This pretreatment resulted in complete inhibition of PGE2-induced phosphorylation of EGFR, ERK, and Akt, while the EGF-induced phosphorylation Dasatinib Sigma of these proteins was not affected (Fig5C and D), indicating that the transactivation is dependent on mechanisms involving ADAM-mediated release of EGFR ligand(s). We also examined the effect of this inhibitor in the primary cultures of rat hepatocytes, and found neither inhibition of PGE2-induced phosphorylation of ERK and Akt in these cells nor any effect on EGF-induced phosphorylation of EGFR, ERK and Akt (Figure5E). Discussion We have shown that in the MH1C1 hepatocarcinoma cells stimulation with PGE2 or PGF2�� causes phosphorylation of the EGFR and an EGFR-dependent phosphorylation of ERK and Akt, indicating that these prostaglandins induced transactivation of EGFR.

Further study of the PGE2 effect suggested that the transactivation was mediated by the Gq-coupled FP receptor and activation of PLC�� with downstream signalling by Ca2+ release, Src, and ADAM-mediated shedding of membrane-bound EGFR ligand precursors. In contrast, in primary hepatocytes, PGE2 did not phosphorylate the EGFR, and gefitinib did not prevent phosphorylation of Akt or ERK after PGE2-stimulation, which lends further support to our previous data suggesting that GPCR agonists do not transactivate the EGFR in normal rat hepatocytes, but rather signal via mechanisms that synergistically enhance the effects of EGF [34,37,38,51,52] (Figure6). Figure 6 Mechanisms by which PGE2 interacts with EGFR-mediated signalling in hepatocytes and MH1C1 hepatocarcinoma cells.

A) In normal rat hepatocytes, PGE2 does not elicit transactivation of EGFR, but induces upregulation of the effectiveness in Ras/ERK and PI3K/Akt … Different receptors and pathways may be involved in mitogenic and tumour-promoting effects of prostaglandins [28]. qRT-PCR analysis showed that the prostaglandin receptors expressed in these cells are EP1, EP4, and FP. No significant increase in cAMP accumulation was detected, in accordance with previous results [53], suggesting either that the EP4 protein levels are low, or that these receptors are functionally uncoupled from adenylyl cyclase. 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 Batimastat antagonist AL8810. PGF2��, which binds to FP receptors with high affinity, mimicked the effects of PGE2.