Cloning and expression of chicken 20-hydroxysteroid dehydrogenase

Abstract

The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20bHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20bHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5a-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3a/b-and 20a/20b-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V max conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined.

Corticosterone metabolism by chicken follicle cells does not affect ovarian reproductive hormone synthesis in vitro

“…Accordingly, Kucka et al Kucka et al (2006) showed 20-HSD activity in ovary, testis, kidney, intestines, liver, brain and oviduct. Also, mRNA expression of 20-HSD was confirmed by Bryndova et al Bryndova et al (2006) in several chicken tissues. In addition to 20β-dihydrocorticosterone, we found that the ovarian tissue also synthesized three other metabolites with higher polarity than corticosterone, which could not be assigned to any of the used standards.…”

Cloning of chicken 11β-hydroxysteroid dehydrogenase type 1 and its tissue distribution

“…One of them is 3,20-hydroxysteroid dehydrogenase, which we have recently cloned and identified in many chicken tissues [29]; the other seems to be 5␣-reductase, which was identified in avian liver by Monder and Lakshmi [8]. The co-existence of several steroid enzymes that need NADPH as a cosubstrate may be of great physiological importance, because liberated NADP + from the reaction catalyzed by 5␣-reductase may hinder 11HSD1 reductase activity.…”

Determinants and significance of corticosterone regulation in the songbird brain

“…While 20 hydroxylase activity or expression has been documented in the chicken (Bryndová et al, 2006; Kučka et al, 2006) and songbird (Katz et al, 2010) brain, 11ß HSD type 3 has only been examined in the periphery of chickens to date (Katz et al 2008). No studies that we know of have investigated this latter enzyme in songbirds, although this is yet another mechanism by which the brain could conceivably regulate local CORT fluctuations and activity.…”