Sterol regulatory element-binding protein-1c activates patatin like phospholipase domain containing 3 gene transcription in rats

Abstract

Objective To explore the effect of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of patatin like phospholipase domain containing 3(PNPLA3) in rat. Methods Seven-week male weight matching Sprague-Dawley rats were assigned to fed ad libitum group(n=3), fasting group(n=3, fasting for 24 h), refeeding group(n=4, fasting for 24 h and refeeding 48 h) and normal control group(n=5), high-fat-diet and streptozotocin-induced diabetic group(n=6). Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of SREBP-1c and PNPLA3 in rats' liver. Three nested deletions of the 5' end of the 1.0 kb PNPLA3 promoter were generated and introduced into the luciferase reporter gene(R-PNPLA3-1, R-PNPLA3-2, R-PNPLA3-3), and then transfected into human normal hepatic cell line LO2 respectively to compare the basal and post-stimulate luciferase activity after transfected with SREBP-1c plasmid. The PNPLA3 promoter which was shown the highest luciferase activity might contain a putative SREBP-1c binding sites (SRE). Then wild-type and mutant SRE reporter vectors were constructed to compare their luciferase activity. Statistical analysis was performed with one-way ANOVA of variance, followed by the Student's t test. Results Compared with the fed ad libitum group, the gene expression of SREBP-1c, PNPLA3 and fatty acid synthase(FAS) in liver decreased significantly in fasting group, and recovered in refeeding group (F=114.14, 334.11, 754.20, all P<0.05). Compared with normal control group, the gene and protein expression of SREBP-1c and PNPLA3 increased markedly in diabetic group(t=-18.39, -30.07 and 4.58, 6.81, all P<0.05). The basal luciferase activity of R-PNPLA3-1 increased for 51.13 folds when compared with that in the control group(t=-28.93, P<0.05), while no notable changes was found with R-PNPLA3-2 and R-PNPLA3 -3; transfection of SREBP-1c plasmid into LO2 cells caused 2.63 folds activation of R-PNPLA3-1(t=-7.64, P<0.01), while no changes in R-PNPLA3-2 and R-PNPLA3-3 groups was found. A putative SRE located in -97/-88 bp of rat PNPLA3 gene promoter, the luciferase activity of mutant SRE reporter vectors(MUT-R-PNPLA3-1) decreased for 40.8% when compared with that in wild-type(R-PNPLA3-1) ( t=4.99, P<0.05). Conclusion SREBP-1c trans-activates rat PNPLA3 via a proximal SRE (-100/-91 bp) of PNPLA3 promoter. Key words: Patatin like phospholipase domain containing 3; Sterol regulatory element-binding protein-1c; Transcriptional regulation