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.