Kulyk W M, Hoffman L M
Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, Canada.
Exp Cell Res. 1996 Mar 15;223(2):290-300. doi: 10.1006/excr.1996.0084.
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, and prostaglandin production that have been implicated as critical regulators 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 ability to promote congenital skeletal deformities in vivo, prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from 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 chondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in 35SO4 incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage 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 alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol on in vitro chondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.
对神经细胞、肝细胞及其他细胞的研究表明，体外乙醇暴露可影响多种与膜相关的信号传导机制。这些机制包括受体激酶磷酸化、腺苷酸环化酶和蛋白激酶C激活以及前列腺素生成等过程，这些过程被认为是胚胎肢体发育过程中软骨细胞分化的关键调节因子。乙醇影响发育中肢体软骨形成信号传导机制的可能性，以及其在体内促进先天性骨骼畸形的已知能力，促使我们研究慢性酒精暴露是否会影响从23 - 25期鸡胚肢体芽分离的软骨前间充质细胞培养物中的软骨分化。我们有了一个新颖且惊人的发现，即乙醇在翻译前和翻译后水平都是体外软骨形成的有效刺激物。在胚胎肢体间充质细胞的高密度培养物中，这些细胞会自发地进行广泛的软骨分化，培养基中乙醇的存在促进了阿尔新蓝阳性软骨基质生成的增加、35SO4掺入基质糖胺聚糖（GAG）的定量增加，以及软骨特征性II型胶原蛋白和聚集蛋白聚糖（软骨蛋白聚糖）mRNA的早熟积累。基质GAG积累的刺激在乙醇浓度为2%（v/v）时最大，尽管低至0.5%乙醇（约85 mM）也能引起显著增加。乙醇似乎直接影响肢体软骨生成祖细胞的分化，因为即使在由24/25期鸡胚翼芽远端亚嵴间充质制备的培养物中，乙醇也能提高软骨形成，而这些培养物中没有成肌前体细胞。当肢体间充质细胞在低密度下培养时，这会抑制自发软骨形成，乙醇暴露会诱导高水平的II型胶原蛋白和聚集蛋白聚糖mRNA的表达，并促进大量软骨基质的形成。这些刺激作用并非乙醇所特有，因为甲醇、丙醇和叔丁醇处理也能增强胚胎肢体间充质培养物中的软骨分化。对乙醇体外软骨形成刺激作用的进一步研究可能会为胚胎发育过程中调节软骨细胞分化的机制以及酒精对骨骼形态发生致畸作用的分子基础提供见解。
