Regulation Of Smad Nuclear Transcription Co-repressor Factor (snon), The Molecular Mechanisms Of Renal Fibrosis

Objectives:Smad transcriptional co-repressor SnoN acts as an antagonist that tightlycontrols the trans-activation of TGF-β/Smad target genes and ensures that ahomeostasis of TGF-βsignal outputs in normal physiologic conditions. Ourprevious studies have demonstrated that SnoN protein is reducedprogressively in the fibrotic kidney after obstructive injury: and hepatocytegrowth factor (HGF) can antagonize the TGF-β1/Smad signaling by inductionof SnoN expression in proximal tubular epithelial cells. However, themechanisms underlying dysregulation of SnoN expression remain unknown.In this study, we investigated the regulation and mechanism of renal SnoNexpression in vivo, as well as the molecular mechanisms underlying the celltype-specific induction of SnoN by HGF. Smad ubiquitination regulatoryfactor-2 (Smurf2) is an E3 ubiqutin ligase that plays a pivotal role inregulating the TGF-βsignaling via selectively targeting key components ofSmad pathway for degradation. TGF-β1 plays a critical role in thedevelopment and progression of human kidney fibrotic diseases. In this study,we have also investigated the regulation of Smurf2 expression, its targetspecificity and the functional implication of its induction in the fibrotickidney.Methods: (1) The animal model of renal disease was induced by unilateral ureteralobstruction (UUO) in male CD-1 mice. Age and weight-matchedsham-operated mice were used as normal controls. Expression levels ofSnoN mRNA and protein were observed by Northern blot, RT-PCR andWestern blots assay. The ubiquitination activity and degradation of SnoNin the obstructed kidney were also assessed by an in vitro assay. Thisstudy initially explored the regulation and mechanism of SnoNexpression in obstructed kidney through detecting renal Smurf2expression and relationship between Smurf2 and SnoN by immunohisto-chemical staining and immunoprecipitation analysis.(2) Using human kidney proximal tubular epithelial cells as an in vitrosystem, this study further explored the molecular basis on the change ofSnoN expression by TGF-β1 and the effect of SnoN on TGF-β1-mediated fibrotic action by Western blot, RT-PCR, plasmidstransfection, immunofluorescence staining and siRNA strategy.(3) We explored the cellular signaling pathway by which HGF mediatesinduction of SnoN expression in HKC cells using methods of Westernblot, RT-PCR and plasmids transfection. Moreover, Chromatin immuno-precipitation assay was used to reveal the molecular mechanism of SnoNinduction by HGF on gene level.(4) The animal model of renal tubular-interstitial disease was induced by UUO in male CD-1 mice. On the day of surgery, mice were also receiveda single intravenous injection of naked HGF expression plasmid(pCMV-HGF) or empty vector pcDNA3. Then, the mechanism of SnoNinduction was further checked in vivo with western blot analysis andimmunohistochemical staining.(5) Smurf2 expression in renal tubules of human kidney biopsies of patientswith different nephropathies was detected by immunohistochemicalstaining.(6) In HKC cells, we explored the effect and mechanism of TGF-β1 onSmurf2 expression by RT-PCR, Western blot.(7) We further observed the effect of Smurf2 on the expression of mediatorsin TGF-β1/Smad signaling pathway by plasmids co-transfection. Then,luciferase assay and Western blot analysis were used to assess the actionof Smurf2 on regulation of gene transcription, E-cadherin and fibronectinby TGF-β1.Results:(1) Whereas SnoN protein was progressively diminished, its mRNA levelsremained relatively constant in the obstructed kidney after ureteralligation. An increased ubiquitination activity and proteasome-dependentdegradation of SnoN was found in obstructed kidney, compared withsham controls. Moreover, Smad ubiquitination regulatory factor-2, an E3 ubiquitin ligase, was induced and formed a complex with SnoN in theobstructive nephropathy.(2) In HKC cells, TGF-β1 promoted SnoN protein degradation, which wasmediated by an ubiquitination and a proteasome-dependent mechanism.SnoN constitutively interacted with another Smad co-repressor, Ski, andthey formed ternary complex with Smad2/3 upon TGF-β1 stimulation.However, ectopic expression of Ski did not alter the degradation rate ofSnoN. Stability of SnoN expression by proteasome inhibitor orknockdown of Smurf2 expression abolished TGF-β1-mediated (x-smoothmuscle actin and fibronectin induction, suggesting that SnoN degradationcould be necessary for TGF-β1 to exert its fibrogenic action.(3) In HKC cells, HGF-mediated SnoN induction is dependent on genetranscription and mediated by extracellular signal-regulated kinase-1 and-2 (Erk-1/2) signaling pathway. HGF rapidly activated both upstream anddownstream signaling of Erk-1/2, which led to the activation of thecAMP response element (CRE) binding protein (CREB). Chromatinimmunoprecipitation assay revealed that activated CREB and Sp 1 boundto their cognate cis-acting elements in SnoN promoter in response toHGF stimulation. Furthermore, HGF selectively induced CREBphosphorylation in HKC cells, but not in mesangial cells and fibroblasts.Moreover, activity of CREB and Spl are required for SnoN induction byHGF. (4) In vivo, administration of HGF gene induced renal Erk-1/2 phospho-rylation, CREB activation and SnoN expression in obstructivenephropathy.(5) Smurf2 was induced in renal tubules of human kidney biopsies ofpatients with different nephropathies by Immunohistochemical staining.In vitro, TGF-β1 can induce upregulation of Smurf2 mRNA and proteinexpression level. Overexpression of SnoN can completely block theinduction of Smurf2 by TGF-β1 in tubular epithelial cells.(6) Under basic condition, overexpression of Smurf2 can induce down-regulation of Smad2 and Smad transcriptional corepressors (SnoN, Ski andTGIF), and this downregulation by Smurf2 is mediated byproteasomal-dependant degradation in tubular epithelial cells. Moreover,overexpression of Smurf2 enhances the TGF-β1-mediated genetranscription, and amplifies the TGF-β1-mediated fibrogenic action intubular epithelial cells.(7) Overexpression of Smad2 and Smad3 can promote induction of fibronectinby TGF-β1, whereas downregulation of Smad2 and Smad3 by siRNAstrategy can block the induction of fibronectin. This result indicates thatSmad2 and Smad3 are required for fibronectin induction by TGF-β1 intubular epithelial cells.Conclusion (1) The downregulation of SnoN expression in the obstructed kidney ismediated by an enhanced ubiquitin-proteasomal-dependent degradation.(2) CREB activation, in concert with Spl, constitutes a molecular switch thatconfers the cell type-specific induction of SnoN in response to HGFstimulation.(3) Expression of Smurf2 is increased in renal tubules of fibrotic kidney, andTGF-β1 can induce Smurf2 expression in vitro by Smad signalingpathway. Our study also indicates that Smurf2 specifically targets bothpositive and negative Smad regulators for destruction in tubular epithelialcells, thereby providing a complex and fine-tuning of the TGF-βsignaling. These results appear that dysregulation of Smurf2 couldcontribute to an aberrant TGF-β/Smad signaling in the pathogenesis ofkidney fibrosis.