Conditional Expression of a Dominant-Negative c-Myb in Vascular Smooth Muscle Cells Inhibits Arterial Remodeling After Injury

American Journal of Physiology-Cell Physiology

Wang

et al. 2003

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. Calcineurinindependent regulation of plasma membrane Ca 2ϩ ATPase-4 in the vascular smooth muscle cell cycle. Am J Physiol Cell Physiol 285: C88-C95, 2003; 10.1152/ajpcell.00518.2002.-Calcineurin mediates repression of plasma membrane Ca 2ϩ -ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45 Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca 2ϩ concentrations ([Ca 2ϩ ]i) increased twofold, and real-time RT-PCR and Western blot revealed a ϳ40% decrease in PMCA4 expression levels from G 0 to G1/S in the cell cycle, where PMCA4 constituted ϳ20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca 2ϩ -independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca 2ϩ ]i through alternate targets. /calmodulin-mediated signals act at multiple points in the cell cycle, including the G 0 /G 1 transition, the initiation of S-phase, and the initiation and completion of M phase (reviewed in Refs. 4,31,38,and 44 ] i ) needed for G 1 -to-S transitions in rat vascular smooth muscle cells (VSMC) (22, 23).We previously showed that the cell cycle-associated repression of PMCA1 expression during G 0 to G 1 /S progression in rat VSMC is mediated by the c-Myb transcription factor (1). However, the mechanism(s) underlying the cell cycle-associated repression of PMCA4 had not been elucidated. Guerini et al. (18) demonstrated in mouse neurons that PMCA4 expression can be repressed by a calcineurin-dependent pathway. Given this result, we hypothesized that G 1 /Sassociated repression of PMCA4 expression in VSMC may also be mediated by calcineurin. To explore these mechanisms in cell culture, we generated a clonal, immortalized mouse VSMC line (MOVAS). Immunostaining for smooth muscle-specific proteins such as SM22␣, calponin, smooth muscle-specific ␣-actin and desmin, as well as SM22␣ promoter-driven enhanced green fluorescent protein (EGFP) expression, confirm the lineage and phenotype of these cells. 45 Ca efflux assays and fura 2-based ratiometric Ca 2ϩ imaging reveal regulated Ca 2ϩ efflux and [Ca 2ϩ ] i at the G 1 /S transition point. Western blot and real time RT-PCR reveal cell cycle-regulated repression of mouse PMCA1 and PMCA4. Drugs in...

Section: Discussionmentioning

confidence: 99%

Calcineurin-independent regulation of plasma membrane Ca²⁺ ATPase-4 in the vascular smooth muscle cell cycle

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American Journal of Physiology-Cell Physiology

Wang

et al. 2003

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“…These mice are referred to as P4KO mice, with their wild-type littermates denoted as P4WT. Protocols for mouse tissue harvest and carotid artery wire denudation injury have been described (15) and were approved by the Animal Care Committee of University Health Network. Primary VSMC cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (Wisent; Montreal, Quebec, Canada), 50 ng/ml rat recombinant PDGF-BB (Sigma), and 1% penicillinstreptomycin in humidified air containing 5% CO 2 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Calcium Efflux Activity of Plasma Membrane Ca2+ ATPase-4 (PMCA4) Mediates Cell Cycle Progression in Vascular Smooth Muscle Cells

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Journal of Biological Chemistry

Khoshbin

et al. 2014

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Background: PMCA4 actions in vascular smooth muscle are not understood. Results: Alternative splicing of PMCA4 changes during vessel injury. Cell cycle arrest and downstream effectors of PMCA4 deletion are rescued by PMCA4a, PMCA4b, and PMCA4b unable to bind PDZ-proteins but not by inactive PMCA4. Conclusion: Ca 2ϩ efflux activity of PMCA4 regulates G 1 progression. Significance: PMCA4 Ca 2ϩ efflux regulates cell cycle.

“…24 Hearts from mice infected with group B coxsackieviruses were used as positive controls. 24 Cardiomyocyte cross-sectional area was measured at the level of the nucleus in 60 to 80 cells from each mouse with an imaging system 22 and Scion Image 4.0.1 software (Scion Corp). Longitudinal or oblique sections were excluded.…”

Section: Histopathologymentioning

confidence: 99%

Conditional Cardiac Overexpression of Endothelin-1 Induces Inflammation and Dilated Cardiomyopathy in Mice

Gros²,

Kabir³

et al. 2004

Circulation

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155

113

Background-Myocardial expression of endothelin-1 (ET-1) and its receptors ET A and ET B is increased in heart failure.However, the role of ET-1 and its signaling pathways in the pathogenesis of myocardial diseases is unclear. Methods and Results-Human ET-1 cDNA was placed downstream of a promoter responsive to a doxycycline (DOX)-regulated transcriptional activator (tTA). This line (ET ϩ ) was bred with one harboring cardiac myocyterestricted expression of tTA (␣MHC-tTA). Myocardial ET-1 peptide levels were significantly increased in binary transgenic (BT, ET ϩ /tTA ϩ ) compared with nonbinary transgenic (NBT, ET ϩ /tTA Ϫ ; ET Ϫ /tTA ϩ ; ET Ϫ /tTA Ϫ ) or DOX-treated BT littermates (40.1Ϯ4.7 versus 2.6Ϯ1.2 fmol/mL, PϽ0.003). BT mice demonstrated progressive mortality between 5 and 11 weeks after DOX withdrawal, associated with left ventricular dilatation and contractile dysfunction (peak ϩdP/dT, 4673Ϯ468 versus 5585Ϯ658 mm Hg/s, PϽ0.05). An interstitial inflammatory infiltrate, including macrophages and T lymphocytes, was evident in the myocardium of BT mice, associated with sequential increases in nuclear factor-B translocation and expression of tumor necrosis factor-␣, interferon-␥, interleukin-1 and interleukin-6. Significant prolongation of survival was observed with the combined ET A /ET B antagonist LU420627 (nϭ8, PϽ0.05) in BT mice but not the ET A -selective antagonist LU135252 (nϭ5, Pϭ0.9), consistent with an important role for ET B in this model. Conclusions-These are the first data to demonstrate that cardiac overexpression of ET-1 is sufficient to cause increased expression of inflammatory cytokines and an inflammatory cardiomyopathy leading to heart failure and death.