Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway.
Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated beta-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-beta (TGF-beta), in view of the high levels of TGF-beta isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-beta1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.
Kim
Kook-Hee
KH
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Republic of Korea.
Park
Geun-Tae
GT
Lim
Young-Bin
YB
Rue
Seok-Woo
SW
Jung
Jae-Chang
JC
Sonn
Jong-Kyung
JK
Bae
Young-Seuk
YS
Park
Jeen-Woo
JW
Lee
Young-Sup
YS
D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D016922:Cellular Senescence / Q000502:physiology*; D055513:Connective Tissue Growth Factor; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004171:Diploidy; D000431:Ethanol / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D005696:Galactosidases / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D020033:Protein Isoforms; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D051785:Smad Proteins; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D015415:Biomarkers; C526317:CCN2 protein, human; C526316:CCN2 protein, rat; C493956:CDKN1A protein, human; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D020033:Protein Isoforms; D051785:Smad Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D016159:Tumor Suppressor Protein p53; D055513:Connective Tissue Growth Factor; D000431:Ethanol; D020122:tert-Butylhydroperoxide; D005696:Galactosidases