ETHANOL EXPOSURE STIMULATES CARTILAGE DIFFERENTIATION BY EMBRYONIC LIMB MESENCHYME CELLS

Studies of neural, hepatic, and other cells have demonstrated that in vitro ethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, an d prostaglandin production that have been implicated as critical regul ators of chondrocyte differentiation during embryonic limb development . The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known abilit y to promote congenital skeletal deformities in vivo prompted us to ex amine whether chronic alcohol exposure could influence cartilage diffe rentiation in cultures of prechondrogenic mesenchyme cells isolated fr om limb buds of stage 23-25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant of in vitro chon drogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously under go extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matr ix production, a quantitative rise in (SO4)-S-35 incorporation into ma trix glycosaminoglycans (GAG), and the precocious accumulation of mRNA s for cartilage-characteristic type II collagen and aggrecan (cartilag e proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The a lcohol appears to directly influence differentiation of the chondrogen ic progenitor cells of the limb, since ethanol elevated cartilage form ation even in cultures prepared from distal subridge mesenchyme of sta ge 24/25 chick embryo wing buds, which is free of myogenic precursor c ells. When limb mesenchyme cells were cultured at low density, which s uppresses spontaneous chondrogenesis, ethanol exposure induced the exp ression of high levels of type II collagen and aggrecan mRNAs and prom oted abundant cartilage matrix formation. These stimulatory effects we re not specific to ethanol, since methanol, propanol, and tertiary but anol treatments also enhanced cartilage differentiation in embryonic l imb mesenchyme cultures. Further investigations of the stimulatory eff ects of ethanol on in vitro chondrogenesis may provide insights into t he mechanisms regulating chondrocyte differentiation during embryogene sis and the molecular basis of alcohol's teratogenic effects on skelet al morphogenesis. (C) 1996 Academic Press, Inc.