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From the Department of Medicine, San Francisco Veterans Affairs Medical Center, University of California, San Francisco; and the Northern California Institute for Research and Education, San Francisco, California
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Abstract |
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These tubular insults create a hypoxic environment that further contributes to matrix production by tubules and fibroblasts.6-8 In addition to stimulating collagen synthesis, hypoxia also stimulates expression of growth factors and cytokines including transforming growth factor-ß1 (TGF-ß1), vascular endothelial growth factor, platelet-derived growth factor, endothelin-1, and angiotensin II, of which TGF-ß1 is the most studied.3,6,9 In cultured renal fibroblasts, TGF-ß1 stimulates production of fibronectin and types I, III, and V collagens, while tubular epithelial cells are stimulated to produce proteoglycans and type I, III, IV, and V collagen.10,11 Numerous studies have demonstrated that renal fibrosis in vivo is associated with elevated TGF-ß1 expression.12 In the Thy1.1 rat model of glomerulonephritis, renal fibrosis was abrogated by administration of anti-TGF-ß1 antibody, antisense oligonucleotides, and decorin, a proteoglycan associated with the interstitial matrix and shown to bind TGF-ß1.13-16
It has become increasing evident that myofibroblasts play a central role in the development of renal interstitial fibrosis. Myofibroblasts are mesenchymal cells that express 
-smooth muscle actin and are thought to be the predominant source of types I and III collagen in fibrosis.17
Increased myofibroblast expression in both human disease and animal models has been associated with matrix accumulation and progression of renal disease.18-28
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 -smooth muscle actin-positive myofibroblasts.29
Tubular cell expression of -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 -smooth muscle actin-positive tubular cells.
Recently, TGF-ß1 was shown to directly induce tubular epithelial-myofibroblast transformation in the NRK-52e normal rat kidney epithelioid cell line in vitro.30 Yang and Liu31 subsequently demonstrated that TGF-ß1-mediated transformation of cultured tubular epithelial cells was temporally associated with a specifically enhanced expression of gelatinase A. Transgenic mice expressing TGF-ß1 develop progressive renal fibrosis with the characteristic features of epithelial-mesenchymal transformation, an event also associated with enhanced synthesis of gelatinase A.32 Other studies have shown that TGF-ß1 stimulates gelatinase A synthesis by cultured fibroblasts and glomerular mesangial cells at both the transcriptional and post-transcriptional levels.33,34
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 1NC1 domain results in epithelial-mesenchymal transformation.35
Song et al36
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-ß1 in vitro. In addition, purified active gelatinase A alone is sufficient to induce epithelial-mesenchymal transformation in these cells without the addition of TGF-ß1. 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.
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Materials and Methods |
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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.
Stimulation Studies
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-ß1 (R&D Systems, Minneapolis, MN). For fluorescence-activated cell sorter (FACS) analysis, fresh serum-free medium containing TGF-ß1 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 al37 Latent gelatinase B was purified to homogeneity by chromatography of conditioned medium over gelatin-Sepharose and Lens culinaris lectin-agarose as reported in detail.38 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.
Inhibition Studies
These studies used a cyclic peptide gelatinase A inhibitor, CTTHWGFTLCGG, isolated by phage display, and a control non-inhibitory peptide, CRAVRALWRCGG.39 Biotin was added during the synthesis to the terminal G residues to permit immunolocalization (see below). To block TGF-ß1-mediated transformation, cells were treated as detailed above in the presence or absence of the indicated concentrations of the inhibitory or control cyclic peptides.
Immunohistochemistry
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 -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 -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--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 -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 -MT1-MMP IgG3 and murine -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 -IgG3 (CalTag, Burlingame, CA) and FITC-conjugated rat -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 -MT1-MMP IgG3 was incubated with FITC-conjugated rat -IgG2a, and murine -E-cadherin IgG2a with biotinylated rat -IgG3 and streptavidin-rhodamine.
Ionomycin Studies
NRK-52e cells were incubated with TGF-ß1 (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 -SMA as described above.
Activation of Latent TGF-ß1 by Gelatinase A
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-ß1 by ELISA (R&D Systems). Data are expressed as means of quadruplicate determinations ± 1 SD of pg active TGF-ß1/100 µg cell layer protein.
Immunohistochemistry of Remnant Kidney
Male Munich Wistar rats (275 to 350 x 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.40
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--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.
FACS Analysis
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--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.
Quantitative Gelatin Zymography
Crude (gelatinase A) and Triton-X114-extracted (MT1-MMP) microsomes from control and TGF-ß1-treated cells were prepared as reported in detail.41 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.34 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.
Transient Transfection with Gelatinase A and MT1-MMP Luciferase Reporter Constructs
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-ß1 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.42 All transfections were performed in quadruplicate and repeated at least three times. Transfection results are graphed and expressed as means ± 1 SD.
Statistical Analyses
Statistical significance was determined for paired comparisons using Student’s t-test or by analysis of variance for multiple comparisons where appropriate.
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Results |
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-smooth muscle actin (-SMA) expression and migration. The effects of TGF-ß1 are time and concentration dependent. As summarized in Figure 1
, treatment of NRK-52e cells with 2 ng/ml TGF-ß1 for 3 days induced -SMA expression and a migratory phenotype (Figure 1, A and B)
. Notably, -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 -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-ß1-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 cellular migration.
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, cells with intact E-cadherin complexes did not express MT1-MMP, while cells with disrupted complexes did.
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. The increases in gelatinase A and MT1-MMP protein synthesis are, at least in part, due to increased rates of transcription following treatment with TGF-ß1. To demonstrate this, NRK-52e cells were transiently transfected with luciferase reporter constructs containing 1686 or 3280 bp of the 5' flanking regions of the gelatinase A or MT1-MMP promoters, respectively (Figure 3B)
. Gelatinase A transcription rates under these conditions were stimulated over twofold by TGF-ß1, while MT1-MMP transcription rates were increased by nearly fivefold.
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-SMA. The results of these studies are summarized in Figure 4
. Concentrations of active gelatinase A as low as 10 nmol/L were sufficient to induce NRK-52e transformation and affected virtually 100% of the cells when a concentration of 50 nmol/L was used (Figure 4, B–D)
. In contrast, 50 nmol/L of latent gelatinase A had no effect on NRK-52e transformation (Figure 4E)
, consistent with a requirement for enzymatic activity. Zymographic analysis of NRK-52e culture supernates indicated that the only other MMP family member synthesized by this cell type is gelatinase B or MMP-9 (not shown). Addition of 50 nmol/L activated purified gelatinase B had no effect on NRK-52e transdifferentiation (Figure 4F)
, indicating a specific requirement for a gelatinase A-specific enzymatic activity for this process.
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-SMA. The results of these experiments are summarized in Figure 5
. Addition of increasing concentrations of the gelatinase A cyclic peptide inhibitor significantly decreased cellular staining for -SMA beginning at concentrations of 25 µmol/L and greater, while the control cyclic peptide at 100 µmol/L concentration had no effect on TGF-ß1-mediated -SMA expression. The concentrations of the cyclic peptide gelatinase A inhibitor required for blockade of -SMA expression are in the same range required for inhibition of enzymatic activity (IC50 
10 µmol/L) as determined in Koivunen et al39
It should be noted in this context that the cyclic peptide gelatinase A inhibitor has no effect on the activity of MT1-MMP.39
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are consistent with a TGF-ß1-mediated coordinated synthesis of MT1-MMP and gelatinase A, membrane assembly of the MT1-MMP/gelatinase A complex, and induction of transformation through the localized proteolytic action of gelatinase A. This suggests that the presentation of active MT1-MMP on the cell surface is a critical regulatory step in the assembly of the complete proteolytic complex. Yu et al45
and Lehti et al46
demonstrated that calcium influx mediated by ionomycin blocked the conversion of pro-MT1-MMP to the active 60 kd form. To evaluate the importance of assembly of the MT1-MMP/gelatinase A complex for induction of transformation, NRK-52e cells were incubated with TGF-ß1 (2 ng/ml) along with ionomycin (0, 100, and 500 nmol/L) for 48 hours, followed by assessment of -SMA expression. The results of these experiments are summarized in Figure 6
and demonstrate that inclusion of ionomycin effectively blocks the development of TGF-ß1-mediated transformation, as assessed by -SMA expression. These experiments demonstrate that TGF-ß1-mediated transformation is dependent on the coordinated presentation of active MT1-MMP on the cell surface, thereby permitting assembly of the MT1-MMP/gelatinase A proteolytic complex.
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-SMA to assess in a quantitative manner the transformation status of the NRK-52e cells. As summarized in Figure 7
, treatment of NRK-52e cells with 50 nmol/L active gelatinase A results in a significant increase in measured -SMA expression, while latent gelatinase A had no effect (Figure 7A)
. This effect of gelatinase A on -SMA expression is concentration-dependent (Figure 7B)
, and could not be reproduced by incubation with 50 nmol/L activated gelatinase B (not shown). Finally, inclusion of 100 µmol/L of the specific gelatinase A cyclic peptide inhibitor completely blocked TGF-ß1-mediated -SMA expression (Figure 7C)
.
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. Thus, gelatinase A may further facilitate the development of the transformation process by the paracrine generation of additional biologically active TGF-ß1 peptide.
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-SMA, gelatinase A, and MT1-MMP was performed using specific monoclonal antibodies. Representative results (n = 6 for each group) are shown in Figures 9–11
. At 10 weeks following sham surgery, there was minimal observable staining for -SMA in renal cortical sections (Figure 9A)
, as well as gelatinase A and MT1-MMP (Figure 9, B and C)
. In contrast, renal cortical sections from rats subjected to 5/6 nephrectomy for 10 weeks revealed dramatic changes in structure, with extensive glomerulosclerosis, interstitial fibrosis, tubular dropout, and dilation of remnant tubular structures. Intense staining for -SMA was evident within the glomeruli and sites of interstitial fibrosis, and was particularly prominent in the tubular epithelial cells lining the dilated tubules (Figure 10A)
. Intense staining in tubular epithelia of dilated tubules was also observed for both MT1-MMP and gelatinase A proteins (Figure 10, B and C)
. Staining for all three proteins was particularly intense at foci of myofibroblastic proliferation and migration through disrupted tubular basement membranes (Figure 11, A–C
, respectively). The results of the histochemical studies were confirmed by Western blots of renal cortical extracts, in which levels of SMA-actin, gelatinase A, and MT1-MMP proteins were greatly increased at 10 weeks as compared to controls (not shown). In conjunction with our in vitro data, the 5/6 nephrectomy studies provide compelling evidence that during the acquisition of the -SMA+ phenotype, tubular epithelial cells co-express MT1-MMP and gelatinase A, thereby driving the transition to the proliferative and migratory myofibroblast.
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Discussion |
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4000) manifested significant changes in expression levels.58
Pathway mapping and functional module assessment demonstrated major changes in the expression levels of genes in signaling cascades, cell-cell and cell-matrix interactions, motility, and mesenchymal development.58
The remarkable complexity of the genetic consequences of TGF-ß1-mediated epithelial-mesenchymal transformation underscores the significance of this study, in which a single, TGF-ß1-dependent gene product, gelatinase A, was found to be both necessary and sufficient for the epithelial-mesenchymal transformation of renal tubular epithelial cells. Enzymatically active gelatinase A was required for the induction of tubular epithelial cell transformation and this effect could not be recapitulated using the closely related gelatinase B (MMP-9). While both enzymes are similar in terms of protein structure, they manifest discrete patterns of substrate specificity. Gelatinase B has a higher degree of activity for types IV and V collagens, while gelatinase A can additionally degrade fibronectin and laminins.59,60 MT1-MMP, first identified as the cell-surface associated activator of gelatinase A, also exhibits catalytic activity against several extracellular matrix proteins, including native types I and III collagens, laminin, and fibronectin.61-66 Notably, recombinant MT1-MMMP has no activity against type IV collagen.67 While all three enzymes cleave the canonical Pro-X-X-/-XHy peptide sequence characteristic of collagenous substrates, Smith and colleagues68-70 have recently demonstrated that gelatinase A, gelatinase B, and MT1-MMP have additional defined substrate recognition motifs unique to each enzyme. These observations suggest that the absolute requirement for gelatinase A in renal tubular epithelial-mesenchymal transformation, as opposed to gelatinase B or MT1-MMP (note the cyclic peptide inhibitor used in this study does not inhibit MT1-MMP), presumably reflects enzyme-specific cleavage of heretofore undefined target substrates. Gelatinase A may also amplify the transformation process in a paracrine manner through the proteolytic generation of active TGF-ß1 peptide.
Treatment of defined extracellular matrix substrates with purified or recombinant matrix metalloproteinases has demonstrated the release of cryptic, biologically active cleavage products. For example, Xu et al71
exposed a cryptic site with type IV collagen by digestion with gelatinase A. The cryptic fragment induced mobility of cultured endothelial cells and co-localized with active gelatinase A at sites of angiogenesis. Giannelli et al60
demonstrated that gelatinase A cleavage of laminin-5, a major component of the tubular basement membrane, releases a cryptic domain from the 
2 subunit that promotes breast epithelial cell migration. Recently, Gilles et al49
found that MT1-MMP was also capable of releasing the 2 subunit fragment. In each of these studies, the 2 subunit stimulated cellular migration without evidence for a complete epithelial-mesenchymal transformation. Thus, one potential mechanism of action for gelatinase A-mediated epithelial-mesenchymal transformation could be the generation of specific biologically active extracellular matrix cleavage products.
Enzymatic disruption of cell-cell or cell-matrix attachments has been suggested as an alternative mechanism for induction of epithelial-mesenchymal transformation. Induction of stromelysin-1 expression in the murine mammary epithelial cell line SCp2 triggers a mesenchymal conversion associated with loss of E-cadherin and disruption of cell-cell contacts.72 In contrast, integrin alterations were relatively insignificant, suggesting that within the cellular context of this system that disruption of cell-matrix interactions is not a major driving force in the transformation process. Ho et al73 used matrix metalloproteinase inhibition to investigate the relative roles of cell-cell and cell-matrix interaction. MMP inhibition resulted in stabilization of cell-cell contracts with redistribution of p125FAK to sites of contact. In addition, MMP inhibition resulted in increased cadherin levels and augmented calcium-dependent cellular aggregation. Based on these findings, it was proposed that MMP inhibitors act to stabilize both cell-cell and cell-matrix interactions.
TGF-ß1 induced the synthesis of both gelatinase A and MT1-MMP, and transcriptional reporter studies indicate that at least one component of the enhanced synthesis of these enzymes is the consequence of transcriptional activation. The proximal promoters of both these genes include functional overlapping Sp1/Sp3 (gelatinase A)74 and Sp1/Egr-1 (MT1-MMP)75 sites and the increases in transcriptional activity of these promoters by TGF-ß1 may be the consequence of the recently reported interaction of Smad3/Smad4 proteins with Sp-1.76-78 This interaction, as a coordinated mechanism for TGF-ß1 transcriptional regulation of these two genes, is currently under investigation.
Notably, only a subset of the NRK52e cells exposed to TGF-ß1 manifested the changes characteristic of the epithelial-mesenchymal transformation. These cells were located on the periphery of the typical epithelial growth islands of the NRK52e cells at sites of active proliferation and had diminished staining for E-cadherin and loss of cell-cell contacts, as opposed to the cells within the middle of the growth islands. E-cadherin has been recently shown to suppress MT1-MMP transcription by suppression of MAP kinase (ERK) activity, suggesting that stabilization of cadherin-dependent cell-cell interaction prevents MT1-MMP expression, gelatinase A activation, and initiation of the epithelial-mesenchymal transformation.79
Given the fact that the epithelial-mesenchymal transformation of tubular epithelial cells displays an absolute requirement for enzymatically active gelatinase A and the recent demonstration that there are uniquely specific cleavage consensus motifs for this enzyme,70 we are examining defined subsets of cell-associated and extracellular matrix proteins for the existence of such motifs to assemble a list of candidate target proteins. These efforts may be expected to provide further insight into the precise means by which gelatinase A triggers the epithelial-mesenchymal transformation. Nonetheless, the determination that the blockade of a single matrix metalloproteinase is sufficient to halt the renal tubular epithelial cell transformation program has major clinical implications. The crystallization of gelatinase A and the determination of enzyme-specific substrates will facilitate the development of specific gelatinase A inhibitors suitable for testing in vitro and in animal models of renal transdifferentiation.70,80 Gelatinase A-specific inhibition may potentially offer a novel approach for the treatment of progressive renal fibrosis.
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Acknowledgements |
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Footnotes |
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Supported by NIH grants DK 39776 to D.H.L. and K08 DK59383–01 to S.C.
Accepted for publication March 5, 2003.
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 T. D. Hewitson, I. Mookerjee, R. Masterson, C. Zhao, G. W. Tregear, G. J. Becker, and C. S. Samuel Endogenous Relaxin Is a Naturally Occurring Modulator of Experimental Renal Tubulointerstitial Fibrosis Endocrinology, February 1, 2007; 148(2): 660 - 669. [Abstract] [Full Text] [PDF]
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E. de Laplanche, K. Gouget, G. Cleris, F. Dragounoff, J. Demont, A. Morales, L. Bezin, C. Godinot, G. Perriere, D. Mouchiroud, et al. Physiological oxygenation status is required for fully differentiated phenotype in kidney cortex proximal tubules Am J Physiol Renal Physiol, October 1, 2006; 291(4): F750 - F760. [Abstract] [Full Text] [PDF]
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S. Cheng, A. S. Pollock, R. Mahimkar, J. L. Olson, and D. H. Lovett Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury FASEB J, September 1, 2006; 20(11): 1898 - 1900. [Abstract] [Full Text] [PDF]
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A. Khandoga, J. S. Kessler, M. Hanschen, A. G. Khandoga, D. Burggraf, C. Reichel, G. F. Hamann, G. Enders, and F. Krombach Matrix metalloproteinase-9 promotes neutrophil and T cell recruitment and migration in the postischemic liver J. Leukoc. Biol., June 1, 2006; 79(6): 1295 - 1305. [Abstract] [Full Text] [PDF]
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R. Nasrallah and R. L. Hebert Prostacyclin signaling in the kidney: implications for health and disease Am J Physiol Renal Physiol, August 1, 2005; 289(2): F235 - F246. [Abstract] [Full Text] [PDF]
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 J. Yamate, Y. Machida, M. Ide, M. Kuwamura, T. Kotani, O. Sawamoto, and J. LaMarre Cisplatin-Induced Renal Interstitial Fibrosis in Neonatal Rats, Developing as Solitary Nephron Unit Lesions Toxicol Pathol, February 1, 2005; 33(2): 207 - 217. [Abstract] [Full Text] [PDF]
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P. J. Margetts, P. Bonniaud, L. Liu, C. M. Hoff, C. J. Holmes, J. A. West-Mays, and M. M. Kelly Transient Overexpression of TGF-{beta}1 Induces Epithelial Mesenchymal Transition in the Rodent Peritoneum J. Am. Soc. Nephrol., February 1, 2005; 16(2): 425 - 436. [Abstract] [Full Text] [PDF]
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 A. Masszi, L. Fan, L. Rosivall, C. A. McCulloch, O. D. Rotstein, I. Mucsi, and A. Kapus 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., December 1, 2004; 165(6): 1955 - 1967. [Abstract] [Full Text] [PDF]
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 P. Stawowy, C. Margeta, H. Kallisch, N. G Seidah, M. Chretien, E. Fleck, and K. Graf Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-{beta}1 involves furin-convertase Cardiovasc Res, July 1, 2004; 63(1): 87 - 97. [Abstract] [Full Text] [PDF]
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C. Maric, K. Sandberg, and C. Hinojosa-Laborde Glomerulosclerosis and Tubulointerstitial Fibrosis are Attenuated with 17{beta}-Estradiol in the Aging Dahl Salt Sensitive Rat J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1546 - 1556. [Abstract] [Full Text] [PDF]
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H. Robertson, S. Ali, B. J. McDonnell, A. D. Burt, and J. A. Kirby Chronic Renal Allograft Dysfunction: The Role of T Cell-Mediated Tubular Epithelial to Mesenchymal Cell Transition J. Am. Soc. Nephrol., February 1, 2004; 15(2): 390 - 397. [Abstract] [Full Text] [PDF]
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Y. Liu Epithelial to Mesenchymal Transition in Renal Fibrogenesis: Pathologic Significance, Molecular Mechanism, and Therapeutic Intervention J. Am. Soc. Nephrol., January 1, 2004; 15(1): 1 - 12. [Abstract] [Full Text] [PDF]
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R. Gong, A. Rifai, E. M. Tolbert, J. N. Centracchio, and L. D. Dworkin Hepatocyte Growth Factor Modulates Matrix Metalloproteinases and Plasminogen Activator/Plasmin Proteolytic Pathways in Progressive Renal Interstitial Fibrosis J. Am. Soc. Nephrol., December 1, 2003; 14(12): 3047 - 3060. [Abstract] [Full Text] [PDF]
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