FUNCTIONAL CHARACTERIZATION OF THE PROMOTER OF THE ACYL-COA SYNTHETASE 4

C. KARLES; J. MILD; A. DUARTE; M. COOKE; A. LAGO; C. MENDEZ; P. MALOBERTI; E.J. PODESTÁ

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

We have described that, in steroidogenic tissues, hormonal action prompts the synthesis of a labile protein, an acyl-CoA synthetase (ACS4), which is involved in the regulation of arachidonic acid (AA) release and is essential for steroidogenesis. We demonstrated that ACS4 protein levels are rapidly induced by steroidogenic hormones and cAMP in Y1 adrenocortical and MA10 Leydig cells. The aim of the present work is to characterize the promoter sequence of ACS4 and to study its transcriptional regulation. In order to determine the minimum sequence required for maximal promoter activity, unidirectional progressive 5´ deletions of the promoter sequence were performed. Obtained constructions were used to measure promoter activity by dual-luciferase assay in transfected MA-10 cells stimulated for 3 h with 8Br-AMPc. The minimum sequence that maintains basal and cAMP-induced promoter activity is contained from base -150. This sequence includes consensus binding sites for CREB, and Sp1 transcriptions factors. Specific binding to CREB and SP1 sites within the promoter sequences was detected by EMSA and binding was augmented when the cells were stimulated by cAMP. We also detected an increase in ACS4 protein and mRNA levels in MA10 cells over-expressing CREB. Our results demonstrate the transcriptional regulation of ACS4 by cAMP.