In order to elucidate mechanisms for the loss of expression of 11β-hydroxylase and 21-hydroxylase, induction of these genes in long-term cultures of bovine adrenocortical cells was reassessed and compared with induction of 17α-hydroxylase. We previously showed that both 11β- and 21-hydroxylases require insulin-like growth factor-I (IGF-I) as well as cAMP for induction; these are the only factors needed by primary cultures. Cells at population doubling level 10 grown on fibronectin-coated polystyrene dishes and incubated with cholera toxin and IGF-I did not express 11β-hydroxylase and 21-hydroxylase. They showed a truncated steroidogenic pathway, converting 25-hy-droxycholesterol to some 11-deoxycortisol but little Cortisol. However, when population doubling level 10 cells were grown for 5 days in extracellular matrix Matrigel, cholera toxin and IGF-I induced a complete steroidogenic pathway to Cortisol. Northern blotting also showed that expression of 11β-hydroxylase messenger RNA (mRNA) after cholera toxin/IGF-I induction was observed only in cultures grown in Matrigel and was undetectable in cultures grown on plastic. 21-Hydroxylase mRNA was observed in cultures grown on plastic but was greatly enhanced by Matrigel; however, 17α-hydroxylase mRNA was induced to a similar extent with and without Matrigel. In other middle passage cultures, whether grown as mass cultures, SV40 T antigen-transfected clones, or normal (nontransfected) clones, cells did not express 11β-hydroxylase except when grown in Matrigel; 21-hydroxylase was low and expression was enhanced by Matrigel, whereas 17α-hydroxylase expression was unaffected. As previously determined, in late-passage cells and clones only side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase activities were detected and 17α-hydroxylase was not expressed. In such cells 11β-hydroxylase and 21-hydroxylase were also not expressed, even in the presence of Matrigel. Thus, prior to the previously described loss of expression of 17α-hydroxylase, Matrigel permits the cholera toxin/IGF-I-induced expression of a complete steroidogenic pathway in bovine adrenocortical cells in long-term culture.
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