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Abstract
Ca2+-transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca2+/HCO3−-sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3′,5′-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca2+/cAMP response element binding protein (CREB). Store-operated Ca2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca2+]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca2+, pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca2+-transcription coupling.
Highlights
In healthy blood vessels, the majority of vascular smooth muscle cells (VSMCs) are quiescent, contractile and proliferate slowly[1]
Phospho-cAMP response element binding protein (CREB) is elevated in neointimal VSMCs that appear after balloon injury of the carotid artery[28], and increased phospho-CREB levels correlate with high proliferation rates in cerebral artery following several forms of vessel injury linked with susceptibility to stroke[29]
Subsequent addition of Ca2+ (1.8 mM) to the extracellular solution induces a rapid rise in cytosolic Ca2+ due to Store-operated Ca2+ entry (SOCE) (Fig. 1a). human coronary artery smooth muscle cells (hCASMCs) transduced with adenoviruses encoding the Förster resonance energy transfer (FRET)-based cyclic AMP biosensor H187 and exposed to the same protocol exhibit changes in intracellular cyclic AMP that essentially mirror the cytosolic Ca2+ increase induced by the addition of thapsigargin and by SOCE (Fig. 1b)
Summary
The majority of vascular smooth muscle cells (VSMCs) are quiescent, contractile and proliferate slowly[1]. Ca2+-induced gene expression can be mediated in VSMCs by the transcription factor Ca2+/cyclic AMP response element binding protein (CREB)[6]. Chronic exposure of animals to lower than normal oxygen levels leads to increased VSMC proliferation in pulmonary vessels and decreased levels of both CREB and phospho-CREB30 This highlights our incomplete understanding of how different environmental cues are integrated by VSMCs to activate gene transcription. The current model for PKA-mediated phosphorylation of CREB in VSMCs is that Ca2+ entering the cytosol binds to calmodulin (CaM) and that the Ca2+/CaM complex activates Ca2+-sensitive transmembrane adenylyl cyclases (tmACs), leading to generation of cyclic AMP and the activation of PKA8. We demonstrate that SOCE activates sAC to generate cyclic AMP and induce phosphorylation of CREB at Ser[133] in human coronary artery smooth muscle cells. This ability to sense and integrate fluctuations in intracellular signals suggests sAC may be an important and overlooked regulatory node in the VSMC Ca2+-transcription pathway
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