title: Cell phenotype-specific down-regulation of Smad3 involves decreased gene activation as well as protein degradation.
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.
authors.affiliation: Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98109, USA. annechr@u.washington.edu
authors.forename: Anne-Christine
authors.initials: AC
authors.lastname: Poncelet
authors.forename: H William
authors.initials: HW
authors.lastname: Schnaper
authors.forename: Ruoyun
authors.initials: R
authors.lastname: Tan
authors.forename: Youhua
authors.initials: Y
authors.lastname: Liu
authors.forename: Constance E
authors.initials: CE
authors.lastname: Runyan
mesh_terms: D000199:Actins / Q000096:biosynthesis; D000818:Animals; D015820:Cadherins / Q000096:biosynthesis; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects*; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005355:Fibrosis; D006801:Humans; D007684:Kidney Tubules / Q000378:metabolism* / Q000473:pathology; D008297:Male; D050527:Mesangial Cells / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D009928:Organ Specificity; D010641:Phenotype; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism*; D051901:Smad4 Protein / Q000378:metabolism; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000199:Actins; D015820:Cadherins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin