This pretreatment resulted in complete inhibition of PGE2-induced phosphorylation of EGFR, ERK, and Akt, while the EGF-induced phosphorylation of these proteins was not affected (Fig 5C 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 LY2835219 of ERK and Akt in these cells nor any effect on EGF-induced phosphorylation of EGFR, ERK and Akt (Figure 5E). Discussion We have shown that in the MH1C1 hepatocarcinoma cells stimulation with PGE2 or PGF2α causes phosphorylation of the EGFR and Copanlisib 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 PLCβ 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 Thiamine-diphosphate kinase mechanisms that synergistically enhance the effects of EGF [34, 37, 38, 51, 52] (Figure 6). selleck chemicals llc Figure 6 Mechanisms by which PGE 2 interacts with EGFR-mediated signalling in hepatocytes and MH 1 C 1 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 pathways downstream of EGFR, leading to an
enhanced mitogenic response to EGF family growth factors [37, 38, 51]. Although not fully clarified, previous studies have indicated that this effect of PGE2 is mediated primarily through EP3 receptors and Gi proteins, requires several hours to develop, and is most likely a result of altered gene expression [34, 37, 38, 51, 52]. B) In MH1C1 rat hepatocarcinoma cells, PGE2 transactivates EGFR and thereby activates the Ras/ERK and PI3K/Akt signalling pathways. The results of the present study suggest that this effect is exerted via FP receptors, Gq proteins, PLCβ, intracellular Ca2+ (but not PKC), Src, and ADAM-mediated release of EGFR ligands. Different receptors and pathways may be involved in mitogenic and tumour-promoting effects of prostaglandins . qRT-PCR analysis showed that the prostaglandin receptors expressed in these cells are EP1, EP4, and FP.