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

Abstract

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-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 11 beta-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 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km 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 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta-HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational processing for activity. 11 beta-HSD2 mRNA (approximately 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 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development.

Role of glucocorticoids in the physiopathology of excessive fat deposition and insulin resistance

“…Its main function is to allow aldosterone access to MRs in the distal nephron, in the face of a large molar excess of circulating glucocorticoids. 42,43 In this review, 11b-HSD-1 only will be considered. Its main physiological and pathological roles stem from studies carried out in genetically altered mice and are summarized by part of Figure 1.…”

11β-Hydroxysteroid dehydrogenase inhibition improves cognitive function in healthy elderly men and type 2 diabetics

“…11␤-HSD1 mRNA was assayed by in situ hybridization histochemistry in frozen sections by using [ 35 S]UTP-labeled antisense cRNA probes transcribed in vitro from a 900-bp HindIII-SstI fragment of ph11␤-HSD1 (39) subcloned in pGEM3. 11␤-HSD2 mRNA was similarly estimated by using an XbaI-linearized pCRIICtBb, which has the 531-bp 11␤-HSD2 insert (corresponds to the 3Ј end of the 11␤-HSD2 coding region, i.e., bases ϩ654 to ϩ1184) (20). Sections were postfixed in 4% paraformaldehyde, acetylated (0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0), washed in PBS, dehydrated through graded alcohols, and airdried.…”

“…The type 2 isozyme (11␤-HSD2) is a potent NADdependent dehydrogenase that catalyzes the rapid inactivation of glucocorticoids. In the periphery, 11␤-HSD2 is confined to aldosterone-selective target tissues such as the distal nephron, colon, and salivary gland, where it acts to prevent illicit occupation by glucocorticoids of intrinsically nonselective mineralocorticoid receptors (19,20). In contrast, the type 1 isozyme (11␤-HSD1) is expressed in key glucocorticoid receptor target tissues such as liver (21,22) and adipose tissue (23).…”

Mechanisms of initiation and reversal of drug-seeking behavior induced by prenatal exposure to glucocorticoids

“…DEX is not biodegraded in the same way as its naturally occurring congeners and crosses the maternal-placental barrier to a greater extent than endogenous GC (9,10); it can thus pose additional risk for the developing brain.…”