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Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in , P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.
© 2017 Copeland, Han, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin-1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C-terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae-associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C-terminus of wild-type CAV1 in caveolae, reduced colocalization of cavin-1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease.
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2(+/-)) similar to those found in the majority of HPAH patients. We show that Bmpr2(+/-) PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2(+/-) PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2(+/-) PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2(+/-) PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.
A challenge in advanced drug delivery is selectively traversing the plasma membrane, a barrier that prohibits the intracellular delivery of most peptide and nucleic acid-based therapeutics. A variety of short amino acid sequences termed protein transduction domains (PTDs) first identified in viral proteins have been utilized for over 20 years to deliver proteins nondestructively into cells, however, the mechanisms by which this occurs are varied and cell-specific. Here we describe the results of live cell imaging experiments with AZX100, a cell-permeable anti-fibrotic peptide bearing an "enhanced" PTD (PTD4). We monitored fluorescently labeled AZX100 upon cell surface binding and subsequent intracellular trafficking in the presence of cellular process inhibitors and various well-defined fluorescently labeled cargos. We conclude that AZX100 enters cells via caveolae rapidly, in a manner that is independent of glycoconjugates, actin/microtubule polymerization, dynamins, multiple GTPases, and clathrin, but is associated with lipid rafts as revealed by methyl-beta-cylodextrin. AZX100 treatment increases the expression of phospho-caveolin (Y14), a critical effector of focal adhesion dynamics, suggesting a mechanistic link between caveolin-1 phosphorylation and actin cytoskeleton dynamics. Our results reveal novel and interesting properties of PTD4 and offer new insight into the cellular mechanisms facilitating an advanced drug delivery tool.
(c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association
Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.
BACKGROUND - In the heart, nitric oxide synthases (NOS) modulate cardiac contraction in an isoform-specific manner, which is critically dependent on their cellular and subcellular localization. Defective NO production by NOS3 (endothelial NOS [eNOS]) in the failing heart may precipitate cardiac failure, which could be reversed by overexpression of NOS3 in the myocardium.
METHODS AND RESULTS - We studied the influence of NOS3 in relation to its subcellular localization on the function of cardiomyocytes isolated from transgenic mice overexpressing NOS3 under the alpha-myosin heavy chain promoter (NOS3-TG). Immunoblot analysis demonstrated moderate (5-fold) NOS3 overexpression in cardiomyocytes from NOS3-TG heterozygotes. Caveolar localization of transgenic eNOS was demonstrated by immunofluorescence, coimmunoprecipitation with caveolin-3, sucrose gradient fractionation, and immunogold staining revealed by electron microscopy. Compared with wild-type littermate, contractility of NOS3-TG cardiomyocytes analyzed by videomicroscopy revealed a lower incidence of spontaneous arrhythmic contractions (n=32, P<0.001); an attenuation of the beta-adrenergic positive inotropic response (isoproterenol, 10(-7) mol/L: 62.1+/-7.8% versus 90.8+/-8.0% of maximum Ca2+ response; n=10 to 17; P<0.05); a potentiation of the muscarinic negative chronotropic response (carbamylcholine, 3.10(-8) mol/L: -63.9+/-14% versus -27.7+/-5.6% of basal rate; n=8 to 10; P<0.05), confirmed by telemetry in vivo; and an attenuation of the accentuated antagonism of beta-adrenergically stimulated contraction (-14.6+/-1.5% versus -3.5+/-1.5; n=7 to 11; P<0.05). Cardiomyocyte NOS inhibition reversed all 4 effects (P<0.05).
CONCLUSIONS - Moderate overexpression of NOS3, targeted to caveolae in murine cardiomyocytes, potentiates the postsynaptic muscarinic response and attenuates the effect of high concentrations of catecholamines. Cardiomyocyte NOS3 may represent a promising therapeutic target to restore the sympathovagal balance and protect the heart against arrhythmia.
Clustering of proteins into membrane microdomains, such as lipid rafts and caveolae, could act as a mechanism for regulating cell signaling and other cellular functions. Certain lipid modifications are hypothesized to target proteins to these domains on the cytoplasmic leaflet of the plasma membrane. This concept has now been tested in living cells using an assay sensitive to the lateral distribution of proteins in membranes over sub-micron distances.
Despite intracellular L-arginine concentrations that should saturate endothelial nitric oxide synthase (eNOS), nitric oxide production depends on extracellular L-arginine. We addressed this 'arginine paradox' in bovine aortic endothelial cells by simultaneously comparing the substrate dependence of L-arginine uptake and intracellular eNOS activity, the latter measured as L-[3H]arginine conversion to L-[3H]citrulline. Whereas the Km of eNOS for L-arginine was 2 microM in cell extracts, the L-arginine concentration of half-maximal eNOS stimulation was increased to 29 microM in intact cells. This increase likely reflects limitation by L-arginine uptake, which had a Km of 108 microM. The effects of inhibitors of endothelial nitric oxide synthesis also suggested that extracellular L-arginine availability limits intracellular eNOS activity. Treatment of intact cells with the calcium ionophore A23187 reduced the L-arginine concentration of half-maximal eNOS activity, which is consistent with a measured increase in L-arginine uptake. Increases in eNOS activity induced by several agents were closely correlated with enhanced L-arginine uptake into cells (r = 0.89). The 'arginine paradox' may be explained in part by regulated L-arginine uptake into a compartment, probably represented by caveolae, that contains eNOS and that is distinct from the bulk cytosolic L-arginine.