise, these cells have to find a way to pass across the tubular basement membrane (TBM) that surrounds the renal tubule and finally enter their newly found home, the interstitial compartments of the kidneys.In this study, we attempt to decipher the key events controlling the tubular epithelial to myofibroblast transition both in vitro and in vivo. Our data suggest that the entire EMT process consists of several key steps that depend on hyperactive transforming growth factor (TGF)-β1 signaling.Materials and MethodsCell Culture and TreatmentHuman proximal tubular epithelial cells (HKC-8) were kindly provided by Dr. L. Racusen of Johns Hopkins University.15Racusen LC Monteil C Sgrignoli A Lucskay M Marouillat S Rhim JG Morin JP Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines.J Lab Clin Med. 1997; 129: 318-329Abstract Full Text PDF PubMed Scopus (202) Google Scholar Cells were cultured in Dulbecco's modified Eagle's medium-F12 medium supplemented with 5% fetal bovine serum (Life Technologies, Inc., Grand Island, NY).16Liu Y Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms.Am J Physiol. 1999; 277: F624-F633PubMed Google Scholar For cytokine treatment, HKC cells were seeded at ∼70% confluence in complete medium containing 5% fetal bovine serum. Twenty-four hours later, the cells were changed to serum-free medium, and recombinant TGF-β1 (R & D Systems, Minneapolis, MN) was added at a final concentration of 4 ng/ml except where otherwise indicated. The cells and conditioned media were collected at different time points for further characterization.Animal ModelMale CD-1 mice weighing 20 to 22 g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Unilateral ureteral obstruction (UUO) was performed using an established procedure.17Hammad FT Wheatley AM Davis G Long-term renal effects of unilateral ureteral obstruction and the role of endothelin.Kidney Int. 2000; 58: 242-250Crossref PubMed Scopus (35) Google Scholar Briefly, under general anesthesia, complete ureteral obstruction was performed by double-ligating the left ureter using 4-0 silk after a midline abdominal incision. Sham-operated mice had their ureters exposed, manipulated but not ligated. Mice were sacrificed at different time points as indicated after surgery, and the kidneys were removed. One part of the kidneys was fixed in 10% phosphate-buffered formalin followed by paraffin embedding for histological and immunohistochemical studies. The remaining kidneys were snap-frozen in liquid nitrogen and stored at −80°C for protein extractions.Western Immunoblot AnalysisHKC cells and cytokine-treated cells were lysed with sodium dodecyl sulfate (SDS) sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromophenol blue). Kidney tissue was homogenized by a polytron homogenizer (Brinkmann Instruments, Westbury, NY) and the supernatant was collected after centrifugation at 13,000 × g at 4°C for 20 minutes, as described previously.18Liu Y Tolbert EM Lin L Thursby MA Sun AM Nakamura T Dworkin LD Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure.Kidney Int. 1999; 55: 442-453Crossref PubMed Scopus (102) Google Scholar After protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma Chemical Co., St. Louis, MO), the tissue lysate was mixed with an equal amount 2× SDS sample buffer. Samples were heated at 100°C for ∼5 to 10 minutes before loading and separated on precasted 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins were electrotransferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL) in transfer buffer containing 48 mmol/L Tris-HCl, 39 mmol/L glycine, 0.037% SDS, and 20% methanol at 4°C for 1 hour. Nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% Carnation nonfat milk in TBS buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20). The membranes were incubated for 16 hours at 4°C with various primary antibodies in TBS buffer containing 5% milk at the dilutions specified by the manufacturers. The monoclonal α-SMA antibody was purchased from Sigma Chemical Co. The antibody for E-cadherin was obtained from Transduction Laboratories (Lexington, KY). The antibody against β-actin was purchased from Santa Cruz Biochemicals (Santa Cruz, CA). The anti-human matrix metalloproteinase (MMP)-2 antibody was purchased from Oncogene Research Products (Cambridge, MA). Binding of primary antibodies was followed by incubation for 1 hour at room temperature with the secondary horseradish peroxidase-conjugated IgG in 1% nonfat milk. The signals were visualized by the enhanced chemiluminescence system (ECL, Amersham), as described previously.18Liu Y Tolbert EM Lin L Thursby MA Sun AM Nakamura T Dworkin LD Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure.Kidney Int. 1999; 55: 442-453Crossref PubMed Scopus (102) Google ScholarImmunostainingIndirect immunofluorescence staining was performed using an established procedure on HKC cells cultured on coverslips. Briefly, control or cytokine-treated HKC cells were washed with cold phosphate-buffered saline (PBS) twice, and fixed with cold methanol:acetone (1:1) for 10 minutes on ice. After extensive washing with PBS containing 0.5% bovine serum albumin, the cells were blocked with 20% normal donkey serum in PBS buffer for 30 minutes at room temperature, and then incubated with specific primary antibodies described above, except the rat monoclonal anti-E-cadherin (clone DECMA-1) that was obtained from Sigma. The cells were then routinely stained with fluorescein isothiocyanate-conjugated secondary antibodies (Sigma). Cells were also stained with 4′,6-diamidino-2-phenylindole, HCl to visualize the nuclei. For visualizing F-actin, cells were stained with tetramethylrhodamine isothiocyanate-conjugated phalloidin (Sigma). Stained cells were mounted with anti-fade mounting medium (Vector Laboratories, Burlingame, CA) and viewed on a Nikon Eclipse E600 Epi-fluorescence microscope (Nikon, Melville, NY). For immunostaining renal tissue, kidney sections from paraffin-embedded tissues were prepared at 4-μm thickness using a routine procedure.10Liu Y Rajur K Tolbert E Dworkin LD Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways.Kidney Int. 2000; 58: 2028-2043Crossref PubMed Google Scholar Immunohistochemical localization was performed using the Vector M.O.M. immunodetection kit (Vector Laboratories). The primary antibodies used were anti-E-cadherin and anti-α-SMA (Sigma), anti-TGF-β1 and anti-TGF-β type I receptor (Santa Cruz Biochemicals). As a negative control, the primary antibody was replaced with either nonimmune mouse or rabbit IgG, corresponding to species of the primary antibodies.Gelatin Zymographic AnalysisZymographic analysis of the MMP proteolytic activity in the supernatant of cultured cells or kidney tissue homogenates was performed according to the method described previously.10Liu Y Rajur K Tolbert E Dworkin LD Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways.Kidney Int. 2000; 58: 2028-2043Crossref PubMed Google Scholar, 19Kim TH Mars WM Stolz DB Michalopoulos GK Expression and activation of pro-MMP-2 and pro-MMP-9 during rat liver regeneration.Hepatology. 2000; 31: 75-82Crossref PubMed Scopus (138) Google Scholar Briefly, an equal number of the HKC cells were seeded on 6-well plates at a density of 4 × 105/well in Dulbecco's modified Eagle's medium-F12 medium containing 5% fetal bovine serum. Twenty-four hours later, culture medium was changed to 0.7 ml of serum-free medium, and TGF-β1 was added to the cultures. At different time points as indicated, conditioned media were collected and centrifuged at 13,000 × g for 5 minutes to remove any cell debris. The protein concentration was determined using a protein assay kit with bovine serum albumin as a standard (Sigma). Kidney tissue homogenates were prepared essentially according to the methods described by Kim and colleagues.19Kim TH Mars WM Stolz DB Michalopoulos GK Expression and activation of pro-MMP-2 and pro-MMP-9 during rat liver regeneration.Hepatology. 2000; 31: 75-82Crossref PubMed Scopus (138) Google Scholar A constant amount of protein from the conditioned media (15 μg) or kidney tissue homogenates (30 μg) was loaded into 10% SDS-polyacrylamide gel containing 1 mg/ml gelatin (Bio-Rad). After electrophoresis, SDS was removed from the gel by incubation in 2.5% Triton X-100 at room temperature for 30 minutes with gentle shaking. The gel was washed well with distilled water to remove detergent and incubated at 37°C for ∼16 to 36 hours in a developing buffer containing 50 mmol/L Tris-HCl, pH 7.6, 0.2 mol/L NaCl, 5 mmol/L CaCl2, and 0.02% Brij 35. The gel was then stained with a solution of 30% methanol, 10% glacial acetic acid, and 0.5% Coomassie Blue G250, followed by destaining in the same solution without dye. Proteinase activity was detected as unstained bands on a blue background representing areas of gelatin digestion.Determination of Basement Membrane Integrity by Bacterial TranslocationBacterial translocation was performed for evaluating TBM integrity using a two-compartment Boyden chamber with transwell filters containing 3-μm diameter pores (Corning Co., Corning, NY). Matrigel (Becton Dickinson Labware, Bedford, MA), a solubilized basement membrane matrix consisting of laminin (56%), collagen IV (31%), entactin (8%), and heparan sulfate proteoglycan (perlecan), was added onto the transwell filters to form matrix gels at 37°C that essentially reconstitute the TBM in vivo.20Vukicevic S Somogyi L Martinovic I Zic R Kleinman HK Marusic M Reconstituted basement membrane (Matrigel) promotes the survival and influences the growth of murine tumors.Int J Cancer. 1992; 50: 791-795Crossref PubMed Scopus (20) Google Scholar Matrigel at a concentration of 22 μg/cm2 produced a matrix gel layer at 15-μm depth, which represents 100-fold thickness of native TBM (150 nm).21Tisher CC Madsen KM Anatomy of the kidney.in: BM Brenner The Kidney. ed 5. W.B. Saunders Co., Philadelphia1996: 3-71Google Scholar Preliminary studies showed that the matrix gel on the transwell filters maintained its structural integrity for >7 days at 37°C that completely blocked bacterial translocation through the gel (data not shown). The transwell filters with the Matrigel were then incubated with the conditioned media (rich in MMP-2) from HKC cells treated with or without TGF-β1 at 37°C for 4 days. Escherichia coli DH5α (Life Technologies) was grown in Luria Broth (LB) medium at 37°C overnight and bacterial concentration was estimated by reading at an optical density of 600 nm with 1 optical density equivalent to 109 bacteria/ml.22Lech K Brent R Escherichia coli, plasmids and bacteriophages.in: Ausubel FM Brent R Kingston RE Moore DD Seidman JG Smith JA Struhl K Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995: 1.0.3-1.2.2Google Scholar Approximately 108 bacteria in 100 μl were added to the upper compartment of the Boyden chamber in a final volume of 400 μl. Aliquots (20 μl) were removed at 0.5 and 2 hours, respectively, from the bottom compartment of the chamber containing 1 ml of media. Dilutions from each aliquot were plated on LB agar plates and incubated at 37°C for 16 hours and colonies were counted. The entire experiments were performed in triplicate for each time points per treatment.Boyden Chamber Motility AssayCell motility and migration were evaluated using Boyden chamber motogenicity assay with tissue culture-treated transwell filters (Costar).23Liu Y Centracchio JN Lin L Sun AM Dworkin LD Constitutive expression of HGF modulates renal epithelial cell phenotype and induces c-met and fibronectin expression.Exp Cell Res. 1998; 242: 174-185Crossref PubMed Scopus (36) Google Scholar HKC cells (1 × 104) were seeded onto the filters (8-μm pore size, 0.33-cm2 growth area) in the top compartment of the chamber. After 2 or 5 days of incubation with or without TGF-β1 at 37°C, filters were fixed with 3% paraformaldehyde in PBS, and stained with 0.1% Coomassie Blue in 10% methanol and 10% actic acid, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. Cells that passed through the pores were counted in five nonoverlapping ×20 fields and photographed with a Nikon microscope.Matrigel Invasion AssayMatrigel (1.43 mg/cm2) was added onto the transwell filters (8-μm pore size, 0.33-cm2 growth area) of the Boyden chamber to form matrix gels at 1.0-mm depth. HKC cells (1 × 104) in a volume of 100 μl were added onto the top of the gels. After 2 and 5 days of incubation with or without TGF-β1 at 37°C, filters were fixed with 3% paraformaldehyde in PBS, and stained with 0.1% Coomassie Blue in 10% methanol and 10% actic acid, and the upper surface of the filters was carefully wiped with a cotton-tipped applicator. Cells that invaded and migrated across the Matrigel and passed the transwell filter pores toward the lower surface of the filters were counted in five nonoverlapping ×10 fields. The experiments were performed in triplicate cultures.Determination of Tissue TGF-β1 Levels by Enzyme-Linked Immunosorbent AssayFor measurement of tissue TGF-β1 level, kidneys were homogenized in the extraction buffer containing 20 mmol/L Tris-HCl, pH 7.5, 2 mol/L NaCl, 0.1% Tween-80, 1 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L phenylmethyl sulfonyl fluoride, and the supernatant was recovered after centrifugation at 19,000 × g for 20 minutes at 4°C. Kidney tissue TGF-β1 level was determined by using a commercial Quantikine TGF-β1 enzyme-linked immunosorbent assay kit in accordance with the protocol specified by the manufacturer (R & D Systems). This kit measures the abundance of active TGF-β1 protein that binds to its soluble type II receptor precoated onto a microplate. The concentration of tissue TGF-β1 in kidneys was expressed as pg/mg total protein.Statistical AnalysisAll data examined were expressed as mean ± SE. For Western blot analysis, quantitation was performed by scanning and analyzing the intensity of the hybridization signals using NIH Imagine software. Statistical analysis of the data were performed by the Student-Newman-Keuls test using SigmaStat software (Jandel Scientific, San Rafael CA). A P value 2 ng/ml.Figure 2 shows the phenotypic conversion of tubular epithelial cells after TGF-β1 treatment. The transformed cells displayed the presence of abundant α-SMA-positive microfilaments in the cytoplasm. Meanwhile, they totally lost the staining of E-cadherin, an epithelial marker that is essential for the structural integrity of renal epithelium (Figure 2). We observed dramatic alteration in the organization of actin cytoskeleton. The transformed cells underwent F-actin reorganization to form long stress fibers (Figure 2). Consistent with these actin reorganizations that often define cell morphology, the transformed cells lost the typical cobblestone pattern of an epithelial monolayer, and displayed a spindle-shape, fibroblast-like morphology (Figure 2). In addition, TGF-β1-treated cells expressed vimentin, a marker of mesenchymal cells, and began to markedly produce fibronectin and collagen I (data not shown). All together, these data suggest that tubular epithelial cells, under appropriate stimulus, undergo a conversion process into myofibroblasts in vitro.Figure 2Tubular epithelial to myofibroblast transition in vitro. HKC cells treated without (left column) or with 4 ng/ml of TGF-β1 (right column) for 72 hours in serum-free medium. The myofibroblast or epithelial cell markers were detected by an indirect immunostaining. The transformed cells acquired α-SMA (A, B), lost E-cadherin (C, D), formed stress fiber by actin reorganization (E, F), and displayed myofibroblast morphology (G, H). Scale bars: 10 μm (A–F), 20 μm (G and H).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Loss of E-Cadherin Expression Is an Early Event during Epithelial to Myofibroblast ConversionTo identify the early event essential for epithelial to myofibroblast conversion, we investigated the time course of the gene expression after TGF-β1 treatment. As shown in Figure 3, we found that loss of E-cadherin expression was an early event that took place as early as 6 hours after TGF-β1 treatment, whereas induction of de novo expression of α-SMA was a delayed response requiring 36 hours of incubation (Figure 3). Other changes in cell phenotype such as induced vimentin and fibronectin expression as well as morphological transformation also required longer periods of persistent incubation with TGF-β1 ranging from 2 to 5 days (data not shown). These results establish that loss of E-cadherin expression probably is an early event, which allows dissociation of structural integrity of renal epithelia and collapse of epithelial polarity.Figure 3Loss of E-cadherin is an early event during epithelial to myofibroblast transition. HKC cells were treated with TGF-β1 for various periods of time as indicated in serum-free medium. The whole cell lysate was immunoblotted with antibodies against α-SMA, E-cadherin, and β-actin, respectively. A: Time-course of E-cadherin expression. B: Time-dependency of α-SMA expression. C: Graphic presentation of the relative abundance of E-cadherin and α-SMA after TGF-β1-induced cell phenotypic transition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Up-Regulation of MMP-2 Expression and Its Role in Disrupting Tubular Basement MembraneTo gain insights into the mechanism underlying the destruction of TBM in vivo, we examined the expression pattern of MMPs during TGF-β1-induced EMT. Zymographic analysis of conditioned media exhibited that TGF-β1 induced a marked increase in MMP-2 expression and secretion in a dose-dependent manner (Figure 4A). TGF-β1 not only induced pro-MMP-2 abundance, but also stimulated activation of pro-MMP-2, as demonstrated by increased abundance of active MMP-2 in the conditioned media. Time-course studies revealed that this induction was also a delayed response that took place after 48 hours of incubation with TGF-β1 (Figure 4B). TGF-β1 also marginally increased MMP-9 activation, as demonstrated by an increase in active MMP-9 abundance in zymographic gels (Figure 4A). The induction of MMP-2 expression by TGF-β1 in tubular epithelial cells was independently confirmed by Western blot analyses of the conditioned media (Figure 4).Figure 4TGF-β1 induces MMP expression in tubular epithelial cells. A and B: Zymographic analysis of the conditioned media derived from HKC cells treated without (control) or with different amounts of TGF-β1 for various periods of time as indicated. Samples equalized for protein content were separated on a 10% SDS-polyacrylamide gel containing 0.1% gelatin. Proteolytic activity is demonstrated by digestion of gelatin, resulting in the bands of clearing. The locations of bands corresponding to pro- and active MMP-2 as well as pro- and active MMP-9 are indicated. C and D: Western blot analyses of the conditioned media from HKC cells treated without (control) or with various concentration of TGF-β1 for different periods of time in the serum-free medium. The location of a 72-kd band corresponding to MMP-2 is indicated. A and C: HKC cells were incubated with different concentrations of TGF-β1 as indicated for 72 hours. B and D: HKC cells were incubated with 2 ng/ml of TGF-β1 for various periods of time as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To directly demonstrate whether the increased expression of MMP-2 is responsible for disrupting the structural and functional integrity of TBM, we examined the ability of conditioned media derived from the transformed cells to degrade Matrigels that essentially reconstitute the native TBM.24Birkedal-Hansen H Proteolytic remodeling of extracellular matrix.Curr Opin Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (974) Google Scholar, 25Borden P Heller RA Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases.Crit Rev Eukaryot Gene Exp. 1997; 7: 159-178Crossref PubMed Scopus (290) Google Scholar, 26Martin J Steadman R Knowlden J Williams J Davies M Differential regulation of matrix metalloproteinase
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