PARENT SESSION
GENE REGULATION AND FUNCTION - A

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(396) IDENTIFICATION OF PROMOTER ELEMENTS RESPONSIBLE FOR HIGH BASAL EXPRESSION AND cAMP-INDUCED REDUCTION OF 3-HYDROXYSTEROID DEHYDROGENASE IN MA-10 LEYDIG TUMOR CELLS.

Xie, Qin¹, Martin, Michelle ², Stalvey, John¹, ¹ Kent State University, Kent, OH² Cleveland Clinic Foundation, Cleveland, OH

ABSTRACT- 3-Hydroxysteroid dehydrogenase/isomerase (3HSD) is a key steroidogenic enzyme. Mouse 3HSD type I is the major isoform expressed in steroidogenic tissues. Previous studies (Payne AH,Sha LL, 1991 Endocrinology) reported high basal expression of mouse 3HSD and that subsequent cAMP treatment (500M) caused a reduced 3HSD mRNA expression in primary mouse Leydig cells. In the current study, a series of mouse 3HSD type I promoter-luciferase constructs, which were serially shortened from the 3' and/or 5' end of mouse 3HSD type I promoter, were transfected into MA-10 Leydig tumor cells or Y-1 adrenal tumor cells. A SV-40 promoter-chloramphenicol acetyltransferase(CAT) reporter gene was cotransfected with mouse 3HSD promoter constructs as a constitutive control to normalize the luc expression among different experiments. Deletion of 5' sequences from -1556 to -963 and 3' sequences from +37 to +292 decreased normalized luc activity by 50-85% of that measured in the full-length 3HSD promoter in mouse MA-10 Leydig cells. A similar decrease was observed in Y-1 adrenal tumor cells transfected with the same constructs. Further 5' deletion, from -963 to -592 and -592 to -359, resulted in luc activity being slightly regained as the result of a sequential 1.5-fold and 1.8-fold increase in the luc expression, respectively, in MA-10 Leydig cells. The treatment for 24 hours with 500M cAMP caused luc activity to decrease to 25% of basal value in each of four constructs. These results suggest that the promoter regions from +37 to +292 and/or -1556 to -963 contain sequences that play a major role in determining high basal expression of mouse 3HSD type I gene and promoter region from -359 to +1 start site contains sequences responsible for cAMP negative regulation for mouse 3HSD type I gene expression.

KEY WORDS: cAMP, luciferase, 3-hydroxysteroid dehydrogenase, promoter