Figure 3 Effect of different prostaglandin receptor inhibitors in MH1C1 cells. A) The EP4 inhibitor L-161982 (10��M) was added 30min prior to stimulation with PGE2 (100��M) for 5min. B) The selleck chemical EP1 inhibitor SC51322 … Evidence of a role for Ca2+, but not PKC, in the PGE2-induced transactivation of EGFR We next tried to determine which pathways downstream of PLC�� are mediating the PGE2-induced transactivation of EGFR. InsP3 and DAG stimulate cytosolic Ca2+ release and protein kinase C (PKC) activity, respectively. Pretreatment of the cells with the PKC inhibitor GF109203X did not prevent the effects of PGE2 on the phosphorylation of the EGFR, ERK, or Akt in the MH1C1 cells (Figure4A).

Furthermore, the data in Figure4B, comparing PGE2 and the direct PKC activator tetradecanoylphorbol acetate (TPA), showed that TPA did not mimic the effect of PGE2 on Akt, and its stimulation of ERK, unlike the effect of PGE2, was blocked by GF109203X. Interestingly, pretreatment of the cells with GF109203X consistently increased basal and PGE2-induced Akt phosphorylation in the cells. This might result from a reduced feedback inhibition by PKC [47]. In contrast to TPA, thapsigargin, which increases the intracellular Ca2+ level by inhibiting the ��sarco/endoplasmic reticulum Ca2+-ATPase�� (SERCA) pump [48], induced gefitinib-sensitive phosphorylation of EGFR, ERK, and Akt (Figure4C). Taken together, these data suggest that Ca2+ rather than PKC mediates the PGE2-induced transactivation of the EGFR in these cells. Figure 4 Role of Ca2+ and PKC in responses to PGE2 in MH1C1 cells.

A) MH1C1 cells were pretreated for 30min with the PKC inhibitor GF109203X (3.5��M) before stimulation with PGE2 (100��M) for 5min. B) MH1C1 cells … Role of Src and metalloproteinases in the transactivation of the EGFR To further elucidate mechanisms involved in transactivation of the EGFR, we investigated the effects of Src inhibitors. As shown in Figure5A, pretreatment of the cells with the Src inhibitor CGP77675 almost completely abolished the PGE2-induced phosphorylation of EGFR and the activation of ERK and Akt, but, in contrast, had little or no effect on the phosphorylation of these proteins elicited by EGF. The Src inhibitor PP2 similarly prevented the phosphorylation of ERK in response to PGE2, while the response to EGF was not significantly affected (Figure5B).

These results suggest an involvement of a Src family kinase in the PGE2-induced transactivation of EGFR in MH1C1 cells. Figure 5 Effect of Src and MMP inhibitors on phosphorylation of EGFR and downstream targets. A) MH1C1 cells were pretreated for 90min with the Src inhibitor CGP 77675 (10��M). Cells were then stimulated with either PGE2 (100��M) … Previous evidence has implicated proteinases of the ��a-disintegrin-and-metalloproteinase�� (ADAM) family in EGFR transactivation by GPCRs GSK-3 in various cells [2,49,50].