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Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2
R W Brown1, K E Chapman, Y Kotelevtsev
1University Department of Medicine, Western General Hospital, Edinburgh, Scotland, UK.
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.
Insights
11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) inactivates glucocorticoids in the placenta and aldosterone target tissues. This study isolated the 11 beta-HSD2 cDNA, characterized its activity, and mapped its expression in human tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) is crucial for regulating glucocorticoid and mineralocorticoid receptor access in target tissues.
- This enzyme plays a protective role by inactivating potent glucocorticoids, such as cortisol and corticosterone, particularly in the placenta and kidney.
Purpose of the Study:
- To isolate and characterize the cDNA encoding human placental 11 beta-HSD2.
- To investigate the enzymatic activity, substrate specificity, and inhibition of 11 beta-HSD2.
- To determine the tissue distribution and developmental expression of 11 beta-HSD2 mRNA and protein.
Main Methods:
- Isolation of a 1897 bp cDNA encoding human placental 11 beta-HSD2 using amino acid sequence.
- Transfection of the cDNA into mammalian (CHO) cells to produce and assay recombinant enzyme activity.
- Characterization of enzyme kinetics, cofactor dependence, and inhibition by specific compounds.
- Generation of rabbit antisera against 11 beta-HSD2 for detection.
- Analysis of 11 beta-HSD2 mRNA expression using Northern blotting and in situ hybridization.
- Immunohistochemical localization of 11 beta-HSD2 protein in human tissues.
Main Results:
- A cDNA encoding human placental 11 beta-HSD2 (predicted M(r) 44126) was isolated.
- Recombinant 11 beta-HSD2 exhibited NAD(+)-dependent inactivation of glucocorticoids with high affinity (Km for corticosterone 12.4 nM) and was inhibited by glycyrrhetinic acid and carbenoxolone.
- 11 beta-HSD2 mRNA was detected in placenta, kidney, parotid, colon, skin, and pancreas.
- Abundant 11 beta-HSD2 expression was localized to the distal nephron in adult kidney and trophoblast in the placenta.
- Fetal kidney expressed 11 beta-HSD2 transcripts by 21-26 weeks, indicating early developmental expression.
Conclusions:
- The isolated cDNA encodes a functional 11 beta-HSD2 enzyme with critical roles in glucocorticoid metabolism.
- The enzyme's activity and tissue-specific expression highlight its importance in protecting tissues from glucocorticoid excess.
- The developmental expression pattern suggests 11 beta-HSD2 establishes its protective function early in human development.