Gelatinase A (MMP-2) Is Necessary and Sufficient for Renal Tubular Cell Epithelial-Mesenchymal Transformation
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64327-1
ISSN1525-2191
Autores Tópico(s)Hemostasis and retained surgical items
ResumoProgressive renal interstitial fibrosis and tubular atrophy represent the final injury pathway for all commonly encountered forms of renal disease that lead to end-stage renal failure. It has been recently recognized that myofibroblastic cells are the major contributors to the deposition of interstitial collagens. While there are several potential cellular sources of myofibroblasts, attention has focused on the transformation of the organized tubular epithelium to the myofibroblastic phenotype, a process potently driven both in vitro and in vivo by transforming growth factor-β1 (TGF-β1). Integrity of the underlying basal lamina provides cellular signals that maintain the epithelial phenotype, and disruption by discrete proteases could potentially initiate the transformation process. We demonstrate that TGF-β1 coordinately stimulates the synthesis of a specific matrix metalloproteinase, gelatinase A (MMP-2), and its activator protease, MT1-MMP (MMP-14), and that active gelatinase A is absolutely required for epithelial-mesenchymal transformation induced by TGF-β1. In addition, purified active gelatinase A alone is sufficient to induce epithelial-mesenchymal transformation in the absence of exogenous TGF-β1. Gelatinase A may also mediate epithelial-mesenchymal transformation in a paracrine manner through the proteolytic generation of active TGF-β1 peptide. MT1-MMP and gelatinase A were co-localized to sites of active epithelial-mesenchymal transformation and basal lamina disruption in the rat remnant kidney model of progressive renal fibrosis. These studies indicate that a discrete matrix metalloproteinase, gelatinase A, is capable of inducing the complex genetic rearrangements that characterize renal tubular epithelial-mesenchymal transformation. Progressive renal interstitial fibrosis and tubular atrophy represent the final injury pathway for all commonly encountered forms of renal disease that lead to end-stage renal failure. It has been recently recognized that myofibroblastic cells are the major contributors to the deposition of interstitial collagens. While there are several potential cellular sources of myofibroblasts, attention has focused on the transformation of the organized tubular epithelium to the myofibroblastic phenotype, a process potently driven both in vitro and in vivo by transforming growth factor-β1 (TGF-β1). Integrity of the underlying basal lamina provides cellular signals that maintain the epithelial phenotype, and disruption by discrete proteases could potentially initiate the transformation process. We demonstrate that TGF-β1 coordinately stimulates the synthesis of a specific matrix metalloproteinase, gelatinase A (MMP-2), and its activator protease, MT1-MMP (MMP-14), and that active gelatinase A is absolutely required for epithelial-mesenchymal transformation induced by TGF-β1. In addition, purified active gelatinase A alone is sufficient to induce epithelial-mesenchymal transformation in the absence of exogenous TGF-β1. Gelatinase A may also mediate epithelial-mesenchymal transformation in a paracrine manner through the proteolytic generation of active TGF-β1 peptide. MT1-MMP and gelatinase A were co-localized to sites of active epithelial-mesenchymal transformation and basal lamina disruption in the rat remnant kidney model of progressive renal fibrosis. These studies indicate that a discrete matrix metalloproteinase, gelatinase A, is capable of inducing the complex genetic rearrangements that characterize renal tubular epithelial-mesenchymal transformation. Progressive fibrosis and tubular atrophy are the key determinants of end-stage renal disease, regardless of the primary disease process. Additionally, it is well established that interstitial fibrosis has a greater impact on the progression of chronic renal disease than glomerulosclerosis.1Mackensen-Haen S Bohle A Christensen J Wehrmann M Kendziorra H Kokot F The consequences for renal function of widening of the interstitium and changes in the tubular epithelium of the renal cortex and outer medulla in various renal diseases.Clin Nephrol. 1992; 37: 70-77PubMed Google Scholar Tubular injury from primary glomerular disease ensues from interstitial microvascular injury, adaptive tubular hypermetabolism, or proteinuria. 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Myofibroblasts are mesenchymal cells that express α-smooth muscle actin and are thought to be the predominant source of types I and III collagen in fibrosis.17Weber KT Fibrosis, a common pathway to organ failure: angiotensin II and tissue repair.Semin Nephrol. 1997; 17: 467-491PubMed Google Scholar Increased myofibroblast expression in both human disease and animal models has been associated with matrix accumulation and progression of renal disease.18Goumenos DS Brown CB Shortland J el Nahas AM Myofibroblasts, predictors of progression of mesangial IgA nephropathy?.Nephrol Dial Transplant. 1994; 9: 1418-1425PubMed Google Scholar, 19Roberts 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 (125) Google Scholar, 20Alpers CE Hudkins KL Gown AM Johnson RJ Enhanced expression of “muscle-specific” actin in glomerulonephritis.Kidney Int. 1992; 41: 1134-1142Crossref PubMed Scopus (263) Google Scholar, 21Alpers CE Hudkins KL Floege J Johnson RJ Human renal cortical interstitial cells with some features of smooth muscle cells participate in tubulointerstitial and crescentic glomerular injury.J Am Soc Nephrol. 1994; 5: 201-209PubMed Google Scholar, 22MacPherson BR Leslie KO Lizaso KV Schwarz JE Contractile cells of the kidney in primary glomerular disorders: an immunohistochemical study using an anti-α-smooth muscle actin monoclonal antibody.Hum Pathol. 1993; 24: 710-716Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 23Diamond JR van Goor H Ding G Engelmyer E Myofibroblasts in experimental hydronephrosis.Am J Pathol. 1995; 146: 121-129PubMed Google Scholar, 24Essawy 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 (181) Google Scholar, 25Zhang G Moorhead P el Nahas A Myofibroblasts and the progression of experimental glomerulonephritis.Exp Nephrol. 1995; 3: 308-318PubMed Google Scholar, 26Hewitson TD Becker GJ Interstitial myofibroblasts in IgA glomerulonephritis.Am J Nephrol. 1995; 15: 111-117Crossref PubMed Scopus (102) Google Scholar, 27Hewitson TD Wu HL Becker GJ Interstitial myofibroblasts in experimental renal infection and scarring.Am J Nephrol. 1995; 15: 411-417Crossref PubMed Scopus (71) Google Scholar, 28Johnson RJ Alpers CE Yoshimura A Lombardi D Pritzl P Floege J Schwartz SM Renal injury from angiotensin II-mediated hypertension.Hypertension. 1992; 19: 464-474Crossref PubMed Scopus (534) Google Scholar Despite their importance, the cellular source(s) of renal interstitial myofibroblasts has not been entirely elucidated. While renal myofibroblasts may derive from the intrinsic fibroblastic population or vascular pericytic cells, considerable attention has been devoted recently to the process of tubular epithelial cell (TEC) transformation. In essence, TEC transformation represents a reversal of the mesenchymal-epithelial cell differentiation process characteristic of nephrogenesis. In a study of 5/6 nephrectomized rats, proximal tubule cells were shown to undergo stepwise transformation into α-smooth muscle actin-positive myofibroblasts.29Ng 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 (351) Google Scholar Tubular cell expression of α-smooth muscle actin was associated with basement membrane disruption and eventual loss of epithelial morphology with migration into the stroma. Myofibroblasts appeared in areas of fibrosis and adjacent to α-smooth muscle actin-positive tubular cells. Recently, TGF-β1 was shown to directly induce tubular epithelial-myofibroblast transformation in the NRK-52e normal rat kidney epithelioid cell line in vitro.30Fan JM Ng YY Hill PA Nikolic-Paterson DJ Mu W Atkins RC Lan HY Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro.Kidney Int. 1999; 56: 1455-1467Crossref PubMed Scopus (483) Google Scholar Yang and Liu31Yang J Liu Y Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar subsequently demonstrated that TGF-β1-mediated transformation of cultured tubular epithelial cells was temporally associated with a specifically enhanced expression of gelatinase A. Transgenic mice expressing TGF-β1 develop progressive renal fibrosis with the characteristic features of epithelial-mesenchymal transformation, an event also associated with enhanced synthesis of gelatinase A.32Kopp JB Factor VM Mozes M Nagy P Sanderson N Bottinger EP Klotman PE Thorgeirsson SS Transgenic mice with increased plasma levels of TGF-β 1 develop progressive renal disease.Lab Invest. 1996; 74: 991-1003PubMed Google Scholar Other studies have shown that TGF-β1 stimulates gelatinase A synthesis by cultured fibroblasts and glomerular mesangial cells at both the transcriptional and post-transcriptional levels.33Overall CM Wrana JL Sodek J Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-β 1 in human fibroblasts: comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression.J Biol Chem. 1991; 266: 14064-14071Abstract Full Text PDF PubMed Google Scholar, 34Marti HP Lee L Kashgarian M Lovett DH Transforming growth factor-β 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase.Am J Pathol. 1994; 144: 82-94PubMed Google Scholar Integrity of the underlying basal lamina is required for the maintenance of a polarized epithelial phenotype, and disruption of type IV collagen lattice assembly by addition of a dominant negative α1NC1 domain results in epithelial-mesenchymal transformation.35Zeisberg M Bonner G Maeshima Y Colorado P Muller GA Strutz F Kalluri R Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation.Am J Pathol. 2001; 159: 1313-1321Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar Song et al36Song W Jackson K McGuire PG Degradation of type IV collagen by matrix metalloproteinases is an important step in the epithelial-mesenchymal transformation of the endocardial cushions.Dev Biol. 2000; 227: 606-617Crossref PubMed Scopus (81) Google Scholar showed that the epithelial-mesenchymal transformation of endocardial cushions was dependent on gelatinase A activity for penetration and disruption of the underlying type IV collagen-rich basal lamina. Taken together, these observations suggest that the degradation and disruption of the underlying basal lamina by specific matrix metalloproteinases, such as gelatinase A, is a critical component of the epithelial-mesenchymal transformation process. In this report we demonstrate that gelatinase A, in association with the membrane-bound MT1-MMP (MMP-14), is absolutely required for the epithelial-mesenchymal transformation of NRK-52e cells induced by TGF-β1 in vitro. In addition, purified active gelatinase A alone is sufficient to induce epithelial-mesenchymal transformation in these cells without the addition of TGF-β1. Finally, in a model of renal epithelial-mesenchymal transformation and fibrosis, we demonstrate the co-localization of gelatinase A and MT1-MMP by the tubular epithelium at sites of ongoing myofibroblast formation and basal lamina disruption. Taken together, these observations indicate that a single matrix metalloproteinase, gelatinase A, is necessary and sufficient for the induction of the complex genetic rearrangements that characterize epithelial-mesenchymal transformation, a finding of considerable therapeutic potential. The NRK-52e normal rat tubular epithelioid cell line was obtained from the American Type Culture Collection andmaintained in DME-H21 (Gibco, Rockville, MD) supplemented with 10% fetal calf serum (FCS; Gibco), 100 units/ml penicillin and 100 μg/ml streptomycin. Subconfluent cultures of NRK-52e cells were washed twice in warm phosphate-buffered saline (PBS) and given fresh DME-H 21 medium supplemented with 0.1% bovine serum albumin (BSA) and the indicated concentrations of TGF-β1 (R&D Systems, Minneapolis, MN). For fluorescence-activated cell sorter (FACS) analysis, fresh serum-free medium containing TGF-β1 was replaced after 3 days. Treated cells were analyzed by immunohistochemistry, FACS analysis, quantitative gelatinase zymography, and transfection with gelatinase A and MT1-MMP luciferase reporter constructs as detailed below. For direct treatment with matrix metalloproteinases, NRK-52e cells were grown to subconfluency, washed with PBS and given fresh serum-free medium containing the denoted concentrations of active and latent gelatinase A or active gelatinase B. Latent gelatinase A was purified to homogeneity from the serum-free conditioned medium of cultured rat glomerular mesangial cells according to the protocol of Okada et al37Okada Y Morodomi T Enghild JJ Suzuki K Yasui A Nakanishi I Salvesen G Nagase H Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts: purification and activation of the precursor and enzymic properties.Eur J Biochem. 1990; 194: 721-730Crossref PubMed Scopus (401) Google Scholar Latent gelatinase B was purified to homogeneity by chromatography of conditioned medium over gelatin-Sepharose and Lens culinaris lectin-agarose as reported in detail.38McMillan JI Riordan JW Couser WG Pollock AS Lovett DH Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy.J Clin Invest. 1996; 97: 1094-1101Crossref PubMed Scopus (144) Google Scholar Latent gelatinases were activated by incubation with 0.5 mmol/L p-aminophenylmercuric acetate (confirmed by zymography), dialyzed against PBS, and used at the indicated concentrations. These studies used a cyclic peptide gelatinase A inhibitor, CTTHWGFTLCGG, isolated by phage display, and a control non-inhibitory peptide, CRAVRALWRCGG.39Koivunen E Arap W Valtanen H Rainisalo A Medina OP Heikkila P Kantor C Gahmberg CG Salo T Konttinen YT Sorsa T Ruoslahti E Pasqualini R Tumor targeting with a selective gelatinase inhibitor.Nature Biotechnol. 1999; 17: 768-774Crossref PubMed Scopus (518) Google Scholar Biotin was added during the synthesis to the terminal G residues to permit immunolocalization (see below). To block TGF-β1-mediated transformation, cells were treated as detailed above in the presence or absence of the indicated concentrations of the inhibitory or control cyclic peptides. For identification of myofibroblasts, cells cultured on etched glass coverslips were fixed for 20 minutes at 4°C with 4% buffered paraformaldehyde and permeabilized in acetone. The slips were blocked with 5% normal goat serum for 30 minutes, rinsed, and blocked with an avidin/biotin kit (Vector, Burlingame, MA). Rinsed coverslips were incubated with primary monoclonal mouse α-smooth muscle actin antibody (Sigma, St. Louis, MO; 6.5 mg/ml) at 1:50 in 0.1% BSA/PBS for 2 hours at room temperature, followed by biotinylated goat anti-mouse IgG (Zymed, San Francisco, CA; 0.4 mg/ml) at 1:20 in 0.1% BSA/PBS for 2 hours at room temperature. Rinsed slips were incubated with either streptavidin-rhodamine or streptavidin-fluorescein (0.5 μg/ml, Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:100 in 0.1% BSA/PBS for 30 minutes. For co-localization of α-SMA and active gelatinase A, cells were fixed in 2% buffered paraformaldehyde for 20 minutes, blocked with avidin/biotin and incubated with the biotinylated cyclic peptides (inhibitory or control) at 5 μg/ml for 1 hour at 4°C, followed by a 1:200 dilution of streptavidin-rhodamine for 30 minutes. Rinsed cells were re-fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 90 seconds, and blocked with 5% normal donkey serum for 30 minutes (Vector). This was followed by monoclonal anti-α-SMA antibody at 1:50 for 2 hours at room temperature and fluorescein-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories) at 1:100 for 2 hours at room temperature. To co-localize active gelatinase A and MT1-MMP, the same protocol as above was followed until after the second fixation. Cells were blocked with 5% normal goat serum, followed by avidin/biotin blockade. Murine monoclonal α-MT1-MMP antibody (Oncogene Research Products, San Diego, CA) was used at 1:50 dilution for 2 hours at room temperature, followed by biotinylated goat anti-mouse IgG (Zymed, 0.4 mg/ml) at 1:200 dilution for 2 hours, and finally with streptavidin-fluorescein (Vector) at 1:100 for 30 minutes at room temperature. For co-localization of MT1-MMP and E-cadherin, cells were fixed with 4% paraformaldehyde, blocked with PBS/CMF with 5% BSA followed by avidin/biotin. Murine monoclonal α-MT1-MMP IgG3 and murine α-E-cadherin IgG2a (Transduction Laboratories, Lexington, KY) were used at 1:50 (2 μg/ml) and 1:50 (2.5 μg/ml), respectively, for 3 hours. The cells were then incubated with biotinylated rat α-IgG3 (CalTag, Burlingame, CA) and FITC-conjugated rat α-IgG2a (Caltag) at 1:200 (2 μg/ml) and 1:50 (8 μg/ml), respectively, followed by streptavidin-rhodamine at 1:200 (2 μg/ml). For control experiments, murine monoclonal α-MT1-MMP IgG3 was incubated with FITC-conjugated rat α-IgG2a, and murine α-E-cadherin IgG2a with biotinylated rat α-IgG3 and streptavidin-rhodamine. NRK-52e cells were incubated with TGF-β1 (2 ng/ml) along with 0, 100, or 500 nmol/L ionomycin (Sigma) for 48 hours in serum-free medium. Cells were then prepared and stained for α-SMA as described above. Eighty percent confluent cultures of NRK-52e cells were incubated with the indicated concentrations of active gelatinase A for 3 days in Optimem (Invitrogen, Carlsbad, CA). The conditioned medium was harvested and measured for active TGF-β1 by ELISA (R&D Systems). Data are expressed as means of quadruplicate determinations ± 1 SD of pg active TGF-β1/100 μg cell layer protein. Male Munich Wistar rats (275 to 350 × g) were subjected to 5/6 renal ablation (n = 6) or a sham operation (n = 6) consisting of laparotomy and manipulation of the renal pedicle as reported.40Lee LK Meyer TW Pollock AS Lovett DH Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney.J Clin Invest. 1995; 96: 953-964Crossref PubMed Scopus (188) Google Scholar Kidneys were harvested for analysis at 10 weeks when segmental sclerosis and tubulointerstitial fibrosis are present. At the time of harvest, animals were anesthetized with pentobarbital, the kidneys perfusion-fixed with 4% paraformaldehyde in PBS, and embedded in paraffin. Deparaffinized 5-micron sections were hydrated, endogenous peroxidase blocked by incubation for 30 minutes with 0.1% H2O2, followed by incubation with 5% normal goat serum and avidin/biotin blocking solution. Monoclonal anti-α-smooth muscle actin antibody (1:400), anti-gelatinase A antibody (1:500, Oncogene Research), or anti-MT1-MMP (1:500, Oncogene Research) were applied for 60 minutes at room temperature, followed by the Vectastain Elite ABC kit (Vector) according to the manufacturer's instructions. Development with DAB/NiCl2 and counterstaining with methyl green were performed using standard methodology. Cells in the respective treatment groups were released with trypsin, fixed with 4% paraformaldehyde for 20 minutes, and permeabilized with 0.2% saponin for 10 minutes at 4°C. Cells were then stained with monoclonal anti-α-SMA antibody and fluorescein-conjugated donkey anti-mouse IgG as detailed above and analyzed by FACS (Becton Dickinson, San Jose, CA) with histograms of 10,000 counts, using excitation of 488 nm and emission of 530 nm. Crude (gelatinase A) and Triton-X114-extracted (MT1-MMP) microsomes from control and TGF-β1-treated cells were prepared as reported in detail.41Turck J Pollock AS Lee LK Marti HP Lovett DH Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation.J Biol Chem. 1996; 271: 15074-15083Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Equal concentrations of microsomal protein (15 μg/lane) were loaded on 7.5% SDS-polyacrylamide gels containing 2 mg/ml gelatin and separated by electrophoresis. Processing of gels was as reported.34Marti HP Lee L Kashgarian M Lovett DH Transforming growth factor-β 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase.Am J Pathol. 1994; 144: 82-94PubMed Google Scholar Experiments were performed in triplicate and repeated at least three times. The major enzymatic activities at 66 (gelatinase A) and 62 (MT1-MMP) kd were quantified by laser densitometry and standardized using serial dilution of known quantities of purified gelatinase A or MT1-MMP. Subconfluent cultures of NRK-52e cells were washed and transfected with FuGene (Roche, Indianapolis, IN) using 1 μg of plasmid DNA from the control pGL2-Basic luciferase reporter plasmid (Promega, Madison, WI), plasmid pT4-Luc1686 (composed of the first 1686 bp of the rat gelatinase A 5′ flanking region cloned into pGL2-Basic), or plasmid pMT1-Luc3280 (composed of the first 3280 bp of the murine MT1-MMP 5′ flanking region). Sixteen to 24 hours after transfection, fresh medium was added in the presence or absence of 2 ng/ml TGF-β1 and the incubation continued for a further 24 hours. Cells were washed, extracted with 400 μl Triton lysis buffer (1% Triton X-100, 1 mmol/L dithiothreitol, 25 mmol/L glycylglycine at pH 7.8, 15 mmol/L MgSO4), and followed by measurement of luciferase and galactosidase activities as reported.42Mertens PR Harendza S Pollock AS Lovett DH Glomerular mesangial cell-specific transactivation of matrix metalloproteinase 2 transcription is mediated by YB-1.J Biol Chem. 1997; 272: 22905-22912Crossref PubMed Scopus (141) Google Scholar All transfections were performed in quadruplicate and repeated at least three times. Transfection results are graphed and expressed as means ± 1 SD. Statistical significance was determined for paired comparisons using Student's t-test or by analysis of variance for multiple comparisons where appropriate. These studies used the well-characterized rat renal tubular epithelial cell line, NRK-52e, which is generally considered to be of proximal tubular origin.43Suzuki E Hirata Y Hayakawa H Omata M Kojima M Kangawa K Minamino N Matsuo H Evidence for C-type natriuretic peptide production in the rat kidney.Biochem Biophys Res Commun. 1993; 192: 532-538Crossref PubMed Scopus (48) Google Scholar, 44Walker C Everitt J Freed JJ Knudson Jr, AG Whiteley LO Altered expression of transforming growth factor-α in hereditary rat renal cell carcinoma.Cancer Res. 1991; 51: 2973-2978PubMed Google Scholar Prior reports by Fan et al30Fan JM Ng YY Hill PA Nikolic-Paterson DJ Mu W Atkins RC Lan HY Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro.Kidney Int. 1999; 56: 1455-1467Crossref PubMed Scopus (483) Google Scholar demonstrated that TGF-β1 induces transdifferentiation of NRK-52e epithelial cells to a mesenchymal phenotype characterized by α-smooth muscle actin (α-SMA) expression and migration. The effects of TGF-β1 are time and concentration dependent. As summarized in Figure 1, treatment of NRK-52e cells with 2 ng/ml TGF-β1 for 3 days induced α-SMA expression and a migratory phenotype (Figure 1, A and B). Notably, α-SMA expression is limited to the subset of cells at the periphery of the epithelial clusters of NRK-52e cells. As detailed in Figure 1, C and D, the subset of α-SMA+ cells also concurrently expressed MT1-MMP protein as determined by immunohistochemistry. Using a biotinylated cyclic peptide, CTTHWGFTLCGG, as a probe for activated gelatinase A, dual immunohistochemical techniques demonstrated the co-localization of the MT1-MMP and active gelatinase A proteins on the cells at the migratory front of the TGF-β1-treated cells (Figure 1, E and F). As shown in Figure 1, G and H, the active gelatinase A protein is clustered at specific sites on the leading and trailing edges of the transdifferentiated cells, consistent with a direct role in cellul
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