Hepatocyte growth factor (HGF) has recently emerged

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1 A Novel Mechanism by which Hepatocyte Growth Factor Blocks Tubular Epithelial to Mesenchymal Transition Junwei Yang,* Chunsun Dai,* and Youhua Liu* *Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Division of Nephrology, Department of Medicine, Nanjing Medical University, Nanjing, China. Hepatocyte growth factor (HGF) is a potent antifibrotic cytokine that blocks tubular epithelial to mesenchymal transition (EMT) induced by TGF- 1. However, the underlying mechanism remains largely unknown. This study investigated the signaling events that lead to HGF blockade of the TGF- 1 initiated EMT. Incubation of human kidney epithelial cells HKC with HGF only marginally affected the expression of TGF- 1 and its type I and type II receptors, suggesting that disruption of TGF- 1 signaling likely plays a critical role in mediating HGF inhibition of TGF- 1 action. However, HGF neither affected TGF- 1 induced Smad-2 phosphorylation and its subsequent nuclear translocation nor influenced the expression of inhibitory Smad-6 and -7 in tubular epithelial cells. HGF specifically induced the expression of Smad transcriptional co-repressor SnoN but not Ski and TG-interacting factor at both mrna and protein levels in HKC cells. SnoN physically interacted with activated Smad-2 by forming transcriptionally inactive complex and overrode the profibrotic action of TGF- 1. In vivo, HGF did not affect Smad-2 activation and its nuclear accumulation in tubular epithelium, but it restored SnoN protein abundance in the fibrotic kidney in obstructive nephropathy. Hence, HGF blocks EMT by antagonizing TGF- 1 s action via upregulating Smad transcriptional co-repressor SnoN expression. These findings not only identify a novel mode of interaction between the signals activated by HGF receptor tyrosine kinase and TGF- receptor serine/threonine kinases but also illustrate the feasibility of confining Smad activity as an effective strategy for blocking renal fibrosis. J Am Soc Nephrol 16: 68 78, doi: /ASN Hepatocyte growth factor (HGF) has recently emerged as a key antifibrotic cytokine that displays a potent therapeutic potential in preventing tissue fibrosis after chronic injury (1,2). The ability of HGF to inhibit tissue fibrotic lesions has been attested extensively in a wide variety of organs, including kidney (3 6). Growing evidence demonstrates that administration of HGF protein or its gene prevents the progressive loss of kidney functions in chronic renal diseases with diverse causes (7 11). Accordingly, blockade of HGF signaling by a neutralizing antibody markedly exacerbates the progression of renal fibrosis and dysfunctions (12,13). These studies establish that supplementation of exogenous HGF may provide an effective strategy for combating chronic renal insufficiency (14 16). Whereas the antifibrotic capacity of HGF has been increasingly recognized, the molecular mechanism underlying its actions remains largely unknown. Earlier studies revealed that administration of HGF markedly inhibits the expression of TGF- 1 (6,13), a well-characterized profibrotic cytokine, in fibrotic tissues in vivo. However, it remains a question whether Received September 29, Accepted September 17, Published online ahead of print. Publication date available at Address correspondence to: Dr. Youhua Liu, Department of Pathology, University of Pittsburgh School of Medicine, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA Phone: ; Fax: ; liuy@upmc.edu this reduction of TGF- 1 in vivo is the primary mechanism mediating HGF s actions or simply reflects a secondary consequence associated with reduced fibrotic lesions. The activity of TGF- 1 is tightly regulated by multiple levels of controlling mechanisms (17 21). Apart from the modulation of its expression, TGF- 1 signal transduction circuit is a subject of regulation by other signal inputs under different circumstances (17,19). Along this line, we hypothesized that HGF may antagonize TGF- 1 action in tubular epithelial cells through intercepting its signal transduction. TGF- 1 signals are transduced by transmembrane type II and type I receptors that contain a cytoplasmic serine/threonine kinase domain (18,22). Receptor activation initiates a cascade of signaling transduction events involving intracellular mediator Smad proteins (23). TGF- 1 specifically initiates Smad-2 and -3 phosphorylation, which in turn bind to the common partner Smad-4 and translocate into cell nuclei, where they control the transcription of TGF- responsive genes. Activated Smads may also assemble complexes with transcriptional co-repressors, making Smads transcriptionally inactive (24 26). Thus, Smad transcriptional co-repressors could be an important regulatory component that confines TGF- 1 signaling (26). Needless to say, disruption of any steps in this cascade of signal transduction processes may potentially disturb TGF- 1 signaling and result in blockage of EMT. In this study, we systematically dissected the signaling events that control HGF blockade of tubular epithelial to mesenchymal transition (EMT) induced by TGF- 1. Our results Copyright 2005 by the American Society of Nephrology ISSN: /

2 J Am Soc Nephrol 16: 68 78, 2005 HGF Blocks EMT by Upregulating SnoN 69 indicate that HGF fails to block Smad activation and nuclear translocation. Rather, HGF specifically upregulates Smad transcriptional co-repressor SnoN expression. Such SnoN induction leads to the formation of transcriptionally inactive SnoN/Smad complex and blocks TGF- 1 s action. These findings define a novel molecular pathway by which HGF antagonizes profibrotic TGF- 1 signaling. Materials and Methods Cell Culture, Cytokine Treatment, and Transient Transfection Human proximal tubular epithelial cells (HKC) were provided by Dr. L. Racusen (Johns Hopkins University, Baltimore, Maryland) (27). Cell culture and cytokine treatments were carried out according to the procedures described previously (7,28). Briefly, HKC cells were seeded in complete medium that contained 10% FBS at approximately 70% confluence. After an overnight incubation, cells were continuously incubated in serum-free medium for another 24 h, before addition of cytokines. Recombinant human TGF- 1 was purchased from R&D Systems (Minneapolis, MN). Recombinant human HGF protein was provided by Genentech Inc. (South San Francisco, CA). Chemical inhibitors PD98059, wortmannin, and SC68376 were purchased from Calbiochem (La Jolla, CA). Transient transfection of HKC cells was carried out by using the Lipofectamine 2000 according to the instructions specified by the manufacturer (Invitrogen, Carlsbad, CA). After transfection with equal amounts of HA-tagged SnoN expression plasmid (29) (provided by Dr. R. Weinberg, Massachusetts Institute of Technology, Cambridge, Massachusetts) or empty vector pcdna3 (Invitrogen), HKC cells were incubated in the absence or presence of 2 ng/ml TGF- 1 for 2 d. Whole-cell lysates were prepared and subjected to Western blot analyses. The stable cell lines overexpressing Smad-7 (HKC psmad7 ) and mock-transfection control (HKC pcdna3 ) were established as described previously (30). Northern Blot Analysis Total RNA isolation and Northern blot analysis for gene expression were carried out by the procedures described previously (31). The cdna probe for TGF- 1 was obtained from American Type Culture collection (ATCC, Manassas, VA). The SnoN cdna probe was provided by R. Weinberg. After autoradiography, membranes were stripped and rehybridized with rat glyceraldehyde-3-phosphate dehydrogenase probe to ensure equal loading of each lane. Western Blot Analysis The preparation of whole-cell lysates and kidney tissue homogenates and Western blot analysis of protein expression were performed according to the procedures described previously (7). The primary antibodies were obtained from following sources: anti -smooth muscle actin (anti -SMA) and anti extracellular signal-regulated kinase-1 and -2 (anti Erk-1/2; Sigma, St. Louis, MO); anti phospho-specific Erk-1/2, phospho-specific and total p38 mitogenactivated protein kinase (MAPK), and phospho-specific and total c-jun N- terminal kinase (Cell Signaling Technology, Inc., Beverley, MA); anti phospho-specific and total Smad-2 (Upstate, Charlottesville, VA); anti TGF- 1 (sc-146), anti TGF- type I receptor (anti-t RI; sc-398), anti TGF- type II receptor (anti-t RII; sc-17792), anti Smad-7 (sc-7004), anti Smad-6 (sc-13048), anti Smad-4 (sc-7066), anti Smad-2/3 (sc-6032), anti-sp1 (sc-420), anti c-ski (sc-9140), anti-snon (sc-9595), anti-tgif (sc-9826), and anti-actin (sc-1616; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and anti-fibronectin (clone 10) and anti E-cadherin (clone 36; BD Biosciences Pharmingen, San Jose, CA). Affinity-purified secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Immunofluorescence Staining Indirect immunofluorescence staining was performed using an established procedure (7). Cells were incubated with the specific primary antibodies described above, followed by staining with FITC- or cyanine dye Cy3-conjugated secondary antibodies. For some samples, cells were double stained with 4,6-diamidino-2-phenylindole, HCl to visualize the nuclei. For immunostaining of kidney sections, cryosections were stained with anti Smad-2/3 and anti-snon antibodies using the Vector M.O.M. immunodetection kit by the protocol specified by the manufacturer (Vector Laboratories, Burlingame, CA). The slides were then stained for the proximal tubular marker with fluorescein-conjugated lectin from Tetragonolobus purpureas (Sigma). Slides were viewed with a Nikon Eclipse E600 Epi-fluorescence microscope equipped with a digital camera (Melville, NY). In each experimental setting, immunofluorescence images were captured with identical light exposure times. Nuclear Protein Preparation HKC cells were subjected to various treatments with various growth factors for 30 min except where otherwise indicated. Cell nuclei were isolated by procedures described previously (32). After being collected by centrifugation at 5000 g for 30 min at 4 C, the nuclei were lysed with SDS sample buffer and subjected to Western blot analysis. Immunoprecipitation Immunoprecipitation was carried out by a procedure described elsewhere (32). HKC cell lysates were immunoprecipitated overnight at 4 C with 1 g of anti Smad-2/3, followed by precipitation with 20 l of protein A/G Plus-Agarose for 3 h at 4 C. After being washed, the immunoprecipitates were boiled for 5 min in SDS sample buffer, followed by immunoblotting with specific antibodies. Animals Male CD-1 mice that weighed approximately 18 to 22 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Unilateral ureteral obstruction (UUO) was performed using an established procedure (6,7). Human HGF expression plasmid (pcmv-hgf) and the empty vector (pcdna3) were administered to mice by rapid injection of naked plasmid solution through the tail vein, as described previously (6). Mice received an injection of plasmids before (day 1) and after (day 3) UUO, respectively. Groups of mice (n 5) were killed at 7 d after UUO, and the kidneys were removed. The therapeutic effects of HGF gene delivery in this mouse model were confirmed and reported previously (6). Statistical Analyses Statistical analysis of the data was performed using SigmaStat software (Jandel Scientific, San Rafael, CA). Comparison between groups was made using one-way ANOVA followed by Student-Newman- Kuels test. P 0.05 was considered significant. Results Regulation of TGF- 1 and Its Receptors by HGF in Tubular Epithelial Cells To gain insights into the mechanism underlying HGF blockade of TGF- 1 initiated tubular EMT, we first investigated the effect of HGF on TGF- 1 expression in tubular epithelial cells. As shown in Figure 1A, HGF did not significantly affect TGF- 1 mrna expression at 1, 6, and 12 h after treatment in HKC cells. At 24 h, moderate inhibition of TGF- 1 mrna level was noticeable, but it returned to the control level at 48 h. The

3 70 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 68 78, 2005 Figure 1. Regulation of TGF- 1 and its receptors by hepatocyte growth factor (HGF) in tubular epithelial cells. (A) Northern blot analysis demonstrates the steady-state level of TGF- 1 mrna in tubular epithelial HKC cells after HGF incubation. HKC cells were treated without or with 20 ng/ml HGF for various periods of time as indicated. Total cellular RNA samples were hybridized with cdna probes for TGF- 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. (B) Western blot analysis shows the protein abundance of TGF- 1 and TGF- type I and type II receptors (T RI and T RII) in tubular epithelial cells after various treatments. Whole-cell lysates derived from the HKC cells that were treated with HGF (20 ng/ml), TGF- 1 (2 ng/ml), or both for various periods of time as indicated were immunoblotted with specific antibodies against TGF- 1, T RI, T RII, and actin, respectively. The size and authenticity of TGF- 1 were confirmed by using the purified TGF- 1 protein in the adjacent lane (data not shown). level of active TGF- 1 in the supernatant of HKC cells was extremely low or undetectable as measured by a specific ELISA, and HGF did not alter the abundance of active TGF- 1 secreted by HKC cells (data not shown). Furthermore, Western blot of whole-cell lysates revealed that HGF did not affect TGF- 1 protein abundance at 24 and 48 h after incubation (Figure 1B). We further examined the potential effect of HGF on TGF- 1 receptor expression in tubular epithelial cells. As demonstrated in Figure 1B, treatment of HKC cells with HGF did not inhibit the abundance of T RI and T RII protein, as illustrated by Western blot analysis. In fact, a trivial increase in T RI level was noticed in HKC cells after stimulation with either HGF or TGF- 1. However, it returned to the control level when the cells were simultaneously incubated with both HGF and TGF- 1 (Figure 1B). Tubular EMT Is Dependent on Intracellular Smad Signaling We next explored the possibility that HGF blocks tubular EMT by interfering TGF- 1 signal transduction. To this end, we initially attempted to identify the TGF- 1 signaling pathway that is important for mediating EMT in HKC cells. A comprehensive screening of the activation profile of various signaling pathways stimulated by TGF- 1 in tubular epithelial cells was carried out by using phospho-specific antibodies. As shown in Figure 2, A through D, TGF- 1 stimulated Smad-2, Akt/protein kinase B, and p38 MAPK phosphorylation in a time-dependent manner. However, TGF- 1 seemed not to activate further Erk- 1/2 (Figure 2D) and c-jun N-terminal kinase (data not shown) in tubular epithelial cells. To determine which signal pathway initiated by TGF- 1 is critical for mediating EMT, we examined the effects of specific blockade of a particular signal pathway on the phenotypic conversion induced by TGF- 1. As shown in Figure 2E, blockade of Akt activation by wortmannin and p38 MAPK activation by SC68376 did not affect TGF- 1 induced suppression of epithelial E-cadherin and de novo expression of -SMA, two hallmarks of tubular EMT. Of note, these chemical inhibitors at the concentrations used were able to specifically block Akt and p38 MAPK activation initiated by TGF- 1 in HKC cells, as reported previously (28,30). We next found that disruption of Smad signaling by overexpressing inhibitory Smad-7 completely abolished TGF- 1 induced tubular EMT. As shown in Figure 2F, in cell line with mock transfection of pcdna3, TGF- 1 caused E-cadherin suppression and -SMA and fibronectin induction in a time-dependent manner, whereas TGF- 1 totally failed to induce the phenotypic conversion in cell line overexpressing Smad-7. Of note, overexpression of Smad-7 inhibits Smad-2 phosphorylation in HKC cells (30). Hence, it seems likely that TGF- 1 induced EMT is dependent on intracellular Smad signaling. HGF Neither Blocks Smad-2 Phosphorylation and Its Association with Smad-4 and Subsequent Nuclear Translocation nor Influences Inhibitory Smads Expression Having established that Smad signaling is crucial in mediating TGF- 1 induced EMT, we reasoned that HGF may block EMT by impairing Smad signal transduction. To test this hypothesis, we examined the effect of HGF on major Smad signaling events in a step-wise manner. As shown in Figure 3, treatment of HKC cells with TGF- 1 induced Smad-2 phosphorylation and activation. However, preincubation of the cells with HGF did not affect TGF- 1 induced Smad-2 phosphorylation (Figure 3A). We next examined whether HGF disturbs the TGF- 1 induced association between Smad-2/3 and Smad-4 by immunoprecipitation. As shown in Figure 3B, after stimulation with TGF- 1, Smad-4 was clearly present in the complexes precipitated by Smad-2/3 antibody, indicating that TGF- 1 induces Smad- 2/3 Smad-4 complex formation in HKC cells. However, HGF also failed to disrupt the physical association between Smad-2/3 and Smad-4 induced by TGF- 1 (Figure 3B). We further investigated whether HGF blocks activated Smad-2 nuclear translocation, because this process has been

4 J Am Soc Nephrol 16: 68 78, 2005 HGF Blocks EMT by Upregulating SnoN 71 Figure 2. Tubular epithelial to mesenchymal transition (EMT) induced by TGF- 1 is dependent on intracellular Smad signaling. (A through D) TGF- 1 triggers various signal events in tubular epithelial cells. HKC cells were incubated with 2 ng/ml TGF- 1 for various durations as indicated, and whole-cell lysates were immunoblotted with either phospho-specific or total Smad-2 (A), Akt (B), p38 mitogen-activated protein kinase (MAPK; C), and extracellular signal-regulated kinase-1 and -2 (Erk-1/2) MAPK (D). (E) Chemical blockade of MAPK and Akt activation does not affect TGF- 1 induced tubular EMT. HKC cells were pretreated with either various chemical inhibitors or vehicle (DMSO) for 30 min, followed by incubating in the absence or presence of 2 ng/ml TGF- 1 for 48 h. Specific inhibitors for phosphoinositide 3-kinase (10 nm wortmannin), Mek1 (10 M PD98059), and p38 MAPK (20 M SC68376) were used. (F) Overexpression of inhibitory Smad-7 completely blocks TGF- 1 induced EMT. Stable cell lines either overexpressing Smad-7 (HKC psmad-7 ) or with mock-transfection of empty vector (HKC pcdna3 ) were treated with 2 ng/ml TGF- 1 for various periods of time as indicated. Whole-cell lysates were immunoblotted with antibodies against Smad-7, E-cadherin, -smooth muscle actin ( -SMA), fibronectin, and actin, respectively. shown to be tightly regulated by HGF in renal interstitial fibroblasts (33). As presented in Figure 3C, HGF did not block activated Smad-2 nuclear translocation in tubular epithelial cells, as the nuclear abundance of phospho-smad-2 was virtually identical in HKC cells that were treated either with TGF- 1 alone or with TGF- 1 plus HGF. To confirm this result further, we used an indirect immunofluorescence staining to visualize the nuclear accumulation of activated Smad-2 in HKC cells after various treatments. Consistent with the biochemical assay, treatment of HKC cells with TGF- 1 induced marked accumulation of phospho-smad-2 in the nuclei (Figure 3E), and HGF apparently had no effect on this TGF- 1 initiated Smad-2 nuclear translocation (Figure 3G). Collectively, this series of experiments demonstrates that HGF blocks TGF- 1 mediated EMT by a mechanism independent of disruption of Smad-2 phosphorylation, its association with Smad-4, and its nuclear translocation. Because overexpression of inhibitory Smad-7 completely abolished TGF- 1 induced tubular EMT (Figure 2), we reasoned that HGF may antagonize TGF- 1 s action by upregulating inhibitory Smads expression. As shown in Figure 4, HGF also did not influence the protein expression of both Smad-6 and -7, two inhibitory members of the Smad protein family, at different time points in HKC cells. In addition, HGF exhibited no effects on common Smad-4 protein expression (Figure 4). HGF Specifically Induces Smad Transcriptional Co-repressor SnoN Expression We next analyzed the expression of Smad transcriptional co-repressors in HKC cells after treatment with HGF. As shown in Figure 5, HGF dramatically induced SnoN protein expression. This induction started as early as 1 h and peaked at 6 h after HGF incubation (Figure 5). Under the same conditions, HGF had little effect on the expression of Ski, a Smad transcriptional co-repressor that shares a high degree of homology with SnoN. The expression of TGIF, another Smad co-repressor that is upregulated by HGF through protein stabilization in mesangial cells (34), was extremely low or undetectable under both basal and HGF-stimulated conditions in HKC cells. Figure 5 also shows the localization of SnoN in tubular epithelial cells after HGF treatment. In control HKC cells, immunofluorescence staining for SnoN protein was essentially negative. However, treatment with HGF dramatically induced SnoN protein expression, which was exclusively localized in

5 72 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 68 78, 2005 Figure 3. HGF does not affect TGF- 1 induced Smad-2 phosphorylation and its binding to Smad-4 and subsequent nuclear translocation in tubular epithelial cells. HKC cells were pretreated with 20 ng/ml HGF for 30 min, followed by incubation with 2 ng/ml TGF- 1 for 45 min. (A) Whole-cell lysates were immunoblotted with antibodies against phospho-specific or total Smad-2, respectively. (B) Cell lysates were immunoprecipitated with anti Smad-2/3 antibody, followed by immunoblotting with antibodies against Smad-4 and Smad-2/3, respectively. (C) Nuclear protein lysates were prepared from HKC cells after various treatments as indicated and immunoblotted with anti phospho-specific Smad-2 antibody. The blot was also probed with antibody against Sp1 for normalization of the nuclear proteins loaded each lane. n.s. indicates nonspecific band. (D through G) Immunofluorescence staining for phospho-specific Smad-2 demonstrates that HGF does not block TGF- 1 mediated Smad-2 nuclear translocation. (D) Control. (E) TGF- 1 (2 ng/ml). (F) HGF (20 ng/ml). (G) TGF- 1 plus HGF. Figure 4. HGF does not affect inhibitory Smad expression in tubular epithelial cells. HKC cells were treated with 20 ng/ml HGF for various periods of time as indicated. Whole-cell lysates were immunoblotted with specific antibodies against Smad-4, Smad-6, Smad-7, and actin, respectively. the nuclei. To study whether SnoN protein induction by HGF is due to an enhanced protein stability or increased gene expression, we examined the SnoN mrna levels after HGF treatment in HKC cells by Northern blot analysis. As illustrated in Figure 5G, four transcripts of SnoN with the sizes of 6.2, 4.4, 3.2, and 2.1 kb were detected in tubular epithelial cells. These transcripts represent differential splicing variants (35). HGF markedly increased the steady-state mrna levels of SnoN in HKC cells. Such induction began as early as 0.5 h and peaked at 1 h after HGF treatment, a kinetics that significantly precedes SnoN protein induction. Hence, SnoN protein induction by HGF is primarily mediated by an increased gene expression in tubular epithelial cells. SnoN Forms Complex with Activated Smad-2 and Overrides the Profibrotic Action of TGF- 1 To understand how increased SnoN blocks TGF- 1 s action in tubular epithelial cells, we investigated whether SnoN can physically interact with activated Smad-2 once it has been translocated to the nuclei after TGF- 1 stimulation. As shown in Figure 6, in HKC cells that were treated with TGF- 1, phosphorylated Smad-2 (green) was translocated to the nuclei (Figure 6B). After treatment with HGF alone, SnoN (red) was induced and localized in the nuclei (Figure 6C). When HKC cells were treated with TGF- 1 plus HGF, both phosphorylated Smad-2 (green) and SnoN (red) were co-localized in the nuclei (yellow; Figure 6D). To reveal a potential interaction between activated Smad-2 and SnoN, we investigated Smad/SnoN complex formation in tubular epithelial cells by co-immunoprecipitation. As shown in Figure 6E, when cell lysates were precipitated with anti-snon antibody, phospho-smad-2 was detectable in the precipitates by Western blotting. There was a profound increase in the association between Smad-2 and SnoN in the cells that were treated with TGF- 1 plus HGF, compared with TGF- 1 or HGF treatment alone. This suggests that HGFinduced SnoN physically binds to the activated Smad-2 in a TGF- 1 dependent manner, which presumably sequesters activated Smad-2 from transactivation of its target genes. Analysis of the whole-cell lysates revealed that TGF- 1 by itself did not induce SnoN expression in HKC cells (Figure 6F). In addition, TGF- 1 did not affect HGF-induced SnoN expression. Identical magnitude of SnoN induction by HGF was observed in the absence or presence TGF- 1 (Figure 6F). To provide direct evidence for SnoN in mediating HGF inhibition of TGF- 1 action, we ectopically transfected SnoN expression vector into tubular epithelial cells, followed by as-

6 J Am Soc Nephrol 16: 68 78, 2005 HGF Blocks EMT by Upregulating SnoN 73 Figure 5. HGF specifically induces Smad transcriptional co-repressor SnoN expression in tubular epithelial cells. (A) Western blot analysis shows that HGF specifically induces the protein abundance of SnoN but not Ski and TG-interacting factor (TGIF) in tubular epithelial cells. HKC cells were treated with 50 ng/ml HGF for various periods of time as indicated. Whole-cell lysates were immunoblotted with specific antibodies against SnoN, Ski, TGIF, and actin, respectively. (B) Graphical presentation of the relative abundance of SnoN after treatment with HGF at various time points as indicated. Relative levels (fold induction over control) are presented after normalized with actin. (C through F) Immunofluorescence staining shows the localization of SnoN in the control (C and D) and HGF-treated cells (E and F). (C and E) Staining for SnoN. (D and F) Nuclear staining with 4,6-diamidino-2-phenylindole, HCl. (G) Northern blot analysis demonstrates SnoN mrna induction by HGF in HKC cells. Total RNA isolated from HKC cells that were treated without or with HGF for various periods of time as indicated was blotted with cdna probes for SnoN and GAPDH, respectively. The sizes of transcripts are indicated on the left. sessing their responsiveness to TGF- 1 stimulation. As shown in Figure 6G, forced expression of exogenous SnoN dramatically blocked TGF- 1 induced E-cadherin suppression and -SMA induction in a dose-dependent manner. However, transfection of empty vector in an identical manner did not affect TGF- 1 initiated tubular EMT. Similar results were obtained by using an indirect immunofluorescence staining approach (Figure 6, H through M). These results indicate that SnoN can effectively override the profibrotic action of TGF- 1. HGF Does not Affect Smad-2 Phosphorylation and Nuclear Accumulation in Tubular Epithelium In Vivo Earlier studies showed that administration of either HGF protein or its gene prevents tubular EMT and renal interstitial fibrosis induced by UUO (7). To investigate the potential mechanism underlying HGF s beneficial effect in the fibrotic kidney, we studied the effect of HGF on Smad signaling in vivo. As shown in Figure 7, there was a dramatic activation of Smad-2, as illustrated by a marked increase in phospho-smad-2 in the homogenates of obstructed kidney, suggesting an active TGF- 1 signaling in diseased kidneys. However, despite HGF s preventing renal EMT and fibrosis in this model, delivery of HGF gene did not block Smad-2 phosphorylation and activation. Figure 7B shows quantitative results derived from five animals per group. This finding is consistent with in vitro data (Figure 3), suggesting that HGF blockade of renal fibrosis in vivo is not mediated by inhibiting Smad activation. We further investigated Smad localization in normal and diseased kidney by an indirect immunofluorescence staining. As demonstrated in Figure 7C, there was abundant Smad-2/3 staining in renal tubular epithelium of sham-operated kidney, when using specific antibody against total Smad-2/3. However, Smads were apparently localized in the cytoplasm of tubular epithelial cells under normal conditions. In the obstructed kidney at 7 d after UUO, Smad-2/3 were predominantly localized in the nuclei of renal tubular epithelial cells, suggesting that Smads were activated and translocated into the nuclei. Administration of HGF did not prevent Smads activation and their nuclear accumulation in the fibrotic kidney (Figure 7E). HGF Restores SnoN Expression in the Fibrotic Kidney In view of the induction of SnoN by HGF in tubular epithelial cells in vitro, we examined the SnoN expression in the fibrotic kidney in vivo. Figure 8 shows SnoN protein expression in the

7 74 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 68 78, 2005 Figure 6. SnoN binds to activated Smad-2 and overrides the profibrotic action of TGF- 1 in tubular epithelial cells. (A through D) Double immunofluorescence staining for phospho-specific Smad-2 (green) and SnoN (red) in HKC cells after various treatments for 24 h. (A) Control. (B) TGF- 1 (2 ng/ml). (C) HGF (50 ng/ml). (D) TGF- 1 plus HGF. (E) Co-immunoprecipitation demonstrates that HGF induces an association between SnoN and activated Smad-2 in tubular epithelial cells. Cell lysates derived from various groups as indicated were immunoprecipitated with anti-snon antibody, followed by immunoblotting with antibodies against phospho-smad-2 and SnoN, respectively. (F) Whole-cell lysates derived from various groups as indicated were immunoblotted with antibodies against Smad-2, SnoN, and actin, respectively. (G) Ectopic expression of SnoN overrides the profibrotic action of TGF- 1 in tubular epithelial cells. HKC cells were transiently transfected with increasing amounts of HA-tagged SnoN expression vector or empty pcdna3 as indicated ( g/well) and followed by incubation in the absence or presence of 2 ng/ml TGF- 1 for 2 d. Whole-cell lysates were immunoblotted with antibodies against HA, E-cadherin, -SMA, and actin, respectively. (H through M) Immunofluorescence staining demonstrates the effects of SnoN overexpression on TGF- 1 mediated E-cadherin suppression and -SMA induction. HKC cells were transfected with either pcdna3 (I and L) or HA-tagged SnoN expression vector (J and M; 5 g/well), followed by incubation with 2 ng/ml TGF- 1 for 2 d. Cells were immunostained with antibodies against E-cadherin (H through J) or -SMA (K through M), respectively. (H and K) Control HKC cells. kidney of various treatment groups. In sham-operated kidney, abundant SnoN protein expression was observed by Western blot analysis of total tissue homogenates (Figure 8A). Immunofluorescence staining displayed that SnoN was primarily localized in the nuclei of tubular epithelium. After obstructive injury for 7 d, intrarenal SnoN protein was dramatically decreased, as described previously (26). After administration of HGF gene, SnoN expression was markedly induced in the obstructed kidney when compared with the pcdna3 controls (Figure 8). Both Western blot analysis and immunofluorescence staining revealed that HGF essentially restored the SnoN protein expression in the fibrotic kidney inhibited by ureteral obstruction. Hence, HGF may elicit its antifibrotic activity in vivo by confining profibrotic TGF- 1 signaling via restoring SnoN expression. Discussion Earlier studies have shown that HGF completely blocks TGF- 1 mediated tubular EMT (7), a phenotypic conversion that is thought to play an imperative role in the generation of the matrix-producing myofibroblasts (36,37). However, how exactly HGF antagonizes the TGF- 1 action in tubular epithelial cells remained unsolved. Thus, the aim of this study was to dissect the signaling events that lead to HGF blockade of TGF- 1 action. We demonstrated that HGF blocks TGF- 1 initiated EMT apparently by a mechanism independent of Smads phosphorylation and their subsequent nuclear translocation. Instead, HGF specifically induces Smad transcriptional co-repressor SnoN expression, which in turn binds to the activated Smads and consequently sequesters their ability to initiate gene transcription in tubular epithelial cells. These actions of HGF are largely recapitulated in the fibrotic kidney induced by obstructive injury in vivo. Therefore, our findings have uncovered a novel molecular pathway by which HGF specifically overrides the profibrotic action of TGF- 1. HGF is a potent antifibrotic factor that prevents the onset and progression of chronic renal disease in a variety of animal models (6,8,14). Although the precise mechanism by which HGF inhibits tissue fibrosis is not fully understood, its interplay with TGF- 1 has been suspected to play a crucial role in mediating its antifibrotic action (7,13). TGF- 1 is shown to inhibit HGF expression in various types of cells, including renal mesangial and endothelial cells (38). However, whether HGF reciprocally suppress TGF- 1 expression remains largely elusive. In

8 J Am Soc Nephrol 16: 68 78, 2005 HGF Blocks EMT by Upregulating SnoN 75 Figure 7. HGF does not inhibit Smad-2 phosphorylation and its nuclear translocation in renal tubular epithelium after obstructive injury in vivo. (A) Western blot demonstrates that delivery of HGF gene does not prevent Smad-2 phosphorylation in the obstructed kidney. Whole-tissue homogenates from different groups as indicated were immunoblotted with antibodies against phospho-specific and total Smad-2, respectively. Numbers (1, 2, and 3) indicate each individual animal in a given group. (B) Graphical presentation of the abundance of phospho-smad-2 after normalization with total Smad-2 in different groups. Relative levels (fold induction over sham control) are presented as means SEM of five animals per group. *P 0.01 versus the sham control. (C through E) Immunofluorescence staining shows the localization of Smad-2/3 in sham and obstructed kidneys. Renal cryosections were immunostained with anti Smad-2/3 antibody, followed by staining with FITC-conjugated lectin from T. purpureas. (C) Sham. (D) Unilateral ureteral obstruction (UUO) with pcdna3. (E) UUO with pcmv-hgf. vivo studies seem to suggest that HGF may inhibit TGF- 1 expression, because administration of HGF reduces TGF- 1 abundance in the fibrotic kidneys (7,13). However, the cause effect relationship of this TGF- 1 reduction in fibrotic kidney remains ambiguous. The present study indicates that HGF only marginally affects TGF- 1 and its receptor expression in tubular epithelial cells, consistent with a recent report indicating that HGF fails to modulate TGF- 1 expression in mesangial cells at basal condition (10). Such a trivial effect of HGF on TGF- 1 and its receptor expression in opposite manners (Figure 1) hardly accounts for its dramatic antifibrotic action. Hence, the primary mechanism for mediating HGF activity may operate through blocking hyperactive TGF- 1 signaling. Because TGF- 1 could induce itself and its receptor expression (39), it is conceivable that HGF blockade of TGF- 1 signaling would indirectly lead to an inhibition of TGF- 1 induction by uncoupling the TGF- 1 positive feedback loop under pathologic settings. In this context, we interpret the reduction of TGF- 1 in the diseased kidney after HGF treatment as a consequence associated with reduced fibrotic lesions. HGF and TGF- 1 transduce their signals by distinct mechanisms. One would expect a cross-talk in the signaling circuits mediated by HGF receptor tyrosine kinase and TGF- receptor serine-threonine kinases, to make it possible for HGF to counteract the profibrotic action of TGF- 1. Such cross-talk could theoretically take place at any point during the cascade of TGF- 1/Smad signaling events (33,40). There are at least three levels of negatively controlling mechanisms that confine TGF- 1 signaling along the Smad signal transduction pathway. In the extracellular compartment, active TGF- 1 can bind to decorin, a proteoglycan associated with the extracellular matrix, which leads to negative regulation of TGF- 1 activity (41). This provides a means of constraining TGF- 1 activity at the prereceptor stage. Smad signaling can also be negatively controlled by inhibitory Smad-7 and -6, which act as functional decoys that compete with the receptorregulated Smads (R-Smads) for binding to the activated type I receptor, leading to an attenuated R-Smad phosphorylation and activation (17,22). This level of regulation takes place in the cytoplasm. Once inside the nuclei, Smad signaling is finally subjected to a third level of negative regulation by Smad transcriptional co-repressors. It is intriguing that HGF seems not to interfere with Smad signaling in tubular epithelial cells until the assembly of Smad transcriptional complexes. Major TGF- 1 signaling events are still operative upon HGF treatment (Figure 3). By upregulating transcriptional antagonist SnoN, HGF receptor signaling leads to sequestering the ability of R-Smads to initiate the transactivation of TGF- 1 target genes. These findings underscore that HGF is capable of precisely targeting a final, decisive step in Smad signaling before the transcription of TGF- 1 responsive genes. Our observations also establish a novel mode of interactions between the signals activated by HGF receptor tyrosine kinase and TGF- receptor serine-threonine kinases ligands. SnoN is abundantly expressed and predominantly localized in the nuclei in normal tubular epithelium (Figure 8) (26).

9 76 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 68 78, 2005 Figure 8. HGF restores SnoN expression in obstructed kidney in vivo. (A) Representative Western blot demonstrates that HGF largely restores SnoN abundances by inducing its expression in the obstructed kidney. Whole-tissue homogenates were immunoblotted with antibodies against SnoN and actin, respectively. Numbers (1, 2, and 3) indicate each individual animal in a given group. (B) Graphical presentation of the relative abundance of SnoN after normalization with actin in different groups. Data are presented as means SEM of five animals per group. *P 0.01 versus sham; P 0.01 versus pcdna3 (n 5). (C through E) Immunofluorescence staining shows the localization of SnoN protein in the obstructed kidney at 7 d after surgery. (C) Sham. (D) UUO with pcdna3. (E) UUO with pcmv-hgf. Under physiologic conditions, a high level of SnoN would be important in preventing the activation of transcription by Smad proteins that may find their way into the nuclei (24). In this respect, SnoN functions as a crucial gatekeeper that tightly constrains Smad signaling under normal conditions. Consistently, knockdown of SnoN by sirna strategy dramatically sensitizes the tubular epithelial cells to respond to TGF- 1 stimulation (26). However, this confining mechanism seems not to operate in the fibrotic kidney, because SnoN protein abundance is significantly reduced (26). Consequently, Smad signal would be transduced without any restraint after TGF- 1 stimulation, leading to an amplified transcriptional activation of TGF- 1 responsive genes. The novel finding that HGF specifically upregulates SnoN expression provides a mechanistic insight into understanding the interplay between antifibrotic HGF and profibrotic TGF- 1 signaling. It becomes clear that the opposite effects of HGF and TGF- 1 in regulating myofibroblast activation and tissue fibrosis are coupled by SnoN expression. By inducing SnoN and restoring its level in the fibrotic kidney, HGF reinstates SnoN transcriptional repressor activity, leading to blockade of tubular EMT and renal fibrosis. Notably, administration of HGF could induce SnoN expression and block renal fibrosis even in the presence of Smad-2 phosphorylation and nuclear accumulation (Figure 7). This observation is significant in that it suggests that the levels of SnoN within the cell but not activated Smad determine an outcome of TGF- 1 response in vivo. In accordance with this, delayed administration of HGF, when the injury was already established, is still effective in reversing EMT and blocking renal fibrosis (9,10,15). In summary, the present study identifies a unique mode of interaction between the signals activated by HGF and TGF- 1 in tubular epithelial cells. It is conceivable that HGF can precisely target the hyperactive Smad signaling through inducing Smad transcriptional antagonist SnoN expression. Such specific interception of Smad signaling by HGF may eventually override the profibrotic action of TGF- 1 in the diseased kidneys, thereby halting the progression of chronic renal fibrosis. Acknowledgments This work was supported by National Institutes of Health grants DK054922, DK061408, and DK (Y.L.). J.Y. and C.D. were supported by postdoctoral fellowships from the American Heart Association Pennsylvania Delaware Affiliate. We thank Dr. R. Weinberg for generously providing plasmid vectors. References 1. Matsumoto K, Nakamura T: Hepatocyte growth factor: Renotropic role and potential therapeutics for renal diseases. Kidney Int 59: , Liu Y: Hepatocyte growth factor in kidney fibrosis: Therapeutic potential and mechanisms of action. Am J Physiol Renal Physiol 287: F7 F16, Mizuno S, Kurosawa T, Matsumoto K, Mizuno- Horikawa Y, Okamoto M, Nakamura T: Hepatocyte growth factor prevents renal fibrosis and dysfunction in a mouse model of chronic renal disease. J Clin Invest 101: , Ueki T, Kaneda Y, Tsutsui H, Nakanishi K, Sawa Y, Mor-

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