Cav-1 Protein Expression Research Articles

Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells

P2X 4 and P2X 7 receptors are abundantly expressed in alveolar epithelial cells, and are thought to play a role in regulating fluid haemostasis. Here, we analyzed the expression and localization of the P2X 4R, and characterized the interaction between Cav-1 and both P2X 4R and P2X 7R in the mouse alveolar epithelial cell line E10. Using the biotinylation assay, we found that only glycosylated P2X 4R is exposed at the cell surface. Triton X-100 solubility experiments and sucrose gradient centrifugation revealed that P2X 4R was partially localized in Cav-1 rich membrane fractions. Cholesterol depletion with Mβ-CD displaced Cav-1 and P2X 4R from the low-density to the high-density fractions. Suppression of Cav-1 protein expression using short hairpin RNAs resulted in a large reduction in P2X 4R levels. Double immunofluorescence showed that P2X 4R and Cav-1 partially colocalize in vitro. Using the GST pull-down assay, we showed that Cav-1 interacts in vitro with both P2X 4R and P2X 7R. Co-immunoprecipitation experiments confirmed the interaction between P2X 7R and Cav-1. ATP stimulation increased the level of P2X 4R in the lipid raft/caveolae fraction, whereas Cav-1 content remained constant. Our results support recent evidence that P2X receptors are present in both raft and non-raft compartments of the plasma membrane and thus exhibit variable ATP sensitivity.

Caveolin expression is essential for NMDA‐mediated ERK1/2 phosphorylation in cultured neurons

Ischemic preconditioning (PC) is a phenomenon whereby various stimuli reduce the vulnerability of the brain to subsequent lethal ischemia. Although the endogenous signaling mechanisms remains unknown, previous work suggests that NMDA receptor (NMDAR) mediated signaling plays a critical role in neuronal PC. Because Src is involved in NMDAR signaling, we hypothesized that neuronal PC is dependent upon transient phosphorylation of Src, the Src scaffolding protein Cav-1, and ERK1/2. Immunoblot analysis of OGDSL (sublethal oxygen glucose deprivation)-treated or NMDA-stimulated neurons revealed a significant enhancement in phosphorylated Cav-1 (P-Cav-1), P-Src, P-ERK1/2, and P-p38 (P<0.05). Cav-1-siRNA treated neurons (72 hr) had reduced Cav-1 protein expression and attenuated NMDA-mediated Src and ERK1/2 phosphorylation (P-ERK1/2) as assessed by immunoblot and immunofluorescence microscopy. NMDA or OGDSL- exposure to neurons from Cav-1 null mice failed to enhance P-Src or P-ERK1/2. In summary, neither Src or ERK1/2 were activated in Cav-1 siRNA treated neurons or neurons from Cav-1 null mice after exposure to NMDA or OGDSL suggesting that Cav-1 expression is required for NMDA and OGDSL-mediated activation of Src and ERK1/2, two pro-survival kinases involved in neuronal preconditioning. (Supported by NIH)

Caveolins in vascular smooth muscle: Form organizing function

Caveolae are becoming increasingly recognized as an important organizational structure for a variety of signal and energy-transducing systems in vascular smooth muscle (VSM). In this review, we discuss the emerging role of the caveolins in organizing and modulating the basic functions of smooth muscle: contraction, growth/proliferation, and the energetic support systems that support these functions. With clear alterations in cell metabolism and function in VSM with altered caveolin-1 (Cav-1) protein expression and with cardiovascular abnormalities associated with Cav-1 null mice, the caveolin family of proteins may play an important role in the function and dysfunction of VSM.

Aortic eNOS expression and phosphorylation in Apo-E knockout mice: Differing effects of rapamycin and simvastatin

BackgroundThe inhibition of nitric oxide (NO) by hypercholesterolemia may be mediated, in part, by interactions with caveolin-1 (Cav-1). Because of the facilitatory effects of statins on endothelial function and the adverse effects of rapamycin (RAPA) on plasma lipids, we compared the effects of simvastatin (SMV) and RAPA on endothelial NO synthase (eNOS) and Cav-1 protein expression and phosphorylation in the aortas of apolipoprotein E (Apo-E) knockout (−/−) mice. MethodsApo-E −/− mice (n=38) fed a high-cholesterol diet were given SMV (100 mg/kg/day po), RAPA (3 mg/kg/day ip), or no treatment for 10 weeks. Blood was drawn for serum lipid analysis, and protein was extracted for Western immunoblotting. Selected aortic specimens from 2 animals in each group were examined by histology and immunohistochemistry. The data are expressed as the mean ± SEM and compared by the Student t test and ANOVA. Significance was established as P < .05. ResultsLipid levels at 10 weeks were similar in the 3 groups except for higher triglyceride levels in RAPA-treated animals. eNOS expression was highest in RAPA-treated mice, but the p-eNOS to eNOS protein expression ratio was significantly greater in the SMV treatment group compared to both RAPA and controls (P < .05). Both Cav-1 and p-Cav-1 expression was significantly lower in the SMV-treated animals (P < .05) compared to mice treated with RAPA. ConclusionsAlthough eNOS expression was greatest in the RAPA-treated mice, the expression of p-eNOS was similar in the RAPA- and SMV-treated animals. The increase in eNOS induced by RAPA and the inverse relationship between p-eNOS and Cav-1 protein expression observed with SMV treatment suggest different mechanisms for the regulation of aortic eNOS expression in Apo-E mice by these 2 agents.

Nerve Growth Factor Blocks the Glucose-induced Down-regulation of Caveolin-1 Expression in Schwann Cells via p75 Neurotrophin Receptor Signaling

Altered neurotrophism in diabetic peripheral neuropathy (DPN) is associated in part with substantial degenerative changes in Schwann cells (SCs) and an increased expression of the p75 neurotrophin receptor (p75NTR). Caveolin-1 (Cav-1) is highly expressed in adult SCs, and changes in its expression can regulate signaling through Erb B2, a co-receptor that mediates the effects of neuregulins in promoting SC growth and differentiation. We examined the hypothesis that hyperglycemia-induced changes in Cav-1 expression and p75NTR signaling may contribute to altered neurotrophism in DPN by modulating SC responses to neuregulins. In an animal model of type 1 diabetes, hyperglycemia induced a progressive decrease of Cav-1 in SCs of sciatic nerve that was reversed by insulin therapy. Treatment of primary neonatal SCs with 20-30 mm d-glucose, but not l-glucose, was sufficient to inhibit transcription from the Cav-1 promoter and decrease Cav-1 mRNA and protein expression. Hyperglycemia prolonged the kinetics of Erb B2 phosphorylation and significantly enhanced the mitogenic response of SCs to neuregulin1-beta1, and this effect was mimicked by the forced down-regulation of Cav-1. Intriguingly, nerve growth factor antagonized the enhanced mitogenic response of SCs to neuregulin1-beta1 and inhibited the glucose-induced down-regulation of Cav-1 transcription, mRNA, and protein expression through p75NTR-dependent activation of JNK. Our data suggest that Cav-1 down-regulation may contribute to altered neurotrophism in DPN by enhancing the response of SCs to neuregulins and that p75NTR-mediated JNK activation may provide a mechanism for the neurotrophic modulation of hyperglycemic stress.

Consequences of a novel caveolin-3 mutation in a large German family.

Mutations in the human caveolin-3 gene (cav-3) on chromosome 3p25 have been described in limb girdle muscular dystrophy, rippling muscle disease, hyperCKemia, and distal myopathy. Here, we describe the genetic, myopathological, and clinical findings in a large German family harboring a novel heterozygous mutation (GAC-->GAA) in codon 27 of the cav-3 gene. This missense mutation causes an amino acid change from asparagine to glutamate (Asp27Glu) in the N-terminal region of the Cav-3 protein, which leads to a drastic decrease of Cav-3 protein expression in skeletal muscle tissue. In keeping with an autosomal dominant mode of inheritance, this novel cav-3 mutation was found to cosegregate with neuromuscular involvement in the reported family. Ultrastructural analysis of Cav-3-deficient muscle showed an abnormal folding of the plasma membrane as well as multiple vesicular structures in the subsarcolemmal region. Neurological examination of all nine subjects from three generations harboring the novel cav-3 mutation showed clear evidence of rippling muscle disease. However, only two of these nine patients showed isolated signs of rippling muscle disease without muscle weakness or atrophy, whereas five had additional signs of a distal myopathy and two fulfilled the diagnostic criteria of a coexisting limb girdle muscular dystrophy. These findings indicate that mutations in the human cav-3 gene can lead to different and overlapping clinical phenotypes even within the same family. Different clinical phenotypes in caveolinopathies may be attributed to so far unidentified modifying factors/genes in the individual genetic background of affected patients.

Blockade of human immunodeficiency virus type 1 expression by caveolin-1.

Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.