Localization In Caveolae Research Articles

Caveolae and caveolin-1 as targets of dietary polyphenols for protection against vascular endothelial dysfunction.

Caveolae, consisting of caveolin-1 proteins, are ubiquitously present in endothelial cells and contribute to normal cardiovascular functions by acting as a platform for cellular signaling pathways as well as transcytosis and endocytosis. However, caveolin-1 is thought to have a proatherogenic role by inhibiting endothelial nitric oxide synthase activity and Nrf2 activation, or by promoting inflammation through NF-κB activation. Dietary polyphenols were suggested to exert anti-atherosclerotic effects by a mechanism involving the inhibition of endothelial dysfunction, by which they can regulate redox-sensitive signaling pathways in relation to NF-κB and Nrf2 activation. Some monomeric polyphenols and microbiota-derived catabolites from monomeric polyphenols or polymeric tannins might be responsible for the inhibition, because they can be transferred into the circulation from the digestive tract. Several polyphenols were reported to modulate caveolin-1 expression or its localization in caveolae. Therefore, we hypothesized that circulating polyphenols affect caveolae functions by altering its structure leading to the release of caveolin-1 from caveolae, and attenuating redox-sensitive signaling pathway-dependent caveolin-1 overexpression. Further studies using circulating polyphenols at a physiologically relevant level are necessary to clarify the mechanism of action of dietary polyphenols targeting caveolae and caveolin-1.

ENOS and Caveolin-1 Gene Polymorphisms Interaction and Intima Media Thickness: A Proof of Concept Study in ESRD Patients

Caveolae are a prominent microdomain in endothelial cells and appropriate localization in caveolae is fundamental for endothelial nitric oxide synthase (eNOS) activity. Since the Glu298Asp variant in the eNOS gene alters caveolar localization of the corresponding enzyme, we tested the interaction between this variant and the rs4730751 polymorphism of the caveolin-1 (CAV-1) gene as related to arterial remodeling in end-stage renal disease (ESRD) patients. One hundred and thirty-three ethnically homogeneous ESRD patients underwent carotid ultrasonographic studies to measure intima-media thickness (IMT) and carotid cross-sectional area (CSA). Genotyping was performed by high-throughput allelic discrimination assays on real-time PCR. Arterial remodeling was associated to the number of G alleles of CAV-1 polymorphism, GG homozygotes displaying an IMT and a CSA that were, respectively, 16% and 21% higher than those in patients without the risk allele (P < 0.012). In multiple linear regression analyses including the CAV-1 and the eNOS polymorphisms and adjusting for classical risk factors and risk factors peculiar to ESRD both polymorphisms were independent correlates of IMT (CAV-1: β = 0.20, P = 0.01; eNOS β = 0.25, P = 0.001) and CSA (CAV-1: β = 0.20, P = 0.01: eNOS β = 0.13, P = 0.09). Furthermore, strong interactions emerged between the two polymorphisms for explaining the variability in IMT (P = 0.001) and in CSA (P = 0.038) in these patients. Overall these findings form preliminary evidence that disturbed interaction between CAV-1 and eNOS may be of relevance for arterial disease in ESRD and perhaps in other human diseases.

Redistribution of caveolae during mitosis

Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis.

Abstract 2091: Nitrooleic acids induce localization of heat shock protein 70 in PAM212 keratinocyte caveolae

Abstract Keratinocytes can be stimulated to express inducible nitric oxide synthase and generate nitric oxide by proinflammatory mediators including γ-interferon and lipopolysaccharide. Once formed, nitric oxide and its oxidation products form signaling molecules via redox mediated nitration reactions. Important targets for nitration in mammalian cells are unsaturated fatty acids. This can result in the production of reactive electrophilic species including 9-nitrooleate (9-NO) and 10-nitrooleate (10-NO). Oxidative stress is known to upregulate expression of heat shock proteins which are important chaperones functioning in protein-protein interactions including folding and trafficking. In the present studies we characterized nitro-fatty acid-induced expression of heat shock protein 70 (hsp70) in PAM212 mouse keratinocytes. Western blot analysis showed low constitutive levels of hsp70. Treatment of keratinocytes with 5-25 μM 9-NO or 10-NO for 6 hr markedly increased hsp70 expression. 10-NO was more active than 9-NO. Constitutive hsp70 was found to be localized in cytosolic fractions of keratinocytes, 9- or 10-NO did not alter constitutive cytosolic hsp70. In contrast, following treatment of keratinocytes with the nitro-fatty acids, hsp70 was selectively upregulated in lipid raft membrane fractions. Both hsp70 and caveolin-1 were co-localized in the lipid raft fractions of the membranes demonstrating that the lipid rafts are caveolae. In mouse keratinocytes, nitro-fatty acids readily stimulate MAP kinase signaling including JNK and p38 MAP kinases. Inhibitors of JNK (SP600125, 20 μM) or p38 (SB203580, 10 μM) had no effect on induction of hsp70 or its localization in caveolae indicating that nitro-fatty acid-induced expression of this protein was MAP kinase-independent. Taken together these data demonstrate that nitro-fatty acids are important in the control of heat shock protein expression. Moreover, selective localization of hsp70 in caveolae suggests that it functions in protecting caveolar proteins from cellular stress. Supported by CA132624, ES004738, ES005022, GM034310 and AR055073. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2011-2091

Abstract 4879: Overexpression of GTP-Cyclohydrolase I in Vascular Endothelium Prevents Oxidative Stress-Induced eNOS Translocation From Caveolae to Cytosol

GTP-Cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis, an essential co-factor required for enzymatic activity of endothelial nitric oxide synthase (eNOS). Recent studies have suggested that endothelium-targeted overexpression of GTPCH I has vasoprotective effects. Therefore, we hypothesized that in endothelium GTPCH I localization in caveolae protects eNOS against oxidative stress. Human umbilical vein endothelial cells (HUVECs) were incubated with recombinant adenovirus encoding the human GTPCH I gene (Ad-GTPCH I, 100 MOI) or control recombinant adenoviral vector (Ad-ΔE1) for 12 hours in serum-free media. Cells were then fed with growth medium for 48 hours prior to analysis. Using sucrose gradient ultracentrifugation we detected both protein expression and enzymatic activity of GTPCH I in caveolar microdomains of HUVECs. Treatment of Ad-ΔE1 transduced endothelial cells for 24 hours with LY83583 (1 μ M), a superoxide anion generator, significantly decreased enzymatic activity of GTPCH I in caveolae-enriched membranes (0.49±0.12 pmol neopterin/mg protein; P&lt;0.05 vs. control: 0.92±0.07 pmol neopterin/mg). Furthermore, LY83583 translocated eNOS from caveolae to cytosol in Ad-ΔE1 treated endothelial cells (P&lt;0.05; n=3). This effect was specific because there was no change in caveolin-1 expression and no decrease in cell viability. Treatment of HUVECs with Ad-GTPCH I significantly increased GTPCH I activity in caveolae-enriched membranes (1.37±0.22 pmol neopterin/mg; P&lt;0.05; n=4). Interestingly, LY83583-induced dislocation of eNOS from caveolar membrane fraction was significantly prevented by overexpression of GTPCH I (P&lt;0.05; n=3). Our results suggest that overexpression of GTPCH I increases its enzymatic activity in caveolae. This effect preserves strategically key localization of eNOS in cell membrane during oxidative stress. This research has received full or partial funding support from the American Heart Association, National Center.

Cleaved Kininogen Inhibits Endothelial Progenitor Cell Differentiation by Interfering with the avb3 Integrin-MMP-2 Pathway.

Circulating endothelial progenitor cells (EPCs) are involved in repairing damaged vasculature and in tumor angiogenesis, thus promoting wide interest in their therapeutic potential in ischemic diseases and cancer. Cleaved high molecular weight kininogen (HKa) potently inhibits endothelial cell (EC) functions including in vivo angiogenesis. We have shown that HKa inhibits differentiation of ECs. We now test a new hypothesis that HKa can inhibit EPC differentiation by blocking matrix metalloprotease-2 (MMP-2) activation. EPCs were isolated from human blood and cultured on collagen type I-coated surface. As detected by flow cytometry and Western-blot, they expressed MMP-2 and membrane-type 1-MMP (MT1-MMP), avb3 integrin, uPAR and caveolin-1, but did not express monocyte markers such as HLA-DR and CD14. A clone-forming assay using retroviral infection indicated that an early single EPC exhibited colony forming property, but mature ECs such as human umbilical vein endothelial cells (HUVECs) did not. Upon stimulation by VEGF, EPCs, but not HUVECs, formed vacuoles and differentiated into capillary networks. Gene silencing of either MT1-MMP or MMP-2 by siRNA completely blocked the vacuole and tube formation by EPCs, suggesting that MT1-MMP and MMP-2 are both essential for differentiation of EPCs. HKa inhibited tube formation by EPCs, as well as the conversion of pro-MMP-2 to MMP-2 as a functioning concentration in the conditioned medium. CS-1 cells expressing endogenous av subunit were transfected with human b3 cDNA. We found that HKa inhibition of MMP-2 activation in these cells is dependent on avb3 heterodimer. The binding of avb3 integrin to MMP-2 was via RGD-dependent and RGD-independent mechanisms, both of which, however, were completely blocked by HKa. Further, we found that HKa inhibited the association of MMP-2 with immunoprecipitated avb3 integrin, and prevented MMP-2 localization in caveolae in EPCs. Since HKa inhibited VEGF-stimulated proliferation of EPCs but not HUVECs on collagen-coated surface, we postulate that EPCs are more sensitive than mature ECs to HKa inhibition. Thus, HKa targets EPCs and inhibits their differentiation via blocking avb3 integrin and MMP-2 interaction. These observations provide novel insight into understanding the interactions between the kallikrein-kinin system and biologic functions of EPCs.

Polarization of caveolins and caveolae during migration of immortalized neurons

During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin-releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl-beta-cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin-1 isoform (cav-1) in the leading processes. In contrast, during two-dimensional migration cav-1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.

Localization of caveolae and mitochondria in adult cardiac myocytes: implications for reductive signaling

Caveolae, membrane invaginations composed of cholesterol and the protein caveolin (Cav), are involved in trafficking and compartmentation of signaling molecules, including endothelial nitric oxide synthase (eNOS) in cardiovascular cells. Recently, heme oxygenase (HO), the rate-limiting enzyme in the oxidative degradation of heme to carbon monoxide (CO), biliverdin and iron, was detected in caveolae in endothelial cells. Here, we used cardiac myocytes (CM) isolated from adult rats and sought to determine whether HO localizes with other enzymes that modulate reactive species in caveolae. Immunoblotting of sucrose density gradient fractions of CM membranes detected enrichment of HO-1, eNOS, cytochrome P450 reductase, and Cu-Zn and Mn superoxide dismutase in buoyant fractions (caveolae); biliverdin reductase was not found in buoyant fractions. Immunoprecipitation studies revealed co-immunoprecipitation of all Cav isoforms and HO-1. Using immunohistochemistry, we found co-localization between Cav-3, HO-1 and cytochrome C, a mitochondrial protein marker, along the sarcolemma. HO-1 and HO-2 co-localized with Cav-3 at both the sarcolemma and intercalated discs. Electron microscopy revealed close apposition of sub-sarcolemmal mitochondria and sarcolemmal caveolae. The localization with caveolin of HO and modulatory enzymes for other reactive species and the close apposition of caveolae and mitochondria suggest that caveolae are microdomains that generate both CO and NO, and may serve as “reductive antioxidant sinks” for free radicals produced by adjacent mitochondria in CM.

CD157 undergoes ligand-independent dimerization and colocalizes with caveolin in CHO and MCA102 fibroblasts

CD157, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, has recently been shown to induce protein tyrosine phosphorylation in monocytes differentiated from HL-60 cells (mHL-60) in a ligand-dependent manner, but in a ligand-independent manner in stable CD157-transfected CHO (CHO/CD157) and MCA102 (MCA/CD157) fibroblasts [Cell Signal. 11 (1999) 891–897.]. Many GPI-anchored proteins need to be clustered by their ligands or antibodies to induce redistribution to caveolae and a concomitant activation of the associated signal-transducing proteins [Nature 387 (1997) 569–572.]. Here, we demonstrate that CD157, independent of antibody crosslinking, undergoes dimerization with disulfide bond formation and localization in caveolae in CHO/CD157 and MCA/CD157 fibroblasts. However, the native CD157 induced in mHL-60 cells remains a monomer form. The structural integrity of caveolae is required for the association of CD157 with caveolin and CD157-mediated tyrosine kinase signalling in the fibroblasts. We propose that an overexpression of CD157 could lead to its dimerization and relocation to caveolae and to further result in the initiation of signalling processes.

Filamin-A Binds to the Carboxyl-terminal Tail of the Calcium-sensing Receptor, an Interaction That Participates in CaR-mediated Activation of Mitogen-activated Protein Kinase

The G protein-coupled, extracellular calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and parathyroid cellular proliferation as well as the functions of diverse other cell types. The CaR resides in caveolae-plasma membrane microdomains containing receptors and associated signaling molecules that are thought to serve as cellular "message centers." An additional mechanism for coordinating cellular signaling is the presence of scaffold proteins that bind and organize components of signal transduction cascades. With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the CaR) as an intracellular binding partner of the CaR's carboxyl (COOH)-terminal tail. A direct interaction of the two proteins was confirmed by an in vitro binding assay. Moreover, confocal microscopy combined with two color immunofluorescence showed co-localization of the CaR and filamin-A within parathyroid cells as well as HEK-293 cells stably transfected with the CaR. Deletion mapping localized the sites of interaction between the two proteins to a stretch of 60 amino acid residues within the distal portion of the CaR's COOH-terminal tail and domains 14 and 15 in filamin-A, respectively. Finally, introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP. Therefore, the binding of the CaR's COOH-terminal tail to filamin-A may contribute to its localization in caveolae, link it to the actin-based cytoskeleton, and participate in CaR-mediated activation of MAPK.

Overexpression of the sarcolemmal calcium pump in the myocardium of transgenic rats.

The plasma membrane calmodulin-dependent calcium ATPase (PMCA) is a calcium-extruding enzyme controlling Ca2+ homeostasis in nonexcitable cells. However, its function in the myocardium is unclear because of the presence of the Na+/Ca2+ exchanger. We approached the question of the physiological function of the calcium pump using a transgenic "gain of function" model. Transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter were established, and expression in the myocardium was ascertained at the mRNA, protein, and functional levels. In vivo hemodynamic measurements in adult homozygous animals showed no differences in baseline and increased cardiac performance recruited by volume overload compared with controls. No differences between transgenic and control cardiomyocytes were found in patch clamp voltage dependence, activation/inactivation behavior of the L-type Ca2+ current, or fast [Ca2+]i transients (assessed by the Fura-2 method). To test whether the PMCA might be involved in processes other than beat-to-beat regulation of contraction/relaxation, we compared growth processes of neonatal transgenic and control cardiomyocytes. A 1.6- and 2.3-fold higher synthesis rate of total protein was seen in cells from transgenic animals compared with controls on incubation with 2% FCS for 24 hours and 36 hours, respectively. An effect of similar magnitude was observed using 20 micromol/L phenylephrine. A 1.4-fold- and 2.0-fold-higher protein synthesis peak was seen in PMCA-overexpressing cardiomyocytes after stimulation with isoproterenol for 12 hours and 24 hours, respectively. Because pivotal parts of the alpha- and beta-adrenergic signal transduction pathways recently have been localized to caveolae, we tested the hypothesis that the PMCA might alter the amplitude of alpha- and beta-adrenergic growth signals by virtue of its localization in caveolae. Biochemical as well as immunocytochemical studies suggested that the PMCA in large part was colocalized with caveolin 3 in caveolae of cardiomyocytes. These results indicate that the sarcolemmal Ca2+-pump has little relevance for beat-to-beat regulation of contraction/relaxation in adult animals but likely plays a role in regulating myocardial growth, possibly through modulation of caveolar signal transduction.

Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium.

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae.

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.