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We isolated a full-length cDNA encoding human caveolin 3, which is 72% and 59% homologous to human caveolin 1 and caveolin 2, respectively. Human caveolin 3 protein contains the 'caveolin signature sequence' and the 33 amino acids spanning intramembrane domain common to all caveolins. Northern blot analysis indicates that the caveolin 3 transcript is 1.6 kb in size and exclusively detectable in muscle tissue.
The inducible-type NO synthase (NOS II; iNOS) is constitutively expressed in slow-twitch skeletal muscle fibres of guinea-pigs [Gath, Closs, Gödtel-Armbrust, Schmitt, Nakane, Wessler and Förstermann (1996) FASEB J. 10, 1614-1620]. Here we studied the expression of NOS II in skeletal muscle of wild-type and NOS II-deficient mice and investigated the molecular basis for the membrane association of this NOS in muscle. A basal expression of NOS II mRNA and protein was detected in skeletal muscle from untreated wild-type mice; expression increased when mice were treated with bacterial lipopolysaccharide (LPS). No NOS II was found in any tissue of untreated or LPS-treated NOS II-deficient mice. Immunoprecipitation experiments were performed with homogenates of gastrocnemius muscle from untreated or LPS-treated wild-type mice. A NOS II-specific antibody precipitated caveolin 3 in all homogenates investigated, the effect being most pronounced in skeletal muscle from LPS-treated animals. Conversely, an antibody against caveolin 3 co-precipitated NOS II in muscle homogenates. Similarly, a weak co-precipitation of NOS II and caveolin 3 was seen in homogenates of untreated murine C2C12 myotubes; co-precipitation was markedly enhanced in cells stimulated with LPS/interferon gamma. The association of NOS II with caveolin 3 might have implications for the regulation of contraction of, and/or glucose uptake by, slow-twitch muscle fibres.
To analyze the molecular mechanism of the increased caveolin 3 activities in dystrophin-deficient muscles, we investigated three-dimensionally the changes in caveolin 3 molecular distribution and density at the sarcolemma of mdx mice by the fracture-label electron microscopic technique. At the sarcolemma of skeletal muscles from mdx mice, the densities of gold particles associated with caveolae, non-associated with caveolae and arranged circularly without caveolae were higher than those in control mice (P<0.01, P<0.01 and P<0.05 by two-tailed t-test), although in mdx mice, the overall arrangement of gold particles appeared to be irregular. These findings may reflect the active process of caveolar formation and the results of the disrupted protein-protein interaction in dystrophin-deficient muscle plasma membrane.
The localization of caveolins in the sinus endothelial cells of the rat spleen has been demonstrated by confocal laser scanning and electron microscopy. Caveolin-3, a muscle-specific caveolin, was detected by Western blot analysis and immunofluorescence microscopy of isolated sinus endothelial cells and tissue cryosections of the spleen. During the immunofluorescence microscopy of isolated endothelial cells, both caveolin-3 and caveolin-1 were found. In tissue cryosections of the spleen, caveolin-3, as well as caveolin-1 and -2, was present in the contours and cytoplasm of the cells. Immunogold electron microscopy of tissue cryosections revealed caveolin-3, -1, and -2 to be present in caveolae in the apical, lateral, and basal plasma membranes and some vesicular profiles in the cytoplasm of sinus endothelial cells. Furthermore, caveolin-3 was colocalized with caveolin-1 in the same caveolae in the apical, lateral, and basal plasma membranes. Stress fibers and tubulovesicular structures were situated in the vicinity of caveolae labeled with anti-caveolin-3, anti-caveolin-1, and anti-caveolin-2 antibodies. It is speculated that caveolae in sinus endothelial cells play an important role in the constriction of stress fibers.
The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.
There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.
The application of subcellular fractionation protocols developed in soft tissues to fibrous organs such as the heart is unsuitable given the substantial differences in subcellular structure these tissues exhibit. The purpose of this study was to develop a simple method for the separation of sarcolemma and endosomes from isolated Langendorff-perfused rat hearts. Hearts were homogenized with either an Ultra-Turrax homogenizer or a hand-held glass tissue grinder. Quantitative immunoblots assessed the enrichment of the sarcolemmal proteins caveolin 3 and the sodium potassium ATPase and the endosomal proteins rab4 and GLUT4 in different membrane fractions. Application of homogenates to sucrose and Percoll density gradients failed to resolve membranes differentially enriched in sarcolemmal or endosomal marker proteins, indicating little difference in density between the sarcolemma and endosomes. However, successive spins of homogenates from a hand-held glass tissue grinder successfully separated the endosomes from the sarcolemma, indicating differences in masses between the two membrane fractions. Approximately 70% of total caveolin 3 and sodium potassium ATPase immunoreactivity was in membrane pellets up to 20,000g and approximately 85% of rab4 and GLUT4 in pellets from 20,000-100,000g. In addition, 86% of ouabain-sensitive ATPase activity (sodium potassium ATPase activity) was in membrane pellets up to 20,000g. Therefore, sarcolemmal membranes were pelleted up to 20,000g, and endosomal membranes between 20,000 and 100,000g. Regional ischemia (40 min) followed by reperfusion (60 min) caused the translocation of GLUT4 (but not rab4) from the endosomal membranes to the sarcolemma in the area of the heart subjected to ischemia.
Hereditary rippling muscle disease (RMD) is an autosomal dominant human disorder characterized by mechanically triggered contractions of skeletal muscle. Genome-wide linkage analysis has identified an RMD locus on chromosome 3p25. We found missense mutations in positional candidate CAV3 (encoding caveolin 3; ref. 5) in all five families analyzed. Mutations in CAV3 have also been described in limb-girdle muscular dystrophy type 1C (LGMD1C; refs. 6,7), demonstrating the allelism of dystrophic and non-dystrophic muscle diseases.
A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. The gel is optimized so that large proteins of more than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be analyzed in the top part, while smaller proteins under 150 kd (eg, calpain 3, the 80-kd fragment of laminin alpha2 chain, all of the sarcoglycans, and caveolin 3) are separated in the lower phase. This basic system could be used for different combinations of antibodies as new muscular dystrophy proteins are identified and require examination. In addition, analysis of the laminin alpha2 chain of merosin showed that this protein was expressed as a doublet or triplet set of bands in many patients with active muscle pathology. This may indicate the existence of an embryonic isoform, which is re-expressed in regenerating fibers.
Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.
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Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogeneous group of myopathies, including autosomal dominant and recessive forms. To date, two autosomal dominant forms have been recognized: LGMD1A, linked to chromosome 5q, and LGMD1B, associated with cardiac defects and linked to chromosome 1q11-21. Here we describe eight patients from two different families with a new form of autosomal dominant LGMD, which we propose to call LGMD1C, associated with a severe deficiency of caveolin-3 in muscle fibres. Caveolin-3 (or M-caveolin) is the muscle-specific form of the caveolin protein family, which also includes caveolin-1 and -2. Caveolins are the principal protein components of caveolae (50-100 nm invaginations found in most cell types) which represent appendages or sub-compartments of plasma membranes. We localized the human caveolin-3 gene (CAV3) to chromosome 3p25 and identified two mutations in the gene: a missense mutation in the membrane-spanning region and a micro-deletion in the scaffolding domain. These mutations may interfere with caveolin-3 oligomerization and disrupt caveolae formation at the muscle cell plasma membrane.
The dystrophin-glycoprotein complex (DGC) serves as a link between cytoplasmic actin, the membrane and the extracellular matrix of striated muscle. Genetic defects in genes encoding a subset of DGC proteins result in muscular dystrophy and a secondary decrease in other DGC proteins. Caveolae are dynamic structures that have been implicated in a number of functions including endocytosis, potocytosis and signal transduction. Caveolin (VIP-21) is thought to play a structural role in the formation of non-clathrin-coated vesicles in a number of different cell types. Caveolin-3, or M-caveolin, was identified as a muscle-specific form of the caveolin family. We show that caveolin-3 co-purifies with dystrophin, and that a fraction of caveolin-3 is a dystrophin-associated protein. We isolated the gene for human caveolin-3 and mapped it to chromosome 3p25. We determined the genomic organization of human caveolin-3 and devised a screening strategy to look for mutations in caveolin-3 in patients with muscular dystrophy. Of 82 patients screened, two nucleotide changes were found that resulted in amino acid substitutions (G55S and C71W); these changes were not seen in a control population. The amino acid changes map to a functionally important domain in caveolin-3, suggesting that these are not benign polymorphisms and instead are disease-causing mutations.
We compared apolipoprotein A-I (apoA-I)-mediated release of cellular cholesterol and phospholipid among several fibroblast cell lines. ApoA-I induced phospholipid release from WI-38, MRC-5, BALB/3T3, L929 and CHO-K1, but not from COS-7, while cholesterol was released only from WI-38, MRC-5 and BALB/3T3 without correlation to the cellular cholesterol content. The reaction consequently generated cholesterol-rich high density lipoprotein (HDL) with WI-38, MRC-5 and BALB/3T3, cholesterol-poor HDL with L929 and CHO-K1, and no HDL from COS-7. In contrast, diffusion-mediated cholesterol efflux to cyclodextrin took place with all the cell lines tested in proportion to the cellular free cholesterol content. While caveolin-1 was expressed in all of these cell lines, ATP-binding cassette transporter (ABC) A1 was detected in all but COS-7. We concluded that (1) fibroblasts were categorized into three groups with respect to the interaction with apoA-I, (i) those to produce cholesterol-rich HDL, (ii) cholesterol-poor HDL and (iii) no HDL; (2) ABCAl was required for the assembly of HDL from phospholipid among the cell lines tested; (3) caveolin-1 alone did not induce cholesterol incorporation into the HDL generated.
Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.
1. The formation of NO from endothelial nitric oxide synthase (eNOS) in rat superior mesenteric artery rings was dependent on extracellular L-arginine, and was optimal at a concentration of L-arginine close to the plasma level (carbachol-stimulated NO: control 15.7+/-0.9, L-arginine 100 micro M 22.8+/-1.3 nM). 2. Enhancement of NO output by L-arginine was stereospecific, required the cationic amino-acid transporter and was dependent on caveolin. 3. Induction of inducible nitric oxide synthase (iNOS) impaired the stimulated NO synthesis from eNOS (100 nM carbachol-stimulated NO: control 5.7+/-0.6, iNOS 0.3+/-0.3 nM). 4. The interaction between iNOS and eNOS was reversed by the superoxide scavenger MnTMPyP. Impairment of eNOS by iNOS was also prevented by L-arginine 100 micro M administered simultaneously with carbachol, but not by L-arginine administered during incubation with lipopolysaccharide. 5. These data provide functional evidence that supplementing L-arginine from the extracellular medium optimises the formation of NO from eNOS and suggests that the impairment of eNOS by iNOS is caused by excess formation of superoxide by NO synthase, which can be prevented by L-arginine. These results provide an explanation for the observations that extracellular L-arginine can enhance endothelium function only when the endothelium is impaired or when iNOS has been induced.
One of the characteristics of senescent cells is unresponsiveness to external stimuli like EGF. Although they have a normal level of receptors and downstream signaling molecules, EGF cannot induce the activation of Erk kinases and DNA synthesis in senescent cells as much as in young cells. Caveolin proteins directly interact with signaling molecules including EGF receptor and suppress the activation of EGFR upon EGF stimulation. We found that Erk activation after EGF stimulation in senescent human diploid fibroblasts was down-regulated. Those senescent cells showed an increased level of three isoforms of caveolin proteins. This change seems to lie in transcriptional control in senescent cells. We also demonstrated up-regulated caveolin proteins were co-localized with EGFR proteins in detergent-insoluble fractions. From these results, we suggest that the up-regulated expression of caveolin might explain the unresponsiveness of senescent fibroblasts to EGF stimulation.
A growing body of evidence suggests that muscle cell caveolae may function as specialized membrane micro-domains in which the dystrophin-glycoprotein complex and cellular signaling molecules reside. Caveolin-3 (Cav-3) is the only caveolin family member expressed in striated muscle cell types (cardiac and skeletal). Interestingly, skeletal muscle fibers from Cav-3 (-/-) knock-out mice show a number of myopathic changes, consistent with a mild-to-moderate muscular dystrophy phenotype. However, it remains unknown whether a loss of Cav-3 affects the phenotypic behavior cardiac myocytes in vivo. Here, we present a detailed characterization of the hearts of Cav-3 knock-out mice. We show that these mice develop a progressive cardiomyopathic phenotype. At four months of age, Cav-3 knock-out hearts display significant hypertrophy, dilation, and reduced fractional shortening, as revealed by gated cardiac MRI and transthoracic echocardiography. Histological analysis reveals marked cardiac myocyte hypertrophy, with accompanying cellular infiltrates and progressive interstitial/peri-vascular fibrosis. Interestingly, loss of Cav-3 expression in the heart does not change the expression or the membrane association of the dystrophin-glycoprotein (DG) complex. However, a marker of the DG complex, alpha-sarcoglycan, was specifically excluded from lipid raft domains in the absence of Cav-3. Because activation of the Ras-p42/44 MAPK pathway in cardiac myocytes can drive cardiac hypertrophy, we next assessed the activation state of this pathway using a phospho-specific antibody probe. We show that p42/44 MAPK (ERK1/2) is hyperactivated in hearts derived from Cav-3 knock-out mice. These results are consistent with previous in vitro data demonstrating that caveolins may function as negative regulators of the p42/44 MAPK cascade. Taken together, our data argue that loss of Cav-3 expression is sufficient to induce a molecular program leading to cardiac myocyte hypertrophy and cardiomyopathy.