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The multifunctional Ca2+/calmodulin-dependent kinase II delta (CaMKIIdelta) controls neointima formation after carotid ligation and vascular smooth muscle cell proliferation through cell cycle regulation by p21.

ABSTRACT: The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) promotes vascular smooth muscle (VSMC) proliferation. However, the signaling pathways mediating CAMKII-dependent proliferative effects in vivo are poorly understood. This study tested the hypothesis that CaMKII? mediates neointimal proliferation after carotid artery ligation by regulating expression and activity of cell cycle regulators, particularly at the G1/S checkpoint. Data herein indicate that 14 days after carotid ligation, C57Bl/6 mice developed a marked neointima with robust CaMKII protein expression. In particular, only the CaMKII isoform ? was increased as demonstrated by quantitative RT-PCR. Genetic deletion of CaMKII ? prevented injury-induced neointimal hyperplasia and cell proliferation in the intima and media. In ligated carotids of control mice, the proliferative cell cycle markers cdk2, cyclin E, and cyclin D1 were activated. In contrast, in CaMKII?(-/-) mice, we detected a reduction in proliferative cell cycle regulators as well as an increase in the cell cycle inhibitor p21. This expression profile was confirmed in cultured CaMKII?(-/-) VSMC, in which cdk2 and cdk4 activity was decreased. Toward understanding how CAMKII? affects p53, a transcriptional regulator of p21, we examined p53 pathway components. Our data indicate that p53 is elevated in CAMKII?(-/-) VSMC, whereas phosphorylation of the p53-specific E3 ligase, Mdm2, was decreased. In conclusion, CaMKII stimulates neointima proliferation after vascular injury by regulating cell proliferation through inhibition of p21 and induction of Mdm-2-mediated degradation of p53.

SUBMITTER: Li W

PROVIDER: S-EPMC3048686 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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The multifunctional Ca2+/calmodulin-dependent kinase II delta (CaMKIIdelta) controls neointima formation after carotid ligation and vascular smooth muscle cell proliferation through cell cycle regulation by p21.

Li Weiwei W Li Hui H Sanders Philip N PN Mohler Peter J PJ Backs Johannes J Olson Eric N EN Anderson Mark E ME Grumbach Isabella M IM

The Journal of biological chemistry 20101230 10

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) promotes vascular smooth muscle (VSMC) proliferation. However, the signaling pathways mediating CAMKII-dependent proliferative effects in vivo are poorly understood. This study tested the hypothesis that CaMKIIδ mediates neointimal proliferation after carotid artery ligation by regulating expression and activity of cell cycle regulators, particularly at the G1/S checkpoint. Data herein indicate that 14 days after carotid ...[more]

PMID: 21193397

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Project description:OBJECTIVE:The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS:The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS:These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.

Project description:Background3,3'Diindolylmethane (DIM), a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs) remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.Methodology/principal findingsDIM dose-dependently inhibited the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4/6 as well as an increase in p27(Kip1) levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptor? (PDGF-R?) and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK)3?, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3). Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.ConclusionThese results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-R? and the activities of downstream signaling pathways. The results suggest that DIM has the potential to be a candidate for the prevention of restenosis.

Dataset Information

The multifunctional Ca2+/calmodulin-dependent kinase II delta (CaMKIIdelta) controls neointima formation after carotid ligation and vascular smooth muscle cell proliferation through cell cycle regulation by p21.

Publications

The multifunctional Ca2+/calmodulin-dependent kinase II delta (CaMKIIdelta) controls neointima formation after carotid ligation and vascular smooth muscle cell proliferation through cell cycle regulation by p21.

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