No abstract

Calcium is a ubiquitous second messenger controlling a broad range of cellular functions including growth and proliferation. Quiescent, hyperthrophic and proliferating cells have different types of calcium signal. In quiescent cells the calcium signal mostly involves elementary calcium events such as sparks and puffs, produced by localized Ca 2+ release via a cluster of intracellular calcium channels, IP3 receptors and ryanodine receptors. This type of calcium signal promotes activation of the transcription factor CREB (cAMP response element binding protein) leading to cell cycle arrest in G1 phase via transactivation of p53/p21 signaling pathways. Proliferation is induced by phosphoinositide-coupled agonists and is associated with a sustained increase in cytosolic calcium due to 1.) enhanced excitability of IP3Rs after IP3 binding; 2.) enhanced activity of store-operated Ca 2+ channels and T-type voltage-operated Ca 2+ channels; 3.) decreased cytosolic Ca 2+ removal due to inhibition of PMCA (plasma membrane Ca 2+ -ATPase) and SERCA (sarco/endoplasmic reticulum Ca 2+ -ATPase) calcium pumps. This type of calcium signal favors activation of the transcription factor NFAT (nuclear factor of activated T lymphocytes) that promotes hypertrophic growth and/or cell cycle progression. We suggest that the two main Ca 2+ -regulated transcription factors, CREB and NFAT, exert opposite control over cell growth and/or proliferation. Therapeutic strategies based on lowering intracellular Ca 2+ or targeting of Ca 2+ -regulated transcription factors seems to be a promising approach to arrest growth and/or proliferation.

Abstract-Proliferation of vascular smooth muscle cells (VSMC) is a primary cause of vascular disorders and is associated with major alterations in Ca 2ϩ handling supported by loss of the sarco/endoplasmic reticulum calcium ATPase, SERCA2a. To determine the importance of SERCA2a in neointima formation, we have prevented loss of its expression by adenoviral gene transfer in a model of balloon injury of the rat carotid artery. Two weeks after injury, the intima/media ratio was significantly lower in SERCA2a-infected than in injured noninfected or injured ␤-galactosidaseinfected carotids (0.29Ϯ0.04 versus 0.89Ϯ0.19 and 0.72Ϯ0.14, respectively; PϽ0.05), and was comparable to that observed in control carotids (0.21Ϯ0.03). The pathways leading to proliferation were analyzed in serum-stimulated VSMC. Forced expression of SERCA2a arrested cell cycle at the G1 phase and prevented apoptosis. SERCA2a inhibits proliferation through inactivation of calcineurin (PP2B) and its target transcription factor NFAT (nuclear factor of activated T-cells) resulting in lowering of cyclin D1 and pRb levels. By using NFAT-competing peptide VIVIT, we showed that NFAT activity is strongly required to promote VSMC proliferation. In conclusion, we provide the first evidence that increasing SERCA2a activity inhibits VSMC proliferation and balloon injury-induced neointima formation. Key Words: vascular smooth muscle cell proliferation Ⅲ gene transfer Ⅲ SERCA2a Ⅲ calcium signaling Ⅲ nuclear factor of activated T-cells C ell hyper-proliferation is an important etiology factor of cardiovascular diseases such as primary atherosclerosis, restenosis, and vein-graft disease. 1,2 The neointimal vascular smooth muscle cell (VSMC) proliferation constitutes a primary cause of vascular disorders 1 but, despite increasing knowledge about the cell-cycle regulation of VSMC, the molecular mechanism governing VSMC proliferation remains elusive. Thus, identifying genetic modifiers of VSMC proliferation is a major focus in cardiovascular biology and medicine. 3 VSMCs have the ability to transition between quiescent differentiated and proliferating phenotypes. Acquisition of proliferating phenotype by VSMC is associated with alterations in Ca 2ϩ handling supported by modification of Ca 2ϩ transporter expression. 4 In quiescent VSMC, the Ca 2ϩ signal consists mainly of localized elementary calcium events. The global increase in Ca 2ϩ concentration is rapidly reduced by calcium pumps, keeping the cytoplasmic Ca 2ϩ concentration low. Spontaneous spark frequency decreases after activation of phosphoinositol-3 kinase (PI3K) and G protein-coupled receptors, probably attributable to inhibition of the ryanodine receptor (RyR). 6,7,14 -16 Several previous studies have suggested that SERCA2a is involved in the control of proliferation and growth: transgenic mice with only one allele of the ATP2a2 gene (SERCA2) develop numerous cancers of the upper digestive tract and skin, and cardiac hypertrophy 17,18 ; low levels of SERCA2a are associated with cardiac hypertrophy bot...