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Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
ABSTRACT
Hepatocyte growth factor (HGF) receptor is a transmembrane receptor tyrosine kinase encoded by the c-met protooncogene. In this study, we demonstrated that c-met expression was upregulated in the kidney after obstructive injury in mice. Because the pattern of c-met induction was closely correlated with transforming growth factor-1 (TGF-1) expression in vivo, we further investigated the regulation of c-met expression in renal tubular epithelial (HKC) cells by TGF-1 in vitro. Real-time RT-PCR and Northern and Western blot analyses revealed that TGF-1 significantly induced c-met expression in HKC cells, which primarily took place at the gene transcriptional level. Overexpression of inhibitory Smad7 completely abolished c-met induction, indicating its dependence on Smad signaling. Interestingly, TGF-1-induced c-met expression was also contingent on a functional Sp1, as ablation of Sp1 binding with mithramycin A abrogated c-met induction in HKC cells. Transfection and sequence analysis identified a cis-acting TGF-1-responsive region in the c-met promoter, in which resided a putative Smad-binding element (SBE) and an adjacent Sp1 site. TGF-1 not only induced Smad binding to the SBE/Sp1 sites in the c-met promoter, but also enhanced the binding of Sp proteins. Furthermore, Sp1 could form a complex with Smads in a TGF-1-dependent fashion. These results suggest a novel regulatory mechanism controlling c-met expression by TGF-1 in renal epithelial cells, in which both Smad and Sp proteins participate and cooperate in activating c-met gene transcription.
transforming growth factor-1; hepatocyte growth factor; gene transcription; unilateral ureteral obstruction; tubular epithelial cells
HEPATOCYTE GROWTH FACTOR (HGF) receptor, the product of the c-met protooncogene, is a member of the receptor tyrosine kinase superfamily (5, 36). The mature c-met receptor is a 190-kDa disulfide-linked heteodimer protein. It contains an extracellular -subunit and a transmembrane -subunit that has an extracellular segment involved in ligand binding, a transmembrane segment, and a cytoplasmic tyrosine kinase domain (27). On binding to HGF, the c-met receptor undergoes autophosphorylation of tyrosine residue in its cytoplasmic domain and initiates cascades of signal transduction events that eventually lead to specific cellular responses (8, 32). It is demonstrated that HGF/c-met signaling plays a vital role in cell proliferation, survival, migration, and differentiation in many tissues including the kidney (20, 26, 41).
As HGF's sole receptor, c-met expression is likely one of the crucial regulatory components in HGF biology. In this regard, earlier studies indicate that it is the c-met level, but not HGF abundance, that correlates with the site of greatest tissue damage and subsequent repair after various injurious stimuli (12, 17). Alteration in the c-met receptor under pathological conditions not only influences the overall activity of HGF but also dictates the site specificity of its action. Copious studies have demonstrated that c-met expression is rapidly and selectively upregulated in the kidney in several models of acute renal failure induced by either ischemia or nephrotoxic agents (17, 24, 38), and such c-met induction after acute injury is believed to play a crucial role in accelerating tubular repair and renal regeneration (9, 38).
Recent studies also implicate the HGF/c-met system in tissue repair and healing after chronic insults to the kidney. HGF expression is upregulated, at least in the early stage, in numerous models of chronic renal diseases, such as in remnant kidney after 5/6 nephrectomy and obstructed kidney after ureteral ligation (22, 23, 47). Delivery of HGF protein or its gene has been shown to ameliorate renal fibrotic lesions and preserve kidney structure and function in animal models of renal fibrotic diseases with different etiologies (15, 16, 33, 43, 44). However, little is known about the regulation of endogenous c-met gene expression in the kidney after chronic injury. Furthermore, the mediators and underlying mechanism controlling renal c-met regulation in vivo remain unknown.
In vitro expression of the c-met gene is tightly controlled by a wide variety of cytokines, growth factors, and extracellular environmental cues, including transforming growth factor-1 (TGF-1) (23, 24, 34). In view of the central role of TGF-1 in the pathogenesis of chronic renal diseases (6, 39), we speculated that TGF-1 may play an important role in the regulation of c-met expression under pathological conditions. Consistent with this view, a rapid and marked induction of both TGF- and its type I receptor is observed in the kidney as early as 1 day after unilateral ureteral obstruction (UUO) (45). TGF-1 initiates its signaling transduction by interacting with its transmembrane serine/threonine kinase receptors. Receptor activation by TGF-1 leads to phosphorylation and activation of intracellular signaling mediators known as Smad2 and Smad3, which subsequently bind to common Smad4 and form the complexes (30). These complexes are then translocated to cell nuclei where they bind to a Smad-binding element (SBE) in the promoter and direct the transcription of the TGF-1-responsive genes (6, 28, 39).
In this study, we examined the expression of c-met in the mouse model of chronic renal disease induced by UUO and investigated the potential mechanism underlying the change in c-met expression under pathological conditions. Our results suggest that c-met expression was markedly induced in a time-dependent manner in the kidney after obstructive injury and that TGF-1 may play an important role in mediating c-met induction. We found that TGF-1-induced c-met expression is completely dependent on both Smad signaling and a functional Sp1 in renal epithelial cells. Sp1 and Smads could physically associate to form complexes, and both of them bind to the c-met promoter in a synergistic way. Therefore, our studies unravel a unique regulatory mechanism by which a general, ubiquitous transcription factor, Sp1, works in concert with Smad to activate c-met gene transcription in response to TGF-1 stimulation.
MATERIALS AND METHODS
Animal model. Male CD-1 mice weighing 2022 g were obtained from Harlan Sprague Dawley (Indianapolis, IN). UUO was performed using an established procedure (46). Briefly, under general anesthesia, complete ureteral obstruction was preformed by double ligating the left ureter with 4-0 silk after a midline abdominal incision. Sham-operated mice had their ureters exposed and manipulated but not ligated. Mice were killed 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 extraction.
Immunohistochemical staining. Localization of c-met receptor protein in the kidney by immunohistochemical staining was performed using a Vector MOM immunodetection kit (Vector Laboratories, Burlingame, CA), as described previously (45). The c-met antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). As a negative control, the primary antibody was replaced with nonimmune rabbit IgG, and no staining occurred.
Cell culture and treatment. Human kidney proximal tubular epithelial cells (HKC) were kindly provided by Dr. L. Racusen of Johns Hopkins University (Baltimore, MD). Cells were cultured in Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM-F-12; 1:1) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). For TGF-1 treatment, cells were seeded at 4050% confluence in complete medium containing 10% FBS. Twenty-four hours later, cells were serum starved for 16 h. For analysis of gene expression at the protein level, the cells were incubated with different concentrations of TGF-1 as indicated for 24 h, except when otherwise specified. For gene expression analysis at the mRNA level by Northern blot, the cells were treated with a different concentration of TGF-1 for 6 h. In some experiments, cells were treated with either various inhibitors at given concentrations or vehicle (0.1% DMSO) 0.5 h before addition of 2 ng/ml TGF-1. PD-98059 (Mek1 inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), and SC-68376 (p38 MAPK inhibitor) were obtained from Calbiochem (La Jolla, CA). Actinomycin D and cycloheximide were purchased from Sigma (St. Louis, MO). For chemical blockade of Sp1 binding, HKC cells were treated with mithramycin A (Sigma) at different concentrations for 16 h before addition of 2 ng/ml TGF-1.
RNA preparation and Northern blot analysis. Total RNA was extracted from the cells, using Ultraspec RNA solution (Biotecx, Houston, TX), according to the instructions specified by the manufacturer. Samples of 20 μg total RNA were electrophoresed on 1.0% formadehyde-agarose gels and then transferred to GeneScreen-plus nylon membrane (DuPont, Boston, MA) by capillary blotting, followed by ultraviolet cross-linking. Membranes were prehybridized for 4 h at 65°C in a buffer containing 6x standard saline citrate (SSC), 5x Denhardt's solution, 1% sodium dodecyl sulfate (SDS), 10% dextran sulfate, and 100 μg/ml denatured salmon sperm DNA. 32P-labeled DNA probes were prepared by a random primer labeling kit (Stratagene, La Jolla, CA) with [-32P]dCTP. The human c-met cDNA probe was generated in our laboratory as described previously (25). Denatured probes were added to the same hybridization buffer at a concentration of 12 x 106 counts?min1 (cpm)?ml1, and hybridization was allowed to proceed at 65°C for 16 h. Membranes were washed and exposed to X-ray film (Eastman Kodak, Rochester, NY) at 80°C with the aid of an intensifying screen as described elsewhere (24, 46). Quantitation was performed by determination of the intensity of the hybridization signals using the NIH Image program.
Determination of mRNA levels by real-time quantitative RT-PCR. Real-time quantitative RT-PCR was performed to determine the steady-state levels of c-met mRNA. Briefly, the first-strand cDNA synthesis was carried out by using a Reverse Transcription System kit according to the instructions of the manufacturer (Promega, Madison, WI). Real-time PCR amplification was performed with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR reaction mixture at a 25-μl volume contained 12.5 μl of 2x SYBR Green PCR Master Mix (Applied Biosystems), 5 μl of diluted RT product (1:20), and 0.5 μM sense and antisense primer sets. The primer sequences were as follow: c-met, 5'-AAGAGGGCATTTTGGTTGTG-3' (sense) and 5'-GATGATTCCCTCGGTCAGAA-3' (antisense); and actin, 5'-AGGCATCCTCACCCTGAAGTA-3' (sense) and 5'-CACACGCAGCTCATTGTAGA-3' (antisense). Each sample was added in duplicate. The PCR reaction was run by using standard conditions. After sequential incubations at 50°C for 2 min and 95°C for 10 min, respectively, the amplification protocol consisted of 50 cycles of denaturing at 95°C for 15 s, annealing, and extension at 60°C for 60 s. The standard curve was made from a series dilutions of template cDNA. Expression levels of c-met mRNA were calculated after normalization with the housekeeping gene -actin.
Western blot analysis. Whole cell lysates were prepared essentially according to the procedures described previously (49). Samples were heated at 100°C for 5 min before loading and separated on precasted 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) under nonreducing conditions. The proteins were transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) in transfer buffer containing 48 mM Tris?HCl, 39 mM glycine, 0.037% SDS, and 20% methanol at 4°C for 2 h. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% nonfat milk in TBS buffer (20 mM Tris?HCl, 150 mM NaCl, and 0.1% Tween 20), and the membrane was then incubated with various primary antibodies at 4°C overnight, followed by incubation for 1 h with a secondary horseradish peroxidase-conjugated IgG in 5% nonfat milk. The specific antibodies against c-met, Smad2/3, Smad4, Sp1, Sp3, and actin were purchased from Santa Cruz Biotechnology. The anti-phosphospecific Smad2 antibody was obtained from Cell Signaling Technology (Beverly, MA). The signals were visualized by the enhanced chemiluminescence system (ECL, Amersham) as described elsewhere (44).
Construction of plasmid, DNA transfection, and luciferase assay. The chimeric plasmids (pGL3-0.1met) containing the 5'-flanking region of the human c-met gene (68 +60) linked to the coding sequence for the firefly luciferase reporter system have been described previously (21). Reporter plasmid pSBE/Sp1-Luc, which contains three copies of the SBE/Sp1 site of the c-met gene, was constructed by subcloning the corresponding oligonucleotide into a luciferase reporter vector containing SV-40 promoter (Promega). For transient transfection, the HKC cells were seeded in six-well plates at 5 x 105 cells/well. The cells were then transfected with pGL3-0.1met or pSBE/Sp1-Luc using Lipofectamine 2000 reagent according to the instructions specified by the manufacturer (Invitrogen). A fixed amount of internal control reporter Renilla reniformis luciferase driven under thymidine kinase (TK) promoter (pRL-TK; Promega) was cotransfected for normalizing the transfection efficiency and correcting firefly luciferase activity. Some samples were cotransfected with a Smad transcriptional corepressor SnoN expression vector (pHA-SnoN, kindly provided by Dr. R. Weinberg, Massachusetts Institute of Technology) and pFlag-Smad3 (provided by Dr. J. Massague). After transfection, the cells were incubated for an additional 48 h in the absence or presence of 2 ng/ml TGF-1 before being harvested for luciferase assay. Luciferase assay was performed using a Dual Luciferase Assay System kit essentially according to the manufacturer's protocols (Promega). Relative luciferase activity of each construct (arbitrary units) was reported as fold-induction over pGL3-0.1met after normalization for transfection efficiency. All experiments were repeated in a minimum of three separate experiments (triple wells/experiment) to assume reproducibility. The values from these experiments were combined and subjected to analysis of variance using SigmaStat statistical software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.
Biotinylated DNA precipitation assay. For detection of proteins bound to the SBE/Sp1 sites of c-met promoter, a biotinylated DNA precipitation approach was employed according to the procedures described previously (13). Briefly, whole cell extracts were prepared by lysing cells in ice-cold PBS containing 0.5% Triton X-100, 5 mM EDTA, 125 μM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail (1:100 dilution), and phosphatase inhibitor cocktail (1:100 dilution) (Sigma). Cell lysates were clarified by centrifugation at 10,000 g for 30 min. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit with bovine serum albumin as a standard (Sigma). The 5'-biotinylated, double-stranded oligonucleotide (oligo) corresponding to the SBE/Sp1 sites of the c-met promoter was mixed with 30 μl of packed streptavidin beads for 3 h at 4°C, followed by incubation with 500 μg of whole cell extract overnight at 4°C. After four washes with ice-cold PBS containing 0.5% Triton X-1005 mM EDTA, proteins were eluted from the beads by the addition of 2x SDS loading buffer, followed by boiling for 5 min. Eluted proteins were analyzed by Western blotting using specific antibodies against p-Smad2, Smad4, Sp1, and Sp3, respectively.
EMSA. The preparation of nuclear protein extracts was carried out according to the procedures reported previously (49). The double-stranded Smad/Sp1 oligo corresponding to the nucleotide sequence of +13 +35 in the c-met promoter, which contains a potential Smad-binding site and a perfect Sp1-binding site, was labeled with [-32P]ATP using T4 kinase (Invitrogen). An oligo containing only Smad-binding sites (designated as F3) and another containing only the Sp1-binding site (designated as F5) were used as competitors. The oligo containing three copies of SBE from the plasminogen activator inhibitor-1 (PAI-1) gene was used as a positive control for Smad binding. The labeled probes were then gel purified and used in EMSA as described previously (49). Four micrograms of poly(dI-dC)-poly(dI-dC) (Amersham) were used as the nonspecific competitor in 10 μl of reaction mixture. The binding reactions were carried out at 37°C for 30 min before being loaded on 5% nondenaturing polyacrylamide (19:1, acrylamide-bisacrylamide) gels. For competition experiments, 100-fold molar excess of unlabeled double-stranded oligo was included in the reaction mixture, except where indicated otherwise. For supershift experiments, specific antibodies against Sp1, Sp3, Egr-1, and normal control IgG (Santa Cruz) were incubated with nuclear protein extracts for 30 min at 37°C before addition of reaction buffer. Gels were run in 0.5x TBE buffer (0.045 M Tris-borate, 0.001 M EDTA) at a constant voltage of 190 V, dried, and autoradiographed with intensifying screens.
Immunoprecipitation. Immunoprecipitation experiments were performed with an identical method as described previously (49). Briefly, HKC cells grown on a 100-mm plate were lysed on ice in 1 ml of RIPA buffer containing 1x PBS, 1% Nonidet P-40, 0.1% SDS, 10 μg/ml PMSF, 1 mM sodium orthovanadate, and 1% protease inhibitor cocktail (Sigma). Whole cell lysates were clarified by centrifugation at 12,000 g for 10 min at 4°C, and the supernatants were transferred into a fresh tube. To preclear cell lysates, 0.25 μg of normal rabbit IgG and 20 μl of protein A/G Plus-Agarose (Santa Cruz) were added into 1 ml of whole cell lysates. After incubation for 1 h at 4°C, supernatants were collected by centrifugation at 1,000 g for 5 min at 4°C. Lysates were immunoprecipitated overnight at 4°C with 1 μg of anti-Sp1 and anti-Smad2/3, respectively, followed by precipitation with 20 μl of protein A/G Plus-Agarose for 3 h at 4°C. The precipitated complexes were separated on SDS-polyacrylamide gels and blotted with various antibodies as described above.
RESULTS
Upregulation of c-met expression in the obstructed kidney after UUO. To investigate the expression of c-met in the kidney after chronic injury, we first examined the c-met protein level by using the Western blotting approach. Figure 1 shows the c-met protein in normal sham-operated kidney and the obstructed kidney in mice. Compared with the normal kidney, c-met protein expression was significantly induced in the obstructed kidney in a time-dependent manner (Fig. 1A). At 3 days after UUO, 28-fold induction of c-met abundance was observed in the obstructed kidney, as determined by quantitatively measuring c-met in Western blots (Fig. 1B). The induction of c-met in the kidney peaked at 3 days after UUO; further obstruction beyond this time point resulted in a moderate decrease in c-met induction (Fig. 1B).
We also examined c-met expression and localization by immunohistochemical staining in normal and obstructed kidneys at 7 days after obstructive injury. As shown in Fig. 1, C and D, c-met protein was detectable in normal kidney, especially in tubular epithelial cells. After obstructive injury, strong c-met staining was observed in renal tubular epithelia. The increase in c-met staining was particularly striking in the dilated, degenerated renal tubules. These results suggest that c-met induction occurs specifically in tubular epithelial cells, the site of injury in this model.
Of note, the pattern of c-met expression in the obstructed kidney appears to be tightly correlated with both TGF-1 and its type I receptor induction in this model, as earlier studies show a rapid and specific increase in the TGF-1 axis in renal tubular epithelia as early as 1 day after ureteral obstruction (44, 45). Such close temporal and spatial association between TGF-1 and c-met induction prompted us to investigate a potential role of TGF-1 in c-met expression by using renal tubular epithelial cells (HKC) as an in vitro model system (see below).
TGF-1 induces c-met expression in renal epithelial cells in vitro. We investigated the regulation of c-met expression by TGF-1 in renal epithelial cells at both mRNA and protein levels by using real-time RT-PCR and Western blot analyses, respectively. As shown in Fig. 2, the steady-state levels of c-met mRNA were increased after TGF-1 treatment in a dose- and time-dependent manner. TGF-1 at the concentration of 2 ng/ml induced a rapid increase in c-met mRNA, which started at 1 h after incubation (Fig. 2B). Similar to the mRNA levels, c-met protein was also dramatically induced after incubation with TGF-1. Western blot analysis revealed that TGF-1 induced c-met protein in a time- and dose-dependent fashion (Fig. 2, C and D). These studies suggest that increased TGF-1 may be, at least partially, responsible for c-met induction in renal tubular epithelial cells after obstructive injury in vivo.
Induction of c-met mRNA by TGF-1 occurs at gene transcriptional level. We next investigated the mechanism leading to c-met induction after TGF-1 treatment in renal epithelial cells. After HKC cells were pretreated with either actinomycin D or cycloheximide to block the gene transcription and translation, respectively, the expression of c-met gene in the presence or absence of TGF-1 was examined by real-time RT-PCR. As shown in Fig. 3, in the absence of gene transcription by pretreatment with actinomycin D, TGF-1 failed to induce c-met mRNA expression in HKC cells. However, pretreatment of HKC cells with cycloheximide appeared not to significantly affect c-met expression induced by TGF-1 (Fig. 3). Similar results were obtained by Northern blot analysis (data not shown). Hence, c-met mRNA induction by TGF-1 primarily takes place at the gene transcriptional level.
Smad signaling is required for c-met induction by TGF-1. To further unravel the mechanism underlying c-met regulation by TGF-1, we attempted to identify a signaling pathway that is necessary and important for mediating TGF-1 induction of c-met expression in renal epithelial cells. It has been demonstrated that TGF-1, upon binding to its specific receptors, elicits diverse cellular activities by initiating multiple signal transduction pathways, including Smad, p38 MAPK, and Akt kinase in renal epithelial cells (9). To address whether Smad signaling is involved in mediating c-met induction, we examined the consequence of blocking Smad signaling on c-met expression by overexpressing inhibitory Smad7. To this end, stable cell lines transfected with Smad7 or empty vector pcDNA3 were established. Previous studies confirmed a complete blockade of the TGF-1-induced Smad2 phosphorylation and activation in HKCSmad7 cells (9). As shown in Fig. 4A, while TGF-1 markedly induced c-met expression in mock-transfected HKCpcDNA3 cells, it failed to induce c-met expression in Smad7-overexpressing HKCSmad7 cells. These results establish that TGF-1-induced c-met expression in renal epithelial cells is dependent on an intact Smad signaling.
Besides Smad signaling, TGF-1 is also capable of stimulating parallel downstream signal pathways that lead to activation of p38 MAPK and protein kinase B/Akt. To examine the potential implication of these pathways in c-met induction, we employed specific chemical inhibitors to block respective signal transduction. As shown in Fig. 4B, specific inhibition of Erk-MAPK activation by PD-98059, p38 MAPK by SC-68376, or Akt activation by wortmannin did not affect c-met induction by TGF-1. Therefore, it is unlikely that these signal transduction pathways play any major role in mediating c-met induction by TGF-1 in renal epithelial cells.
Induction of c-met expression by TGF-1 also depends on Sp transcription factors. We defined the TGF-1-responsive region in the c-met promoter by transiently transfecting promoter-luciferase reporter constructs into HKC cells. Both transfection results and sequence analysis revealed that a region at the nucleotide positions 68 +60 in the c-met promoter contained a cis-acting element responsible for TGF-1 stimulation (Fig. 5, A and B). Within this region, there was a putative SBE with a nucleotide sequence of 5'-AGACAGACA-3' in the c-met promoter (Fig. 5A). Of interest, there was a Sp1-binding site in the close proximity of SBE in this region (Fig. 5A).
Given the fact that Sp proteins are crucial in establishing c-met constitutive expression in renal cells (49, 50), we reasoned that Sp1 may also play a role in mediating c-met induction by TGF-1. To test this hypothesis, we investigated the function of Sp1 in c-met expression induced by TGF-1 by interrupting Sp1 binding with the specific chemical inhibitor mithramycin A. As shown in Fig. 5B, when the reporter construct pGL3-0.1met, which contained Smad and Sp1-binding elements (SBE/Sp1), was transiently transfected into HKC cells, TGF-1 was able to induce the c-met promoter activity. However, interruption of Sp1 binding with mithramycin A not only suppressed c-met promoter activity at basal conditions but also completely abolished the inducibility of luciferase activities elicited by TGF-1 (Fig. 5B), indicating that c-met induction by TGF-1 in renal epithelial cells is also contingent on a functional Sp1.
We further investigated the effect of ablation of Sp1 binding by mithramycin A on endogenous c-met expression in renal epithelial cells. Consistent with the promoter activities described above (Fig. 5B), treatment with mithramycin A resulted in a decrease in c-met mRNA under basal conditions and complete abrogation of c-met induction in response to TGF-1 stimulation (Fig. 5C). Similarly, Western blot analysis showed that mithramycin A also abolished c-met protein expression induced by TGF-1 in renal epithelial cells (Fig. 5D). Of note, mithramycin A exhibited no influence on 18S RNA and actin expression in HKC cells (Fig. 5, C and D), illustrating the specificity of its action. Therefore, a functional Sp1 is crucial in mediating c-met induction by TGF-1 in renal epithelial cells.
Both Sp1 and Smad participate in enhancing c-met gene transcription. To confirm the involvement of both Sp1 and Smad in regulating c-met gene transcription, we subcloned the SBE/Sp1 site into a luciferase reporter vector containing heterologous SV-40 promoter. When pSBE/Sp1-Luc, a luciferase reporter plasmid containing three copies of the c-met SBE/Sp1 site in front of the SV-40 promoter (Fig. 6 A), was transfected into HKC cells, it conferred the responsiveness to TGF-1 stimulation (Fig. 6B). However, disruption of Sp1 binding with mithramycin A markedly reduced the luciferase activity under both basal and TGF-1-stimulated conditions. Similarly, overexpression of the Smad transcriptional corepressor SnoN also inhibited reporter activity.
With cotransfection of the Smad3 expression vector, luciferase activity elicited by pSBE/Sp1-Luc plasmid in HKC cells was dramatically increased, suggesting that exogenous Smad3 enhanced SBE/Sp1-mediated gene transcription. Under this condition, either ablation of Sp1 binding with mithramycin A or suppression of Smad activity with SnoN inhibited luciferase activity (Fig. 6B). Therefore, the enhancer activity of SBE/Sp1 of the c-met gene is dependent on the participation and cooperation of both Sp1 and Smad.
TGF-1 enhances the binding of Smad and Sp proteins to the SBE/Sp1 sites of c-met promoter. To delineate the mechanism underlying the necessity of both Smad signaling (Fig. 4) and Sp1 binding (Fig. 5) in TGF-1-mediated c-met induction, we studied the proteins bound to the SBE/Sp1 sites of the c-met promoter by using a biotinylated DNA precipitation approach. After biotinylated oligos containing the SBE/Sp1 sites corresponding to nucleotide position +13 +35 in c-met promoter were incubated with the HKC cell lysate, proteins bound to DNA were precipitated with streptavidine-agarose beads, followed by analysis by immunoblotting. As shown in Fig. 7, TGF-1 induced phosphorylated Smad2 binding to the SBE/Sp1 sites of the c-met promoter in a time-dependent fashion. Dramatic induction of activated Smad2 binding to SBE/Sp1 was observed in HKC cells at 0.5 and 1 h after TGF-1 treatment. As negative control, no binding occurred when the biotinylated oligo was omitted in the experiments. Similarly, TGF-1 also increased Smad4 binding to the SBE/Sp1 sites of the c-met promoter. Of interest, although both Sp1 and Sp3 proteins bound to the SBE/Sp1 sites under basal conditions, treatment of TGF-1 significantly enhanced their binding. These results suggest that TGF-1 stimulation not only induces Smad binding to SBE/Sp1 sites but also promotes the interaction between general transcription factors Sp1 and Sp3 and their cognate cis-acting element.
Comparable results were obtained when the EMSA approach was used. As shown in Fig. 8, when SBE/Sp1 oligo (F1) was incubated with nuclear protein extract derived from HKC cells at basal conditions, multiple DNA-protein complexes were formed with retarded migration in the polyacrylamide gel under nondenaturing conditions. TGF-1 stimulation not only strengthened the formation of these complexes, such as C1, but also induced three new shifted bands (C2 C4) located above C1 (Fig. 8A). Of note, the double bands below C1 (Fig. 8A) appeared to be specific; however, their abundance was not significantly altered after TGF-1 stimulation. In a competition assay, the complexes were completely abrogated in the presence of 100-fold molar excess of SBE/Sp1 oligo (F1). When F3 oligo containing only the SBE site or F5 oligo containing only the Sp1 site was used as a competitor, the majority of these complexes also disappeared (Fig. 8B). A supershift assay with specific antibodies revealed that both Sp1 and Sp3 were involved in the formation of these complexes (Fig. 8C).
Physical interaction between Sp1 and Smads. To assess a potential protein-protein interaction among Sp and Smad proteins, we preformed coimmunoprecipitation (IP) experiments to demonstrate any physical interactions between Smads and Sp1 in HKC cells after TGF-1 treatment. As presented in Fig. 9A, when the cell lysates derived from HKC cells treated without or with TGF-1 were immunoprecipitated with Sp1 antibody, Smad4 protein was detectable in the precipitated complexes, suggesting a physical interaction between Sp1 and Smad4. Such association between Sp1 and Smad was apparently TGF-1 dependent, because in the absence of TGF-1, there was no Smad4 in the precipitates by Sp1 antibody (Fig. 9A, lane 1). In the reciprocal experiments, when Smad2/3 was immunoprecipitated, Sp1 was detected in the precipitated complexes. Moreover, such association between Sp1 and Smad2/3 was also TGF-1 dependent (Fig. 9B, lanes 24). As expected, Smad2/3 and Smad4 could form complexes after TGF-1 incubation. Hence, Sp1 can interact with Smad proteins to form complexes in renal epithelial cells after TGF-1 stimulation.
DISCUSSION
Given the essential role of the HGF/c-met signaling system in such diverse cellular processes as cell survival, proliferation, migration, and differentiation, it is not difficult to appreciate that c-met expression is strictly regulated in various physiological and pathological conditions. Previous studies from our laboratory demonstrate a crucial role for the Sp family of transcription factors in controlling c-met constitutive expression in kidney as well as in various types of renal cells (28). The importance of Sp1 in c-met regulation in renal tubular epithelial cells is also illustrated by the observation that the downregulation of c-met after oxidative stress is mediated by Egr-1 sequestration of Sp1 as an activator of c-met gene transcription (29). In this report, we have demonstrated that c-met expression is markedly induced in the kidney after UUO; and such induction of c-met expression in diseased kidneys is likely mediated by a hyperactive TGF-1 signaling. Of interest, c-met induction by TGF-1 not only depends on Smad signaling, as one may expect, but also necessitates the ubiquitous Sp1 transcription factor for participation and cooperation. These observations suggest a novel regulatory mechanism dictating c-met expression, in which Smad and Sp1 can physically interact in a TGF-1-dependent manner and work in concert to enhance c-met transcription in renal epithelial cells. Together with previous findings, our studies provide a unique example to demonstrate how ubiquitous transcription factors such as Sp1 play a crucial role in regulating a particular gene’s expression in the kidney in multiple ways under different circumstances.
In response to chronic injuries that damage the kidney, renal cells initially undergo hypertrophy, hyperplasia, dedifferentiation, and/or apoptosis, adaptive changes that are important in an attempt to compensate for the lost functions. However, these compensatory alterations eventually fail to restore kidney functions and have been linked to progressive renal diseases. Both HGF and TGF-1 signaling systems are believed to play critical roles in the pathogenesis of chronic renal diseases (14, 40). Whereas HGF is demonstrated to promote injury repair and regeneration, TGF-1 is generally considered to be detrimental in promoting cell apoptosis and the tubular epithelial-to-mesenchymal transition (EMT) that gives rise to matrix-producing myofibroblasts (6, 9, 19, 45). Therefore, the observation that TGF-1 induces c-met expression as shown in this report illustrates a different picture of TGF-1 in chronic renal diseases, in which TGF-1 is linked through transcriptional regulation to a HGF/c-met signaling system that often displays beneficial effects. Of note, a previous report also demonstrated an increased c-met expression in obstructive nephropathy, although the role of TGF-1 in c-met induction was unknown in that study (15). We speculate that by inducing c-met expression, TGF-1 acts as a part of the tissue repair and wound- healing processes after injurious stimuli. This notion is consistent with the pleiotropic activities of TGF-1 in many developmental and pathological courses by influencing cell proliferation, apoptosis, and motility. Thus the initial activation of TGF-1 after tissue injury could be beneficial by reparation of damages, promotion of healing, and restoration of function. However, it is plausible to envision that a persistent, chronic exposure to hyperactive TGF-1 signaling will eventually overwhelm the system in favor of profibrotic effects, the hideous sides of TGF-1 actions. In accordance with this, c-met induction is significantly reduced in time points beyond 3 days after obstructive injury (Fig. 1), despite a sustained activation of TGF-1 in this model.
TGF-1 initiates its signaling transduction by interacting with its transmembrane serine/threonine kinase receptors, which leads to phosphorylation and activation of Smad transcription factors (28). When activated Smads enter into the nuclei, they recognize and bind to the SBE found in their target genes. There are two different consensus sequences for Smad binding [GTCTAGAC and AG(C/A)CAGACAC] (11, 35); both of them contain the core motif AGAC. This motif is present in the promoter of numerous TGF- target genes, including PAI-1 (10, 11, 37), Smad7 (35), p15Ink4B (14), and 2(I) collagen (48). Besides Smad signaling, many reports indicate that TGF-1 may also elicit its activity by activating several MAPKs, including p38 MAPK, Erk1/2, JNK in different cell systems (9, 29, 31, 42). However, c-met induction by TGF-1 in renal epithelial cells appears primarily dependent on Smad but not on MAPK pathways (Fig. 4). This conclusion is supported by several observations. First, a putative SBE (5'-AGACAGACAC-3') is found in the c-met promoter (Fig. 5A) that is functionally responsive to TGF-1 stimulation (Fig. 5B). Second, overexpression of Smad 7, an inhibitory Smad that competes with Smad2/3 to bind to the TGF- receptor and consequently blocks Smad signaling, completely abolishes c-met induction initiated by TGF-1 (Fig. 4A). Third, expression of the Smad transcriptional corepressor SnoN blocks SBE/Sp1-mediated gene transcription (Fig. 6). Finally, endogenous Smads in renal epithelial cells bind to the SBE of the c-met promoter upon TGF-1 stimulation (Fig. 7).
One of the interesting findings in this report is that c-met induction by TGF-1 is also totally dependent on proper binding and function of a ubiquitous transcription factor, Sp1, as ablation of Sp1 binding abrogates TGF-1-induced c-met promoter activity and mRNA and protein expression in renal epithelial cells (Fig. 5). The dependency of c-met induction on Sp1 is consistent with the observation that there is only a single copy of SBE identified in the c-met promoter. Because of low-affinity binding to DNA, Smads may have to cooperate with other DNA-binding proteins such as activator protein-1, TEF3, Sp1/Sp3 or p300/CBP (29) to bind efficiently to the SBE and initiate gene transcription. Studies elsewhere indicate that a single copy of SBE is not sufficient to induce TGF-1 target gene transcription. In many TGF-1-responsive genes such as PAI-1, multiple copies of SBE exist to confer TGF- inducibility. It is well known that multiplication of a weak cis-acting element would produce a substantial response. For example, multiplication of SBE (5'-AGACAGACAC-3') into nine copies in the reporter construct induces reporter luciferase activity by fourfold (11). Therefore, in the native c-met gene, cooperation of Sp1 with Smads is necessary for avid binding to the single copy of SBE and, thereby, for inducing c-met expression in response to TGF-1 stimulation.
The fact that the Sp1 site is located in close proximity to SBE in the c-met promoter provides ready access and availability for Sp1 and Smads to physically interact. Such interaction among members of Smad and Sp families of transcription factors are clearly exhibited by immunoprecipitation studies (Fig. 9). Of note, this type of cooperation between Sp1 and Smad for gene regulation is not without precedent. In the promoters of numerous TGF-1-responsive genes, such as PAI-1 (10), Smad7 (7), 5-integrin (18), and 2(I) collagen (37, 48), there are both SBE- and Sp1-binding motifs. Along this line, cooperation between Sp1 and Smad may represent a general regulatory mechanism for conferring the TGF-1 inducibility of many genes.
The interaction between Sp and Smad proteins is also highlighted by the finding that they bind to the DNA elements in a synergistic way. Under basal conditions with absence of TGF-1, the binding of Sp1/Sp3 to the SBE/Sp1 sites of the c-met promoter was low, as demonstrated by biotinylated DNA precipitation assay as well as EMSA (Figs. 7 and 8). The ability of Sp proteins to bind its cognate site in the c-met promoter is clearly enhanced after TGF-1 treatment (Figs. 7 and 8), suggesting that Smad activation may augment Sp1-binding capacity. Reciprocally, Smad binding to the SBE of the c-met promoter appears also to be dependent on Sp1 binding, as ablation of Sp1 binding with mithramycin A also abolished Smad binding in EMSA (Zhang X and Liu Y, unpublished observations). These results underscore that both Sp1 and Smad can promote the other's binding to the SBE/Sp1 sites of the c-met promoter in a mutually stimulating fashion. Given the fact that Sp1 and Smad are capable of forming complexes via protein-protein interaction in a TGF-1-dependent way, questions remain whether their interaction is contingent on their binding to DNA elements, respectively. However, regardless of the mechanism involved, the augmentation of binding to respective DNA elements by both Sp1 and Smad in a reciprocal, mutually stimulating manner highlights a potential mechanism by which Sp and Smad proteins cooperate in c-met induction after TGF-1 stimulation.
It should be pointed out that we were unable to detect Smad binding to c-met promoter by supershift assay in EMSA, despite the fact that Smad binding is clearly evident in biotinylated DNA precipitation assay (Fig. 7) and in competition experiments in EMSA (Fig. 8B). This phenomenon is also observed in the 2(I) collagen gene (37, 48). Poncelet and Schnaper (37) speculated that the failure of anti-Smad antibody to form the supershift in EMSA could be due to a relatively low abundance of Smad in these protein-DNA complexes. However, this may not readily explain the lack of Smad supershift in EMSA in the c-met promoter, because strong Smad bands that bound to DNA were detected in biotinylated DNA precipitation assay. One possible explanation could be that the interaction between Sp1/Sp3 and Smad changes the conformation of Smad and impairs the recognition domain in Smad protein for antibody appreciation.
Although c-met mRNA induction apparently occurs at the gene transcriptional level (Fig. 3), we cannot rule out the possibility that posttranscriptional regulation may also play a significant role in c-met expression. The latter is likely, particularly in view of the observation that the magnitude of c-met mRNA induction by TGF-1 is smaller than its protein (Fig. 2).
Studies in this report, together with previous observations (49, 50), have established that a general, ubiquitous transcription factor Sp1 plays a central role in c-met regulation in renal cells under different circumstances. Under basal conditions, Sp proteins are essential for establishing c-met constitutive expression in different types of renal cells through alterations in their cellular abundance (49). Furthermore, Sp1 mediates c-met suppression after oxidative stress by limiting its availability to c-met promoter through Egr-1-mediated sequestration (50). Herein, we have demonstrated that Sp1 is also crucial for c-met induction triggered by TGF-1 through interaction with Smad proteins. These studies not only set a foundation for unraveling the mechanism governing the transcriptional regulation of the c-met gene under different physiological and pathological conditions, but also suggest a unique model for understanding how the ubiquitous transcription factor Sp1 participates in controlling the constitutive, suppressive, and inducible expression of a single gene. Clearly, further studies using site-directed mutagenesis are needed to better characterize the multifaceted interactions among the trans-acting factors and cis-acting elements and to understand their functional role in controlling c-met gene expression in various conditions.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-054922, DK-061408, and DK-064005.
ACKNOWLEDGMENTS
We thank Drs. J. Massague and R. Weinberg for generously providing various plasmid vectors.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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