Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2.
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.
Brown
R W
RW
University Department of Medicine, Western General Hospital, Edinburgh, Scotland, UK.
Chapman
K E
KE
Kotelevtsev
Y
Y
Yau
J L
JL
Lindsay
R S
RS
Brett
L
L
Leckie
C
C
Murad
P
P
Lyons
V
V
Mullins
J J
JJ
Edwards
C R
CR
Seckl
J R
JR
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005333:Fetus / Q000201:enzymology; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010920:Placenta / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases