Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Feb 1;313(Pt 3):1007–1017. doi: 10.1042/bj3131007

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

R W Brown 1, K E Chapman 1, Y Kotelevtsev 1, J L Yau 1, R S Lindsay 1, L Brett 1, C Leckie 1, P Murad 1, V Lyons 1, J J Mullins 1, C R Edwards 1, J R Seckl 1
PMCID: PMC1216963  PMID: 8611140

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.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agarwal A. K., Mune T., Monder C., White P. C. NAD(+)-dependent isoform of 11 beta-hydroxysteroid dehydrogenase. Cloning and characterization of cDNA from sheep kidney. J Biol Chem. 1994 Oct 21;269(42):25959–25962. [PubMed] [Google Scholar]
  2. Albiston A. L., Obeyesekere V. R., Smith R. E., Krozowski Z. S. Cloning and tissue distribution of the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol. 1994 Nov;105(2):R11–R17. doi: 10.1016/0303-7207(94)90176-7. [DOI] [PubMed] [Google Scholar]
  3. Albrecht E. D., Pepe G. J. Placental steroid hormone biosynthesis in primate pregnancy. Endocr Rev. 1990 Feb;11(1):124–150. doi: 10.1210/edrv-11-1-124. [DOI] [PubMed] [Google Scholar]
  4. Beitins I. Z., Bayard F., Ances I. G., Kowarski A., Migeon C. J. The metabolic clearance rate, blood production, interconversion and transplacental passage of cortisol and cortisone in pregnancy near term. Pediatr Res. 1973 May;7(5):509–519. doi: 10.1203/00006450-197305000-00004. [DOI] [PubMed] [Google Scholar]
  5. Benediktsson R., Lindsay R. S., Noble J., Seckl J. R., Edwards C. R. Glucocorticoid exposure in utero: new model for adult hypertension. Lancet. 1993 Feb 6;341(8841):339–341. doi: 10.1016/0140-6736(93)90138-7. [DOI] [PubMed] [Google Scholar]
  6. Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
  7. Brown R. W., Chapman K. E., Edwards C. R., Seckl J. R. Human placental 11 beta-hydroxysteroid dehydrogenase: evidence for and partial purification of a distinct NAD-dependent isoform. Endocrinology. 1993 Jun;132(6):2614–2621. doi: 10.1210/endo.132.6.8504762. [DOI] [PubMed] [Google Scholar]
  8. Brown R. W., Chapman K. E., Murad P., Edwards C. R., Seckl J. R. Purification of 11 beta-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique. Biochem J. 1996 Feb 1;313(Pt 3):997–1005. doi: 10.1042/bj3130997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng K. C., White P. C., Qin K. N. Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase. Mol Endocrinol. 1991 Jun;5(6):823–828. doi: 10.1210/mend-5-6-823. [DOI] [PubMed] [Google Scholar]
  10. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  11. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  12. Edwards C. R., Benediktsson R., Lindsay R. S., Seckl J. R. Dysfunction of placental glucocorticoid barrier: link between fetal environment and adult hypertension? Lancet. 1993 Feb 6;341(8841):355–357. doi: 10.1016/0140-6736(93)90148-a. [DOI] [PubMed] [Google Scholar]
  13. Edwards C. R., Stewart P. M., Burt D., Brett L., McIntyre M. A., Sutanto W. S., de Kloet E. R., Monder C. Localisation of 11 beta-hydroxysteroid dehydrogenase--tissue specific protector of the mineralocorticoid receptor. Lancet. 1988 Oct 29;2(8618):986–989. doi: 10.1016/s0140-6736(88)90742-8. [DOI] [PubMed] [Google Scholar]
  14. Emini E. A., Hughes J. V., Perlow D. S., Boger J. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol. 1985 Sep;55(3):836–839. doi: 10.1128/jvi.55.3.836-839.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feldman D., Funder J. W., Edelman I. S. Evidence for a new class of corticosterone receptors in the rat kidney. Endocrinology. 1973 May;92(5):1429–1441. doi: 10.1210/endo-92-5-1429. [DOI] [PubMed] [Google Scholar]
  16. Funder J. W., Pearce P. T., Smith R., Smith A. I. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science. 1988 Oct 28;242(4878):583–585. doi: 10.1126/science.2845584. [DOI] [PubMed] [Google Scholar]
  17. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  18. Ghosh D., Pletnev V. Z., Zhu D. W., Wawrzak Z., Duax W. L., Pangborn W., Labrie F., Lin S. X. Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution. Structure. 1995 May 15;3(5):503–513. doi: 10.1016/s0969-2126(01)00183-6. [DOI] [PubMed] [Google Scholar]
  19. Ghosh D., Weeks C. M., Grochulski P., Duax W. L., Erman M., Rimsay R. L., Orr J. C. Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10064–10068. doi: 10.1073/pnas.88.22.10064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Israel D. I. A PCR-based method for high stringency screening of DNA libraries. Nucleic Acids Res. 1993 Jun 11;21(11):2627–2631. doi: 10.1093/nar/21.11.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  23. Lakshmi V., Monder C. Purification and characterization of the corticosteroid 11 beta-dehydrogenase component of the rat liver 11 beta-hydroxysteroid dehydrogenase complex. Endocrinology. 1988 Nov;123(5):2390–2398. doi: 10.1210/endo-123-5-2390. [DOI] [PubMed] [Google Scholar]
  24. Low S. C., Chapman K. E., Edwards C. R., Seckl J. R. 'Liver-type' 11 beta-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells. J Mol Endocrinol. 1994 Oct;13(2):167–174. doi: 10.1677/jme.0.0130167. [DOI] [PubMed] [Google Scholar]
  25. Marchesi V. T., Furthmayr H., Tomita M. The red cell membrane. Annu Rev Biochem. 1976;45:667–698. doi: 10.1146/annurev.bi.45.070176.003315. [DOI] [PubMed] [Google Scholar]
  26. Marks A. R., McIntyre J. O., Duncan T. M., Erdjument-Bromage H., Tempst P., Fleischer S. Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart. J Biol Chem. 1992 Aug 5;267(22):15459–15463. [PubMed] [Google Scholar]
  27. Monder C., Shackleton C. H., Bradlow H. L., New M. I., Stoner E., Iohan F., Lakshmi V. The syndrome of apparent mineralocorticoid excess: its association with 11 beta-dehydrogenase and 5 beta-reductase deficiency and some consequences for corticosteroid metabolism. J Clin Endocrinol Metab. 1986 Sep;63(3):550–557. doi: 10.1210/jcem-63-3-550. [DOI] [PubMed] [Google Scholar]
  28. Murphy B. E., Clark S. J., Donald I. R., Pinsky M., Vedady D. Conversion of maternal cortisol to cortisone during placental transfer to the human fetus. Am J Obstet Gynecol. 1974 Feb 15;118(4):538–541. doi: 10.1016/s0002-9378(16)33697-3. [DOI] [PubMed] [Google Scholar]
  29. Murphy B. E. Ontogeny of cortisol-cortisone interconversion in human tissues: a role for cortisone in human fetal development. J Steroid Biochem. 1981 Sep;14(9):811–817. doi: 10.1016/0022-4731(81)90226-0. [DOI] [PubMed] [Google Scholar]
  30. Naville D., Keeney D. S., Jenkin G., Murry B. A., Head J. R., Mason J. I. Regulation of expression of male-specific rat liver microsomal 3 beta-hydroxysteroid dehydrogenase. Mol Endocrinol. 1991 Aug;5(8):1090–1100. doi: 10.1210/mend-5-8-1090. [DOI] [PubMed] [Google Scholar]
  31. Nishikawa K. Assessment of secondary-structure prediction of proteins. Comparison of computerized Chou-Fasman method with others. Biochim Biophys Acta. 1983 Oct 28;748(2):285–299. doi: 10.1016/0167-4838(83)90306-0. [DOI] [PubMed] [Google Scholar]
  32. Náray-Fejes-Tóth A., Rusvai E., Denault D. L., St Germain D. L., Fejes-Tóth G. Expression and characterization of a new species of 11 beta-hydroxysteroid dehydrogenase in Xenopus oocytes. Am J Physiol. 1993 Dec;265(6 Pt 2):F896–F900. doi: 10.1152/ajprenal.1993.265.6.F896. [DOI] [PubMed] [Google Scholar]
  33. Náray-Fejes-Tóth A., Rusvai E., Fejes-Tóth G. Is the renal type III corticosteroid-binding site the collecting duct-specific isoform of 11 beta-hydroxysteroid dehydrogenase? Endocrinology. 1994 Apr;134(4):1671–1675. doi: 10.1210/endo.134.4.8137730. [DOI] [PubMed] [Google Scholar]
  34. Pawlowski J. E., Huizinga M., Penning T. M. Cloning and sequencing of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. J Biol Chem. 1991 May 15;266(14):8820–8825. [PubMed] [Google Scholar]
  35. Pepe G. J., Waddell B. J., Stahl S. J., Albrecht E. D. The regulation of transplacental cortisol-cortisone metabolism by estrogen in pregnant baboons. Endocrinology. 1988 Jan;122(1):78–83. doi: 10.1210/endo-122-1-78. [DOI] [PubMed] [Google Scholar]
  36. Persson B., Krook M., Jörnvall H. Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur J Biochem. 1991 Sep 1;200(2):537–543. doi: 10.1111/j.1432-1033.1991.tb16215.x. [DOI] [PubMed] [Google Scholar]
  37. Päbo S., Bhat B. M., Wold W. S., Peterson P. A. A short sequence in the COOH-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum. Cell. 1987 Jul 17;50(2):311–317. doi: 10.1016/0092-8674(87)90226-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rodin A., Thakkar H., Taylor N., Clayton R. Hyperandrogenism in polycystic ovary syndrome. Evidence of dysregulation of 11 beta-hydroxysteroid dehydrogenase. N Engl J Med. 1994 Feb 17;330(7):460–465. doi: 10.1056/NEJM199402173300703. [DOI] [PubMed] [Google Scholar]
  39. Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
  40. Rysavy F. R., Bishop M. J., Gibbs G. P., Williams G. W. The UK Human Genome Mapping Project online computing service. Comput Appl Biosci. 1992 Apr;8(2):149–154. doi: 10.1093/bioinformatics/8.2.149. [DOI] [PubMed] [Google Scholar]
  41. Seckl J. R., Brown R. W. 11-beta-hydroxysteroid dehydrogenase: on several roads to hypertension. J Hypertens. 1994 Feb;12(2):105–112. [PubMed] [Google Scholar]
  42. Shin J., Dunbrack R. L., Jr, Lee S., Strominger J. L. Signals for retention of transmembrane proteins in the endoplasmic reticulum studied with CD4 truncation mutants. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1918–1922. doi: 10.1073/pnas.88.5.1918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Soro A., Ingram M. C., Tonolo G., Glorioso N., Fraser R. Evidence of coexisting changes in 11 beta-hydroxysteroid dehydrogenase and 5 beta-reductase activity in subjects with untreated essential hypertension. Hypertension. 1995 Jan;25(1):67–70. doi: 10.1161/01.hyp.25.1.67. [DOI] [PubMed] [Google Scholar]
  44. Stewart P. M., Murry B. A., Mason J. I. Type 2 11 beta-hydroxysteroid dehydrogenase in human fetal tissues. J Clin Endocrinol Metab. 1994 Jun;78(6):1529–1532. doi: 10.1210/jcem.78.6.8200959. [DOI] [PubMed] [Google Scholar]
  45. Tannin G. M., Agarwal A. K., Monder C., New M. I., White P. C. The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J Biol Chem. 1991 Sep 5;266(25):16653–16658. [PubMed] [Google Scholar]
  46. Ulick S., Levine L. S., Gunczler P., Zanconato G., Ramirez L. C., Rauh W., Rösler A., Bradlow H. L., New M. I. A syndrome of apparent mineralocorticoid excess associated with defects in the peripheral metabolism of cortisol. J Clin Endocrinol Metab. 1979 Nov;49(5):757–764. doi: 10.1210/jcem-49-5-757. [DOI] [PubMed] [Google Scholar]
  47. Wu L., Einstein M., Geissler W. M., Chan H. K., Elliston K. O., Andersson S. Expression cloning and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20 alpha-hydroxysteroid dehydrogenase activity. J Biol Chem. 1993 Jun 15;268(17):12964–12969. [PubMed] [Google Scholar]
  48. Yang K., Yu M. Evidence for distinct isoforms of 11 beta-hydroxysteroid dehydrogenase in the ovine liver and kidney. J Steroid Biochem Mol Biol. 1994 Jun;49(2-3):245–250. doi: 10.1016/0960-0760(94)90016-7. [DOI] [PubMed] [Google Scholar]
  49. Yau J. L., Kelly P. A., Sharkey J., Seckl J. R. Chronic 3,4-methylenedioxymethamphetamine administration decreases glucocorticoid and mineralocorticoid receptor, but increases 5-hydroxytryptamine1C receptor gene expression in the rat hippocampus. Neuroscience. 1994 Jul;61(1):31–40. doi: 10.1016/0306-4522(94)90057-4. [DOI] [PubMed] [Google Scholar]
  50. Zhao H. F., Labrie C., Simard J., de Launoit Y., Trudel C., Martel C., Rhéaume E., Dupont E., Luu-The V., Pelletier G. Characterization of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues. J Biol Chem. 1991 Jan 5;266(1):583–593. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES