Thromboxane A2 receptor-mediated epidermal growth factor receptor transactivation: involvement of PKC-delta and PKC-epsilon in the shedding of epidermal growth factor receptor ligands.

We examined thromboxane A(2) receptor (TP)-mediated transactivation of epidermal growth factor receptor (EGFR) through the shedding of EGFR ligands. A TP agonist U46619 caused the phosphorylation of EGFR in 1321N1 human astrocytoma cells, which was inhibited by an EGFR selective inhibitor AG1478 and by a disintegrin and metalloproteinase (ADAM) inhibitor TAPI-2, indicating TP stimulation caused the EGFR transactivation through the EGFR ligand shedding. Since 1321N1 cells expressed heparin-binding EGF (HB-EGF) mRNA, the mechanism of TP-mediated EGFR transactivation was examined in HEK293 cells expressing alkaline phosphatase-conjugated HB-EGF and TP. U46619 caused the shedding of HB-EGF in a time- and concentration-dependent manner. The TP-mediated shedding was inhibited by a furin inhibitor CMK, TAP-2, dominant-negative G alpha(q), a G(q/11) inhibitor YM254890, and also by a non-selective PKC inhibitor GF109203X and PKC down-regulation, but not by a conventional PKC inhibitor Gö6976. Furthermore, siRNAs of PKC-delta and PKC-epsilon inhibited U46619-induced HB-EGF shedding. Although BAPTA/AM had no effect on U46619-induced shedding of HB-EGF, EGTA inhibited it. These results suggest that TP-mediated EGFR transactivation is partially caused by shedding of HB-EGF, which involves furin and ADAM via novel types of PKCs (PKC-delta and PKC-epsilon) through G alpha(q/11) proteins in an extracellular Ca(2+)-dependent manner.