TNFa- Regulation of cAMP Stimulated 3b-Hydroxysteroid Dehydrogenase Type 2 (3b-HSDII) Promoter Activity

SARACO NORA; PEPE CAROLINA; BAQUEDANO MARÍA SONIA; RIVAROLA MARCO A.; BELGOROSKY ALICIA

Boston, Massachusetts, USA.

Congreso; The Endocrine Society's 88th Annual Meeting; 2006

Endocrine Society (ENDO)

TNF- is a cytokine produced in the adrenal cortex. An important role of TNF- in human adrenal physiology has been suggested, i. e., to have an effect in reducing cortisol production and in enhancing DHAS synthesis under ACTH stimulation. The enzyme 3 -HSDII is essential for adrenal steroid biosynthesis and its expression is inversely associated with adrenal androgen production suggesting a key role in adrenarche. The aims of this study were to analyze the effect of TNF- on cAMP stimulated 3 -HSDII promoter activity and the mechanisms involved.Three 5 deletion mutants of the 1057 to +41 bp fragment of the 3 -HSDII gene were generated and inserted into a luciferase reporter plasmid (CI:-1057;CII:-297;CIII:-107 bp). The constructs were transfected into the H295R adrenal cell line and luciferase activity was evaluated by Dual Luciferase Reporter Assay System. Assays were carried out at least in triplicates. Under cAMP (0.5mM) luciferase activity significantly increased (CI: 3.0; CII: 2.6; CIII: 1.8 fold over basal) and TNF- (10 ng/ml) reduced cAMP stimulated luciferase activity in the three constructs (CI: 19.2 %; CII: 61.3%; CIII: 77.2% reduction of cAMP stimulation). By site-directed mutagenesis a known SF1 responsive element (NR5A1-RE) was mutated in CII (MCII) and CIII (MCIII). Under cAMP stimulation luciferase activity increased in both mutated constructs (MCII: 2.7; MCIII: 2.1 fold over basal) and TNF- reduced cAMP stimulated activity (MCII: 60.6%; MCIII: 99% reduction of cAMP stimulation). Site-directed mutagenesis on a possible NFkB responsive element of CIII did not affect cAMP stimulation of luciferase activity (1.83 fold over basal) nor TNF- action (80.7 % reduction of cAMP stimulation).These results show that TNF- acts on the 3 -HSDII promoter reducing cAMP induced luciferase activity. The NR5A1-RE seems not to be involved in the TNF- action nor the cAMP stimulation of 3 -HSDII promoter. TNF- does not seem to display a direct interaction with NFkB on responsive element, in the CIII. It is concluded that TNF- , by inhibiting cAMP stimulation of 3 -HSDII, might favor DHEAS secretion in zona reticularis cells.