Relation of TGF-beta 2 to inhibition of limb bud chondrogenesis by retinoid in rats.

A newly synthesized retinoid, Am-80 (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl] carbamoxyl) benzoic acid, induced limb reduction defects in rats in vivo. The present study was designed to investigate the mechanisms of its teratogenic action and also to examine whether transforming growth factor-beta (TGF-beta) is involved in limb reduction defects by using a rat limb bud cell culture system. Am-80 inhibited DNA synthesis and chondrogenesis of limb bud cells from day 12 embryos at concentrations of 1-4 ng/ml in a concentration-dependent manner. Quantities of TGF-beta 1 and 2 in culture medium from limb bud cells treated with Am-80 were evaluated using sandwich ELISAs. Although TGF-beta 1 was not detected in conditioned medium with or without Am-80, TGF-beta 2 increased in a concentration-dependent manner at drug concentrations that inhibited DNA synthesis and chondrogenesis. In addition, when TGF-beta 2 was exogenously added to limb bud cell culture medium, the synthesis of glycosaminoglycan (GAGs) was reduced even without Am-80 treatment. Neutralization of TGF-beta 2 with antibody prevented the inhibition of GAGs synthesis induced by Am-80. These results suggest that TGF-betas, especially beta 2, are involved in the inhibition of chondrogenesis caused by Am-80 in limb bud cells.