We examined the effect of TGF-β1 on cPLA2
α phosphorylation and protein expression in three human hepatocellular carcinoma cell lines (Hep3B, Huh7 and HepG2). Treatment of these cells with TGF-β1 (5 ng/ml) induced a rapid phosphorylation of cPLA2
α, occurring within 15 minutes (). In contrast, TGF-β1 treatment had no effect on the expression of cPLA2
α and COX-2 proteins (Supplementary Figure 1
). Consistent with its effect on cPLA2
α phosphorylation, TGF-β1 treatment significantly increased AA release and PGE2
production in these cells (). These results show that TGF-β1 activates cPLA2
α phosphorylation, increase AA release and PGE2
synthesis in these cells.
TGF-β1 activates arachidonic acid signaling cascade through cPLA2α phosphorylation in transformed human hepatocytes
α is phosphorylated by protein kinases including p38 MAPK and ERK1/2 (p44/42 MAPK), we also examined the effect of TGF-β1 on p38 MAPK and ERK1/2 activation in these cells. TGF-β1 treatment induced the phosphorylation of p38 MAPK and ERK1/2 as well as Smad2/3 (). These findings, along with the significant increase of Smad2/3 reporter activity by TGF-β1 (), indicate intact TGF-β-initiated signaling in these cells. The involvement of p38 MAPK and ERK1/2 in TGF-β1-induced cPLA2
α phosphorylation is demonstrated by the fact that TGF-β1-induced cPLA2
α phosphorylation in these cells was inhibited by the p38 MAPK inhibitor SB203580 and by the MEK1/2 inhibitor PD98059 (). Consistent with this, the TGF-β1-induced AA release and PGE2
production was also inhibited by SB203580, PD98059, and by the cPLA2
α inhibitor pyrrolidine (). These findings demonstrate the role of p38 MAPK and ERK1/2-mediated cPLA2
α activation in TGF-β1-induced AA release and PGE2
synthesis. In addition, the TGF-β1-induced PGE2
production was also inhibited by the selective COX-2 inhibitor NS-398 (), although the level of COX-2 expression was not altered (Supplementary Figure 1
). Taken together, these findings suggest that TGF-β1 induces AA release for PGE2
production via p38 MAPK and ERK1/2-mediated cPLA2
The effect of PPARγ overexpression and ligands on p3TP and PPRE reporter activities
Further experiments were performed to determine whether cPLA2α overexpression or PGE2 treatment could prevent TGF-β1-induced inhibition of cell growth. As shown in , overexpression of cPLA2α in Hep3B cells prevents TGF-β1-induced inhibition of growth; PGE2 treatment of Hep3B cells as well as rat primary hepatocytes also prevented TGF-β1-induced inhibition of cell growth. The observation that cPLA2α overexpression prevents TGF-β1-induced caspase-3 cleavage in Hep3B cells suggests the role of cPLA2α for prevention of TGF-β-induced apoptosis. These data indicate that cPLA2α signaling pathway is able to counteract the growth-inhibitory effect of TGF-β.
cPLA2α signaling prevents TGF-β1-induced inhibition of cell growth
We next investigated whether the cPLA2α-mediated AA release might influence Smad transcriptional activity. Hep3B cells were transiently transfected with the cPLA2α expression plasmid or the control plasmid MT-2 with cotransfection of the p3TP-Lux reporter construct (containing Smad2/3-responsive element) and the cell lysates were obtained to determine the luciferase reporter activity. As shown in , overexpression of cPLA2α significantly inhibited Smad2/3 transcriptional activity. Accordingly, depletion of cPLA2α by siRNA significantly enhanced Smad2/3 transcriptional activity (). These findings reveal an important role of cPLA2α for modulation of Smad2/3 transcription activity.
The effect of cPLA2α on p3TP and PPRE reporter activities
PPARγ is a ligand-activated nuclear transcription factor regulating the expression of target genes by binding to specific peroxisome proliferator response elements (PPRE) or by interacting with other intracellular signaling molecules(26
). The activity of PPAR-γ is regulated by several ligands, including thiazolidinediones, 15-deoxy-Δ12,14
, and arachidonic acid, among others. Consistent with our previous study showing that cPLA2
α is able to activate PPAR-γ in other cells(17
α overexpression was found to increase PPRE reporter (containing PPAR response element) activity in all three hepatocellular carcinoma cell lines used in this study (Hep3B, Huh7 and HepG2) (). Since PPAR-γ is known to bind and inhibit Smad3 in vitro
) and cPLA2
α is able to activate PPAR-γ, we postulated that cPLA2
α might inhibit Smad3 through activation of PPAR-γ.
To document the direct effect of PPAR-γ on Smad activation, Hep3B, HepG2 and Huh7 cells were co-transfected with the PPAR-γ expression plasmid and the p3TP-Lux reporter construct containing the Smad2/3 response element. As shown in , overexpression of PPAR-γ partially inhibited Smad transcriptional activity in those cells. Accordingly, activation of PPAR-γ by its ligands (ciglitazone and piglitazone) significantly inhibited TGF-β1-induced Smad activation in Hep3 cells; this effect was observed with or without Smad3 overexpression (). In contrast, siRNA inhibition of PPAR-γ augmented TGF-β1-mediated Smad transcription (). In the transfection experiments with a reporter construct containing PPRE, we observed approximately 2 fold increase of PPRE reporter activity in Hep3B cells after PPAR-γ ligand treatment (ciglitazone and piglitazone) (), suggesting that the endogenous PPAR-γ protein in these cells is functional.
The effect of PPARγ depletion on p3TP reporter activity and cell growth
To further evaluate the role of cPLA2α and PPAR-γ in Smad activation and hepatic cell growth, additional experiments were performed to determine whether depletion of endogenous cPLA2α and PPAR-γ might inhibit cell growth via Smad2/3. As shown in , depletion of either cPLA2α or PPAR-γ significantly reduced cell growth and this effect was blocked by Smad2/Smad3 siRNA. These findings suggest the involvement of Smad2/3 in cPLA2α/PPAR-γ depletion-induced inhibition of cell growth. Further, the cPLA2α product, arachidonic acid, and the PPAR-γ ligands, ciglitazone and piglitazone, inhibited TGF-β1-induced binding of Smad 3 to its DNA response element (). Therefore, Smad3 is a downstream target of cPLA2α/PPAR-γ in hepatic cells.
AA and PPARγ ligands block TGF-β1-induced Smad 3 binding to its DNA response element. Comparison of cPLA2α and PPARγ expression in different cell lines
We have found that cPLA2α activates PPAR-γ in all the three hepatic cell lines used in this study. However, these cell lines respond differently to TGF-β treatment – whereas TGF-β1 significantly inhibited the growth of Hep3B cells, it had minimal growth inhibitory effect in Huh7 or HepG2 cells under the same experimental condition (). The exact mechanism for such a differential effect among different cell lines is complex; but it is possible that this may partly relate to the low level of PPAR-γ expression in Hep3B cells (hence sensitive to TGF-β-induced inhibition of growth) and the high level of PPAR-γ in Huh7 and HepG2 cells (hence resistant to TGF-β-induced inhibition of growth).
To further address the role of cPLA2α in TGF-β-induced hepatocyte growth regulation, we generated transgenic mice with targeted expression of the cPLA2α in the liver () and the produced animals were utilized to determine TGF-β-induced inhibition of hepatocyte growth. Primary hepatocytes were isolated from the cPLA2α transgenic or wild type mice and the cultured cells were treated with different concentrations of TGF-β1 in serum-free medium to determine [3H]-thymidine incorporation. As shown in , although TGF-β1 significantly inhibited the growth of hepatocytes from wild type mice, this effect was attenuated in cPLA2α overexpressed hepatocytes. Thus, overexpression of cPLA2α in hepatocytes renders the cells resistant to TGF-β1-induced inhibition of growth.
Overexpression of cPLA2α in hepatocytes prevents TGF-β1-induced mitoinhibition