Cell Phenotype-specific Down-regulation of Smad3 Involves Decreased Gene Activation as Well as Protein Degradation

Anne-Christine Poncelet,Youhua Liu,Constance E Runyan,H William Schnaper,Ruoyun Tan

doi:10.1074/jbc.m701991200

Abstract

Signaling by transforming growth factor-beta (TGF-beta), a regulator of several biological processes, including renal fibrosis, is mediated, in part, by the Smad proteins. Tight control of Smad level and activity is critical for proper TGF-beta biological functions. Here, we have investigated the mechanisms involved in regulating Smad3 expression. In human glomerular mesangial cells, Smad3 protein levels were specifically reduced by 24 h of TGF-beta1 treatment, whereas Smad2 and Smad4 levels were not. TGF-beta1 increased endogenous Smad3 ubiquitination, and proteasome inhibitor treatment blocked TGF-beta1-mediated Smad3 down-regulation resulting in accumulation of ubiquitinated Smad3. These data support the concept that Smad3 down-regulation occurs via degradation by the ubiquitin/proteasome machinery. However, changes in Smad3 protein levels were also paralleled by changes in Smad3 mRNA expression. TGF-beta1 did not decrease Smad3 mRNA stability, but it significantly inhibited Smad3 promoter activity. In renal tubular epithelial cells, decreased Smad3 levels were observed only after exposure to TGF-beta1 for longer time periods (5-7 days) that paralleled epithelial-to-mesenchymal transition, as determined by increased expression of smooth muscle alpha-actin and decreased expression of E-cadherin. Decline in Smad3 expression also occurred in kidneys after unilateral ureteral obstruction, a model of tubulointerstitial fibrosis associated with TGF-beta up-regulation and epithelial-to-mesenchymal transition. Our data show for the first time that TGF-beta1 modulates the expression of a receptor-activated Smad at both the protein and transcriptional level. Smad3 down-regulation could represent a feedback loop controlling TGF-beta signaling in a cell phenotype-specific manner.

Highlights

Glomerulosclerosis and tubulointerstitial fibrosis are the consequences of excessive extracellular matrix (ECM)2 accumulation and lead to end-stage renal failure [1]
Role of the Ubiquitin-Proteasome Pathway—Because polyubiquitination is generally considered to be a marker of protein degradation by the proteasome pathway [27], we examined the extent of Smad3 ubiquitination in response to Transforming growth factor-␤ (TGF-␤)1
Because Smad3 plays a critical role in wound healing and the pathogenesis of fibrosis, determining how Smad3 is regulated is essential to understanding the mechanisms of these processes

Summary

Introduction

Glomerulosclerosis and tubulointerstitial fibrosis are the consequences of excessive extracellular matrix (ECM)2 accumulation and lead to end-stage renal failure [1]. Regulation of Smad3 Promoter Activity—Our results in Fig. 4B suggest that TGF-␤1 inhibits mesangial cell Smad3 expression by a transcriptional mechanism. These data suggest that, in addition to decreasing Smad3 protein stability via the ubiquitin-proteasome pathway (Fig. 2), TGF-␤1 inhibits Smad3 gene transcription.

Results

Conclusion