title: Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts.
abstract: OBJECTIVE
  Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor beta (TGFbeta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGFbeta signaling, in scleroderma fibroblasts.
  
  
  METHODS
  Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGFbeta was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGFbeta blockade on SMAD subcellular distribution was determined using anti-TGFbeta antibodies as well as a dominant-negative TGFbeta receptor type II (TGFbetaRII) vector to disrupt TGFbeta responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.
  
  
  RESULTS
  Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGFbeta, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGFbeta signaling with antibodies or by expression of dominant-negative TGFbetaRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFbeta receptors. The activity of a SMAD-responsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts.
  
  
  CONCLUSIONS
  This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGFbeta/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.
authors.affiliation: University of Illinois at Chicago College of Medicine, Chicago, IL 60607, USA.
authors.forename: Yasuji
authors.initials: Y
authors.lastname: Mori
authors.forename: Shu-Jen
authors.initials: SJ
authors.lastname: Chen
authors.forename: John
authors.initials: J
authors.lastname: Varga
mesh_terms: D000328:Adult; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005355:Fibrosis; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology*; D012867:Skin / Q000166:cytology; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta