Czajkowski_2015_Steroids_104_118

The effect of C/EBPalpha on the expression of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase (HSL) was investigated in Y-1 CCL79 cells. It was found that transfection of these cells with the vector overexpressing C/EBPalpha increased both the level of LIPE transcript, measured by RT-qPCR, and the luminesce emitted by luciferase reporter gene fused to the -2150 fragment of LIPE promoter. Activation of adenylyl cyclase by forskolin resulted in 2.5-fold increase in the intensity of luminescence and over 3-fold increase in luminescence was observed when the cells were cotransfected with the vector overexpressing C/EBP. The incubation of C/EBP-cotransfected cells with forskolin caused over 6-fold increase in the intensity of luminescence, suggesting that the effects of C/EBPalpha and forskolin are additive. The analysis of sequence of the proximal LIPE promoter showed multiple binding sites for various transcription factors including C/EBPalpha site, which is located between nucleotides -46bp and -59bp. When the Y-1 cells were transfected with the recombinant vector containing -60bp fragment of LIPE promoter fused to the luciferase reporter gene and were cotransfected with the vector overexpressing C/EBPalpha, the luminescence increases about 9-fold indicating that C/EBPalpha stimulates the expression of LIPE by reacting with its response element. The results indicate that C/EBPalpha stimulates the expression of LIPE independently of the PKA pathway by binding to a response element situated within the -60bp fragment of LIPE promoter. This suggests that C/EBPalpha might be involved in the regulation of LIPE expression and thus cholesterol supply for steroid hormone synthesis.