Transcriptional regulation of an acyl-CoA synthetase (ACS4), an enzyme involved in the intracellular control of free arachidonic acid levels.

MALOBERTI PAULA; MILD JESICA; DUARTE ALEJANDRA; KARLES CRISTINA; COOKE MARIANA; LAGO AMPARO; PAPADOPOULOS VASSILIOS; PODESTÁ ERNESTO J

Bariloche, Río Negro, Argentina

Congreso; Combined Meetings: "Gene Expression and RNA Processing" (ICGEB) and "Cell Biology, Signaling and Alternative Splicing" (EURASNET); 2007

Intracellular levels of arachidonic acid (AA) are regulated in answer to different physiological agonists in different tissues. We have previously demonstrated the existence of a novel mechanism aimed at the compartmentalization of AA release, a mechanism that could determine whether cells proliferate or commit to apoptosis. That mechanism involves the concerted action of two enzymes, an acyl-CoA synthetase (ACS4) and a mitochondrial acyl-CoA thioesterase (Acot2). ACS4 is reportedly over-expressed in some tumors and we have demonstrated its over-expression in a highly aggressive human breast cell line. The aim of the present work was to isolate the promoter region of the ACS4 gene and to study its transcriptional regulation. By means of semi quantitative RT-PCR, we have observed a significant increase in the abundance of ACS4 mRNA in MA10 cells (a cell line derived from tumoral Leydig cells), in response to 8Br-cAMP. Transcription initiation sites are located in the same region within 50 bases for both splicing variants, placed at 387 and 220 bases from the ATG codon of splicing variants 1 and 2 of ACS4 respectively, as determined by RNA ligase-mediated 5’RACE. ACS4’s promoter was amplified by PCR resulting in a 1.7 kb fragment that was cloned in the pGL3-BASIC vector. This vector was then used to transfect MA-10 cells and transcriptional activity measured in the presence or absence of 8Br-cAMP by the dual luciferase system. We observed a significant basal promoter activity as compared to empty vector (8-fold) and this activity resulted increased in the presence of cAMP (3-fold). Sequence analysis of the promoter region unveiled the presence of a consensus binding sites for Sp1 factor in -51 and -55 positions as well as consensus sites for CREB, Gata, SF-1 and C/EBP, whereas no typical or TATA box-homologous consensus site was present within the promoter region.