TEC promoted PPARγ binding to the Bsep promoter region and up-regulated its expression
In vitro studies have found that thiazolidinedione compounds (TZDs) can increase the expression of Bsep, but the exact molecular mechanism is still not fully understood[37]. Consistent with our current research results, TEC regulated the expression of Bsep independent of NFκb activity in hepatocytes (Figure 5D). Western blotting analysis showed that TEC increased the expression of Bsep, but not PPARγ in HepG2 cells (Figure 6A). When HepG2 cells were treated with TEC and GW1929 or TEC and GW9662, TEC did not further regulate the expression of Bsep compared with GW1929 or GW9662 treatment alone (Figure 6B). Further analysis revealed that TEC regulation of Bsep was completely dependent on PPARγ, as PPARγ deficiency blocked the regulation of Bsep by TEC (Figure 6C). Importantly, the luciferase assay showed that TEC enhanced the transcriptional activity of PPARγ in a dose-dependent manner (Figure 6D).
Thus, we hypothesized that TEC increased the binding of PPARγ to the Bsep promoter and promoted its transcription. To gain further insight into the molecular mechanism of the regulation of Bsep by TEC, a series of Bsep promoter deletion mutants were cloned into the luciferase reporter system, and it was found that overexpression of PPARγ enhanced Bsep activity only in the Luc1 constructs, suggesting that the PPARγ binding site within the Bsep promoter was between the –2948 and –2235 base pairs (bp) (Figure 6E). Chromatin immunoprecipitation (ChIP) assay was used to confirm that PPARγ was recruited to the –2948 and –2235 bp (Figure 6F). The promoter analysis tool JASPAR predicted putative PPARγ binding motif sequences (-2560 bp to -2541 bp: ttggtccacagtgacctcca) based on the PPARγ consensus sequence outlined (Figure 6G). These results collectively indicated that TEC treatment increased the binding of PPARγ to the Bsep promoter region, thereby upregulating its expression.