In contrast, PGE2 stimulated accumulation of inositol phosphates. Pretreatment with the EP4 antagonist L161982 or the EP1 antagonist SC51322, had no effect on the PGE2-induced

phosphorylation of EGFR, ERK, or Akt, while the phosphorylation of these proteins were markedly inhibited by the FP antagonist AL8810. PGF2α, which binds to FP receptors with high affinity, mimicked the effects of PGE2. Together, these results suggest that in contrast to the normal rat hepatocytes, where the effect of PGE2 seems YAP-TEAD Inhibitor 1 chemical structure to be mediated primarily through the EP3 receptor [37, 52, 54], the MH1C1 cells, which do not express EP3 receptors, respond to PGE2 through FP receptors, Gq, and PLCβ. It is of interest that expression of EP3 receptors has been found to be suppressed or absent in colon cancer in vivo and see more in vitro, as compared to normal mucosa [55]. PLCβ can regulate cellular functions via two distinct pathways, involving DAG-mediated activation of PKC and InsP3-induced release and elevation of cytosolic Ca2+, respectively. Our findings suggest that in the MH1C1 cells, the effect of PGE2 was mediated through Ca2+, since it was not mimicked by TPA and not inhibited by a PKC blocker, while thapsigargin, which elevates intracellular Ca2+, mimicked the PGE2 effect, inducing a gefitinib-sensitive phosphorylation of EGFR. In other cells, both ligand-dependent

and ligand-independent mechanisms have been found to mediate EGFR transactivation [5]. Ligand-dependent mechanisms involve the release of EGFR agonists by cleavage and shedding of membrane-associated precursors by proteinases of the ADAM family [2, 49]. Ligand-independent mechanisms have been suggested to involve intracellular

molecules Mirabegron including Src family kinases and Pyk2 [1, 3, 56, 57]. Han et al. reported that in Hep3B cells, PGE2 induced phosphorylation of the EGFR through EP1 receptors and an intracellular mechanism involving Src [57]. Itabashi et al. demonstrated that in some hepatocarcinoma cell lines EGFR transactivation triggered by angiotensin II stimulation was mediated through release of EGFR ligand by members of the ADAM family [58]. In the MH1C1 cells, we observed that Src inhibitors abolished PGE2-stimulated phosphorylation of the EGFR, ERK, and Akt, but in contrast, only slightly affected the response to EGF, suggesting a role of Src in the transactivation in these cells. We also found evidence for the involvement of ligand shedding in the transactivation of EGFR after PGE2 stimulation, since pretreatment of the cells with the metalloproteinase inhibitor GM6001 almost completely prevented PGE2-induced, but not EGF-induced, phosphorylation of EGFR, Akt and ERK. GM6001 did not affect the effects of PGE2 in the normal hepatocytes. The lack of transactivation in response to PGE2 in these cells could be due to the low expression of metalloproteinases in hepatocytes as compared to hepatocarcinoma cells [59].

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