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

TitleCloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2.
Publication TypeJournal Article
Year of Publication1996
AuthorsBrown WR, Chapman KE, Kotelevtsev Y, Yau JL, Lindsay RS, Brett L, Leckie C, Murad P, Lyons V, Mullins JJ, Edwards CR, Seckl JR
JournalBiochem J
Volume313 ( Pt 3)
Pagination1007-17
Date Published1996 Feb 1
ISSN0264-6021
Keywords11-beta-Hydroxysteroid Dehydrogenases, Adult, Amino Acid Sequence, Animals, Antibodies, Base Sequence, CHO Cells, Cloning, Molecular, Cricetinae, DNA Primers, DNA, Complementary, Female, Fetus, Humans, Hydroxysteroid Dehydrogenases, In Situ Hybridization, Male, Molecular Sequence Data, Molecular Structure, Placenta, Polymerase Chain Reaction, Pregnancy, Rabbits, RNA, Messenger, Sequence Homology, Amino Acid, Tissue Distribution, Transfection
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.

Alternate JournalBiochem. J.
PubMed ID8611140
PubMed Central IDPMC1216963
Grant List / / Wellcome Trust / United Kingdom