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. 2013 Feb;54(2):379–385. doi: 10.1194/jlr.M030304

Fig. 1.

Fig. 1.

Effect of phospholipase D2 gain of function on human BSEP promoter activity. UPS cells were transfected with expression constructs for PLD2 and/or FIC1. The human BSEP promoter was used as a readout of FXR activity. Activity in untreated UPS cells was set at 100% and all results were normalized to renilla luciferase from a pRL-TK construct as a control for transfection efficiency. The scale of the y axis is logarithmic. Error bars represent the SD for nine measurements. A: Effect of PLD2 and FIC1. Human BSEP promoter activity was significantly increased by overexpression of PLD2 and/or FIC1 with synergistic activation by both PLD2 and FIC1. All comparisons were significant at P < 0.001. B: Effect of PKCζ inhibition on PLD2-mediated signaling. UPS cells were transfected with no PLD2 construct (control) or a wild-type PLD2 expression construct. PKCζ was inhibited by either siRNA silencing (siPKCζ) or by treatment with 100 µM myristolated pseudosubstrate inhibitor (PSI). PLD2 activated the BSEP promoter in the untreated UPS cells but not in the cells where PKCζ was either silenced or inhibited. C: Effect of FXR inhibition on PLD2-mediated signaling. UPS cells were transfected with no PLD2 construct (control) or a wild-type PLD2 expression construct. FXR was inhibited by either siRNA silencing (siFXR) or overexpression of a dominant negative FXR (dn FXR). Basal activity of the BSEP promoter was markedly reduced and not significantly increased when FXR was inhibited. D: Effect of an FXR response element mutant BSEP promoter on PLD2 mediated signaling. UPS cells were transfected with a wild-type human BSEP promoter reporter construct or one in which the FXR binding cis-element was mutated (FXREµ). Media for these studies included 0.5% charcoal fetal calf serum to minimize the effect of bile acids in fetal calf serum. Cells were untreated (control), treated with 50 µM CDCA and/or transfected with wild-type PLD2. Treatment with CDCA and/or PLD2 significantly increased the wild-type human BSEP promoter activity, but not the FXR response element mutant. Synergistic activation of wild-type human BSEP promoter was seen when CDCA and PLD2 were combined. All comparisons of the wild-type BSEP promoter were significant at a P < 0.001. There were no significant differences for any of the mutant promoter responses.