The signalling pathways that link G-protein-coupled receptors to mitogen-activated protein kinases involve receptor and non-receptor tyrosine kinases and protein kinase C (PKC). We explored the pathways that are implicated in the thromboxane (TX) A2-dependent activation of extracellular-signal-regulated protein kinase (ERK) and the role of the two TX receptor (TP) isoforms, TPα and TPβ. ERK activation by IBOP, a TX analogue, was dependent on epidermal-growth-factor receptor (EGFR) in TPα- or TPβ-transfected cells and in human aortic smooth muscle cells (hASMCs), since AG1478, a selective inhibitor of tyrosine phosphorylation of the EGFR, strongly blocked ERK and EGFR phosphorylation. In addition, EGFR transactivation leading to ERK activation involved matrix metalloproteinases (MMPs), since BB2516, an inhibitor of MMP, decreased ERK and EGFR phosphorylation in TPα- or TPβ-transfected cells. Moreover, we showed that both isoforms activate ERK phosphorylation in an Src-kinase-dependent manner, whereas PKC was mainly implicated in ERK activation and EGFR phosphorylation by TPβ. In hASMCs, we showed that ERK activation depended on both pertussis-sensitive and -insensitive Gα-proteins. We demonstrated further that EGFRs, PKC, Src kinase and MMPs are involved in ERK activation by TX. The results of the present study highlight a role for MMPs and PKC in EGFR transactivation triggered by the TPs and demonstrate this mechanism for the first time in primary cells, i.e. hASMCs.
- protein kinase C (PKC)
- thromboxane receptor
- growth-factor-receptor transactivation
- vascular cell
↵1 These authors contributed equally to this work.
Abbreviations used: ADAM, a disintegrin and metalloproteinase-like; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular-signal-regulated kinase; FGF-2, fibroblast growth factor 2; GPCR, G-protein-coupled receptor; hASMC, human aortic smooth muscle cell; HB-EGF, heparin-binding EGF; HEK-293-TPα/β, HEK-293 cells stably overexpressing TPα or TPβ; IBOP, [1S-[1α,2α(Z),3β(1E,3S),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; JNK, c-Jun N-terminal kinase; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinases; MEK1, MAPK/ERK kinase 1; MMP, matrix metalloproteinase; PDGFR, platelet-derived-growth-factor receptor; PKA, protein kinase A; PKC, protein kinase C; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine; PTX, pertussis toxin; RTK, receptor tyrosine kinase; SmGM, smooth-muscle-cell growth medium; SQ 29548, [(1S)1α,2β(5Z)3α,4β]-7-[3-[2-(phenylamino)carbonyl-hydrazino-methyl]-7-oxabicyclo[2.2.1]-hept-2-yl]-5-heptenoic acid; TP, TX receptor; TX, thromboxane.
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