Caveolar Protein Research Articles

Caveolar Fatty Acids and Acylation of Caveolin-1

PurposeCaveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.MethodsCaveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.ResultsIn contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×107 cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.ConclusionCaveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.

Adipocyte size and cellular expression of caveolar proteins analyzed by confocal microscopy

Caveolae are abundant in adipocytes and are involved in the regulation of lipid accumulation, which is the main volume determinant of these cells. We have developed and applied a confocal microscopic technique for measuring individual cellular expression of the caveolar proteins cavin-1 and caveolin-1 along with the size of individual adipocytes. The technique was applied on collagenase isolated adipocytes from ad libitum fed Sprague-Dawley rats of different age (4-26 wk) and weight (103-629 g). We found that cellular expression of caveolar proteins was variable (SD of log expression in the range from 0.25 to 0.65). Regression analysis of protein expression on adipocyte size revealed that the expression of the caveolar proteins cavin-1 and caveolin-1 on adipocytes from individual rats was tightly related to adipocyte cell surface area (mean coefficient of regression was 0.83 for cavin and 0.77 for caveolin), indicating that caveolar density was the same in membranes from all cells within a biopsy. This intrinsic relation remained unchanged with animal age, but adipocytes from animals with increasing age showed a decrease in mean expression of caveolar proteins per unit cell surface. The different relation between adipocyte size and cellular expression levels of caveolar proteins within and between individuals of different age shows that caveolar density is an age-sensitive characteristic of adipocytes.

Caveolin-1 and polymerase I and transcript release factor: new players in insulin-like growth factor-I receptor signaling.

Caveolae are plasma membrane regions enriched in Caveolin proteins which regulate vesicular transport, endocytosis, and cell signaling. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates Caveolin-1 (Cav-1), the most represented caveolar protein. Cav-1 participates to IGF-IR internalization and signaling directly interacting with IGF-IR and its substrates. Recently, polymerase I and transcript release factor (PTRF) or Cavin-1, has been identified in the caveolar backbone. PTRF does not play a Cav-1 ancillary role and emerging data support a direct role of PTRF in IGF-IR signaling. PTRF and Cav-1 can bind IGF-IR and regulate IGF-IR internalization and plasma membrane replacement, mechanisms frequently deregulated in cancer cells. Although the exact roles of Cav-1 and IGF-IR in human cancer continue to be a matter of some debate, there is a strong evidence for an association between Cav-1 and IGF-IR in cancer development. With the discovery of IGF-IR interaction with PTRF in caveolae, new insight emerged to understand the growing functions of these domains in IGF-I action.

FSHD Myotubes with Different Phenotypes Exhibit Distinct Proteomes

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric region. This deletion induces epigenetic modifications that affect the expression of several genes located in the vicinity. In each D4Z4 element, we identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription factor that plays a major role in the development of FSHD through the initiation of a large gene dysregulation cascade that causes myogenic differentiation defects, atrophy and reduced response to oxidative stress. Because miRNAs variably affect mRNA expression, proteomic approaches are required to define the dysregulated pathways in FSHD. In this study, we optimized a differential isotope protein labeling (ICPL) method combined with shotgun proteomic analysis using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56+ FSHD myoblasts were found to fuse into myotubes presenting various proportions of an atrophic or a disorganized phenotype. To better understand the FSHD myogenic defect, our improved proteomic procedure was used to compare predominantly atrophic or disorganized myotubes to the same matching healthy control. FSHD atrophic myotubes presented decreased structural and contractile muscle components. This phenotype suggests the occurrence of atrophy-associated proteolysis that likely results from the DUX4-mediated gene dysregulation cascade. The skeletal muscle myosin isoforms were decreased while non-muscle myosin complexes were more abundant. In FSHD disorganized myotubes, myosin isoforms were not reduced, and increased proteins were mostly involved in microtubule network organization and myofibrillar remodeling. A common feature of both FSHD myotube phenotypes was the disturbance of several caveolar proteins, such as PTRF and MURC. Taken together, our data suggest changes in trafficking and in the membrane microdomains of FSHD myotubes. Finally, the adjustment of a nuclear fractionation compatible with mass spectrometry allowed us to highlight alterations of proteins involved in mRNA processing and stability.

Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid‐rafts and its internalization in human endometrial cells

Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the G(M1)-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.

Caveolin-1 knockout mice exhibit airway hyperreactivity

Caveolae are flask-shaped plasma membrane invaginations expressing the scaffolding caveolin proteins. Although caveolins have been found in endothelium and epithelium (where they regulate nitric oxide synthase activity), their role in smooth muscle is still under investigation. We and others have previously shown that caveolae of human airway smooth muscle (ASM), which express caveolin-1, contain Ca(2+) and force regulatory proteins and are involved in mediating the effects of inflammatory cytokines such as TNF-α on intracellular Ca(2+) concentration responses to agonist. Accordingly, we tested the hypothesis that in vivo, absence of caveolin-1 leads to reduced airway hyperresponsiveness, using a knockout (KO) (Cav1 KO) mouse and an ovalbumin-sensitized/challenged (OVA) model of allergic airway hyperresponsiveness. Surprisingly, airway responsiveness to methacholine, tested by use of a FlexiVent system, was increased in Cav1 KO control (CTL) as well as KO OVA mice, which could not be explained by a blunted immune response to OVA. In ASM of wild-type (WT) OVA mice, expression of caveolin-1, the caveolar adapter proteins cavins 1-3, and caveolae-associated Ca(2+) and force regulatory proteins such as Orai1 and RhoA were all increased, effects absent in Cav1 KO CTL and OVA mice. However, as with WT OVA, both CTL and OVA Cav1 KO airways showed signs of enhanced remodeling, with high expression of proliferation markers and increased collagen. Separately, epithelial cells from airways of all three groups displayed lower endothelial but higher inducible nitric oxide synthase and arginase expression. Arginase activity was also increased in these three groups, and the inhibitor nor-NOHA (N-omega-nor-l-arginine) enhanced sensitivity of isolated tracheal rings to ACh, especially in Cav1 KO mice. On the basis of these data disproving our original hypothesis, we conclude that caveolin-1 has complex effects on ASM vs. epithelium, resulting in airway hyperreactivity in vivo mediated by altered airway remodeling and bronchodilation.

Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1

Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.

Decreased caveolin‐3 and increased GlcNAcation in cardiac myocyte caveolae during diabetes mellitus

Cardiomyopathy that is independent of dysfunctional vasculature occurs in patients with diabetes mellitus (DM), thus suggesting that factors within cardiac myocytes contribute to DM‐associated cardiac remodeling. Caveolin‐3 (Cav‐3), a membrane structural protein, can modulate cardiac remodeling where mice with knockout of Cav‐3 have a cardiomyopathic phenotype and overexpressors are protected. To test the hypothesis that post‐translational modification of caveolar proteins and mitochondrial dysfunction occur in DM, we assessed hearts from control or streptozotocin‐induced DM mice using fractionation/immunoblot analysis, electron microscopy (EM), and mitochondrial function assays. We found that diabetic hearts have reduced Cav‐3 expression and increased O‐GlcNAcation in Cav‐3 enriched membranes. EM revealed that diabetic hearts had disruption of cytoarchitecture with disorganized, rounded mitochondria and fewer caveolar invaginations. Functional studies showed that mitochondrial respiration in DM myocytes is impaired and oxidative stress enhanced. Therapies designed to enhance Cav‐3/caveolae expression thus are potentially novel approaches to limit DM‐associated cardiac remodeling.

Caveolin‐1 orchestrates fibroblast growth factor 2 signaling control of angiogenesis in placental artery endothelial cell caveolae

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.

Caveolae, caveolins, cavins, and endothelial cell function: new insights.

Caveolae are cholesterol and glycosphingolipid-rich flask-shaped invaginations of the plasma membrane which are particularly abundant in vascular endothelium and present in all other cell types of the cardiovascular system, including vascular smooth-muscle cells, macrophages, cardiac myocytes, and fibroblasts. Caveolins and the more recently discovered cavins are the major protein components of caveolae. When caveolae were discovered, their functional role was believed to be limited to transport across the endothelial cell barrier. Since then, however, a large body of evidence has accumulated, suggesting that these microdomains are very important in regulating many other important endothelial cell functions, mostly due to their ability to concentrate and compartmentalize various signaling molecules. Over the course of several years, multiple studies involving knockout mouse and small interfering RNA approaches have considerably enhanced our understanding of the role of caveolae and caveolin-1 in regulating many cardiovascular functions. New findings have been reported implicating other caveolar protein components in endothelial cell signaling and function, such as the understudied caveolin-2 and newly discovered cavin proteins. The aim of this review is to focus primarily on molecular and cellular aspects of the role of caveolae, caveolins, and cavins in endothelial cell signaling and function. In addition, where appropriate, the possible implications for the cardiovascular and pulmonary physiology and pathophysiology will be discussed.

Genetic variation in the mouse model of Niemann Pick C1 affects female, as well as male, adiposity, and hepatic bile transporters but has indeterminate effects on caveolae

We have previously shown that male Npc1 heterozygous mice ( Npc1 +/− ), as compared to homozygous wild-type mice ( Npc1 +/+ ), both maintained on the “lean” BALB/cJ genetic background, become obese on a high fat but not on a low fat diet. We have now extended this result for female heterozygous mice. When fed high-fat diet, the Npc1 +/− white adipose weight is also increased in females, therefore following the same trend as males. Bile transporters which had previously been found to be altered in Npc1 −/− mice on a high fat diet, showed related, but small, changes in mRNA levels but large changes in protein expression. We have addressed the possible role of caveolae in these differences. It has long been known that caveolin 1 is increased in the liver (sex not specified) of Npc1 +/− (compared to Npc1 +/+ and Npc1 −/− ) mice and in heterozygous cultured skin fibroblasts of NPC1 carriers. We now find that caveolin 1 is increased in male, but not female liver and female, but not male adipose tissue. The caveolin 1 increase was not accompanied by changes in another caveolar protein, polymerase1 and transcript release factor (Ptrf). The numbers of caveolae in female adipose cells could not be correlated with levels of caveolae. Thus, we conclude that Npc1 affects female as well as male obesity and bile transporters but that effects on caveolin 1 are not discernible.

Comparative Properties of Caveolar and Noncaveolar Preparations of Kidney Na+/K+-ATPase

To evaluate previously proposed functions of renal caveolar Na+/K+-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na+/K+-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na+/K+-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na+/K+-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na+/K+-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na+/K+-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na+/K+-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na+/K+-ATPase, our findings suggest that interacting caveolar Na+/K+-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

Pacsin 2 is recruited to caveolae and functions in caveolar biogenesis

The pacsin (also termed syndapin) protein family is well characterised structurally. They contain F-BAR domains associated with the generation or maintenance of membrane curvature. The cell biology of these proteins remains less understood. Here, we initially confirm that EHD2, a protein previously shown biochemically to be present in caveolar fractions and to bind to pacsins, is a caveolar protein. We go on to report that GFP-pacsin 2 can be recruited to caveolae, and that endogenous pacsin 2 partially colocalises with caveolin 1 at the plasma membrane. Analysis of the role of pacsin 2 in caveolar biogenesis using small interfering RNA (siRNA) reveals that loss of pacsin 2 function results in loss of morphologically defined caveolae and accumulation of caveolin proteins within the plasma membrane. Overexpression of the F-BAR domain of pacsin 2 (but not the related F-BAR domains of CIP4 and FBP17) disrupts caveolar morphogenesis or trafficking, implying that pacsin 2 interacts with components required for these processes. We propose that pacsin 2 has an important role in the formation of plasma membrane caveolae.

Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity

Mice and humans lacking functional caveolae are dyslipidemic and have reduced fat stores and smaller fat cells. To test the role of caveolins/caveolae in maintaining lipid stores and adipocyte integrity, we compared lipolysis in caveolin-1 (Cav1)-null fat cells to that in cells reconstituted for caveolae by caveolin-1 re-expression. We find that the Cav1-null cells have a modestly enhanced rate of lipolysis and reduced cellular integrity compared with reconstituted cells as determined by the release of lipid metabolites and lactic dehydrogenase, respectively, into the media. There are no apparent differences in the levels of lipolytic enzymes or hormonally stimulated phosphorylation events in the two cell lines. In addition, acute fasting, which dramatically raises circulating fatty acid levels in vivo, causes a significant upregulation of caveolar protein constituents. These results are consistent with the hypothesis that caveolae protect fat cells from the lipotoxic effects of elevated levels fatty acids, which are weak detergents at physiological pH, by virtue of the property of caveolae to form detergent-resistant membrane domains.

Compartmentalizing Proximal FGFR1 Signaling in Placental Artery Endothelial Cell Caveolae.

Fibroblast growth factor-2 (FGF2) signaling via FGF receptor 1 (FGFR1) is critical for fetal placental angiogenesis. We have recently shown that the flask-shaped plasma membrane microdomain caveolae function as a platform for compartmentalizing ERK1/2 and AKT pathways activated by FGF2 and VEGF in placental artery endothelial cells. However, whether the proximal FGFR1 signaling events are also compartmentalized in the caveolae has not been explored. Proximal FGFR1 signaling is unique in that it is mediated by direct binding to and phosphorylating an adaptor protein FGFR substrate 2 (FRS2); this results in Grb2-mediated ERK2/1 activation and Gab1-mediated PI3K/Akt signaling. The objective of this study was to determine if the proximal FGFR1 signaling events are compartmentalized in placental endothelial cell caveolae. In ovine fetoplacental artery endothelial cells (oFPAEC), FGF2 treatment induced FRS2 phosphorylation on Tyr196 in a time- and dose-dependent fashion. Co-immunoprecipitation (Co-IP) experiments demonstrated that FGF2 rapidly and transiently increased the levels of FGFR1 bound FRS2 and the formation of a ternary complex FRS2-Gab1-Grb2 in oFPAEC. Co-IP and double fluorescence labeling experiments showed that FGFR1 and FRS2 was associated with the major caveolar structural protein caveolin-1 and co-purified with caveolin-1 in caveolae membranes. In oFPAEC treated with the cholesterol depletant methyl-β-cyclodextrin (MβCD) to disrupt caveolae, FGF2-induced FRS2 phosphorylation was inhibited and FGF2-induced formation of FRS2-Gab1-Grb2 complex was greatly attenuated. Subsequently, FGF2 was no longer able to activate the ERK/2 and Akt signaling pathways. Thus, integral caveolae compartmentalizes the proximal FGFR1 signaling via FRS2 and the formation of a ternary complex FRS2-Gab1-Grb2 (Supported by RO1 HL74947). (poster)

Ouabain activates the Na-K-ATPase signalosome to induce autosomal dominant polycystic kidney disease cell proliferation

The Na-K-ATPase is part of a cell signaling complex, the Na-K-ATPase signalosome, which upon activation by the hormone ouabain regulates the function of different cell types. We previously showed that ouabain induces proliferation of epithelial cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Here, we investigated the signaling pathways responsible for mediating the effects of ouabain in these cells. Incubation of ADPKD cells with ouabain, in concentrations similar to those found in blood, stimulated phosphorylation of the epidermal growth factor receptor (EGFR) and promoted its association to the Na-K-ATPase. In addition, ouabain activated the kinase Src, but not the related kinase Fyn. Tyrphostin AG1478 and PP2, inhibitors of EGFR and Src, respectively, blocked ouabain-dependent ADPKD cell proliferation. Treatment of ADPKD cells with ouabain also caused phosphorylation of the caveolar protein caveolin-1, and disruption of cell caveolae with methyl-β-cyclodextrin prevented Na-K-ATPase-EGFR interaction and ouabain-induced proliferation of the cells. Downstream effects of ouabain in ADPKD cells included activation of B-Raf and MEK and phosphorylation of the extracellular regulated kinase ERK, which translocated into the ADPKD cell nuclei. Finally, ouabain reduced expression of the cyclin-dependent kinase inhibitors p21 and p27, which are suppressors of cell proliferation. Different from ADPKD cells, ouabain showed no significant effect on B-Raf, p21, and p27 in normal human kidney epithelial cells. Altogether, these results identify intracellular pathways of ouabain-dependent Na-K-ATPase-mediated signaling in ADPKD cells, including EGFR-Src-B-Raf-MEK/ERK, and establish novel mechanisms involved in ADPKD cell proliferation.

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

Differences in the Expression of Multi‐Drug Resistant Protein 5 (MRP5) and Regulation of cGMP Levels in Phasic and Tonic Smooth Muscles

Previous studies have identified differences in the expression of contractile proteins and signaling pathways that regulate myosin light chain phosphorylation and contraction in phasic and tonic smooth muscle. cGMP plays a critical role in smooth muscle relaxation and the intracellular levels of cGMP are regulated by its hydrolysis via phosphodiesterase 5 (PDE5) and efflux via MRP5. AIM To determine the role of MRP5 in the regulation of cGMP levels in proximal versus distal stomach. METHODS Expression of MRP5 was analyzed by quantitative RT-PCR and western blot. Increase in cGMP levels in response to sodium nitroprusside (SNP) was measured in the presence of the PDE5 inhibitor, zaprinast. RESULTS Both mRNA and protein expression of MRP5 was 4-fold higher in muscle cells from proximal stomach than distal stomach. MRP5 was co-immunoprecipitated with caveolin-1, the main caveolar protein. In both proximal and distal stomach, SNP caused an increase in intra-and extracellular cGMP levels. The increase in extracellular cGMP was significantly higher than intracellular cGMP in muscle cells of proximal stomach, whereas extra- and intracellular cGMP levels were not significantly different in muscle cells of distal stomach. CONCLUSION Higher expression and function of MRP5, which provides a rapid mechanism for regulation of cGMP levels, may further contribute to the tonic contractile phenotype of proximal stomach.

Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation

Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that "caveosomes," previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.

Cavin proteins: New players in the caveolae field

Caveolae are specialized lipid microdomains, forming small invaginations in the plasma membrane, known to be implicated in multiple functions including lipid storage, cell signaling and endocytosis. Formation of these wide flask-shaped invaginations is dependent on the expression of a caveolar coat protein, namely caveolin. Until now, the accepted paradigm was that caveolin was the sole and only structural protein of caveolae since its expression was necessary and sufficient to drive caveolae biogenesis. The recent characterizations of PTRF/cavin-1 and subsequently other cavin family members in caveolae formation have highlighted additional levels of complexity in the biogenesis of these plasma membrane invaginations. In this review, recent advances on the role of the different cavin family members in the regulation of caveolae structures as well as potential new functions will be discussed.