Caveolar Protein Research Articles

Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma

Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation.

Redefining caveolae as signalling platforms: specificity of interactions on endocytic caveolae in response to stress

Caveolae are small invaginations of the plasma membrane, which can expand upon stimulation and be internalized as endocytic vesicles. In addition to their role in clathrin‐independent endocytosis, caveolae play a role in many different signalling pathways.The major caveolar membrane protein, caveolin‐1 (CAV1) forms a dense coat of approx. 150 monomers assembled at the surface of the caveolae. CAV1 is believed to possess a “scaffolding domain” capable of interacting directly with numerous signalling proteins; its high oligomeric state amplifies its potency as a signalling platform.Here we have conducted a comprehensive in vitro screen of potential interacting partners to verify this “caveolin signalling” hypothesis. We have developped a binding assay using co‐expression of interactors and CAV1 in a cell‐free system which mimics the folding and insertion of caveolin in native membranes. Electron tomography shows that CAV1 generates membrane domains of uniform size and curvature and that we can re‐create small decorated vesicles that closely resemble endocytic caveolae.We measured binding of 40 potential interactors on these “reconstituted” caveolae using single‐molecule fluorescence and screened for interaction at the monomer level (non‐inserted CAV1) using a binding assay (AlphaScreen). We reveal exquisite selectivity of the assembled caveolae compared to monomeric CAV1. CAV1 phosphorylation, as found in cellular stress, showed amplification of binding to a subset of interactors. We further characterized binding sites by testing different mutations and truncations that alter the structure and oligomerisation state of CAV1. The results were confirmed in cells by proximity ligation assay, validating protein‐protein interactions and the effect of protein posttranslational modifications.Taken together, our findings create a novel picture of the role of caveolae in cell signalling and stress‐response.

Caveolae protect endothelial cells from membrane rupture during increased cardiac output

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1(-/-) mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.

P35. Novel interaction of placental caveolin-1 expression with markers of oxidative stress and the renin-angiotensin system (RAS) in pre-eclampsia

Introduction Caveolin-1 (cav-1) is one of the major protein components of caveolae required for the coordination of some signalling pathways, including that of angiotensin II (AngII) with its Type 1 receptor (AT1R). We have previously reported decreased cav-1 and increased AT1R protein expression in placentae from women with pre-eclampsia (PE). Within the PE placenta, an increase in AngII acting via AT1R may lead to increased vasoconstriction, but possible functional effects of altered cav-1, and interactions with components of RAS and markers of oxidative stress have not been investigated. Objective To establish mechanistic interactions of placental cav-1 with RAS components and surrogate markers of oxidative stress and antioxidant concentrations. Methods Immunohistochemistry was performed on paraffin-embedded serial placental sections from 24 normotensive (NC) and 19 women with PE, using antibodies to cav-1, prorenin receptor (PRR), angiotensinogen (AGT), AT1R, AngII type 2 receptor (AT2R) and eNOS. Protein expression was semi-quantitatively assessed and also analysed with respect to previously measured maternal plasma TBARS (Thiobarbituric acid-reactive substances) concentration and placental glutathione peroxidase (GPx) activity. Results Positive correlations were observed between placental expression of cav-1 and the AT2R ( P = 0.003) and PRR ( P P > 0.7, P > 0.5). However, cav-1 was inversely correlated with eNOS expression in both NC and PE placentae ( P P = 0.003). No significant correlations were observed with AGT or AT1R. A negative correlation was observed between maternal TBARS and cav-1 protein ( P = 0.003), and between placental GPx activity and cav-1 protein ( P = 0.027) only in the PE placentae. Conclusions A complex cascade links activation of the AT1R with NADPH oxidase activation, generation of reactive oxygen species (ROS), the uncoupling of eNOS and subsequent modification of redox-sensitive caveolar proteins in vascular smooth muscle outside pregnancy. Our findings suggest that this cascade is altered in hypertensive pregnancy, in a direction which would lead to enhanced local vasoconstriction.

Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.

Heterozygous Null Bone Morphogenetic Protein Receptor Type 2 Mutations Promote SRC Kinase-dependent Caveolar Trafficking Defects and Endothelial Dysfunction in Pulmonary Arterial Hypertension

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.

Caveolin-1 mediated uptake via langerin restricts HIV-1 infection in human Langerhans cells

BackgroundHuman Langerhans cells (LCs) reside in foreskin and vaginal mucosa and are the first immune cells to interact with HIV-1 during sexual transmission. LCs capture HIV-1 through the C-type lectin receptor langerin, which routes the virus into Birbeck granules (BGs), thereby preventing HIV-1 infection. BGs are langerin-positive organelles exclusively present in LCs, however, their origin and function are unknown.ResultsHere, we not only show that langerin and caveolin-1 co-localize at the cell membrane and in vesicles but also that BGs are langerin/caveolin-1-positive vesicles are linked to the lysosomal degradation pathway in LCs. Moreover, inhibition of caveolar endocytosis in primary LCs abrogated HIV-1 sequestering into langerin+ caveolar structures. Notably, both inhibition of caveolar uptake and silencing of caveolar structure protein caveolin-1 resulted in increased HIV-1 integration and subsequent infection. In contrast, inhibition of clathrin-mediated endocytosis did not affect HIV-1 integration, even though HIV-1 uptake was decreased, suggesting that clathrin-mediated endocytosis is not involved in HIV-1 restriction in LCs.ConclusionsThus, our data strongly indicate that BGs belong to the caveolar endocytosis pathway and that caveolin-1 mediated HIV-1 uptake is an intrinsic restriction mechanism present in human LCs that prevents HIV-1 infection. Harnessing this particular internalization pathway has the potential to facilitate strategies to combat HIV-1 transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0123-7) contains supplementary material, which is available to authorized users.

ALTERED GLYCOSYLATION AND DISTRIBUTION OF THE β2 ADRENOCEPTOR IN HEART FAILURE

Heart failure (HF) is a serious problem within the developed world. β-adrenergic receptors (βAR) undergo dramatic remodelling in HF characterised by a reduction in β1AR expression and decreased sympathetic responsiveness....

Damage control: cellular mechanisms of plasma membrane repair.

When wounded, eukaryotic cells reseal in a few seconds. Ca(2+) influx induces exocytosis of lysosomes, a process previously thought to promote repair by 'patching' wounds. New evidence suggests that resealing involves direct wound removal. Exocytosis of lysosomal acid sphingomyelinase (ASM) triggers endocytosis of lesions followed by intracellular degradation. Characterization of injury-induced endosomes revealed a role for caveolae, sphingolipid-enriched plasma membrane invaginations that internalize toxin pores and are abundant in mechanically stressed cells. These findings provide a novel mechanistic explanation for the muscle pathology associated with mutations in caveolar proteins. Membrane remodeling by the ESCRT complex was also recently shown to participate in small-wound repair, emphasizing that cell resealing involves previously unrecognized mechanisms for lesion removal that are distinct from the patch model.

Abstract 177: Promotion Of Nitroso-redox Balance By Beta 3 Adrenoceptor Agonism: Therapeutic Implications For Cardiovascular Complications Of Diabetes

Rationale: Disrupted balance between NO and O2.- is central in pathobiology of diabetes-induced cardiomyopathy and vascular dysfunction. We examined if stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re-establish NO/O2.- balance, relieve oxidative inhibition of key caveolar proteins and protect against diabetes-induced cardiovascular dysfunction. Methods/Results: A hyperglycemic, hyperinsulinemic state was established in male White New Zealand rabbits by infusion of the insulin receptor antagonist S961 (12 μg/kg/h). Diabetes induced NADPH oxidase-dependent glutathionylation (GSS-) of the caveolar proteins Na+-K+ pump’s β1 subunit and eNOS in cardiac myocytes and aorta, an oxidative modification that inhibits the pump and uncouples eNOS. Consistent with this, diabetes was associated with reduced electrogenic Na+-K+ pump current in voltage-clamped cardiac myocytes and impaired endothelium-dependent vasorelaxation. Selective β3 AR agonist CL316243 (CL, 40 μg/kg/h) restored NO levels analysed by spin-trapping of NO-Fe(DETC)2 complexes; decreased diabetes-induced elevation in O2.- measured by HPLC analysis of dihydroethidium oxidation products, improved endothelium-dependent vasorelaxation, and restored the Na+-K+ pump function in cardiac myocytes. These effects were mediated by CL abolishing diabetes-induced increase in eNOS-GSS and β1-GSS through a decrease in forward reaction rate for glutathionylation by suppressing diabetes-induced NADPH oxidase activation, which was further amplified by promotion of de-glutathionylation via enhancement in association of glutaredoxine-1, the enzyme catalysing de-glutathionylation, with eNOS and Na+-K+ pump. Conclusion: β3 AR activation re-established nitroso-redox balance and relieved oxidative inhibition of key caveolar proteins in diabetes. β3 AR agonists are promising in treatment of diabetes-induced cardiovascular complications.

Cavin-3 knockout mice show that cavin-3 is not essential for caveolae formation, for maintenance of body composition, or for glucose tolerance.

The cavins are a family of proteins associated with caveolae, cavin-1, -2 and -3 being widely expressed while cavin-4 is restricted to striated muscle. Deletion of cavin-1 results in phenotypes including metabolic changes consistent with adipocyte dysfunction, and caveolae are completely absent. Deletion of cavin-2 causes tissue-specific loss of caveolae. The consequences of cavin-3 deletion are less clear, as there are divergent data on the abundance of caveolae in cavin-3 null mice. Here we examine the consequences of cavin-3 deficiency in vivo by making cavin-3 knockout mice. We find that loss of cavin-3 has minimal or no effects on the levels of other caveolar proteins, does not appear to play a major role in formation of protein complexes important for caveolar morphogenesis, and has no significant effect on caveolae abundance. Cavin-3 null mice have the same body weight and fat mass as wild type animals at ages 8 through 30 weeks on both normal chow and high fat diets. Likewise, the two mouse strains exhibit identical glucose tolerance tests on both diets. Microarray analysis from adipose tissue shows that the changes in mRNA expression between cavin-3 null and wild type mouse are minimal. We conclude that cavin-3 is not absolutely required for making caveolae, and suggest that the mechanistic link between cavin-3 and metabolic regulation remains uncertain.

The interaction of enolase-1 with caveolae-associated proteins regulates its subcellular localization

Cell-surface-associated proteolysis plays a crucial role in embryonic development, monocyte/macrophage recruitment and tumour cell invasion. The glycolytic enzyme ENO-1 (enolase-1) is translocated from the cytoplasm to the cell surface, where it binds PLG (plasminogen) to enhance pericellular plasmin production and cell motility. In the present study, ENO-1 was found to localize to a specialized subset of lipid rafts called caveolae as demonstrated by fluorescence confocal microscopy and sucrose gradient ultracentrifugation. Co-immunoprecipitation studies revealed that ENO-1 interacts with Cav-1 (caveolin-1), but not with Cav-2, via the CSD (Cav-scaffolding domain). Moreover, an evolutionarily conserved CBM (Cav-binding motif) F296DQDDWGAW304 was identified within ENO-1. The point mutation W301A within the ENO-1 CBM was, however, not sufficient to disrupt ENO-1-Cav-1 interaction, whereas the mutations F296A and W304A markedly affected ENO-1 protein expression. Furthermore, ENO-1 was found associated with Annx2 (annexin 2), representing another caveolar protein, and this interaction was dependent on Cav-1 expression. Knockdown of Cav-1 and Annx2 markedly decreased cell surface expression of ENO-1. ENO-1 overexpression increased cell migration and invasion in a Cav-1-dependent manner. Thus the differential association of ENO-1 with caveolar proteins regulates ENO-1 subcellular localization and, consequently, ENO-1-dependent cell migration and invasion.

Endocytic Crosstalk: Cavins, Caveolins, and Caveolae Regulate Clathrin-Independent Endocytosis

Author SummaryEndocytosis is the process that allows cells to take up molecules from the environment. Several endocytic pathways exist in mammalian cells. While the best understood endocytic pathway uses clathrin, recent years have seen a great increase in our understanding of clathrin-independent endocytic pathways. Here we characterize the crosstalk between caveolae, flask-shaped specialized microdomains present at the plasma membrane, and a second clathrin-independent pathway, the CLIC/GEEC Cdc42-regulated endocytic pathway. These pathways are segregated in migrating cells with caveolae at the rear and CLIC/GEEC endocytosis at the leading edge. Here we find that specific caveolar proteins, caveolins and cavins, can also negatively regulate the CLIC/GEEC pathway. With the help of several techniques, including quantitative electron microscopy analysis and real-time live-cell imaging, we demonstrate that expression of caveolar proteins affects early carrier formation, causes cellular lipid changes, and changes the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. The functional consequences of loss of caveolar proteins on the CLIC/GEEC pathway included inhibition of polarized cell migration and increased endocytosis in tissue explants.

Cytoskeleton Modification and Cholesterol Depletion Affect Membrane Properties and Caveolae Positioning of CHO Cells

The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-β-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.

A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype.

Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells.

Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca2+ ([Ca2+]i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca2+]i regulation, which is further enhanced by TNFα. Whether caveolae and CD38 are functionally linked in mediating TNFα effects is unknown. In this regard, whether the related cavin proteins (cavin-1 and -3) that maintain structure and function of caveolae play a role is also not known. In the present study, we hypothesized that TNFα effects on CD38 expression and function in human ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expression was upregulated by exposure to 20ng/ml TNFα (48h). ASM cells expressed cavin-1 and cavin-3, which were also upregulated by TNFα. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantly reduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNFα. Furthermore, caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca2+]i responses to histamine under control conditions, and blunted the enhanced [Ca2+]i responses in TNFα-exposed cells. These data demonstrate that CD38 is expressed within caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The link between caveolae and CD38 is further enhanced during airway inflammation demonstrating the important role of caveolae in regulation of [Ca2+]i and contractility in the airway.

Low-dose Endothelial Monocyte-activating Polypeptide-II Increases Permeability of Blood–tumor Barrier by Caveolae-mediated Transcellular Pathway

Low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase blood-tumor barrier (BTB) permeability via the paracellular pathway. The role of the transcellular pathway in this process is unclear. This study was conducted to evaluate the potential involvement of the transcellular pathway in EMAP-II-induced opening of the BTB and to identify the associated mechanisms. Evans blue extravasation test was used to measure changes in BTB permeability after EMAP-II (80ng/kg) administration in a rat model of C6 glioma. Changes in the quantity of pinocytotic vesicles in rat brain microvascular endothelial cells (BMECs) were observed using transmission electron microscopy. Reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry assays were performed to detect the expression of the caveolar structural proteins, caveolin-1 and caveolin-2, in BMECs. Alterations in the expression of phospho (p)-Src, p-caveolin-1, and p-caveolin-2 and the activity of RhoA also were measured. Effects of tyrosine kinase inhibition on EMAP-II-induced RhoA/Rho kinase activations and tyrosine kinase, RhoA, or Rho kinase inhibition on EMAP-II-induced caveolin-1 and caveolin-2 phosphorylation were determined by inhibition studies. One hour after EMAP-II administration, the quantity of pinocytotic vesicles in BMECs increased markedly, consistent with changes in BTB permeability. The expression levels of caveolin-1, caveolin-2, p-caveolin-1, and p-caveolin-2 in BMECs also were significantly increased at 1h. The peak expression level of p-Src and the peak activity of RhoA occurred at 0.25 and 0.5h, respectively. Inhibition of tyrosine kinase significantly diminished the activities of RhoA and Rho kinase induced by EMAP-II. In addition, EMAP-II-induced phosphorylation of caveolin-1 and caveolin-2 was completely blocked by inhibition of tyrosine kinase, RhoA, or Rho kinase. We suggest that low-dose EMAP-II can induce BTB hyperpermeability via the transcellular pathway, which is associated with phosphorylation and upregulation of caveolin-1 and caveolin-2 and involves the tyrosine kinase/RhoA/Rho kinase signaling pathway.

Membrane Glucocorticoid Receptor Activation Induces Proteomic Changes Aligning with Classical Glucocorticoid Effects

Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function.

Hyperglycemia-Induced Protein Kinase C β2 Activation Induces Diastolic Cardiac Dysfunction in Diabetic Rats by Impairing Caveolin-3 Expression and Akt/eNOS Signaling

Protein kinase C (PKC)β2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCβ2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocalization of PKCβ2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-β-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCβ2 phosphorylation. Inhibition of PKCβ2 activation by compound CGP53353 or knockdown of PKCβ2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCβ2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2−, nitrotyrosine, Cav-1, and iNOS expression. In conclusion, hyperglycemia-induced PKCβ2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCβ2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling.

Abstract 546: Negative Regulation of Reactive Oxygen Species From Nadph Oxidases by Caveolar Proteins

The importance of caveolar proteins, including caveolin-1 (Cav1) have been widely explored in cardiovascular disease. Cav1 binds to numerous signaling proteins via interactions with its scaffolding domain, and caveolin-2 (Cav2) and Cavin1 depend on Cav1 for stability and expression. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress. Loss of cav1 has been shown to increase vascular ROS production and can promote cardiovascular diseases such as pulmonary arterial hypertension. However, the ability of Cav1 to regulate Nox enzymes is not known. Here, we found that increased expression of Cav1 by gene transfer or by administration of its scaffolding domain peptide, potently inhibited ROS production from Nox1, Nox2, Nox4 and Nox5. In contrast, inhibition of endogenous Cav1 with small interfering RNA increased ROS production from human vascular cells. The Cav1 scaffolding domain mutant, Cav1 F16A, failed to inhibit Nox activity. In addition, Cav2 and Cavin-1 can also decreased ROS production from Nox enzymes. To further explore the underlying mechanism, we found Cav1 did not modify intracellular calcium but reduced ROS output in an isolated enzyme assay. Furthermore, we found increased Cav1, Cav2 and Cavin-1 expression robustly decreased the expression level of Nox proteins. Given the similar intracellular location of Cav1 and Nox and the presence of highly conserved Cav1 binding sequence in Nox enzymes, we predict that caveolin proteins may directly bind with Nox and promote their degradation. It is also possible that degradation is mediated by Cav1 regulated Nox protein ubiquitylation. Together, these results suggest that caveolin proteins negatively regulate NADPH oxidases to ameliorate cellular stress and are an important regulatory pathway in the cardiovascular system.