Expression of 11 beta-hydroxylase and 21-hydroxylase in long-term cultures of bovine adrenocortical cells requires extracellular matrix factors.
In order to elucidate mechanisms for the loss of expression of 11 beta-hydroxylase and 21-hydroxylase, induction of these genes in long-term cultures of bovine adrenocortical cells was reassessed and compared with induction of 17 alpha-hydroxylase. We previously showed that both 11 beta- 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 11 beta-hydroxylase and 21-hydroxylase. They showed a truncated steroidogenic pathway, converting 25-hydroxycholesterol 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 11 beta-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, 17 alpha-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 11 beta-hydroxylase except when grown in Matrigel; 21-hydroxylase was low and expression was enhanced by Matrigel, whereas 17 alpha-hydroxylase expression was unaffected. As previously determined, in late-passage cells and clones only side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase activities were detected and 17 alpha-hydroxylase was not expressed. In such cells 11 beta-hydroxylase and 21-hydroxylase were also not expressed, even in the presence of Matrigel. Thus, prior to the previously described loss of expression of 17 alpha-hydroxylase, Matrigel permits the cholera toxin/IGF-I-induced expression of a complete steroidogenic pathway in bovine adrenocortical cells in long-term culture.
Cheng
C Y
CY
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912.
Hornsby
P J
PJ
D000302:Adrenal Cortex / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D000305:Adrenal Cortex Hormones / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D003094:Collagen; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000502:physiology*; D006854:Hydrocortisone / Q000096:biosynthesis; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007700:Kinetics; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000378:metabolism*
D000305:Adrenal Cortex Hormones; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D007334:Insulin-Like Growth Factor I; D003094:Collagen; D002772:Cholera Toxin; D013255:Steroid 21-Hydroxylase; D013252:Steroid 11-beta-Hydroxylase; D006854:Hydrocortisone