Identification of the 11 β-hydroxysteroid Dehydrogenase Type 1 mRNA and Protein in Human Mononuclear Leukocytes

Abstract

The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11β-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11β-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11β-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11β-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11β-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [³ H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15–1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18β-Glycyrrhetinic acid, an inhibitor of 11 β-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11β-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation.

Key words

11β-hydroxysteroid dehydrogenase type 1 - human mononuclear leukocytes - inflammation - oxidative stress - receptors - glucocorticoids

References

• 1 Albiston AL, Obeyesekere VR, Smith RE. et al . Cloning and tissue distribution of the human 11 beta- hydroxysteroid dehydrogenase type 2 enzyme.  Mol Cell Endocrinol. 1994;  105 R11-R17
• 2 Armanini D, Strasser T, Weber PC. Characterization of aldosterone binding sites in circulating human mononuclear leucocytes.  Am J Physiol. 1985;  248 E388-E390
• 3 Caló LA, Zaghetto F, Pagnin E. et al . Effect of aldosterone and glycyrrhetinic acid on the protein expression of PAI-1 and p22(phox) in human mononuclear leukocytes.  J Clin Endocrinol Metab. 2004;  89 1973-1976
• 4 Christ M, Meyer C, Sippel K. et al . Rapid aldosterone signaling in vascular smooth muscle cells: involvement of phospholipase C, diacylglycerol and protein kinase Cα.  Biochem Biophys Res Comm. 1995;  213 123-129
• 5 Dandona P, Aljada A, Ghanim H. et al . Acute suppressive effect of hydrocortisone on p47^phox subunit of nicotinamide adenine dinucleotide phosphate oxidase.  Metabolism. 2001;  50 548-552
• 6 Draper N, Stewart PM. 11beta-hydroxysteroid dehydrogenase and the pre-receptor regulation of corticosteroid hormone action.  J Endocrinol. 2005;  186 251-271
• 7 Freeman L, Hewison M, Hughes SV. et al . Expression of 11beta-hydroxysteroid dehydrogenase type 1 permits regulation of glucocorticoid bioavailability by human dendritic cells.  Blood. 2005;  106 2042-2049
• 8 Johnstone JF, Bocking AD, Unlugedik E. et al . The effects of chorioamnionitis and betamethasone on 11beta hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor in preterm human placenta.  J Soc Gynecol Investig. 2005;  12 238-245
• 9 Kasuya Y, Ishimaru H, Shibasaki H. et al . Stable isotope methodology for kinetic studies of interconversion of cortisol and cortisone in a human subject.  Steroids. 1998;  63 122-129
• 10 Lio D, Candore G, Crivello A. et al . Opposite effects of interleukin 10 common gene polymorphisms in cardiovascular diseases and in successful ageing: genetic background of male centenarians is protective against coronary heart disease.  J Med Genet. 2004;  41 790-794
• 11 Murphy VE, Clifton VL. Alterations in human placental 11beta-hydroxysteroid dehydrogenase type 1 and 2 with gestational age and labour.  Placenta. 2003;  24 739-744
• 12 Mziaut H, Korza G, Hand AR. et al . Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11β-hydroxysteroid dehydrogenase and the 50-kDa esterase.  J Biol Chem. 1999;  274 14122-14129
• 13 Odermatt A, Atanasov AG, Balazs Z. et al . Why is 11 β-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11 β-HSD1.  Mol Cell Endocrinol. 2006;  248 15-23
• 14 Preisler MT, Weber PSD, Tempelman RJ. et al . Glucocorticoid receptor expression profiles in mononuclear leukocytes of periparturient holstein cows.  J Dairy Sci. 2000;  83 38-47
• 15 Refojo D, Liberman AC, Holsboer F. et al . Transcription factor-mediated molecular mechanisms involved in the functional cross-talk between cytokines and glucocorticoids.  Immunol Cell Biol. 2001;  79 385-394
• 16 Rook GA. Glucocorticoids and immune function.  Baillieres Best Pract Res Clin Endocrinol Metab. 1999;  13 567-581
• 17 Schweizer RA, Zurcher M, Balazs Z. et al . Rapid hepatic metabolism of 7-ketocholesterol by 11β-hydroxysteroid dehydrogenase Type 1: species-specific differences between the rat, human and hamster enzyme.  J Biol Chem. 2004;  279 18415-18424
• 18 Seckl JR. 11β-hydroxysteroid dehydrogenases: changing glucocorticoid action.  Curr Opin Pharmacol. 2004;  4 597-602
• 19 Thieringer R, Le Grand CB, Carbin L. et al . 11 Beta-hydroxysteroid dehydrogenase type 1 is induced in human monocytes upon differentiation to macrophages.  J Immunol. 2001;  167 30-35
• 20 Thieringer R, Le Grand CB, Mundt SS. et al . 11β-hydroxysteroid dehydrogenase type 1 is expressed in human monocytes upon differentiation to macrophages as well as in THP-1 cells, a human macrophage-like cell line.  , 82nd Annual Meeting of the Endocrinology Society, Toronto, ON, Canada, 2000: 21–24
• 21 Tomlinson JW, Moore J, Cooper MS. et al . Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines.  Endocrinology. 2001;  142 1982-1989
• 22 Wehling M, Armanini D, Strasser T. et al . Effect of aldosterone on sodium and potassium concentration in human mononuclear leucocytes.  Am J Physiol. 1987;  252 E505-E508
• 23 Zhang TY, Ding X, Daynes RA. The expression of 11 beta-hydroxysteroid dehydrogenase type I by lymphocytes provides a novel means for intracrine regulation of glucocorticoid activities.  J Immunol. 2005;  174 879-889

1 These authors contributed equally.

Correspondence