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


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.


CONCLUSIONS
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.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Gemma E
GE
White
Thomas C C
TC
Tan
Alison E
AE
John
Carl
C
Whatling
William L
WL
McPheat
David R
DR
Greaves
D017209:Apoptosis / Q000502:physiology*; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D054428:Chemokine CX3CL1 / Q000235:genetics / Q000378:metabolism*; D003331:Coronary Vessels / Q000166:cytology; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D066260:Epiregulin; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008934:Mitogens / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; C587880:EREG protein, human; D066260:Epiregulin; D008934:Mitogens; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases