Sphingosine 1-phosphate induces CREB activation in rat cerebral artery via a protein kinase C-mediated inhibition of voltage-gated K + channels

Abstract

Sphingosine 1-phosphate (S1P) is a potential mitogenic stimulus for vascular smooth muscle. S1P promotes an increase in the intracellular calcium concentration ([Ca 2+] i) in cerebral arteries, however S1P effects on regulation of gene expression are not known. Activation of the Ca 2+-dependent transcription factor, cAMP response element-binding protein (CREB), is associated with smooth muscle proliferation. The aim of this study was to examine the Ca 2+-dependent mechanisms involved in S1P-induced CREB activation in cerebral artery. Western blotting and immunofluorescence with a phospho-CREB antibody were used to detect CREB activation in Sprague–Dawley rat cerebral arteries. Whole-cell patch clamp recording and single cell imaging of [Ca 2+] i were performed on freshly isolated cerebral artery myocytes. S1P increased activation of CREB in the nucleus of cerebral arteries. This activation was mediated by Ca 2+/calmodulin-dependent protein kinase and was dependent on an increase in [Ca 2+] i via two mechanisms: (i) intracellular Ca 2+ release via an inositol 1,4,5-trisphosphate (InsP 3)-dependent pathway and (ii) Ca 2+ entry through voltage-dependent Ca 2+ channels (VDCC). Activation of the VDCC occurred through S1P-induced inhibition (approximately 50%) of the voltage-gated potassium (K +) current. This inhibition was via a protein kinase C-mediated pathway resulting in tyrosine phosphorylation of at least one isoform of the Kv channel (Kv 1.2). These results demonstrate that S1P can activate the transcription factor CREB through different Ca 2+-dependent pathways including intracellular Ca 2+ release and inhibition of voltage-gated K + channels leading to Ca 2+ influx. Our findings suggest a potential role for S1P in regulation of gene expression in vascular smooth muscle.