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BASIC SCIENCE |
1 Signaling
Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Correspondence to Dr. Youhua Liu, Department of Pathology, University of Pittsburgh School of Medicine, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: 412-648-1916; E-mail: liuy{at}upmc.edu
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Abstract |
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1 is a profibrotic cytokine that plays a central role in the onset and progression of chronic renal diseases. The activity of TGF-1 is tightly controlled by multiple mechanisms, in which antagonizing Smad-mediated gene transcription by co-repressors is an important regulatory component. This study examined the expression of Smad transcriptional co-repressors in the fibrotic kidney and investigated their potential functions in controlling TGF-1 response. Western blot analysis demonstrated that the protein levels of Smad transcriptional co-repressors SnoN and Ski were progressively reduced in a time-dependent manner in the fibrotic kidney induced by unilateral ureteral obstruction in mice, whereas renal Smad abundance was relatively unaltered. Consistently, SnoN and Ski staining was diminished in the nuclei of renal tubular epithelium and interstitium after obstructive injury. In vitro, knockdown of SnoN expression by RNA interference in tubular epithelial cells dramatically sensitized their responsiveness to TGF-1 stimulation. Conversely, ectopic expression of exogenous SnoN or Ski after transfection conferred tubular epithelial cell resistance to TGF-1–induced epithelial to myofibroblast transition. Both SnoN and Ski could block Smad-mediated activation of TGF-1–responsive promoter and exhibited additive effect in abrogating the profibrotic actions of TGF-1. These results indicate that as a result of loss of Smad transcriptional co-repressors, the profibrotic TGF-1 signaling in diseased kidney is markedly amplified in a magnitude much greater than previously thought. Therefore, new strategy aimed to increase Smad transcriptional co-repressors expression may be effective in antagonizing TGF-1 signaling and thereby blocking the progression of chronic renal fibrosis. |
Introduction |
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1 is a well-characterized profibrotic cytokine that is believed to play a crucial role in tissue fibrogenesis in a wide variety of organs (1–3). Extensive studies suggest that TGF-1 perhaps is one of the most important factors implicated in the onset and progression of various chronic renal diseases with diverse causes (4,5). Aberrant expression of TGF-1 and its receptors is reported in virtually all animal models of chronic renal diseases and in patients with chronic renal insufficiency (6,7). Overexpression of TGF-1 gene in transgenic mice leads to development of chronic renal fibrotic lesions (8,9). Conversely, inhibition of TGF-1 with different strategies in animal models ameliorates, at least partially, the fibrotic lesions and prevents progressive loss of renal functions (10–13).
Because TGF-1 plays an essential role in such diverse cellular processes as proliferation, differentiation, apoptosis, and extracellular matrix homeostasis (14,15), its activity is tightly regulated by multiple levels of controlling mechanisms (16). As one may expect, TGF-1 expression is meticulously regulated by diverse stimuli, including cytokines, growth factors, and extracellular environmental cues. TGF-1 protein is synthesized as an inactive precursor that includes a latency-associated peptide; hence, the release of active TGF-1 from latent complexes in pericellular compartment is also subjected to regulation by multiple factors, including plasmin and thrombospondin (3). In addition, the expression and activity of TGF-1 receptors are modulated under the pathologic conditions (17,18). Apart from these, recent studies demonstrate that the signal transduction processes of TGF-1 are also influenced by multiple inputs from other signaling pathways (19–21).
TGF-1 signals are transduced by transmembrane serine/threonine kinase type I and type II receptors and intracellular mediators known as Smad (22,23). Upon TGF-1 stimulation, Smad-2 and -3 are phosphorylated at serine residues in the carboxyl termini by the type I receptor (24). Such phosphorylation of Smad-2/3 induces their association with common partner Smad-4, and they subsequently translocate into the nuclei, where they control the transcription of TGF-1–responsive genes (1,16). Once inside the nuclei, activated Smad can interact with general transcriptional co-activators, resulting in transcriptional activation. Alternatively, they form transcriptionally inactive complexes with co-repressors (25,26). Thus, the relative levels of Smad transcriptional co-repressors present within the cell in a given circumstance may determine the ultimate outcome of a TGF-1 response.
Three Smad transcriptional co-repressors, namely Ski (Sloan-Kettering Institute proto-oncogene), SnoN (Ski-related novel gene, non Alu-containing), and TGIF (TG-interacting factor), have been identified (25,27–29). They exert transcriptional repression by numerous mechanisms (30–33). Clearly, Smad transcriptional co-repressors are unique regulatory components within the nuclei during the final stage of TGF-1 signaling; hence, their abundance and activity allow the cell to make the decision whether to proceed with the transcription of TGF-1–responsive genes. However, despite the obvious importance of Smad transcriptional co-repressors in controlling TGF-1 signaling, the regulation and relative significance of their expression in fibrotic kidney are completely unknown.
In this study, we demonstrate that the abundance of Smad transcriptional co-repressors SnoN and Ski are markedly reduced in the fibrotic kidney induced by unilateral ureteral obstruction (UUO) in mice. Such reduction of Smad antagonists greatly sensitizes renal epithelial cell responsiveness to TGF-1 stimulation. These results unravel a new level of controlling mechanism leading to amplification of profibrotic TGF-1 signaling in diseased kidney.
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Materials and Methods |
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Western Blot Analysis
Protein expression in kidney tissue and cultured cells was analyzed by Western blotting according to the procedures described previously (34). The primary antibodies used were as follows: anti–phospho-specific and total Smad-2 (Upstate, Charlottesville, VA), anti–c-Ski (sc-9140), anti-SnoN (sc-9595), anti-TGIF (sc-9826), anti–Smad-7 (sc-7004), anti–Smad-6 (sc-13048), anti–Smad-4 (sc-7966), anti–Smad-2/3 (sc-6032), anti-actin (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA), anti–E-cadherin (clone 36), anti-fibronectin (clone 10; Transduction Laboratories, Lexington, KY), and anti–
-smooth muscle actin (-SMA; clone 1A4; Sigma, St. Louis, MO).
Immunofluorescence Staining
Indirect immunofluorescence staining was performed using an established procedure (34). Briefly, kidney cryosections were prepared and mounted onto poly L-lysine–coated slides and fixed in 4% paraformaldehyde in PBS for 30 min. The slides were stained for Ski, SnoN, and Smad-2/3 (Santa Cruz Biotechnology) 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 tubules with Fluorescein-conjugated lectin from Tetragonolobus purpureas (Sigma). For visualizing the nuclei, slides were double-stained with 4',6'-diamidino-2-phenylindole, HCl. Stained sections were mounted with Vectashield anti-fade mounting medium (Vector Laboratories) and viewed with a Nikon Eclipse E600 Epi-fluorescence (Melville, NY).
Generation of Small Interfering RNA
Generation of sequence-specific small interfering RNA (siRNA) duplexes was carried out by using Silencer siRNA construction kit essentially according to the manufacturer’s instructions (Ambion, Austin, TX). Two siRNA oligonucleotide templates for targeting SnoN gene were designed and chemically synthesized by a commercial source (Invitrogen, Carlsbad, CA). Both sense and antisense templates contain 21 nt targeted gene-specific sequence and 8 nt leader sequence 5'-CCTGTCTC-3' that is complementary to the T7 promoter primer. An unrelated siRNA template (with no sequence homology) was also used to generate negative control siRNA. After siRNA templates were hybridized to a T7 promoter primer, the 3' ends were extended by the Klenow fragment of DNA polymerase to create double-stranded siRNA transcription templates, followed by in vitro transcription using T7 polymerase. The leader sequences were removed by digestion with a single-strand specific ribonuclease. After being purified by glass fiber filter binding and elusion, the resulting 21-mer siRNA were quantified by determination of ultraviolet absorbance at 260 nm and used for transfection into renal tubular epithelial cells.
Cell Culture and Transfection
Human proximal tubular epithelial cells (HKC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium supplemented with 10% FBS, as described previously (34). HKC cells were seeded at approximately 70% confluence in complete medium containing 10% FBS. Twenty-four hours later, the cells were subjected to transfection with various expression plasmids or siRNA. Transient transfection of HKC cells with HA-tagged Ski or SnoN expression vectors (provided by Dr. R. Weinberg, Massachusetts Institute of Technology), empty vector pcDNA3, or specific siRNA duplexes was carried out by using Lipofectamine 2000 according to the instructions specified by the manufacturer (Invitrogen). After transfection, HKC cells were incubated in the absence or presence of TGF-1 at the concentrations as indicated. Recombinant human TGF-1 was purchased from R & D Systems (Minneapolis, MN). Whole-cell lysates were prepared and subjected to various analyses.
Reporter Construct, Transfection, and Luciferase Assay
The reporter construct p3TP-Lux was provided by Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York, NY). For transient transfection, the HKC cells were seeded in six-well plates at 2 x 105 cells per well. The cells were then transfected with p3TP-Lux (1.0 µg), with or without Smad-2 (0.5 µg) and Smad-3 (0.5 µg) expression vectors, and pHA-hSnoN (0.5 µg) or pHA-hSki (0.5 µg) expression vectors. The amounts of plasmid DNA were equalized for each transfection by adding the empty vector pcDNA3. A fixed amount (0.5 µg) of internal control reporter Renilla reniformis luciferase driven under thymidine kinase (TK) promoter (pRL-TK; Promega, Madison, WI) was also co-transfected for normalizing the transfection efficiency. After transfection with Lipofectamine 2000 reagent, the cells were incubated for an additional 48 h in the absence or presence of 2 ng/ml TGF-1. Luciferase assay was performed using the Dual Luciferase Assay System kit essentially according to the manufacturer’s protocols (Promega). Relative luciferase activity of each construct (arbitrary unit) was reported as fold induction after normalizing for transfection efficiency.
Statistical Analyses
All data examined were expressed as mean ± SEM. Quantification of the Western blot data was performed by measuring the intensity of the hybridization signals using NIH Image analysis program. Statistical analysis of the data was performed using SigmaStat software (Jandel Scientific, San Rafael, CA). Comparison among groups was made using one-way ANOVA followed by Student-Newman-Kuels test. P < 0.05 was considered significant.
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Results |
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1 signaling, we used an RNA interference (RNAi) approach to knock down SnoN expression in cultured tubular epithelial (HKC) cells. This posttranscriptional gene silencing strategy has been demonstrated to knock down specific gene expression by using sequence-specific siRNA (36–38). Therefore, double-stranded siRNA were transiently transfected into HKC cells. Figure 5 shows the results of SnoN levels in HKC cells by Western blot analysis. Quantitative determination revealed that the SnoN protein levels were reduced by approximately 40 to 50%, when compared with control HKC cells or cells transfected with control siRNA. Under the same conditions, expression of unrelated genes such as actin (Figure 5b) and c-met receptor (data not shown) was unaltered, suggesting the specificity of SnoN inhibition by this RNAi strategy. Of note, such a moderate reduction of SnoN protein by RNAi inhibition was primarily due to a low efficiency of transient transfection in HKC cells, because only approximately 30 to 60% of the cell population was typically transfected by examining green fluorescent protein (GFP) expression in HKC cells using the same transfection method (Figure 5c).
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1 Stimulation1 stimulation in tubular epithelial cells with reduced endogenous SnoN. Hence, HKC cells after transient transfection with sequence-specific siRNA were treated with different concentrations of TGF-1. Loss of epithelial E-cadherin and induction of myofibroblast marker -SMA, two hallmarks of tubular epithelial myofibroblast transition (EMT) (17,39), were used to evaluate tubular cell responsiveness to TGF-1 stimulation. As shown in Figure 6, HKC cells after transfection with SnoN-specific siRNA displayed a greater responsiveness to TGF-1 stimulation, when compared with the parent HKC cells with mock transfection or the cells transfected with unrelated control siRNA. At the concentration as low as 0.01 ng/ml, TGF-1 suppressed E-cadherin expression by >60% in the cells transfected with SnoN siRNA, whereas the same concentration of TGF-1 displayed no effect on E-cadherin expression in the control cells (Figure 6, a and b).
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-SMA and fibronectin induction by TGF-1. As demonstrated in Figure 6, transfection of SnoN siRNA into HKC cells markedly sensitized their responsiveness to TGF-1 stimulation to induce de novo expression of -SMA. TGF-1 at the concentration of 0.01 ng/ml was capable of inducing significant -SMA expression in the HKC cells transfected with SnoN siRNA, whereas approximately 0.1 to 0.5 ng/ml TGF-1 was required to induce a similar level of -SMA expression in the control cells (Figure 6, a and c). Of interest, despite a moderate reduction of SnoN level by RNAi (Figure 5), the sensitivity of the cells to TGF-1 stimulation was increased by approximately 10-fold (Figure 6, b and c). This suggests that downregulation of SnoN dramatically lowers the threshold of TGF-1 responsiveness and amplifies TGF-1 signaling in tubular epithelial cells.
Ectopic Expression of SnoN and Ski Confers Resistance to TGF-1–Induced Tubular EMT
To confirm further the importance of Smad transcriptional co-repressors in controlling TGF-1 action, we used a reverse strategy by examining tubular cell responsiveness to TGF-1 after forced expression of exogenous SnoN or Ski. As shown in Figure 7, ectopic expression of SnoN by transfection conferred tubular epithelial cells resistance to TGF-1–induced epithelial to myofibroblast phenotypic transition, as illustrated by E-cadherin suppression and -SMA induction. TGF-1 at a concentration of 1 ng/ml induced E-cadherin suppression and -SMA expression in the control HKC cells transfected with empty vector pcDNA3. However, under the same conditions, TGF-1 essentially was unable to induce similar phenotypic conversion in HKC cells overexpressing SnoN protein. Even at a concentration as high as 10 ng/ml, TGF-1 was not capable of completely suppressing E-cadherin and inducing -SMA expression in SnoN-overexpressing HKC cells. Similar results were obtained when HKC cells were transfected with Ski expression vector (Figure 7, a and b). Figure 7b shows the dose-response curves of E-cadherin suppression in response to TGF-1 in HKC cells after transfection with pcDNA3, SnoN, or Ski expression vectors, respectively.
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1 in a dose-dependent manner. In the presence of the same concentration of TGF-1 (2 ng/ml), transfection of increasing amounts of SnoN or Ski expression vectors in HKC cells rendered a dose-dependent resistance to TGF-1–mediated E-cadherin suppression and -SMA induction. Thus, ectopic expression of SnoN or Ski can functionally override TGF-1’s action in tubular epithelial cells.
SnoN and Ski Block Smad-Mediated Gene Transcription
To elucidate whether SnoN and Ski elicit their repressive activity of TGF-1 by antagonizing Smad, we examined the effects of SnoN and Ski on the Smad-mediated gene transcription of TGF-1–responsive genes. To this end, we used luciferase reporter construct p3TP-Lux that has been widely used in the study of TGF-1 responsiveness in a variety of cell lines (40,41). When the p3TP-Lux plasmid was transfected into HKC cells, luciferase activity was increased by more than threefold after stimulation with TGF-1 (Figure 8a). However, co-transfection with either SnoN or Ski expression vectors dramatically repressed endogenous Smad-mediated induction of luciferase activity in HKC cells (Figure 8a). Of note, the relative fold induction of the luciferase activities after TGF-1 treatment under each condition was similar (P > 0.05; Figure 8a). Figure 8b shows that forced expression of exogenous Smad-2/3 in HKC cells increased the promoter activity in the absence or presence of TGF-1. Such exogenous Smad-mediated induction of the promoter activity could also be repressed by co-transfection with either SnoN or Ski (Figure 8b). Similar results were observed when Smad-2 and Smad-3 expression vectors were transfected separately (Figure 8c).
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1 Activity1 signaling. As shown in Figure 9, co-transfection of SnoN and Ski expression vectors exhibited an additive effect in restoring E-cadherin expression repressed by TGF-1. Likewise, SnoN and Ski also displayed an additive effect in abolishing TGF-1–induced -SMA and fibronectin expression in HKC cells (Figure 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 signaling in the pathogenesis of chronic renal diseases has been widely recognized (1,2,42), previous studies largely focused on TGF-1 expression in the diseased kidney. In the present study, we demonstrated a dramatic reduction of Smad transcriptional co-repressors Ski and SnoN in the fibrotic kidney after obstructive injury. This occurs in vivo on top of the well-documented TGF-1 and its receptor induction in this model (17,43). Because SnoN and Ski function as Smad transcriptional antagonists, their loss represents a critical step in eliminating a crucial negative controlling mechanism for TGF-1 action, which would lead to TGF-1 signal transduction without any restraint in the kidney under pathologic conditions. These studies provide the first demonstration that loss of Smad co-repressors is an important mechanism that amplifies the profibrotic TGF-1 signaling and perhaps plays a crucial role in the pathogenesis of chronic renal fibrosis.
The regulation of TGF-1 activity is operated at both prereceptor and postreceptor stages through a multitude of regulatory components (1,16), which include the TGF-1 gene expression, latent TGF-1 activation and its receptors expression, and the postreceptor Smad signaling. Dysregulation of TGF-1 expression and its posttranslational activation and receptors expression has been implicated in the pathogenesis of the fibrotic lesions in a wide variety of chronic renal diseases (1,7,42). However, little was known about the role of different components of Smad signaling in the development of chronic renal insufficiency. Upon examination, it was found that the expression levels of various types of Smad exhibit no significant changes at different time points in obstructive nephropathy. These observations underscore that despite its progressive nature, renal fibrogenesis after obstructive injury is not associated with significant alterations in Smad expression. Rather, the major change in various regulatory components of TGF-1 signaling is the loss of Smad transcriptional co-repressors SnoN and Ski. It is interesting that such loss of Smad co-repressors takes place progressively, which closely imitates the course of renal fibrosis in this model. At present, it remains ambiguous what triggers the downregulaton of Smad co-repressors in the obstructed kidney. Earlier in vitro studies suggested that TGF-1 itself may play a role in the degradation of Smad co-repressors (28,29). However, the possibility remains that SnoN and Ski expression may be transcriptionally suppressed under pathologic conditions. Regardless of the mechanism involved, diminishing of Smad co-repressors in the diseased kidney would create an ideal "profibrotic" environment in which TGF-1 signal is transduced in such a way without any constraint and perhaps is out of control, a scenario much akin to the loss of tumor suppressor gene during tumorigenesis.
Antagonizing Smad-mediated gene transcription by co-repressors is likely an effective way to confine TGF-1 signaling. In normal kidney, abundant SnoN and Ski are present in the nuclei of renal cells (Figure 2), which could safeguard and prevent unwanted TGF-1 response, in case Smad find their way into the nuclei. Unlike other regulatory mechanisms, including the modulation of TGF-1 expression and its pericellular activation, suppression of transcriptional activation by Smad co-repressors is unique in that it occurs inside the nuclei during the final stage of TGF-1 signaling, right before the transcription of TGF-1 target genes. The strategic position of Smad co-repressors in the regulatory circuit of TGF-1 signaling renders them a matchless capacity to dictate an ultimate response of the cells to TGF-1 stimulation. This speculation has been validated in this study by using two different approaches. Despite a moderate reduction of SnoN (<50%) in tubular epithelial cells by RNAi strategy, it greatly lowers the threshold of TGF-1 response, enabling the cells to respond to TGF-1 at a concentration as low as 0.01 ng/ml (Figure 6). Conversely, ectopic expression of SnoN via transient transfection lifts up the threshold, conferring the cells’ resistance to TGF-1 at a concentration as high as 10 ng/ml (Figure 7). It should be emphasized that the ability of Smad transcriptional co-repressors to antagonize TGF-1 action is clearly underestimated in this study, because only approximately 30 to 60% of tubular epithelial cells were actually transfected by using this transient transfection protocol (Figure 5c). Hence, the real impact of Smad co-repressor abundance to TGF-1 response is possibly even greater in vivo. In view of the facts that both SnoN and Ski are reduced by 90% in the kidney at 14 d after obstructive injury (Figure 1) and that SnoN and Ski exhibit an additive effect in antagonizing TGF-1 action (Figure 9), it is reasonable to postulate that the loss of Smad transcriptional co-repressors in the fibrotic kidney is one of the key events that have greatest influence on the ultimate outcome of TGF-1 signaling and renal fibrogenesis.
Several mechanisms have been proposed to explain how co-repressors block Smad-mediated gene transcription (30–33). It is reported that the co-repressor proteins can physically bind to and interact with activated Smad in the nuclei. By doing so, the transactivation capacity of Smad is sequestered by co-repressors (31). In addition, SnoN and Ski may displace the general transcriptional coactivators in the transcriptional complexes formed between Smad and coactivators. Furthermore, members of Smad co-repressor family proteins such as TGIF have the ability to interact directly with other transcriptional repressors such as mSin3 and to recruit it to a TGF-–activated Smad complex, leading to repression of a TGF- transcriptional response (33). Irrespective of the mechanism, our observation on downregulation of SnoN and Ski after obstructive injury sheds new light on understanding the precise physiologic and pathologic roles of Smad co-repressors in the setting of chronic renal fibrosis. By virtue of eliminating a confining mechanism, it becomes clear that the profibrotic TGF-1 signaling is dramatically amplified in the diseased kidney in a magnitude much greater than previously envisioned. This may explain why it is so difficult to blunt completely TGF-1 signaling and halt the progression of chronic renal fibrosis.
The present study may have significant implications in designing future therapeutic strategies for the treatment of chronic renal diseases. Given the vital role of hyperactive TGF-1 signaling in chronic renal fibrosis, substantial efforts have been made to develop therapeutic strategies targeted to TGF-1 expression and/or its activity by diverse methods such as using neutralizing antibody or antisense inhibition (5,10–13). However, the results of the present study suggest that because of the almost complete eradication of the repressors for Smad signaling, kidney cells in chronic disease settings are extremely susceptible to TGF-1 stimulation. As the threshold of TGF-1 response is markedly reduced, only a minimal amount of TGF-1 is necessary and sufficient to trigger a full-scale TGF-1 response under chronic disease conditions. Therefore, to normalize completely TGF-1 signaling in the diseased kidney, one would need to decrease TGF-1 to a level much lower than that in normal kidney, to compensate for the loss of Smad co-repressors. Such a task may be impossible to accomplish. In this respect, restoring Smad transcriptional co-repressors may be more effective than inhibiting TGF-1 itself, because slight modulation of Smad co-repressor levels would have enormous impacts on the transcription of TGF-1–responsive genes. Along this line, future studies are warranted to identify the factors and agents that can increase the threshold of TGF-1 response by upregulating Smad transcriptional co-repressor expression in vivo.
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Acknowledgments |
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We thank Drs. J. Massague and R. Weinberg for generously providing various expression vectors.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 signaling in renal disease. J Am Soc Nephrol 13: 2600–2610, 20021 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling. J Biol Chem 278: 12537–12545, 2003/Smad signaling by oncogenic Ras. Genes Dev 13: 804–816, 1999This article has been cited by other articles:
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  S. Kroening, S. Solomovitch, M. Sachs, B. Wullich, and M. Goppelt-Struebe Regulation of connective tissue growth factor (CTGF) by hepatocyte growth factor in human tubular epithelial cells Nephrol. Dial. Transplant., October 1, 2008; (2008) gfn530v1. [Abstract] [Full Text] [PDF]
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 R. Tan, W. He, X. Lin, L. P. Kiss, and Y. Liu Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1076 - F1083. [Abstract] [Full Text] [PDF]
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P. Boor, K. Sebekova, T. Ostendorf, and J. Floege Treatment targets in renal fibrosis Nephrol. Dial. Transplant., December 1, 2007; 22(12): 3391 - 3407. [Full Text] [PDF]
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 Y. Li, C. Dai, C. Wu, and Y. Liu PINCH-1 Promotes Tubular Epithelial-to-Mesenchymal Transition by Interacting with Integrin-Linked Kinase J. Am. Soc. Nephrol., September 1, 2007; 18(9): 2534 - 2543. [Full Text] [PDF]
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R. Tan, X. Zhang, J. Yang, Y. Li, and Y. Liu Molecular Basis for the Cell Type Specific Induction of SnoN Expression by Hepatocyte Growth Factor J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2340 - 2349. [Abstract] [Full Text] [PDF]
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M. Yoshikawa, K. Hishikawa, T. Marumo, and T. Fujita Inhibition of Histone Deacetylase Activity Suppresses Epithelial-to-Mesenchymal Transition Induced by TGF-beta1 in Human Renal Epithelial Cells J. Am. Soc. Nephrol., January 1, 2007; 18(1): 58 - 65. [Abstract] [Full Text] [PDF]
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R. Tan, J. Zhang, X. Tan, X. Zhang, J. Yang, and Y. Liu Downregulation of SnoN Expression in Obstructive Nephropathy Is Mediated by an Enhanced Ubiquitin-Dependent Degradation J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2781 - 2791. [Abstract] [Full Text] [PDF]
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 K. Okano, H. W. Schnaper, K. Bomsztyk, and T. Hayashida RACK1 Binds to Smad3 to Modulate Transforming Growth Factor-beta1-stimulated {alpha}2(I) Collagen Transcription in Renal Tubular Epithelial Cells J. Biol. Chem., September 8, 2006; 281(36): 26196 - 26204. [Abstract] [Full Text] [PDF]
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 J.-L. Xia, C. Dai, G. K. Michalopoulos, and Y. Liu Hepatocyte Growth Factor Attenuates Liver Fibrosis Induced by Bile Duct Ligation Am. J. Pathol., May 1, 2006; 168(5): 1500 - 1512. [Abstract] [Full Text] [PDF]
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J. Yang, C. Dai, and Y. Liu A Novel Mechanism by which Hepatocyte Growth Factor Blocks Tubular Epithelial to Mesenchymal Transition J. Am. Soc. Nephrol., January 1, 2005; 16(1): 68 - 78. [Abstract] [Full Text] [PDF]
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Y. Liu Hepatocyte growth factor in kidney fibrosis: therapeutic potential and mechanisms of action Am J Physiol Renal Physiol, July 1, 2004; 287(1): F7 - F16. [Abstract] [Full Text] [PDF]
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Y. Liu Epithelial to Mesenchymal Transition in Renal Fibrogenesis: Pathologic Significance, Molecular Mechanism, and Therapeutic Intervention J. Am. Soc. Nephrol., January 1, 2004; 15(1): 1 - 12. [Abstract] [Full Text] [PDF]
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P < 0.01 versus pcDNA3 group in the presence of TGF-
) or presence (**) of TGF-