Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling

Fractalkine (CX(3)CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX(3)CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX(3)CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX(3)CL1. We sought to determine the effect of CX(3)CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. CX(3)CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX(3)CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX(3)CL1. We describe a novel and specific small molecule antagonist of CX(3)CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX(3)CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX(3)CL1 involving release of a soluble EGFR ligand. Specifically, CX(3)CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX(3)CL1. We have demonstrated two novel and important functions of CX(3)CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.