An arachidonic acid-preferring acyl-CoA synthetase is a hormone-dependent and obligatory protein in the signal transduction pathway of steroidogenic hormones.

We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, (35)S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis.StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.