Interrenal steroid 21-hydroxylase in eels: primary structure, progesterone-specific activity and enhanced expression by ACTH

Abstract

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 17 -hydroxyprogesterone to 11-deoxycortisol, although the latter is a preferred substrate for mammalian P450c21. To examine whether 21-hydroxylated progesterone is actually 17 -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 17 -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.

Proteasomal Degradation of Human CYP1B1: Effect of the Asn453Ser Polymorphism on the Post-Translational Regulation of CYP1B1 Expression

“…To assess the impact of CYP1B1 allelic variations on the expression level of five common CYP1B1 polymorphic variants, we have employed transient expression in mammalian COS-1 cells, which have been extensively employed for the characterization of P450s Jinno et al, 2003;Li et al, 2003;Szczesna-Skorupa et al, 2003), including CYP1A1 (Meyer et al, 2002), CYP1A2 (Black et al, 1997), and CYP1B1 itself (McLellan et al, 2000). The data represented in Fig.…”

Steroidogenic Acute Regulatory Protein in Eels: cDNA Cloning and Effects of ACTH and Seawater Transfer on Its mRNA Expression

“…The double-stranded cDNA pool was prepared from 0.5 µ g poly (A) + -RNA using SMART cDNA Library Construction Kit (Clontech Laboratories, Palo Alto, CA) as described previously (Inoue et al ., 2003). Degenerate primer S1 [5'-GCAACHTTCAAAYTGT-GYGC-3'] and A1 [5'-TTCCASTCVCCCATYTGYTCCAT-3'] ( Fig.…”

Mineralocorticoid Receptors and Hormones: Fishing for Answers

“…1; Ref. 12) and, although data are sparse, the compound itself appears to be present in fish blood at levels that are not only measurable but are comparable to the concentrations required to activate the trout MR (13). In suggesting the potential for 11-deoxycorticosterone to function as a fish mineralocorticoid hormone, Sturm et al (6) have presented an intriguing challenge to the research community.…”

“…Schematic of the probable pathways involved in cortisol and 11-deoxycorticosterone biosynthesis in fish. Solid black arrows indicate that the enzyme required for that step has been cloned in fish and displays appropriate activity (3,12); unfilled arrows indicate steps for which confirmation of enzyme activity is required. The gray arrow indicates that the enzyme required for that step has been cloned in fish but did not display the appropriate activity (12).…”