Caveolin-1 Functions as a Scaffolding Protein for Phosphofructokinase in the Metabolic Organization of Vascular Smooth Muscle†

Biochemistry ◽  
2004 ◽  
Vol 43 (51) ◽  
pp. 16224-16232 ◽  
Author(s):  
Johana Vallejo ◽  
Christopher D. Hardin
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Scaffolding Protein ◽  
Caveolin 1 ◽  
Metabolic Organization

Metabolic organization in vascular smooth muscle: distribution and localization of caveolin-1 and phosphofructokinase

2004 ◽  
Vol 286 (1) ◽  
pp. C43-C54 ◽  
Author(s):  
Johana Vallejo ◽  
Christopher D. Hardin
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Vascular Smooth Muscle ◽  
Cultured Cells ◽  
Cell Types ◽  
Similar Distribution ◽  
Isolated Cells ◽  
Caveolin 1 ◽  
A7r5 Cells ◽  
Metabolic Organization

We have shown that a compartmentation of glycolysis and gluconeogenesis exists in vascular smooth muscle (VSM) and that an intact plasma membrane is essential for compartmentation. Previously, we observed that disruption of the caveolae inhibited glycolysis but stimulated gluconeogenesis, suggesting a link between caveolae and glycolysis. We hypothesized that glycolytic enzymes specifically localize to caveolae. We used confocal microscopy to determine the localization of caveolin-1 (CAV-1) and phosphofructokinase (PFK) in freshly isolated VSM cells and cultured A7r5 cells. Freshly isolated cells exhibited a peripheral (membrane) localization of CAV-1 with 85.3% overlap with PFK. However, only 59.9% of PFK was localized with CAV-1, indicating a wider distribution of PFK than CAV-1. A7r5 cells exhibited compartmentation of glycolysis and gluconeogenesis and displayed two apparent phenotypes distinguishable by shape (spindle and ovoid shaped). In both phenotypes, CAV-1 fluorescence overlapped with PFK fluorescence (83.1 and 81.5%, respectively). However, the overlap of PFK with CAV-1 was lower in the ovoid-shaped (35.9%) than the spindle-shaped cells (53.7%). There was also a progressive shift in pattern of colocalization from primarily the membrane in spindle-shaped cells (both freshly isolated and cultured cells) to primarily the cytoplasm in ovoid-shaped cells. Overall, cellular colocalization of PFK with CAV-1 was significant in all cell types (0.68 ≥ R2 ≤ 0.77). Coimmunoprecipitation of PFK with CAV-1 further validated the possible interaction between the proteins. We conclude that a similar distribution of one pool of PFK with CAV-1 contributes to the compartmentation of glycolysis from gluconeogenesis.

Urokinase-receptor-mediated phenotypic changes in vascular smooth muscle cells require the involvement of membrane rafts

2009 ◽  
Vol 423 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Julia Kiyan ◽  
Graham Smith ◽  
Hermann Haller ◽  
Inna Dumler
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Lipid Rafts ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Caveolin 1 ◽  
Phenotypic Changes ◽  
Dependent Cell

The cholesterol-enriched membrane microdomains lipid rafts play a key role in cell activation by recruiting and excluding specific signalling components of cell-surface receptors upon receptor engagement. Our previous studies have demonstrated that the GPI (glycosylphosphatidylinositol)-linked uPAR [uPA (urokinase-type plasminogen activator) receptor], which can be found in lipid rafts and in non-raft fractions, can mediate the differentiation of VSMCs (vascular smooth muscle cells) towards a pathophysiological de-differentiated phenotype. However, the mechanism by which uPAR and its ligand uPA regulate VSMC phenotypic changes is not known. In the present study, we provide evidence that the molecular machinery of uPAR-mediated VSMC differentiation employs lipid rafts. We show that the disruption of rafts in VSMCs by membrane cholesterol depletion using MCD (methyl-β-cyclodextrin) or filipin leads to the up-regulation of uPAR and cell de-differentiation. uPAR silencing by means of interfering RNA resulted in an increased expression of contractile proteins. Consequently, disruption of lipid rafts impaired the expression of these proteins and transcriptional activity of related genes. We provide evidence that this effect was mediated by uPAR. Similar effects were observed in VSMCs isolated from Cav1−/− (caveolin-1-deficient) mice. Despite the level of uPAR being significantly higher after the disruption of the rafts, uPA/uPAR-dependent cell migration was impaired. However, caveolin-1 deficiency impaired only uPAR-dependent cell proliferation, whereas cell migration was strongly up-regulated in these cells. Our results provide evidence that rafts are required in the regulation of uPAR-mediated VSMC phenotypic modulations. These findings suggest further that, in the context of uPA/uPAR-dependent processes, caveolae-associated and non-associated rafts represent different signalling membrane domains.

Abstract P110: EBP50 Regulates Vascular Smooth Muscle Cell Growth by Skp2-Mediated Degradation of p21/cip1

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Gyun Jee Song ◽  
Stacey Barrick ◽  
Kristen L Leslie ◽  
Nathalie M Fiaschi-Taesch ◽  
Alessandro Bisello
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Mrna Levels ◽  
Neointima Formation ◽  
Vsmc Proliferation ◽  
Genetic Ablation ◽  
Skp2 Expression

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-binding phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout (KO) mice. The molecular mechanisms underlying EBP50 actions in VSMC are unknown. Genetic ablation of EBP50 reduced VSMC proliferation and was associated with increased (5-fold) expression of the cell cycle inhibitor p21cip1 both in vessels and in primary cells. No differences in mRNA levels of p21cip1 were observed in WT and KO cells. However, the half-life of p21cip1 in KO VSMC was significantly longer than in WT VSMC (80 min vs. 45 min) and p21cip1 levels were similar in WT and KO VSMC treated with the proteasome inhibitor MG132. These observations suggest that EBP50 regulates post-translational degradation of p21cip1. The S-phase kinase-associated protein 2 (skp2) is a component of the E3 ligase complex that degrades p21cip1. The C-terminal four amino acids of skp2 (ProSerCysLeu) are a canonical PDZ-binding sequence. Indeed, co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and skp2. Mutation of the C-terminal Leu to Ala (L424A-skp2) abrogated the interaction with EBP50. Skp2 expression was significantly lower in KO than in WT cells and inhibition of EBP50 expression by an shRNA lentivirus decreased skp2 expression in WT cells. Moreover, expression of skp2, but not of the mutant L424A-skp2, in WT cells reduced p21cip1 levels. Therefore, EBP50 regulates both expression and activity of skp2 with attendant effects on p21cip1 and VSMC proliferation. Collectively, these experiments show that EBP50, by regulating skp2 and p21cip1 expression, controls VSMC proliferation and the progression of neointima formation. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide a mechanistic basis for the remarkable effect of this scaffolding protein on vascular remodeling.

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