In inhibitor expert agreement with the inhibition of AP 1 protein Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries expression, AG1478 also dramatically suppressed LPA stimulated AP 1 DNA binding and transcriptional activities in Caov 3 cells. Similar results were obtained from the SKOV 3 and Dov 13 ovarian cancer cell lines. To confirm that AG1478 indeed specifically inhibited EGFR instead of toxic or non specific interference with other targets, we expressed a truncated form of EGFR lacking the cytosolic domain and thereby functioning as a dominant negative mutant through dimerization with wild type EGFR. EGFR DN was introduced into Caov 3 cells using adenovirus expressing EGFR DN or by transient transfection of pcDNA3 EGFR DN with Amaxa nuocleofector Kit T that yield high transfection efficiency in ovarian cancer cell lines as we described previously.

As shown Inhibitors,Modulators,Libraries in Fig. 3B, expression of EGFR DN indeed inhibited LPA induced expression of AP 1 proteins. These results demonstrated that an intact EGFR is indispensable for LPA activation of AP 1. To address whether EGFR, Inhibitors,Modulators,Libraries instead of other RTKs, is specifically required for GPCR signaling to AP 1, we co stimulated Caov 3 cells with LPA and HGF, an agonist of c Met, in the presence of the EGFR blocker AG1478. As shown in Fig. 3A, the inhibitory effect of AG1478 on AP 1 proteins was reversed by co stimulation of the cells with LPA and HGF. LPA induced AP 1 DNA bind ing activity was also restored by HGF in the presence of AG1478. The impact of HGF was not due to activation of AP 1 proteins by HGF itself as the effect of HGF on AP 1 was marginal compared to that of LPA.

These results indicate that the activity of a RTK, not necessarily EGFR, provides a permissive input to allow transmission of GPCR signals to AP 1 although such an input itself is not sufficient to induce full AP 1 activation. EGFR independent activation of NF B by LPA The role of EGFR in LPA induction of AP 1 activity raises the possibility that Inhibitors,Modulators,Libraries EGFR might be required ubi quitously for GPCR actions. This could be due to the requirement of a RTK activity for overall functioning of GPCR. However, if the RTK input is implicated in acti vation of the specific intracellular signaling processes instead of GPCR itself, certain LPA signaling pathways may be exceptional to this requirement. To distinguish these possibilities, we examined LPA induced activation of NF B, another prominent transcription factor criti cally involved in activation of many LPA target genes.

In Caov 3 treated with AG1478 or overexpressing EGFR DN, LPA induced NF B p65 phosphorylation, I Ba phosphorylation and I Ba degradation at levels comparable to those detected in control cells with intact INCB018424 EGFR. Similarly, LPA stimulated NF B DNA binding activity was not compromised by AG1478 as measured by EMSA. Nor was LPA driven NF B transcriptional activity significantly affected by incubation of cells with AG1478 as analyzed by the NF B responsive luciferase reporter assay.