Involvement of the constitutive complex formation of c-Ski/SnoN with Smads in the impaired negative feedback regulation of transforming growth factor beta signaling in scleroderma fibroblasts.
OBJECTIVE
The principal effect of transforming growth factor beta1 (TGFbeta1) on mesenchymal cells is its stimulation of extracellular matrix synthesis. Previous reports indicated the significance of the autocrine TGFbeta loop in the pathogenesis of scleroderma. The aim of this study was to examine c-Ski and SnoN, principal molecules in the negative regulation of TGFbeta signaling, to further understand the autocrine TGFbeta loop in scleroderma.


METHODS
Levels of expression of c-Ski/SnoN on cultured normal and scleroderma fibroblasts were determined by Western blotting, Northern blotting, and immunohistochemical staining. To determine the protein-protein interaction between c-Ski/SnoN, Smads, and p300, immunoprecipitation was performed. A transient transfection assay was performed to measure promoter activity of the alpha2(I) collagen gene and the 3TP-Lux plasmid construct.


RESULTS
Scleroderma fibroblasts exhibited increased c-Ski/SnoN levels compared with normal fibroblasts, both in vivo and in vitro. Although c-Ski/SnoN constitutively formed a complex with Smads by immunoprecipitation, the inhibitory effect of c-Ski/SnoN on the promoter activity of human alpha2(I) collagen and 3TP-Lux was impaired in scleroderma fibroblasts. Immunoprecipitation analyses also revealed that overexpressed c-Ski/SnoN could not compete with p300 in these cells.


CONCLUSIONS
These results indicate that impaired competition with p300 is the possible cause of dysfunction of c-Ski/SnoN in scleroderma fibroblasts and that this might contribute to maintenance of the autocrine TGFbeta loop in this disease.
Jinnin
Masatoshi
M
University of Tokyo, Tokyo, Japan.
Ihn
Hironobu
H
Mimura
Yoshihiro
Y
Asano
Yoshihide
Y
Tamaki
Kunihiko
K
D000328:Adult; D016022:Case-Control Studies; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D050881:E1A-Associated p300 Protein / Q000502:physiology; D025461:Feedback, Physiological / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000473:pathology / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D012595:Scleroderma, Systemic / Q000235:genetics / Q000473:pathology / Q000503:physiopathology*; D015398:Signal Transduction / Q000502:physiology*; D051900:Smad3 Protein / Q000235:genetics / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
D024042:Collagen Type I; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011518:Proto-Oncogene Proteins; C060615:SKIL protein, human; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C060614:SKI protein, human; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human