CaM kinase IIδ2-dependent regulation of vascular smooth muscle cell polarization and migration

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKIIδ2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKIIδ2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKIIδ2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKIIδ2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKIIδ2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

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Chlamydia pneumoniae infection induces vascular smooth muscle cell migration via Rac1 activation

Journal of Medical Microbiology ◽

10.1099/jmm.0.065359-0 ◽

2014 ◽

Vol 63 (2) ◽

pp. 155-161 ◽

Cited By ~ 2

Author(s):

Junxia Zhang ◽

Haiwei Wang ◽

Lijun Zhang ◽

Tengteng Zhang ◽

Beibei Wang ◽

...

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Botulinum Toxin ◽

Vascular Smooth Muscle ◽

Chlamydia Pneumoniae ◽

Leading Edge ◽

Primary Role ◽

Phosphatidylinositol 3 Kinase ◽

Smooth Muscle Cell Migration

Chlamydia pneumoniae infection has been shown to be associated with the development of atherosclerosis by promoting the migration of vascular smooth muscle cells (VSMCs). However, how C. pneumoniae infection induces VSMC migration is not fully understood. A primary role of Ras-related C3 botulinum toxin substrate 1 (Rac1) is to generate a protrusive force at the leading edge that contributes to cell migration. Whether Rac1 activation plays a role in C. pneumoniae infection-induced VSMC migration is not well defined. In the present study, we therefore examined Rac1 activation in C. pneumoniae-infected rat primary VSMCs and the role of Rac1 activation in C. pneumoniae infection-induced VSMC migration. Glutathione S-transferase pull-down assay results showed that Rac1 was activated in C. pneumoniae-infected rat primary VSMCs. A Rac1 inhibitor, NSC23766 (50 µM,) suppressed Rac1 activation stimulated by C. pneumoniae infection, and thereby inhibited C. pneumoniae infection-induced VSMC migration. In addition, C. pneumoniae infection-induced Rac1 activation in the VSMCs was blocked by LY294002 (25 µM), an inhibitor of phosphatidylinositol 3-kinase (PI3K). Taken together, these data suggest that C. pneumoniae infection promotes VSMC migration, possibly through activating Rac1 via PI3K.

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CEMIP regulates the proliferation and migration of vascular smooth muscle cells in atherosclerosis through the WNT–beta-catenin signaling pathway

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0249 ◽

2020 ◽

Vol 98 (2) ◽

pp. 249-257

Author(s):

Qiang Xue ◽

Xiaoli Wang ◽

Xiaohui Deng ◽

Yue Huang ◽

Wei Tian

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Matrix Metalloproteinase ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Signaling Pathway ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

And Migration ◽

Proliferation And Migration

In this study we investigated the regulatory role of cell-migration-inducing and hyaluronan-binding protein (CEMIP) in the proliferation and migration of vascular smooth muscle cells (VSMCs). The mRNA and protein levels of CEMIP were upregulated in the plasma samples from patients with atherosclerosis, and in VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB), compared with plasma from healthy subjects and untreated VSMCs. Silencing CEMIP suppressed PDGF-BB-induced cell migration and proliferation in VSMCs, as determined using a Cell Counting Kit-8 assays, 5-ethynyl-2′-deocyuridine (EDU) assays, flow cytometry, wound healing assays, and Transwell assays. Overexpression of CEMIP promoted the proliferation and migration of VSMCs via activation of the Wnt–β-catenin signaling pathway and the upregulation of its target genes, including matrix metalloproteinase-2, matrix metalloproteinase-7, cyclin D1, and c-myc, whereas CEMIP deficiency showed the opposite effects. The knockdown of CEMIP in ApoE−/− mice by intravenous injection of lentiviral vector expressing si-CEMIP protected against high-fat-diet-induced atherosclerosis, as shown by the reduced aortic lesion areas, aortic sinus lesion areas, and the concentration of blood lipids compared with mice normally expressing CEMIP. These results demonstrated that CEMIP regulates the proliferation and migration of VSMCs in atherosclerosis by activating the WNT–β-catenin signaling pathway, which suggests the therapeutic potential of CEMIP for the management of atherosclerosis.

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Abstract 374: Inhibition of Mitochondrial CaMKII Reduces Vascular Smooth Muscle Migration and Neointima Formation and Halts Mitochondrial Mobility

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.374 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Emily Nguyen ◽

Olha Koval ◽

Isabella Grumbach

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Mitochondrial Fission ◽

Leading Edge ◽

Neointima Formation ◽

Vsmc Proliferation ◽

Mitochondrial Motility ◽

And Migration

Background: Restenosis after angioplasty for coronary vascular disease remains a critical problem in cardiovascular medicine. Vascular smooth muscle cell (VSMC) migration and proliferation cause restenosis through neointima formation. Mitochondrial motility is likely necessary for cell proliferation and migration, and is inhibited in microdomains with increased Ca 2+ . The Ca 2+ /calmodulin-dependent kinase II (CaMKII) in mitochondria (mtCaMKII) is proposed to control mitochondrial matrix Ca 2+ uptake through mitochondrial Ca 2+ uniporter (MCU). Thus, we hypothesized that blocking mtCaMKII decreases VSMC migration and neointima formation by decreasing mitochondrial motility. Methods: mtCaMKII was inhibited by expression of the mitochondria-targeted CaMKII inhibitor peptide (CaMKIIN) in a novel transgenic mouse model in smooth muscle only (SM-mtCaMKIIN) or delivered by adenoviral transduction (Ad-mtCaMKIIN). Results: In our models, mtCaMKIIN was detected selectively in mitochondria of VSMC. mtCaMKIIN significantly reduced mitochondrial Ca 2+ current and Ca 2+ content compared to WT in vivo and in vitro. SM-mtCaMKIIN mice showed significantly reduced neointimal area 28 days after endothelial injury (n=8, p<0.05) and fewer proliferating neointimal cells by PCNA staining. In vitro, Ad-mtCaMKIIN mildly reduced VSMC proliferation and mitochondrial ROS production without altering maximal respiration after PDGF treatment. Ad-mtCaMKIIN abolished VSMC migration, as did mitoTEMPO and MCU inhibitor Ru360. Ad-mtCaMKIIN blocked mitochondrial mobility towards the leading edge, while relocation of mitochondria was seen in WT cells 6 h after PDGF treatment. Mitochondrial redistribution was also inhibited by Ru360, but not by mitoTEMPO or cytoplasmic CaMKII inhibition. Mitochondrial fission promotes cell migration. Accordingly, PDGF increased mitochondrial particles in WT VSMC, while mitochondria in Ad-mtCaMKIIN cells were fragmented and unresponsive to PDGF treatment. Conclusions: mtCaMKIIN prevents mitochondrial distribution to the leading edge and reduces VSMC migration and neointima formation. These data suggest mitochondrial Ca 2+ regulation plays an important role in VSMC migration by altering mitochondrial location.

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Migration of human vascular smooth muscle cells involves serum-dependent repeated cytosolic calcium transients

Journal of Cell Science ◽

10.1242/jcs.113.4.653 ◽

2000 ◽

Vol 113 (4) ◽

pp. 653-662 ◽

Cited By ~ 3

Author(s):

A. Scherberich ◽

M. Campos-Toimil ◽

P. Ronde ◽

K. Takeda ◽

A. Beretz

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Cell Polarization ◽

Free Calcium ◽

Type I ◽

Serum Dependent

Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca(2+)](i)were observed in single human VSMCs migrating on type I collagen. Such [Ca(2+)](i)transients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca(2+)](i) with BAPTA led to accumulation of (beta)1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca(2+)](i) transients in the rapid, serum-dependent migration of VSMCs. These [Ca(2+)](i)transients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB.

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Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01014.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. H688-H698 ◽

Cited By ~ 52

Author(s):

Hui Li ◽

Weiwei Li ◽

Arun K. Gupta ◽

Peter J. Mohler ◽

Mark E. Anderson ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Muscle Hypertrophy ◽

Central Feature ◽

Ang Ii ◽

Downstream Signaling ◽

And Migration ◽

Medial Hypertrophy

Despite our understanding that medial smooth muscle hypertrophy is a central feature of vascular remodeling, the molecular pathways underlying this pathology are still not well understood. Work over the past decade has illustrated a potential role for the multifunctional calmodulin-dependent kinase CaMKII in smooth muscle cell contraction, growth, and migration. Here we demonstrate that CaMKII is enriched in vascular smooth muscle (VSM) and that CaMKII inhibition blocks ANG II-dependent VSM cell hypertrophy in vitro and in vivo. Specifically, systemic CaMKII inhibition with KN-93 prevented ANG II-mediated hypertension and medial hypertrophy in vivo. Adenoviral transduction with the CaMKII peptide inhibitor CaMKIIN abrogated ANG II-induced VSM hypertrophy in vitro, which was augmented by overexpression of CaMKII-δ2. Finally, we identify the downstream signaling components critical for ANG II- and CaMKII-mediated VSM hypertrophy. Specifically, we demonstrate that CaMKII induces VSM hypertrophy by regulating histone deacetylase 4 (HDAC4) activity, thereby stimulating activity of the hypertrophic transcription factor MEF2. MEF2 transcription is activated by ANG II in vivo and abrogated by the CaMKII inhibitor KN-93. Together, our studies identify a complete pathway for ANG II-triggered arterial VSM hypertrophy and identify new potential therapeutic targets for chronic human hypertension.

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17β-Estradiol attenuates PDGF signaling in vascular smooth muscle cells at the postreceptor level

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00240.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. H538-H546 ◽

Cited By ~ 20

Author(s):

Kai Kappert ◽

Evren Caglayan ◽

Michael Huntgeburth ◽

Anselm T. Bäumer ◽

Jan Sparwel ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Premenopausal Women ◽

Cellular Responses ◽

Dominant Negative ◽

Vsmc Proliferation ◽

Downstream Signaling ◽

Pdgf Signaling ◽

17Β Estradiol ◽

And Migration

Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17β-estradiol (E2) on β-PDGF receptor (β-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 μM–0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 ± 15.8% and 79.4 ± 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter β-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the β-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-γ, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the β-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.

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A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics

eLife ◽

10.7554/elife.67078 ◽

2021 ◽

Vol 10 ◽

Author(s):

Elizabeth M Steenkiste ◽

Jason D Berndt ◽

Carissa Pilling ◽

Christopher Simpkins ◽

Jonathan A Cooper

Keyword(s):

Cell Migration ◽

Phosphorylation Site ◽

Leading Edge ◽

Ubiquitin Proteasome System ◽

Sh2 Domain ◽

Downstream Signaling ◽

Regulatory Circuit ◽

Positive Feedback Circuit ◽

Ubiquitin Proteasome ◽

And Migration

Integrin adhesion complexes regulate cytoskeletal dynamics during cell migration. Adhesion activates phosphorylation of integrin-associated signaling proteins, including Cas (p130Cas, BCAR1), by Src-family kinases. Cas regulates leading-edge protrusion and migration in cooperation with its binding partner, BCAR3. However, it has been unclear how Cas and BCAR3 cooperate. Here, using normal epithelial cells, we find that BCAR3 localization to integrin adhesions requires Cas. In return, Cas phosphorylation, as well as lamellipodia dynamics and cell migration, requires BCAR3. These functions require the BCAR3 SH2 domain and a specific phosphorylation site, Tyr 117, that is also required for BCAR3 downregulation by the ubiquitin-proteasome system. These findings place BCAR3 in a co-regulatory positive-feedback circuit with Cas, with BCAR3 requiring Cas for localization and Cas requiring BCAR3 for activation and downstream signaling. The use of a single phosphorylation site in BCAR3 for activation and degradation ensures reliable negative feedback by the ubiquitin-proteasome system.

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Abstract 732: The P2Y 2 Receptor Selectively Binds to Filamin A to Regulate Spreading and Migration of Vascular Smooth Muscle Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_138-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Ningpu Yu ◽

Gerardo E Fernandez ◽

Gary A Weisman ◽

Cheikh I Seye

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Actin Cytoskeleton ◽

Vascular Smooth Muscle Cells ◽

Filamin A ◽

Wild Type ◽

Adenoviral Infection ◽

And Migration

The functional expression of the G protein-coupled P2Y 2 nucleotide receptor has been associated with the development of intimal lesions. Activation of this receptor also stimulates actin cytoskeleton reorganization and migration of vascular smooth muscle cells (SMCs). Since cell migration has been linked to the dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, the aim of the present study was to identify cytoskeletal proteins that bind to the P2Y 2 receptor and potentially regulate SMC migration. Using the yeast two-hybrid system, we isolated filamin A, a filamentous actin-cross linking protein that interacts with the C-terminal domain of the P2Y 2 receptor and we used deletion mapping to identify amino acid deletion in the P2Y 2 receptor that led to selective loss of filamin A binding. Ex-vivo treatment of aortic explants with the P2Y 2 receptor agonist UTP (10 μmol/L) significantly promoted migration of SMCs in wild type but not in P2Y 2 receptor −/− mice. Likewise, using a Transwell migration assay we showed that UTP increased migration of SMCs in wild type (3.5 folds, P<0.01 vs. untreated cells) but not P2Y 2 receptor −/− mice (P<0.4 vs. untreated cells). Adenoviral infection of the full length P2Y 2 receptor restored UTP-mediated cell migration in P2Y 2 receptor −/− mice whereas infection of a mutant P2Y 2 receptor that does not bind filamin A did not. UTP-induced migration was preceded by a rapid phosphorylation of filamin A that was not observed either in P2Y 2 receptor −/− SMCs or in P2Y 2 receptor −/− SMCs infected with the mutant P2Y 2 receptor that does not bind filamin A. Treatment of SMCs from wild type mice with UTP (10 μmol/L) caused a 4-fold increase in spreading to collagen I as compared to unstimulated cells. The UTP-mediated increase of SMC spreading was not found in P2Y 2 receptor −/− SMCs but was restored by adenoviral infection of the full length P2Y 2 receptor cDNA into these cells. However, adenoviral delivery of a mutant receptor −/− which does not bind to filamin A did not restore UTP-mediated spreading of P2Y 2 receptor SMCs to collagen. This study demonstrates that P2Y 2 -dependent modulation of the actin cytoskeleton selectively regulates spreading and migration of SMC.

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