Involvement of alphavbeta5 integrin-mediated activation of latent transforming growth factor beta1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts.
OBJECTIVE
To confirm the involvement of alphavbeta5 in the self-activation system in systemic sclerosis (SSc) fibroblasts.


METHODS
Levels of alphavbeta5 expression were analyzed by immunoprecipitation. The promoter activity of the human alpha2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor beta (TGFbeta) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.


RESULTS
Levels of alphavbeta5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-alphavbeta5 antibody or beta5 antisense oligonucleotide significantly reduced human alpha2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFbeta1 antisense oligonucleotide, the exogenous latent TGFbeta1 stimulation significantly increased human alpha2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-alphavbeta5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with beta5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of SSc fibroblasts.


CONCLUSIONS
Up-regulated expression of alphavbeta5 contributes to the establishment of autocrine TGFbeta signaling in SSc fibroblasts through activation of endogenous latent TGFbeta1.
Asano
Yoshihde
Y
University of Tokyo, Tokyo, Japan.
Ihn
Hironobu
H
Yamane
Kenichi
K
Jinnin
Masatoshi
M
Mimura
Yoshihiro
Y
Tamaki
Kunihiko
K
D019138:Antibodies, Blocking / Q000494:pharmacology; D002452:Cell Count; D002454:Cell Differentiation; D002478:Cells, Cultured; D020405:Dermis / Q000166:cytology / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D016023:Integrins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D053779:Latent TGF-beta Binding Proteins; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D019030:Receptors, Vitronectin / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D012595:Scleroderma, Systemic / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D019138:Antibodies, Blocking; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D016376:Oligonucleotides, Antisense; D019030:Receptors, Vitronectin; D016212:Transforming Growth Factor beta; C064660:integrin alphaVbeta5