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) conta ining the transforming growth factor-beta (TGF-beta) response element was s ynthesized. Rat fetal lung fibroblasts were stably transfected with the Col Cat 3.6 plasmid, which contains a portion of the 5'-flanking region of the pro alpha 1(l) collagen gene linked to the chloramphenicol acetyltransferas e (CAT) gene. The cells were transiently transfected with the modified olig odcoxynucleotides in both the presence and absence of bleomycin, a fibrogen ic antineoplastic agent. At 50 mu g ssPT, the bleomycin-induced increase in CAT activity was abrogated. The ability of ssPT to inhibit collagen synthe sis in rat fetal lung fibroblasts was determined. Single-stranded PTs inhib ited both collagen synthesis and noncollagen protein synthesis induced by T GF-beta 1, the mediator of the bleomycin fibrogenic effect. Inflamed granul ation tissue fibroblasts were prepared from polyvinyl alcohol sponges impla nted in the backs of rats. These fibroblasts were treated with various dose s of ssPTs in the presence and absence of TGF-beta 1. Single-stranded PTs a lso blocked both the TGF-beta 1-induced increase in collagen synthesis and noncollagen synthesis in these fibroblasts. However, the TGF-beta 1-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 prote in synthesis in rat lung fibroblasts or in granuloma derived fibroblasts. S ince dexamethasone was also able to block the TGF-beta 1-induced increase i n collagen and noncollagen protein synthesis (Meisler et al., [1997] J. Inv est. Dermatol. 108:285-289), these data indicate that phosphorothioate olig odeoxynucleotide antifibrotic agents mimic the inhibitory effect of glucoco rticoids on collagen synthesis without the untoward side effects of these s teroids. J. Cell. Biochem. 75:196-205, 1999. Published 1999 Wiley-Liss, Inc .dagger.