24 These results provided evidence that the PPARβ subtype interacted with the MAT2A PPRE sequence. Whether this interaction had a functional effect was determined in subsequent experiments. PPARβ exhibited enhanced binding to Selleckchem GSK1120212 PPREs 1, 2, 4, and 6 in activated HSCs from BDL livers compared with their quiescent counterparts from sham controls (Fig. 7A,B). PPARβ showed strong interaction with PPRE-5 in quiescent HSCs, and this binding was not enhanced

further during HSC activation (Fig. 7A,B). Knockdown of PPARβ in activated BSC cells (Fig. 7C,D, left panel) and primary rat HSCs (Fig. 7C,D, right panel) lowered the expression of both MAT2A mRNA and protein by 1.6- to two-fold. This also inhibited MAT2A promoter activity by two-fold compared with a negative control siRNA in activated BSC cells

(Fig. 7E). These results showed that in activated HSCs, PPARβ promoted MAT2A transcription. Forced expression of MAT2A vector resulted in a three- to four-fold increase Ceritinib of MAT2A protein in RSG-treated cells (Fig. 8A) that is comparable to endogenous expression of MAT2A in activated HSCs.15 This further resulted in a 55%-60% decrease in PPARγ and C/EBPβ protein expression (Fig. 8A) as well as a decrease in PPARγ mRNA (Fig. 8B) but not C/EBPβ (data not shown). The protein levels of PPARβ and α-SMA increased by two- to three-fold in MAT2A HSCs compared with vector control (Fig. 8A). A significant increase in α-SMA (Fig. 8B) but not PPARβ mRNA (data not shown) was observed after MAT2A overexpression. Lowering C/EBPβ reserves in RSG-treated BSC cells Depsipeptide mouse by siRNA resulted in a modest 1.3- to 1.4-fold increase in MAT2A mRNA and protein expression (Supporting Fig. 1A,B) and a similar increase in MAT2A promoter activity (Supporting Fig. 1C). However, overexpression of C/EBPβ in activated cells did not significantly alter MAT2A expression or promoter activity (data

not shown). MAT1A and MAT2A genes exhibit differential expression within various cell types of the liver. Hepatocytes mainly express MAT1A, and Kupffer cells and endothelial cells express MAT1A with trace amounts of MAT2A, whereas normal HSCs exclusively express MAT2A.14 Despite the predominant expression of MAT1A in the differentiated liver, the small percentage of quiescent HSCs uses MAT2A rather than MAT1A for SAM biosynthesis.14 In rapidly dividing and dedifferentiated liver, a significant induction of MAT2A has been observed along with silencing of the MAT1A gene.9, 10, 20 It is intriguing that the pattern of MAT expression in HSCs from adult differentiated liver resembles that of actively growing hepatocytes, which have high MAT2A and low MAT1A expression. In light of these facts, it is logical to hypothesize that MAT2A would be tightly regulated in quiescent HSCs.