Promoter competitors as novel antifibrotics that inhibit transforming growth factor-beta induction of collagen and noncollagen protein synthesis in fibroblasts.
A single-stranded 27-mer phosphorothioate oligodeoxynucleotide (ssPT) containing the transforming growth factor-beta (TGF-beta) response element was synthesized. Rat fetal lung fibroblasts were stably transfected with the ColCat 3.6 plasmid, which contains a portion of the 5'-flanking region of the proalpha1(I) collagen gene linked to the chloramphenicol acetyltransferase (CAT) gene. The cells were transiently transfected with the modified oligodeoxynucleotides in both the presence and absence of bleomycin, a fibrogenic antineoplastic agent. At 50 microg ssPT, the bleomycin-induced increase in CAT activity was abrogated. The ability of ssPT to inhibit collagen synthesis in rat fetal lung fibroblasts was determined. Single-stranded PTs inhibited both collagen synthesis and noncollagen protein synthesis induced by TGF-beta1, the mediator of the bleomycin fibrogenic effect. Inflamed granulation tissue fibroblasts were prepared from polyvinyl alcohol sponges implanted in the backs of rats. These fibroblasts were treated with various doses of ssPTs in the presence and absence of TGF-beta1. Single-stranded PTs also blocked both the TGF-beta1-induced increase in collagen synthesis and noncollagen synthesis in these fibroblasts. However, the TGF-beta1-induced increase in collagen and noncollagen protein synthesis was not blocked by ssPTs containing a mutated TGF-beta response element. In addition, ssPT did not significantly alter the basal levels of collagen and noncollagen protein synthesis in rat lung fibroblasts or in granuloma derived fibroblasts. Since dexamethasone was also able to block the TGF-beta1-induced increase in collagen and noncollagen protein synthesis (Meisler et al., [1997] J. Invest. Dermatol. 108:285-289), these data indicate that phosphorothioate oligodeoxynucleotide antifibrotic agents mimic the inhibitory effect of glucocorticoids on collagen synthesis without the untoward side effects of these steroids.
Meisler
N T
NT
Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Chiu
J F
JF
Cutroneo
K R
KR
D000818:Animals; D001761:Bleomycin / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects*; D005786:Gene Expression Regulation; D016296:Mutagenesis; D011347:Procollagen / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D020218:Response Elements / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D011347:Procollagen; D011500:Protein Synthesis Inhibitors; D013873:Thionucleotides; D016212:Transforming Growth Factor beta; D001761:Bleomycin; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase