Cloning and production of antisera to human placental 11β-hydroxysteroid dehydrogenase type 2

Abstract

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11β-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11β-HSD2 activity which is NAD+-dependent, is without reductase activity, avidly metabolizes glucocorticoids (K(m) values 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 recognizing 11β-HSD2 have been raised to an 11β-HSD2-(370-383)-peptide-carrier conjugate. Recombinant 11β-HSD2, like native human placental 11β-HSD2, is detectable with affinity labelling and anti-11β-HSD2 antisera, and appears to require little post-translational processing for activity. 11β-HSD2 mRNA (~ 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11β-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11β-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11β-HSD2 distribution resembling that in the adult is established by this stage in human development.