Tubulointerstitial fibrosis is a final common pathway leading to end-stage renal failure regardless of the initial cause of the disease, and it is a major determinant of progressive renal injury (1). Tubulointerstitial fibrosis is histologically characterized by tubular loss and the accumulation of extracellular matrix (ECM) proteins, including fibronectin and collagens (types I, III, IV, V, and VII) (1). ECM is thought to be produced mainly by myofibroblasts, although tubular epithelial cells (TEC) can also synthesize a variety of ECM proteins in response to stimuli such as transforming growth factor–β (TGF-β) (2). It has been shown that TEC can express fibroblast specific protein–1 (FSP-1) (3). Furthermore, we have recently demonstrated that TEC are profibrotic cells capable of transdifferentiation into ECM-producing myofibroblasts (α-smooth muscle actin [α-SMA+]) and that they play an active role in the development of tubulointerstitial fibrosis (4).
TGF-β is a secreted signaling molecule with fibrotic properties, and it regulates a diverse range of cellular responses, including proliferation, differentiation, migration and apoptosis (5,6). There is increasing evidence that TGF-β is a key mediator of fibrosis in both experimental and human kidney diseases (5,6). This is clearly illustrated by the finding that renal fibrosis can be induced by the deliberate overexpression of TGF-β1 within the normal kidney (7). TGF-β stimulates ECM deposition by both increasing the synthesis of ECM proteins and by inducing the production of protease inhibitors, which block their degradation (6). In vitro, TGF-β has been shown to stimulate collagen synthesis and induce expression of FSP-1 in cultured TEC (8). Most recently, we demonstrated that TGF-β is able to induce normal TEC to transform into a myofibroblast phenotype (9). Tubular epithelial cells expressing a myofibroblast-like phenotype have also been identified in human glomerulonephritis with progressive tubulointerstitial fibrosis (10). These studies suggest that TGF-β may play an important role in the pathogenesis of tubulointerstital fibrosis.
Recent studies have suggested a pivotal role of Smads as intracellular effector molecules of the TGF-β family members (11,12). TGF-β exerts its biologic effects by signaling through a heteromeric receptor complex of the type I and type II serine/threonine kinase receptors, TβRI and TβRII (11,12). In the absence of TGF-β, TβRI and TβRII form a latent receptor complex. Upon TGF-β binding, the receptors rotate relatively within the complex (11,12), resulting in phosphorylation of TβRI by the constitutively active and autophosphorylated TβRII and thereby activate TβRI. The activated TβRI then directly signals to downstream intracellular substrates, Smad2 and Smad3 (R-Smads). Activated R-Smads heteroligomerize with the common partner Smad4 (Co-Smad) and these complexes are translocated into the nucleus to regulate target gene expression. Activation of the TGF-β signaling pathway can also result in the expression of inhibitory Smads (I-Smads), including Smad6 and Smad7. These inhibitory Smads appear to act by specifically inhibiting Smad2 and Smad3 phosphorylation by blocking their access to TβRI (11,12).
The involvement of Smads in TGF-β–mediated renal fibrosis has been demonstrated by the groups of Poncelet et al. (13,14) and Schiffer et al. (15), with the findings that Smads may play a role in TGF-β–induced collagen matrix synthesis in human mesangial cells and podocyte apoptosis during glomerulosclerosis. In this study, we have examined the role of Smad signaling in the fibrogenic response of TEC (NRK52E) to TGF-β. We report that TEC stimulated with TGF-β caused activation of Smad2 (phosphorylation and nuclear localization) with the de novo expression of collagens I, III, and IV and the transformation of TEC into a “myofibroblast” phenotype (α-SMA+) with the loss of E-cadherin. We also report that overexpression of Smad7 blocks Smad2 activation and prevents collagen synthesis and myofibroblast transformation. Thus, this study demonstrates the actual role for Smad signaling in regulating (Smad2) and counter-regulating (Smad7) tubular fibrogenesis in response to TGF-β.
Materials and Methods
Establishing Doxycycline-Regulated Smad7-Expressing TEC Cell Lines
The Doxycycline (Dox)–regulated Smad7-expressing cell line was established as described previously (16), but with a well-characterized normal rat kidney TEC line (NRK52E). Mouse Smad7 cDNA with a flag tag (m2) at its NH2 terminus in pcDNA3 was a generous gift from Dr. Peter ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden). To subclone the flag-tagged Smad7 cDNA into a tetracycline (Tet)–inducible vector, pTRE (Clontech, Palo Alto, CA), to obtain pTRE-Smad7, a BamHI-XbaI fragment encoding full-length Smad7 and flag tag at its NH2 terminus, and an XhoI-BamHI fragment from pTRE were ligated into pTRE at its XhoI and XbaI sites. An improved pTet-on vector (Clontech), pEFpurop-Tet-on, was generously provided by Dr. Gino Vario (Cerylid, Melbourne, Australia). Briefly, the gene encoding the “reverse” Tet repressor was subcloned into a pEF-BOS vector, pEFr-PGKpuropAv18, which confers puromycin resistance. Thus, the EF-1 promoter drives the expression of the reverse Tet repressor, and stable cell lines can be selected by puromycin. To obtain Dox (a Tet derivative)–induced Smad7-expressing NRK52E cell lines, pTRE-Smad7 and pEFpurop-Tet-on were cotransfected into NRK52E cells by electroporation, and then the stable transfected cells were selected in the presence of puromycin (2 μg/ml). Positive clones were confirmed by their ability to express Smad7 in the presence of Dox by Western blot analyses and immunohistochemistry using an anti-flag antibody m2 (IBI; Eastman Kodak, Rochester, NY). Three clones of stable Smad7–expressing NRK52E cell lines were obtained, and clone S7-7 was used in this study.
The stable, Dox-regulated Smad7–expressing NRK52E cells were grown in a 5% CO2 atmosphere at 37°C in Dulbecco’s modified Eagle’s medium (Life Technologies BRL, Gaithersburg, MD) containing 0.5% fetal bovine serum, 60 μg/ml penicillin, and 100 μg/ml streptomycin in 6-well plastic plates or eight-chamber glass slides (Nunc, Naperville, CT). A recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) at concentrations of 0, 0.615, 1.25, 2.5, 5, and 10 ng/ml were added into the cell culture for periods of 0, 5, 15, 30, 60, and 120 min and 1, 3, and 5 d. To induce overexpression of Smad7, cells were treated with Dox at designated concentrations of 0, 0.125, 0.25, 0.5, 1, and 2 μg/ml for 24 h. TGF-β1 was then added at the designated concentrations for periods as described above. At least four experiments were performed throughout the study.
Reverse Transcriptase–PCR Analyses
Total RNA was isolated using the High Pure RNA Isolation Kit according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). Contaminated DNA was removed by treating the samples with RNAase-free DNAase Ι (Promega, Madison, WI). Reverse transcriptase–PCR (RT-PCR) was performed using a ThermoScrip RT-PCR Kit following the manufacturer’s instruction (Life Technologies BRL). The first-strand cDNA was synthesized by using oligonucleotide primers and M-MLV reverse transcriptase (Promega) before PCR amplification (35 cycles) using primers specific for mouse Smad2 (5′-GTTCAATCCAGCAAGGAGTAC-3′ and 5′-CTCATGCGGTGCACATTC-3′), Smad7 (5′-CATACTGGGAGGAGAAGA-3′ and 5′-CTGTTGAAGATGACCTCTAGCC-3′), rat collagen type I (5′-TGCCGTGACCTCAAGATGTG–3′ and 5′-CACAAGCGTGCTGTAGGTGA-3′), collagen type III (5′-CTG GAC CAA AAG GTG ATG CTG-3′ and 5′-TGC CAG GGA ATC CTC GAT GTC-3′), collagen type IV (5′-CTC AGG TCT CTG CTC AGA GCC-3′ and 5′-CTG CGC TCC TCG TGG AGC AGA AG-3′), α-SMA (5′-GAT CAC CAT CGG GAA TGA ACG C-3′ and 5′-CTT AGA AGC ATT TGC GGT GGA C-3′), E-cadherin (5′-CTC AGT GTT TGC TCG GCG TTT GC-3′ and 5′-GCT CTG GGT TGG ATT CAG AG-3′), and GAPDH (5′-TCC GCC CCT TCC GCT GAT G-3′ and 5′-CAC GGA AGG CCA TGC CAG TGA-3′). All samples were subjected to RT-PCR for housekeeping gene GAPDH as a positive control and as an internal standard. Afterward, RT-PCR products were resolved on 1.5% agarose gels in 1× Tris-borate-EDTA (TBE) buffer, visualized by ethidium bromide, photographed using a gel 1000 ultraviolet documentation system (Bio-Rad, Hercules, CA), and analyzed by densitometry.
Western Blot Analyses
NRK52E cells grown in six-well plates with or without TGF-β1 or Dox were analyzed by Western blotting as described previously (9). Cells were washed in phosophate-buffered saline (PBS) and then lysed in 1 ml of 1% Nonidet P-40, 25 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, pH 8.0, containing a 1:50 dilution of a protease inhibitor cocktail (P2714; Sigma, St. Louis, MO) for 30 min on ice. Samples were centrifuged at 14,000 g for 5 min to pellet cell debris. Samples (20 μg) were mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled for 5 min, electrophoresed on a 10% SDS polyacrylamide gel, and electroblotted onto Hybond-ECL nitrocellulose membrane (Amersham International, Buckinghamshire, UK). The membrane was blocked in PBS containing 5% skimmed milk powder and 0.02% Tween 20. To detect phosphorylated Smad2 (p-Smad2), total Smad2 and Smad7, collagen types I, III, and IV protein synthesis, and tubular epithelial-myofibroblast transformation (TEMT) with the de novo expression of α-SMA and the loss of E-cadherin, the membrane was incubated for 1 h with mouse monoclonal (mAb) to Smad2 (Transduction Laboratories, Lexington, KY) or polyclonal antibodies (Ab) to Smad7 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), p-Smad 2 (Upstate Biotechnology, Lake Placid, NY), rabbit polyclonal antibodies to collagen types I, III, and IV (Santa Cruz Biotechnology), mAb to α-SMA (1A4, Sigma), and anti–E-cadherin antibody (Santa Cruz Biotechnology). After washing, the membrane was incubated with a 1:20,000 dilution of peroxidase-conjugated goat anti-mouse IgG or swine anti-rabbit IgG in PBS containing 1% normal goat serum and 1% fetal calf serum. The blot was then developed using the ECL detection kit (Amersham International) to produce a chemiluminescence signal, which was captured on x-ray film.
NRK52E cells were cultured in eight-chamber glass slides in the presence or absence of TGF-β1 or Dox as described above. Cells were analyzed for activation of Smad2 using the anti–p-Smad2 antibody. Briefly, cells were fixed in 2% paraformaldehyde and preincubated with 10% fetal calf serum and 10% normal sheep serum to block nonspecific binding. Cells were then incubated with the anti–p-Smad2 Ab or an irrelevant isotype control rabbit IgG at 4°C overnight. After inactivation of endogenous peroxidase, cells were incubated with peroxidase-conjugated goat anti-mouse IgG or swine anti-rabbit IgG and then by the mouse or rabbit peroxidase anti-peroxidase complexes. After being washed, slides were developed with diaminobenzidine to produce a brown color, and the cell nucleus was counterstained with hematoxylin.
Microwave-based two-color immunostaining was used to examine TGF-β–induced TEC-myofibroblast transformation and the inhibition of this process by Dox-induced overexpression of m2-Smad7 (9,17). Briefly, after color developing with the anti–α-SMA mAb as described above, cells were treated with 10 min of microwave oven heating in 10 mM sodium citrate (pH 6.0) at 2450 MHz and 800 watts of power to block antibody cross-reactivity and to facilitate antigen retrieval. Cells were then incubated with the anti-m2 mAb (IBI, Eastman Kodak Co.) or control mAb (73.5, which recognized human CD45R antigen) for 60 min and then with alkaline phosphatase-conjugated goat anti-mouse IgG and mouse alkaline phospatase anti-alkaline phosphatase complexes. Finally, sections were developed with Fast Blue BB Base (Sigma) and coverslipped in an aqueous mounting medium. All procedures were performed at room temperature.
For quantitative analyses of p-Smad2 transnuclear location, Smad7 expression, and α-SMA, the percentage of positive cells in eight-chamber slides stained for various Abs as described above was determined by counting at least 1000 cells under high power (×400) in each well. All scoring was performed blind on coded slides.
Data obtained from this study are expressed as the mean ± SD. Statistical analyses were performed using GraphPad Prism 3.0 (GraphPad Software, Inc. San Diego, CA). Differences in p-Smad2, Smad7, ECM, and E-cadherin expression were assessed by one-way ANOVA or by t test.
TGF-β1 Induces Activation of Smad2 in Renal Tubular Epithelial Cells
To examine the kinetics of TGF-β–induced Smad2 activation in TEC, NRK52E cells were cultured with the TGF-β1 for periods of time and examined for Smad2 activation. As shown in Figure 1A, immunocytochemical analyses demonstrated that TGF-β1 (10 ng/ml) induced a rapid transnuclear location of p-Smad2 in a time-dependent manner, being evident at 5 min (20%), peaking over 15 to 30 min (85%), and then declining to normal levels by 2 h (5 to 10%). Consistent with these observations, Western blot analyses showed that TGF-β1–induced Smad2 phosphorylation in both a time-dependent and dose-dependent fashion (Figure 1B).
Smad7 Inhibits TGF-β–Induced Smad2 Phosphorylation and Nuclear Localization
Smad7 has been reported as an intracellular negative regulator of TGF-β signaling, particularly in R-Smad activation (11,12). To investigate the counter-regulating role of Smad7 on TGF-β–induced Smad2 activation in renal TEC, we established a cell line in which the expression of Smad7 in NRK52E cells was tightly regulated by Dox. We first examined the ability of Dox to induce Smad7 expression. As shown in Figure 2A, cells cultured in the presence of Dox for 24 h exhibited a strong Smad7 expression in a dose-dependent manner, with maximal expression at 2 μg/ml. We then examined the counter-regulating role of Smad7 in the inhibition of TGF-β–induced Smad2 activation. As demonstrated by Western blot analyses, TGF-β–induced Smad2 phosphorylation was inhibited by the addition of Dox in both a time-dependent and dose-dependent fashion, with marked inhibition of Smad2 phosphorylation at a dose of 2 μg/ml of Dox (Figure 2, B and C). This inhibitory effect is on the activation (phosphorylation), because Dox-induced Smad7 overexpression did not alter Smad2 expression at both protein (unphosphorylated) and mRNA levels (Figure 2, D and E). Immunostaining also showed that inducing overexpression of Smad7 by the addition of Dox caused a marked inhibition of Smad2 transnuclear location in a dose-dependent manner (Figure 2F). These data demonstrate that overexpression of Smad7 is able to negatively regulate TGF-β–induced Smad2 activation in NRK52E cells.
Regulating Role of Smad Signaling on the Fibrogenic Effects of TGF-β on Renal Tubular Epithelial Cells
We previously reported that TGF-β stimulates fibrotic transdifferentiation of renal tubular epithelial cells into myofibroblasts (9). We now examined whether this TGF-β–induced fibrotic process is regulated by Smad signaling. Using a Dox-regulated Smad7–expressing NRK52E cells, TGF-β1 (in the absence of Dox) induced NRK52E cells to transform into a myofibroblast phenotype as evidenced by de novo synthesis of α-SMA mRNA (Figure 3A) and protein (Figure 3B), a myofibroblast marker, and by the loss of the epithelial marker, E-cadherin mRNA and protein, a molecule that plays a key role in maintaining the integrity of epithelial cells (Figure 3, A and B). Furthermore, TGF-β1 also induced the synthesis of ECM mRNA (Figure 3A) and protein (Figure 3B) by TEC, including collagen types I, III, and IV in a dose-dependent manner. RT-PCR showed that a significant increase in α-SMA and collagen (types I, III, and IV) mRNA expression was evident at day 1 (P < 0.001) and steadily increased with time, and this was associated with the loss of E-cadherin mRNA expression (Figure 4, A and C). We then examined whether TGF-β–induced fibrotic effects on TEC can be blocked by Smad7. As shown in Figures 4, A(i) and C(i), and 5A, TGF-β induced low levels of endogenous Smad7 mRNA and protein expression in a time-dependent manner, being significant at day 3. Strikingly, the addition of Dox for 24 h before the addition of TGF-β (termed day 0) induced marked Smad7 mRNA and protein expression in a time-dependent and/or dose-dependent manner (Figures 4, B(i) and C(i), and 5A). When overexpression of Smad7 was induced, TGF-β–induced TEC transdifferentiation into ECM-producing myofibroblasts was significantly inhibited. This was demonstrated by inhibition of de novo α-SMA mRNA and protein synthesis, the maintenance of E-cadherin expression, and inhibition of collagen mRNA and protein synthesis (Figures 4, B and C, and 5). The suppressive effect of TGF-β–induced TEC-myofibroblast transformation and ECM production by Smad7 was time-dependent (Figure 4, B and C) and dose-dependent (Figure 5). These results were further supported by two-color immunostaining in which cells expressing Smad7 (induced by Dox) did not express the myofibroblast marker (α-SMA+), preventing TEC-myofibroblast transformation (Figure 6, A through C). Semiquantitative analyses showed that the number of Smad7-expressing cells inversely correlated with the number of α-SMA+ cells (Figure 6D).
In this study, we have established a Dox-inducible Smad7 expressing normal rat tubular epithelial cell line (NRK52E), which provides a valuable model for the in vitro study of the intracellular mechanism of TGF-β in renal fibrosis. Using this model, we have found that TGF-β activates Smad2 in renal TEC in association with ECM production and myofibroblast transformation and that overexpression of Smad7 blocks this process.
TGF-β has long been considered to play a pathogenic role in renal fibrosis. The recent discovery of the TGF-β signaling pathway, Smads, allows us to delineate the exact mode of TGF-β action in this process. It is well documented that TGF-β binds to TβRII, resulting in the activation of TβRI, which causes the phosphorylation of specific Smad proteins, called R-Smads, including Smad2 and Smad3, that initiate TGF-β signaling transduction (18–21). On the other hand, activated TβRI also induces the activation of inhibitory Smad7 that function to antagonize TGF-β signaling by preventing TGF-β receptor–mediated phosphorylation of Smad2 and Smad3 (22,23) and by increasing ubiquitin-mediated degradation of the TGF-β type I receptor itself (24). In this study, we found that the fibrotic effects of TGF-β on renal TEC, in terms of collagen production and myofibroblast transformation, may signal through the activation of Smad2. Indeed, blockade of Smad2 activation by inducing overexpression of its inhibitor, Smad7, results in inhibition of TGF-β–induced TEC-myofibroblast transformation and collagen production. The tight association between Smad2 activation and de novo synthesis of α-SMA (a marker of myofibroblasts) and collagen types I, III, and IV mRNA and protein indicates that the phosphorylated Smad2 may act on the target genes to regulate TEC to become collagen-producing myofibroblasts at the transcriptional level when it is translocated into the nucleus. This is further demonstrated by the finding that overexpression of Smad7 inhibits TGF-β–induced Smad2 phosphorylation and nuclear localization, subsequent TEC-myofibroblast transformation, and collagen matrix production. Thus, activation of Smad2 may be required for TGF-β–induced fibrotic effects on TEC. This also implies that activation of Smad2 may be one intracellular mechanism by which TGF-β mediates renal fibrosis. These observations are consistent with the recent finding that TGF-β induces collagen matrix in human mesangial cells by a process involving the activation of Smad proteins (13,14).
It is also possible that the activation of Smad3, another R-Smad protein, may be required for TGF-β–induced fibrotic process in TEC. Indeed, TGF-β induced Smad3 nuclear localization was also evident in this study (data not shown). Activation of Smad3 has been shown to promote TGF-β–induced α2(I) collagen gene expression by human mesangial cells, which is enhanced by Sp1 (14). There are some functional differences between Smad2 and Smad3. Smad3 is involved in activation of the Smad7 gene promoter (25), whereas Smad2 has a functional role in TGF-β1-induced the matrix metalloproteinase MMP-2 (26). Smad3-deficient mice show accelerated wound healing and die from chronic inflammation (27,28) and colon carcinomas between 4 and 6 mo of age (29), whereas Smad2-deficient mice are embryonic lethal (30). However, the functional role of Smad3 in TGF-β–induced TEC-myofibroblast transformation and collagen production by TEC remains to be defined.
Smad7 and Smad6 belong to an inhibitory subfamily of Smads that block both TGF-β and bone morphogenetic protein signaling, whereas Smad6 is able to inhibit bone morphogenetic protein signaling and partially inhibit TGF-β signaling (22,23,31,32). Endogenous Smad7 is rapidly induced by TGF-β and, in turn, downregulates TGF-β signaling through a negative feedback loop to prevent R-Smad from phosphorylation, an early step of TGF-β signaling (11,12,31). Smad7 may act by competition for binding of R-Smad2/3 to TβRI, thereby blocking Smad2 and Smad3 from interacting with the receptor after TGF-β stimulation (11,12,31,33), or by increasing ubiquitin-mediated degradation of TGF-β receptor (24). It has been shown that Smad7, but not Smad6, is a TGF-β–induced attenuator of Smad2/3-mediated inhibition of embryonic morphogenesis (34,35). Overexpression of Smad7, but not Smad6, inhibits TGF-β–induced heme oxygenase-1 by human TEC (36). Expression of Smad7 transgene also blocks Smad2 phosphorylation induced by bleomycin in mouse lung, and gene transfer of Smad7, but not Smad6, prevents bleomycin-induced lung fibrosis (37). In this study, we have investigated the negative regulatory role of Smad7 in the profibrotic effects of TGF-β on renal TEC using an improved Tet–inducible system in which the expression of Smad7 is under the tight control of Dox. We found that TGF-β induced endogenous Smad7 expression, but that the low grade of endogenous Smad7 expression was unable to overcome the fibrogenic effect of TGF-β on renal TEC associated with the activation of R-Smads. We found, however, that inducing overexpression of Smad7 transgene by addition of Dox blocked the activation of Smad2 and prevented the TGF-β–induced TEC-myofibroblast transformation and stimulation of collagen types I, III, and IV mRNA and protein synthesis. Thus, overexpression of Smad7 is able to alter the physiopathologic balance between R-Smads and I-Smads in TEC in response to TGF-β and blocks TGF-β–induced fibrosis.
Although inhibition of Smad2 and Smad3 may block TGF-β–mediated fibrosis, recent studies in Smad2 and Smad3 knockout mice have demonstrated that absence of Smad2 and Smad3 may also impair the immune system and embryonic development (27–30). In contrast, overexpression of Smad7 may also cause cell death through apoptosis (15,38). Therefore, it is critical to maintain a physiologic balance between R-Smads and I-Smads when attempting to target TGF-β signaling. In this study, the degree of expression of Smad7 could be controlled by varying the concentrations of Dox. These in vitro data implicate that it may be more safe and advantageous to use a Dox-regulated Smad7, rather than naked Smad7 gene alone, to prevent or treat renal fibrosis in vivo.
TGF-β is a major mediator in renal fibrosis and acts to both regulate the transdifferentiation of TEC into myofibroblasts and to stimulate their synthesis of ECM proteins (4–10,39,40). In this study, we demonstrate that the fibrogenic effect of TGF-β in TEC involves activation of Smad2 and that overexpression of Smad7 can block Smad2 activation and the fibrogenic process. Thus, this study demonstrates the complex interplay between R-Smads (Smad2) and I-Smads (Smad7) in TGF-β–induced tubular fibrogenesis.
This work was supported by grants from the Juvenile Diabetes Foundation (JDRF 1–2001–596), National Health and Medical Research Council (164812), Australia, and the Hong Kong University Research Committee (CRCG 98–33704 and 99–05485).
1. Eddy AA: Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 7: 2495–2508, 1996
2. Creely JJ, Dimari SJ, Howe AM, Haralson MA: Effects of transforming growth factor-beta on collagen synthesis by normal rat kidney epithelial cells. Am J Pathol 140: 45–55, 1992
3. Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomaszewski JE, Neilson EG: Identification and characterization of a fibroblast marker: FSP1. J Cell Biol 130: 393–405, 1995
4. Ng YY, Huang TP, Yang WC, Chen ZP, Yang AH, Mu W, Nikolic-Paterson DJ, Atkins RC, Lan HY: Evidence for tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephretomized rats. Kidney Int 54: 864–876, 1998
5. Border WA, Noble NA: Mechanisms of Disease: Transforming growth factor (beta) in tissue fibrosis. N Eng J Med 331: 1286, 1994
6. Border WA, Noble NA: TGF-beta in kidney fibrosis: A target for gene therapy. Kidney Int 51: 1388–1396, 1997
7. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E: Glomerulosclerosis induced by in vivo transfection of transforming growth factor-beta or platelet-derived growth factor gene into the rat kidney. J Clin Invest 92: 2597–2601, 1993
8. Okada H, Danoff TM, Kalluri R, Neilson EG: Early role of Fsp1 in epithelial-mesenchymal transformation. Am J Physiol 273: F563–F574, 1997
9. Fan JM, Ng YY, Hill P, Nikolic-Paterson DJ, Mu W, Atkins RC, Lan HY: TGF-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Kidney Int 56: 1455–1467, 1999
10. Jinde K, Nikolic-Paterson DJ, Huang XR, Sakai H, Kurokawa K, Atkins RC, Lan HY: Tubular phenotypic changes in tubulointerstitial fibrosis in human glomerulonephritis. Am J Kid Dis 38: 761–769, 2001
11. Kretzschmar M, Massague J: Smads: Mediators and regulators of TGF-β signaling. Curr Opin Genet Dev 8: 103–111, 1998
12. Zimmerman CM, Padgett RW: Transforming growth factor B signaling mediators and regulators. Gene 249: 17–30, 2000
13. Poncelet AC, de Caestecker MP, Schnaper HW: The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells. Kidney Int 56: 1354–1365, 1999
14. Poncelet AC, Schnaper HW: Sp1 and Smad proteins cooperate to mediate transforming growth factor-beta 1-induced alpha 2(I) collagen expression in human glomerular mesangial cells. J Biol Chem 276: 6983–6992, 2001
15. Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, Bottinger EP: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 108: 807–816, 2001
16. Zhu HJ, Iaria j, Sizeland AM: Smad7 differentially regulates transforming growth factor β-mediated signaling pathways. J Biol Chem 274: 32258–32264, 1999
17. Lan HY, Mu W, Nikolic-Paterson DJ, Atkins RC: A novel, simple, sensitive, and reliable method for multiple immunoenzyme staining. The use of microwave oven heating technique for blocking of antibody crossreactivities and retrieval of antigens. J Histochem Cytochem 47: 95–102, 1995
18. Nakao A, Roijer E, Imamura T, Souchelnytskyi S, Stenman G, Heldin CH, ten Dijke P: Identification of Smad2, a human Mad-related protein in the transforming growth factor beta signaling pathway. J Biol Chem 272: 2896–2900, 1997
19. Liu X, Sun Y, Constantinescu SN, Karam E, Weinberg RA, Lodish HF: Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc Natl Acad Sci USA 94: 10669–10674, 1997
20. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki k, Hanai J, Heldin C, Miyazono K, ten Dijke P: TGF-beta: Receptor-mediated signaling through Smad2, Smad3 and Smad4. EMBO J 16: 5353–5362, 1997
21. Massague J, Chen YG: Controlling TGF-beta signaling. Genes Dev 14: 627–644, 2000
22. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA, Jr Wrana J, Falb D: The MAD-related protein Smad7 associates with the TGF-β receptor and functions as an antagonist of TGF-β signaling. Cell 89: 1165–1173, 1997
23. Topper JN, Cai J, Qiu Y, Anderson KR, Xu YY, Deeds JD, Feeley R, Gimeno CJ, Woolf EA, Tayber O, Mays GG, Sampson BA, Schoen FJ, Gimbrone MA Jr, Falb D: Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium. Proc Natl Acad Sci USA 94: 9314–9319, 1997
24. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL: Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 6: 1365–1375, 2000
25. von Gersdorff G, Susztak K, Rezvani F, Bitzer M, Liang D, Bottinger EP: Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. J Biol Chem 275: 11320–11326, 2000
26. Piek E, Ju WJ, Heyer J, Escalante-Alcalde D, Stewart CL, Weinstein M, Deng C, Kucherlapati R, Bottinger EP, Roberts AB: Functional characterization of transforming growth factor beta signaling in smad2- and smad3-deficient fibroblasts. J Biol Chem 276: 19945–19953, 2001
27. Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H, Roberts AB, Deng C: Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β. EMBO J 18: 1280–1291, 1999
28. Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JL, Mizel DE, Anzano M, Greenwell-Wild T, Wahl SM, Deng C, Robertss AB: Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol 1: E117–E119, 1999
29. Zhu Y, Richardson JA, Parda LF, Graff JM: SMAD3 mutant mice develop metastatic colorectal cancer. Cell 94: 703–714, 1998
30. Nomura M, Li E: Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature 393: 737–739, 1998
31. Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, ten Dijke P: Identification of Smad7, a TGF beta-inducible antagonist of TGF-beta signaling. Nature 389: 631–635, 1997
32. Hata A, Lagna G, Massagué J, Hemmati-Brivanlou A: Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. Genes Dev 2: 186–197, 1998
33. Itoh S, Landström M, Hermansson A, Itoh F, Heldin CH, Heldin NE, ten Dijke P: Transforming Growth Factor-β1 Induces Nuclear Export of Inhibitory Smad7. J Biol Chem 44: 29195–29201, 1998
34. Zhao J, Shi W, Chen H, Warburton D: Smad7 and Smad6 differentially modulate transforming growth factor beta -induced inhibition of embryonic lung morphogenesis. J Biol Chem 275: 23992–23997, 2000
35. Zhao J, Crowe DL, Castillo C, Wuenschell C, Chai Y, Warburton D: Smad7 is a TGF-beta-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis. Mech Dev 93: 71–81, 2000
36. Hill-Kapturczak N, Truong L, Thamilselvan V, Visner GA, Nick HS, Agarwal A: Smad7-dependent regulation of heme oxygenase-1 by transforming growth factor-beta in human renal epithelial cells. J Biol Chem 275: 40904–40909, 2000
37. Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto I: Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 104: 5–11, 1999
38. Landstrom M, Heldin NE, Bu S, Hermansson A, Itoh S, ten Dijke P, Heldin CH: Smad7 mediates apoptosis induced by transforming growth factor in prostatic carcinoma calls. Current Biol 10: 535–538, 2000
39. Ng YY, Fan JM, Mu W, Nickolic-Paterson DJ, Yang WC, Huang DP, Atkins RC, Lan HY: Glomerular epithelial-myofibroblast transdifferentiation in the evolution of glomerular crescent formation. Nephrol Dial Transplant 14: 2860–2872, 1999
40. Hay ED, Zuk A: Transformations between epithelium and mesenchyme: Normal, pathological, and experimentally induced. Am J Kid Disease 26: 678–690, 1995