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 inhib- itory 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, 2005. doi: 10.1681/ASN.2003090795 epatocyte growth factor (HGF) has recently emerged this reduction of TGF-␤1 in vivo is the primary mechanism as a key antifibrotic cytokine that displays a potent mediating HGF’s actions or simply reflects a secondary conse- therapeutic potential in preventing tissue fibrosis af- quence associated with reduced fibrotic lesions. The activity of H ␤ ter chronic injury (1,2). The ability of HGF to inhibit tissue TGF- 1 is tightly regulated by multiple levels of controlling fibrotic lesions has been attested extensively in a wide variety mechanisms (17–21). Apart from the modulation of its expres- of organs, including kidney (3–6). Growing evidence demon- sion, TGF-␤1 signal transduction circuit is a subject of regula- strates that administration of HGF protein or its gene prevents tion by other signal inputs under different circumstances the progressive loss of kidney functions in chronic renal dis- (17,19). Along this line, we hypothesized that HGF may antag- eases with diverse causes (7–11). Accordingly, blockade of HGF onize TGF-␤1 action in tubular epithelial cells through inter- signaling by a neutralizing antibody markedly exacerbates the cepting its signal transduction. progression of renal fibrosis and dysfunctions (12,13). These TGF-␤1 signals are transduced by transmembrane type II and studies establish that supplementation of exogenous HGF may type I receptors that contain a cytoplasmic serine/threonine provide an effective strategy for combating chronic renal insuf- kinase domain (18,22). Receptor activation initiates a cascade of ficiency (14–16). signaling transduction events involving intracellular mediator Whereas the antifibrotic capacity of HGF has been increas- Smad proteins (23). TGF-␤1 specifically initiates Smad-2 and -3 ingly recognized, the molecular mechanism underlying its ac- phosphorylation, which in turn bind to the common partner tions remains largely unknown. Earlier studies revealed that Smad-4 and translocate into cell nuclei, where they control the administration of HGF markedly inhibits the expression of transcription of TGF-␤–responsive genes. Activated Smads TGF-␤1 (6,13), a well-characterized profibrotic cytokine, in fi- may also assemble complexes with transcriptional co-repres- brotic tissues in vivo. However, it remains a question whether sors, making Smads transcriptionally inactive (24–26). Thus, Smad transcriptional co-repressors could be an important reg- ulatory component that confines TGF-␤1 signaling (26). Need- Received September 29, 2003. Accepted September 17, 2004. less to say, disruption of any steps in this cascade of signal transduction processes may potentially disturb TGF-␤1 signal- Published online ahead of print. Publication date available at www.jasn.org. ing and result in blockage of EMT. Address correspondence to: Dr. Youhua Liu, Department of Pathology, Univer- In this study, we systematically dissected the signaling sity of Pittsburgh School of Medicine, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: 412-648-1916; events that control HGF blockade of tubular epithelial to mes- E-mail: [email protected] enchymal transition (EMT) induced by TGF-␤1. Our results Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1601-0068 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 Immunofluorescence Staining translocation. Rather, HGF specifically upregulates Smad tran- Indirect immunofluorescence staining was performed using an es- scriptional co-repressor SnoN expression. Such SnoN induction tablished procedure (7). Cells were incubated with the specific primary leads to the formation of transcriptionally inactive SnoN/Smad antibodies described above, followed by staining with FITC- or cyanine complex and blocks TGF-␤1’s action. These findings define a dye Cy3-conjugated secondary antibodies. For some samples, cells were double stained with 4Ј,6-diamidino-2-phenylindole, HCl to visu- novel molecular pathway by which HGF antagonizes profi- alize the nuclei. For immunostaining of kidney sections, cryosections brotic TGF-␤1 signaling. 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 Materials and Methods manufacturer (Vector Laboratories, Burlingame, CA). The slides were Cell Culture, Cytokine Treatment, and Transient then stained for the proximal tubular marker with fluorescein-conju- Transfection gated lectin from Tetragonolobus purpureas (Sigma). Slides were viewed Human proximal tubular epithelial cells (HKC) were provided by Dr. with a Nikon Eclipse E600 Epi-fluorescence microscope equipped with L. Racusen (Johns Hopkins University, Baltimore, Maryland) (27). Cell a digital camera (Melville, NY). In each experimental setting, immuno- culture and cytokine treatments were carried out according to the fluorescence images were captured with identical light exposure times. procedures described previously (7,28). Briefly, HKC cells were seeded in complete medium that contained 10% FBS at approximately 70% Nuclear Protein Preparation confluence. After an overnight incubation, cells were continuously HKC cells were subjected to various treatments with various growth incubated in serum-free medium for another 24 h, before addition of factors for 30 min except where otherwise indicated. Cell nuclei were cytokines. Recombinant human TGF-␤1 was purchased fromR&D isolated by procedures described previously (32). After being collected Systems (Minneapolis, MN). Recombinant human HGF protein was by centrifugation at 5000 ϫ g for 30 min at 4°C, the nuclei were lysed provided by Genentech Inc. (South San Francisco, CA). Chemical in- with SDS sample buffer and subjected to Western blot analysis. hibitors 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 instruc- Immunoprecipitation tions specified by the manufacturer (Invitrogen, Carlsbad, CA). After Immunoprecipitation was carried out by a procedure described else- transfection with equal amounts of HA-tagged SnoN expression plas- where (32). HKC cell lysates were immunoprecipitated overnight at 4°C ␮ ␮ mid (29) (provided by Dr. R. Weinberg, Massachusetts Institute of with 1 g of anti–Smad-2/3, followed by precipitation with 20 lof Technology, Cambridge, Massachusetts) or empty vector pcDNA3 (In- protein A/G Plus-Agarose for3hat4°C. After being washed, the vitrogen), HKC cells were incubated in the absence or presence of 2 immunoprecipitates were boiled for 5 min in SDS sample buffer, fol- ng/ml TGF-␤1 for 2 d. Whole-cell lysates were prepared and subjected lowed by immunoblotting with specific antibodies. to Western blot analyses. The stable cell lines overexpressing Smad-7 (HKCpSmad7) and mock-transfection control (HKCpcDNA3) were estab- Animals lished as described previously (30). Male CD-1 mice that weighed approximately 18 to 22 g were pur- chased from Harlan Sprague Dawley (Indianapolis, IN). Unilateral Northern Blot Analysis ureteral obstruction (UUO) was performed using an established proce- Total RNA isolation and Northern blot analysis for gene expression dure (6,7). Human HGF expression plasmid (pCMV-HGF) and the were carried out by the procedures described previously (31). The empty