Muscular dystrophy associated mutations in caveolin-1 induce neurotransmission and locomotion defects in Caenorhabditis elegans

Mutations in human caveolin-3 are known to underlie a range of myopathies. The cav-1 gene of Caenorhabditis elegans is a homologue of human caveolin-3 and is expressed in both neurons and body wall muscles. Within the body wall muscle CAV-1 localises adjacent to neurons, most likely at the neuromuscular junction (NMJ). Using fluorescently tagged CAV-1 and pre- and post-synaptic markers we demonstrate that CAV-1 co-localises with UNC-63, a post-synaptic marker, but not with several pre-synaptic markers. To establish a model for human muscular dystrophies caused by dominant-negative mutations in caveolin-3 we created transgenic animals carrying versions of cav-1 with homologous mutations. These animals had increased sensitivity to levamisole, suggesting a role for cav-1 at the NMJ. Animals carrying a deletion in cav-1 show a similar sensitivity. Sensitivity to levamisole and locomotion were also perturbed in animals carrying a dominant-negative cav-1 and a mutation in dynamin, which is a protein known to interact with caveolins. Thus, indicating an interaction between CAV-1 and dynamin at the NMJ and/or in neurons.     

Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.     

Mutual regulation between metabotropic glutamate type 1alpha receptor and caveolin proteins: from traffick to constitutive activity

In this paper, a molecular and functional interaction between metabotropic glutamate receptor type 1alpha (mGlu1alpha receptor) and caveolin-1 or caveolin-2beta is described. An overlapping pattern of staining for mGlu1alpha receptor with caveolin-1 and caveolin-2 by confocal laser microscopy in transiently transfected HEK-293 cells is observed. The presence of mGlu1alpha receptor in caveolin-enriched membrane fractions was demonstrated by flotation gradient analysis in the absence of detergents and the interaction between mGlu1alpha receptor with caveolin-1 and with caveolin-2beta was demonstrated by coimmunoprecipitation experiments. In HEK-293 cells, caveolin-2beta accumulates surrounding lipid droplets when single expressed but coexpression with mGlu1alpha receptor changed dramatically the subcellular localization of caveolin-2beta, directing it from lipid droplets to the cell surface. At the membrane level, the interaction between caveolin-1 and mGlu1alpha receptor could abrogate the constitutive activity exhibited by mGlu1alpha receptor. Overall, these results show that mGlu1alpha receptor interacts with caveolins and that this interaction is physiologically relevant for receptor function. Interestingly, we provide evidence that caveolin-1 is not just acting as a scaffolding protein for the mGlu1alpha receptor but that also regulates mGlu1alpha receptor constitutive activity.     

Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells

It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-deficient mouse embryonic fibroblasts, in which we have stably expressed caveolin-1, -2, or -3, we find that caveolin-1 interacts with caveolin-2 in this setting, whereas caveolin-3 does not, in agreement with most published observations. Next, we used a transfected L6 myoblast cell system expressing all three caveolin proteins. Surprisingly, we found that caveolin-1, -2, and -3 all coimmunoprecipitate in this cell type, suggesting that this interaction is muscle cell specific. Similar results were obtained when the skeletal muscle of caveolin-1 transgenic animals was analyzed for caveolin-1 and caveolin-3 coimmunoprecipitation. Thus we conclude that all three caveolins can interact to form a discrete hetero-oligomeric complex, but that such complex formation is clearly muscle specific. caveolae; caveolin-1; caveolin-2; caveolin-3     

Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone.

Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.     

Caveolin-3 directly interacts with the C-terminal tail of beta -dystroglycan. Identification of a central WW-like domain within caveolin family members

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with beta-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and co-immunoprecipitates with a fusion protein containing the cytoplasmic tail of beta-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of beta-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within beta-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with beta-dystroglycan. In this regard, interaction of caveolin-3 with beta-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy.     

Caveolae-associated proteins in cardiomyocytes: caveolin-2 expression and interactions with caveolin-3

Caveolin-3 the muscle-specific caveolin isoform, acts like the more ubiquitously expressed caveolin-1 to sculpt caveolae, specialized membrane microdomains that serve as platforms to organize signal transduction pathways. Caveolin-2 is a structurally related isoform that alone does not drive caveolae biogenesis; rather, caveolin-2 cooperates with caveolin-1 to form caveolae in nonmuscle cells. Although caveolin-2 might be expected to interact in an fashion analogous to that of caveolin-3, it generally has not been detected in cardiomyocytes. This study shows that caveolin-2 and caveolin-3 are detected at low levels in ventricular myocardium and increase dramatically with age or when neonatal cardiomyocytes are placed in culture. In contrast, flotillins (caveolin functional homologs) are expressed at relatively constant levels in these preparations. In neonatal cardiac cultures, caveolin-2 and -3 expression is not influenced by thyroid hormone (a postnatal regulator of other cardiac gene products). The further evidence that caveolin-2 coimmunoprecipitates with caveolin-3 and floats with caveolin-3 by isopycnic centrifugation in cardiomyocyte cultures suggests that caveolin-2 may play a role in caveolae biogenesis and influence cardiac muscle physiology.     

Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C). Retention of LGMD-1C caveolin-3 mutants within the golgi complex.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Autosomal dominant limb girdle muscular dystrophy (LGMD-1C) in humans is due to mutations within the caveolin-3 gene: (i) a 9-base pair microdeletion that removes three amino acids within the caveolin scaffolding domain (DeltaTFT) or (ii) a missense mutation within the membrane spanning domain (P --> L). The molecular mechanisms by which these two mutations cause muscular dystrophy remain unknown. Here, we investigate the phenotypic behavior of these caveolin-3 mutations using heterologous expression. Wild type caveolin-3 or caveolin-3 mutants were transiently expressed in NIH 3T3 cells. LGMD-1C mutants of caveolin-3 (DeltaTFT or P --> L) were primarily retained at the level of a perinuclear compartment that we identified as the Golgi complex in double-labeling experiments, while wild type caveolin-3 was efficiently targeted to the plasma membrane. In accordance with these observations, caveolin-3 mutants formed oligomers of a much larger size than wild type caveolin-3 and were excluded from caveolae-enriched membrane fractions as seen by sucrose density gradient centrifugation. In addition, these caveolin-3 mutants were expressed at significantly lower levels and had a dramatically shortened half-life of approximately 45-60 min. However, caveolin-3 mutants were palmitoylated to the same extent as wild type caveolin-3, indicating that targeting to the plasma membrane is not required for palmitoylation of caveolin-3. In conclusion, we show that LGMD-1C mutations lead to formation of unstable high molecular mass aggregates of caveolin-3 that are retained within the Golgi complex and are not targeted to the plasma membrane. Consistent with its autosomal dominant form of genetic transmission, we demonstrate that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the retention of wild type caveolin-3 at the level of the Golgi. These data provide a molecular explanation for why caveolin-3 levels are down-regulated in patients with this form of limb girdle muscular dystrophy (LGMD-1C).     

Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.     

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.     

Differential expression of caveolin-1 and caveolin-3: potential marker for cardiac toxicity subsequent to chronic ozone inhalation

Earlier studies from our laboratory have shown myocardial dysfunction subsequent to chronic O(3) exposure in rats may be associated with a decrease in antioxidant reserve and increased activity of inflammatory mediators. The present study tested the hypothesis that O(3)-induced cardiac dysfunction in healthy adult rats may be due to changes in caveolin-1 and caveolin-3 levels. Sprague-Dawley rats were exposed 8?h/day for 28 and 56?days to filtered air or 0.8?ppm O(3). In order to assess the chronic effects to O(3), in vivo cardiac function was assessed by measuring LVDP, 24?h after termination of O(3) exposure. Compared to rats exposed to filtered air, LVDP values significantly decreased in all O(3)-exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-α levels and decreased myocardial activities of superoxidase dismutase. Progressive increases in the expression of myocardial TNF-α in 28?days and 56?days O(3)-exposed animals were followed by decreases in cardiac caveolin-1 levels. On the other hand, differential changes in the expression of caveolin-3 in hearts from 28 and 56?days O(3)-exposed animals were independent of intra-cardiac TNF-α levels. These novel findings suggest the interesting possibility that a balance between caveolin-1 and caveolin-3 may be involved in O(3)-mediated cardiac toxicity.     

Loss of caveolin-1 in bronchiolization in lung fibrosis.

Bronchiolization is a key process in fibrosing lung in which the proliferative status of bronchiolar epithelium changes, leading to abnormal epithelial morphology. Within the context that caveolin-1 acts to suppress epithelial proliferation, we postulated that stimulating epithelial injury would lead to caveolin-1 downregulation and encourage proliferation. The present study evaluates the expression of caveolin-1, especially in bronchiolization, in C57BL/6J mice with bleomycin-induced lung fibrosis and in various types of re-epithelialization in human interstitial pneumonias (IPs). Immunohistochemically, levels of caveolin-1 decreased in the bronchiolar epithelium of mice treated with bleomycin. Levels of caveolin-1 mRNA in the whole lung were decreased at 7 and 14 days. Caveolin-1 mRNA was also decreased in laser-capture microdissection- retrieved bronchiolar epithelial cells at 7 days. Among patients with 12 IPs, including four usual IPs (UIPs) and eight nonspecific IPs (NSIPs), whole lung caveolin-1 was significantly decreased compared with 12 controls at both mRNA and protein levels. By scoring immunointensity, caveolin-1 was significantly reduced in bronchiolization and squamous metaplasia as well as in bronchiolar epithelium in 23 IPs (12 UIPs and 11 NSIPs) compared with bronchiolar epithelium from seven controls. These data suggested that loss of caveolin-1 is associated with abnormal re-epithelialization in lung fibrosis.     

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneider's and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.     

Phenotypic behavior of caveolin-3 R26Q,a mutant associated with hyperCKemia,distal myopathy,and rippling muscle disease

Four different phenotypes have been associated with CAV3 mutations: limb girdle muscular dystrophy-1C (LGMD-1C), rippling muscle disease (RMD), and distal myopathy (DM), as well as idiopathic and familial hyperCKemia (HCK). Detailed molecular characterization of two caveolin-3 mutations (P104L and TFT), associated with LGMD-1C, shows them to impart a dominant-negative effect on wild-type caveolin-3, rendering it dysfunctional through sequestration in the Golgi complex. Interestingly, substitution of glutamine for arginine at amino acid position 26 (R26Q) of caveolin-3 is associated not only with RMD but also with DM and HCK. However, the phenotypic behavior of the caveolin-3 R26Q mutation has never been evaluated in cultured cells. Thus we characterized the cellular and molecular properties of the R26Q mutant protein to better understand how this mutation can manifest as such distinct disease phenotypes. Here, we show that the caveolin-3 R26Q mutant is mostly retained at the level of the Golgi complex. The caveolin-3 R26Q mutant formed oligomers of a much larger size than wild-type caveolin-3 and was excluded from caveolae-enriched membranes. However, caveolin-3 R26Q did not behave in a dominant-negative fashion when coexpressed with wild-type caveolin-3. Thus the R26Q mutation behaves differently from other caveolin-3 mutations (P104L and TFT) that have been previously characterized. These data provide a possible explanation for the scope of the various disease phenotypes associated with the caveolin-3 R26Q mutation. We propose a haploinsufficiency model in which reduced levels of wild-type caveolin-3, although not rendered dysfunctional due to the caveolin-3 R26Q mutant protein, are insufficient for normal muscle cell function. muscle cell caveolae; caveolin-3; muscular dystrophy     

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.     

Caveolin-3 Is Associated with the T-Tubules of Mature Skeletal Muscle Fibers

Caveolae are abundant in skeletal muscle and their coat contains a specific isoform of caveolin, caveolin-3. It has been suggested that during muscle development, caveolin-3 is associated with the T-tubules, but that in adult muscle it is found on the plasma membrane only. We have studied the distribution of caveolin-3 in single skeletal muscle fibers from adult rat soleus by confocal immunofluorescence and by immunogold electron microscopy. We found that caveolin-3 occurs at the highest density on the plasma membrane but is also present in the core of the fibers, at the I-band/A-band interface where it is associated with the T-tubules. In neither domain of the muscle surface does caveolin-3 colocalize with the glucose transporter GLUT4 and there is no evidence for internalization of the caveolae in muscle.     

Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation.

Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.     

Angiotensin II induces nephrin dephosphorylation and podocyte injury: role of caveolin-1

Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10⁻⁶ M) pretreated with or without losartan (10⁻⁵ M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342.Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.     

Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae

The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar.These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae.