Regulation of Extracellular Signal-Regulated Kinase Cascades by α- and β-Isoforms of the Human Thromboxane A2Receptor

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Abstract
Thromboxane A2 (TXA2) stimulates mitogenic growth of vascular smooth muscle. In humans, TXA2 signals through two TXA2 receptor (TP) isoforms, termed TPα and TPβ. To investigate the mechanism of TXA2-mediated mitogenesis, regulation of extracellular signal-regulated kinase (ERK) signaling was examined in human embryonic kidney 293 cells stably overexpressing the individual TP isoforms. The TXA2 mimetic 9,11-dideoxy-9α,11α-methano epoxy prostaglandin F2α (U46619) elicited concentration- and time-dependent activation of ERK1 and -2 through both TPs with maximal TPα- and TPβ-mediated ERK activation observed after 10 and 5 min, respectively. U46619-mediated ERK activation was inhibited by the TP antagonist [1S-[1α,2β-(5Z)-3β,4α-]]-7-[3-[[2-(phenylamino)carbonyl]hydrazine] methyl]-7-oxabicyclo[-2,2,1-]hept-2yl]-5-heptenoic acid (SQ29,548), and by the mitogen-activated protein kinase kinase inhibitor 2′-amino-3′-methoxyflavone (PD 98059). Although ERK activation through TPα was dependent on 2-[1-(dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X)-sensitive protein kinase (PK) Cs, ERK activation through TPβ was only partially dependent on PKCs. ERK activation through both TPα and TPβ was dependent on PKA and phosphoinositide 3-kinase (PI3K) class 1A, but not class 1B, and was modulated by Harvey-Ras, A-Raf, c-Raf, and Rap1B/B-Raf and also involved transactivation of the epidermal growth factor receptor. Additionally, PKB/Akt was activated through TPα and TPβ in a PI3K-dependent manner. In conclusion, we have defined the key components of TXA2-mediated ERK signaling and have established that both TPα and TPβ are involved. TXA2-mediated ERK activation through the TPs is a complex event involving PKC-, PKA-, and PI3K-dependent mechanisms in addition to transactivation of the EGF receptor. TPα and TPβ mediate ERK activation through similar mechanisms, although the time frame for maximal ERK activation and PKC dependence differs.