Interrenal steroid 21-hydroxylase in eels: primary structure, progesterone-specific activity and enhanced expression by ACTH.
Cytochrome P450 21-hydroxylase (P450c21) is a key enzyme for corticosteroidogenesis. To understand the regulatory mechanisms of cortisol production in fish, we have cloned a cDNA encoding P450c21, for the first time in non-mammalian vertebrates, from the head kidney of the eel (Anguilla japonica). The overall similarity of the deduced P450c21 sequence was modest (41-44% amino acid identity) between the eel and mammals. However, the functional domains for steroid-binding, heme-binding and proton-transfer sites were well conserved (74-100% identity). The eel P450c21 mRNA was expressed abundantly in the anterior quarter of the head kidney, but was undetectable in the remaining three-quarters or in other tissues including the gill, heart, liver, intestine, kidney, immature gonad and skeletal muscle. Functional expression of the cDNA clone in non-steroidogenic COS-1 cells produced a protein with high 21-hydroxylase activity to convert progesterone to 11-deoxycortisterone but not 17alpha-hydroxyprogesterone to 11-deoxycortisol, although the latter is a preferred substrate for mammalian P450c21. To examine whether 21-hydroxylated progesterone is actually 17alpha-hydroxylated in the eel interrenal, 11-deoxycorticosterone and (3)H-corticosterone were respectively incubated with the interrenal-containing anterior quarter of the head kidney. The separation of the steroids produced by two HPLC systems revealed that cortisol was produced from both substrates, showing the 17alpha-hydroxylation of 11-deoxycorticosterone and corticosterone in the eel interrenal. ACTH infused at 3 pmol/kg per min for 5 h consistently increased plasma cortisol levels and interrenal P450c21 mRNA levels in seawater eels. These results showed that the interrenal-specific eel P450c21 cloned in this study is involved in cortisol production through conversion of progesterone to 11-deoxycorticosterone in the interrenal-containing anterior quarter of eel head kidney. Furthermore, ACTH stimulates cortisol production in part through enhanced P450c21 expression in the eel interrenal.
D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D018076:DNA, Complementary; D003900:Desoxycorticosterone / Q000378:metabolism; D004524:Eels / Q000235:genetics* / Q000378:metabolism; D006854:Hydrocortisone / Q000096:biosynthesis / Q000097:blood; D007401:Interrenal Gland / Q000201:enzymology* / Q000378:metabolism; D007668:Kidney / Q000187:drug effects; D008969:Molecular Sequence Data; D011374:Progesterone / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects; D013255:Steroid 21-Hydroxylase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D003900:Desoxycorticosterone; D011374:Progesterone; D000324:Adrenocorticotropic Hormone; D013255:Steroid 21-Hydroxylase; D006854:Hydrocortisone