CLONING AND PRODUCTION OF ANTISERA TO HUMAN PLACENTAL 11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-2

By inactivating potent glucocorticoid hormones (cortisol and corticost erone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) pla ys an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by control ling ligand access to co-localized glucocorticoid and mineralocorticoi d receptors. Amino acid sequence from homogeneous human placental 11 b eta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (pred icted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is wi thout reductase activity, avidly metabolizes glucocorticoids (K-m valu es for corticosterone, cortisol and dexamethasone of 12.4+/-1.5, 43.9/-8.5 and 119+/-15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recogn izing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370-383)-pepti de-carrier conjugate. Recombinant 11 beta-HSD2, like native human plac ental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational p rocessing for activity. 11 beta-HSD2 mRNA (similar to 1.9 kb transcrip t) is expressed in placenta, aldosterone target tissues (kidney, parot id, colon and skin) and pancreas. In situ hybridization and immunohist ochemistry localize abundant 11 beta-HSD2 expression to the distal nep hron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liv er or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribut ion resembling that in the adult is established by this stage in human development.