Aims Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor
CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is
expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis
towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate
the signalling mechanisms involved.
Methods
and results
CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining
and Annexin-V flow cytometry. Furthermore, CX3CL1 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 CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1
(AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 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 CX3CL1 involving release of a soluble EGFR
ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold
within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1.
Conclusion We have demonstrated two novel and important functions of CX3CL1 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.