Download PDF
Horm Metab Res 2010; 42(7): 528-534
DOI: 10.1055/s-0030-1249629
Original Basic

© Georg Thieme Verlag KG Stuttgart · New York

Etomidate Unmasks Intraadrenal Regulation of Steroidogenesis and Proliferation in Adrenal Cortical Cell Lines

S. Hahner1 , [*] , A. Stürmer1 , [*] , M. Fassnacht1 , R. W. Hartmann2 , K. Schewe2 , S. Cochran1 , M. Zink1 , A. Schirbel3 , B. Allolio1
  • 1Endocrinology & Diabetes Unit, Department of Medicine I, University of Würzburg, Würzburg, Germany
  • 2Pharmaceutical and Medicinal Chemistry, University of Saarland, Saarbrücken, Germany
  • 3Department of Nuclear Medicine, University of Würzburg, Würzburg, Germany
Further Information

Publication History

received 20.07.2009

accepted 22.02.2010

Publication Date:
29 March 2010 (online)

Also available at
    Permissions and Reprints

Abstract

To characterize intraadrenal adaptations for inhibition of cortisol synthesis, we analyzed the effects of etomidate (ETO) on steroid hormone secretion and expression of key regulators of steroidogenesis and proliferation in human NCI-h295 adrenocortical cancer cells. Etomidate potently blocked 11β-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2), and side chain cleavage enzyme (CYP11A1). This inhibition of steroidogenesis was associated with increased expression of steroidogenic acute regulatory protein (StAR), and CYP11A1 and 17α-hydroxylase/17, 20-lyase (CYP17A1) protein levels, but not of the respective mRNA levels. Promoter activity of CYP11A1 and melanocortin 2 receptor (MC2R) was not increased by etomidate in treated cells compared to controls. The increase in protein levels was partially reversed by cycloheximide suggesting post-transcriptional mechanisms but also protein stabilization as underlying cause. Furthermore, ETO exhibited antiproliferative activity paralleled by a decrease in phosphorylation of MEK and ERK1, 2. In summary, ETO exhibits pleiotropic effects on adrenal function in vitro. Inhibition of steroidogenesis is followed by increased levels of steroidogenic key proteins and reduced proliferation. These changes reflect adaptations to maintain steroidogenesis at the cost of adrenal proliferation.

Key words

etomidate - steroidogenic enzymes - StAR - CYP11A1 - adrenostatic compounds - proliferation

References

  • 1 de Jong FH, Mallios C, Jansen C, Scheck PA, Lamberts SW. Etomidate suppresses adrenocortical function by inhibition of 11 beta-hydroxylation.  J Clin Endocrinol Metab. 1984;  59 1143-1147
    Search in Google Scholar
  • 2 Kenyon CJ, Young J, Gray CE, Fraser R. Inhibition by etomidate of steroidogenesis in isolated bovine adrenal cells.  J Clin Endocrinol Metab. 1984;  58 947-949
    Search in Google Scholar
  • 3 Varga I, Racz K, Kiss R, Futo L, Toth M, Sergev O, Gláz E. Direct inhibitory effect of etomidate on corticosteroid secretion in human pathologic adrenocortical cells.  Steroids. 1993;  58 64-68
    Search in Google Scholar
  • 4 Wagner RL, White PF. Etomidate inhibits adrenocortical function in surgical patients.  Anesthesiology. 1984;  61 647-651
    Search in Google Scholar
  • 5 Wagner RL, White PF, Kan PB, Rosenthal MH, Feldman D. Inhibition of adrenal steroidogenesis by the anesthetic etomidate.  N Engl J Med. 1984;  310 1415-1421
    Search in Google Scholar
  • 6 Wada A, Ohnishi T, Nonaka Y, Okamoto M. Inhibition of bovine adrenocortical mitochondrial cytochrome P-450(11)beta-mediated reactions by imidazole derivatives and mineralocorticoid analogs.  J Steroid Biochem. 1988;  31 803-808
    Search in Google Scholar
  • 7 Lamberts SW, Bons EG, Bruining HA, de Jong FH. Differential effects of the imidazole derivatives etomidate, ketoconazole and miconazole and of metyrapone on the secretion of cortisol and its precursors by human adrenocortical cells.  J Pharmacol Exp Ther. 1987;  240 259-264
    Search in Google Scholar
  • 8 Fassnacht M, Hahner S, Beuschlein F, Klink A, Reincke M, Allolio B. New mechanisms of adrenostatic compounds in a human adrenocortical cancer cell line.  Eur J Clin Invest. 2000;  30 (S 03) 76-82
    Search in Google Scholar
  • 9 Allolio B, Stuttmann R, Fischer H, Leonhard W, Winkelmann W. Long-term etomidate and adrenocortical suppression.  Lancet. 1983;  2 (8350) 626
    Search in Google Scholar
  • 10 Krakoff J, Koch CA, Calis KA, Alexander RH, Nieman LK. Use of a parenteral propylene glycol-containing etomidate preparation for the long-term management of ectopic Cushing's syndrome.  J Clin Endocrinol Metab. 2001;  86 4104-4108
    Search in Google Scholar
  • 11 Schulte HM, Benker G, Reinwein D, Sippell WG, Allolio B. Infusion of low dose etomidate: correction of hypercortisolemia in patients with Cushing's syndrome and dose-response relationship in normal subjects.  J Clin Endocrinol Metab. 1990;  70 1426-1430
    Search in Google Scholar
  • 12 Bergstrom M, Juhlin C, Bonasera TA, Sundin A, Rastad J, Akerstrom G, Långström B. PET imaging of adrenal cortical tumors with the 11beta-hydroxylase tracer 11C-metomidate.  J Nucl Med. 2000;  41 275-282
    Search in Google Scholar
  • 13 Bergstrom M, Sorensen J, Kahn TS, Juhlin C, Eriksson B, Sundin A, Sundin A, Bonasera TA, Fasth K, Långström B. PET with[11C]-Metomidate for the Visualization of Adrenocortical Tumors and Discrimination from Other Lesions.  Clin Positron Imaging. 1999;  2 339
    Search in Google Scholar
  • 14 Hennings J, Lindhe O, Bergstrom M, Langstrom B, Sundin A, Hellman P. [11C] metomidate positron emission tomography of adrenocortical tumors in correlation with histopathological findings.  J Clin Endocrinol Metab. 2006;  91 1410-1414
    Search in Google Scholar
  • 15 Khan TS, Sundin A, Juhlin C, Langstrom B, Bergstrom M, Eriksson B. 11C-metomidate PET imaging of adrenocortical cancer.  Eur J Nucl Med Mol Imaging. 2003;  30 403-410
    Search in Google Scholar
  • 16 Minn H, Salonen A, Friberg J, Roivainen A, Viljanen T, Langsjo J, Salmi J, Välimäki M, Någren K, Nuutila P. Imaging of adrenal incidentalomas with PET using (11)C-metomidate and (18)F-FDG.  J Nucl Med. 2004;  45 972-979
    Search in Google Scholar
  • 17 Zettinig G, Mitterhauser M, Wadsak W, Becherer A, Pirich C, Vierhapper H, Niederle B, Dudczak R, Kletter K. Positron emission tomography imaging of adrenal masses: (18)F-fluorodeoxyglucose and the 11beta-hydroxylase tracer (11)C-metomidate.  Eur J Nucl Med Mol Imaging. 2004;  31 1224-1230
    Search in Google Scholar
  • 18 Fassnacht M, Beuschlein F, Vay S, Mora P, Allolio B, Reincke M. Aminoglutethimide suppresses adrenocorticotropin receptor expression in the NCI-h295 adrenocortical tumor cell line.  J Endocrinol. 1998;  159 35-42
    Search in Google Scholar
  • 19 Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, La Rocca RV. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis.  Cancer Res. 1990;  50 5488-5496
    Search in Google Scholar
  • 20 Denner K, Doehmer J, Bernhardt R. Cloning of CYP11B1 and CYP11B2 from normal human adrenal and their functional expression in COS-7 and V79 Chinese hamster cells.  Endocr Res. 1995;  21 443-448
    Search in Google Scholar
  • 21 Denner K, Vogel R, Schmalix W, Doehmer J, Bernhardt R. Cloning and stable expression of the human mitochondrial cytochrome P45011B1 cDNA in V79 Chinese hamster cells and their application for testing of potential inhibitors.  Pharmacogenetics. 1995;  5 89-96
    Search in Google Scholar
  • 22 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 (5259) 680-685
    Search in Google Scholar
  • 23 Huang MC, Miller WL. Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system.  Endocrinology. 2001;  142 2569-2576
    Search in Google Scholar
  • 24 Sharp S, Barker EV, Coughtrie MW, Lowenstein PR, Hume R. Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans.  Eur J Biochem. 1993;  211 539-548
    Search in Google Scholar
  • 25 Bumke-Vogt C, Bahr V, Diederich S, Herrmann SM, Anagnostopoulos I, Oelkers W, Quinkler M. Expression of the progesterone receptor and progesterone-metabolising enzymes in the female and male human kidney.  J Endocrinol. 2002;  175 349-364
    Search in Google Scholar
  • 26 Ehmer PB, Bureik M, Bernhardt R, Muller U, Hartmann RW. Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells.  J Steroid Biochem Mol Biol. 2002;  81 173-179
    Search in Google Scholar
  • 27 Ulmschneider S, Muller-Vieira U, Mitrenga M, Hartmann RW, Oberwinkler-Marchais S, Klein CD, Bureik M, Bernhardt R, Antes I, Lengauer T. Synthesis and evaluation of imidazolylmethylenetetrahydronaphthalenes and imidazolylmethyleneindanes: potent inhibitors of aldosterone synthase.  J Med Chem. 2005;  48 1796-1805
    Search in Google Scholar
  • 28 Zwermann O, Beuschlein F, Lalli E, Klink A, Sassone-Corsi P, Reincke M. Clinical and molecular evidence for DAX-1 inhibition of steroidogenic factor-1-dependent ACTH receptor gene expression.  Eur J Endocrinol. 2005;  152 769-776
    Search in Google Scholar
  • 29 Clark BJ, Soo SC, Caron KM, Ikeda Y, Parker KL, Stocco DM. Hormonal and developmental regulation of the steroidogenic acute regulatory protein.  Mol Endocrinol. 1995;  9 1346-1355
    Search in Google Scholar
  • 30 Stocco DM, Clark BJ. Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis.  Biochem Pharmacol. 1996;  51 197-205
    Search in Google Scholar
  • 31 Caron KM, Ikeda Y, Soo SC, Stocco DM, Parker KL, Clark BJ. Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein.  Mol Endocrinol. 1997;  11 138-147
    Search in Google Scholar
  • 32 Clark BJ, Combs R, Hales KH, Hales DB, Stocco DM. Inhibition of transcription affects synthesis of steroidogenic acute regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor cells.  Endocrinology. 1997;  138 4893-4901
    Search in Google Scholar
  • 33 Liu J, Heikkila P, Kahri AI, Voutilainen R. Expression of the steroidogenic acute regulatory protein mRNA in adrenal tumors and cultured adrenal cells.  J Endocrinol. 1996;  150 43-50
    Search in Google Scholar
  • 34 Dardis A, Miller WL. Dexamethasone does not exert direct intracellular feedback on steroidogenesis in human adrenal NCI-H295A cells.  J Endocrinol. 2003;  179 131-142
    Search in Google Scholar
  • 35 Christenson LK, McAllister JM, Martin KO, Javitt NB, Osborne TF, Strauss 3rd JF. Oxysterol regulation of steroidogenic acute regulatory protein gene expression Structural specificity and transcriptional and posttranscriptional actions.  J Biol Chem. 1998;  273 30729-30735
    Search in Google Scholar
  • 36 King SR, Matassa AA, White EK, Walsh LP, Jo Y, Rao RM, Stocco DM, Reyland ME. Oxysterols regulate expression of the steroidogenic acute regulatory protein.  J Mol Endocrinol. 2004;  32 507-517
    Search in Google Scholar
  • 37 Reyland ME, Evans RM, White EK. Lipoproteins regulate expression of the steroidogenic acute regulatory protein (StAR) in mouse adrenocortical cells.  J Biol Chem. 2000;  275 36637-36644
    Search in Google Scholar
  • 38 Gonzalez FJ, Ueno T, Umeno M, Song BJ, Veech RL, Gelboin HV. Microsomal ethanol oxidizing system: transcriptional and posttranscriptional regulation of cytochrome P450, CYP2E1.  Alcohol Alcohol Suppl. 1991;  1 97-101
    Search in Google Scholar
  • 39 Kuhn-Velten WN, Lohr JB. Ligand dependence of cytochrome P450c17 protection against proteolytic inactivation: structural, methodological and functional implications.  FEBS Lett. 1996;  388 21-25
    Search in Google Scholar
  • 40 Granot Z, Geiss-Friedlander R, Melamed-Book N, Eimerl S, Timberg R, Weiss AM, Hales KH, Hales DB, Stocco DM, Orly J. Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins.  Mol Endocrinol. 2003;  17 2461-2476
    Search in Google Scholar
  • 41 Fassnacht M, Hahner S, Hansen IA, Kreutzberger T, Zink M, Adermann K, Jakob F, Troppmair J, Allolio B. N-terminal proopiomelanocortin acts as a mitogen in adrenocortical tumor cells and decreases adrenal steroidogenesis.  J Clin Endocrinol Metab. 2003;  88 2171-2179
    Search in Google Scholar
  • 42 Schulte DM, Shapiro I, Reincke M, Beuschlein F. Expression and spatio-temporal distribution of differentiation and proliferation markers during mouse adrenal development.  Gene Expr Patterns. 2007;  7 72-81
    Search in Google Scholar
  • 43 Joannard F, Rissel M, Gilot D, Anderson A, Orfila-Lefeuvre L, Guillouzo A, Atfi A, Lagadic-Gossmann D. Role for mitogen-activated protein kinases in phenobarbital-induced expression of cytochrome P450 2B in primary cultures of rat hepatocytes.  Toxicol Lett. 2006;  161 61-72
    Search in Google Scholar
  • 44 Sewer MB, Waterman MR. CAMP-dependent protein kinase enhances CYP17 transcription via MKP-1 activation in H295R human adrenocortical cells.  J Biol Chem. 2003;  278 8106-8111
    Search in Google Scholar

1 Both authors contributed equally to this work.

Correspondence

B. AllolioMD 

Endocrinology & Diabetes Unit

Department of Medicine

University of Würzburg

Oberdürrbacher-Straße 6

97080 Würzburg

Germany

Phone: +49/931/201 39020

Fax: +49/931/201 639200

Email: allolio_b@medizin.uni-wuerzburg.de