Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase: targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (TYR-14).

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

Influence of caveolin-1 on cellular cholesterol efflux mediated by high-density lipoproteins.

Caveolin-1 is a principal structural component of caveolae membranes. These membrane microdomains participate in the regulation of signaling, transcytosis, and cholesterol homeostasis at the plasma membrane. In the present study, we determined the effect of caveolin-1 expression on cellular cholesterol efflux mediated by high-density lipoprotein (HDL). We evaluated this effect in parental NIH/3T3 cells as well as in two transformed NIH/3T3 cell lines in which caveolin-1 protein levels are dramatically downregulated. Compared with parental NIH/3T3 cells, these two transformed cell lines effluxed cholesterol more rapidly to HDL. In addition, NIH/3T3 cells harboring caveolin-1 antisense also effluxed cholesterol more rapidly to HDL. However, this effect was not due to changes in total cellular cholesterol content. We further showed that chronic HDL exposure reduced caveolin-1 protein expression in NIH/3T3 cells. HDL exposure also inhibited caveolin-1 promoter activity, suggesting a direct negative effect of HDL on caveolin-1 gene transcription. Moreover, we showed that HDL-induced downregulation of caveolin-1 prevents the uptake of oxidized low-density lipoprotein in human endothelial cells. These data suggest a novel proatherogenic role for caveolin-1, i.e., regarding the uptake and/or transcytosis of modified lipoproteins.

Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level.

Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress.

Environmental stressors have been recently shown to activate intracellular mitogen-activated protein (MAP) kinases, such as p38 MAP kinase, leading to changes in cellular functioning. However, little is known about the downstream elements in these signaling cascades. In this study, we show that caveolin-1 is phosphorylated on tyrosine 14 in NIH 3T3 cells after stimulation with a variety of cellular stressors (i.e. high osmolarity, H2O2, and UV light). To detect this phosphorylation event, we employed a phosphospecific monoclonal antibody probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Since p38 MAP kinase and c-Src have been previously implicated in the stress response, we next assessed their role in the tyrosine phosphorylation of caveolin-1. Interestingly, we show that the p38 inhibitor (SB203580) and a dominant-negative mutant of c-Src (SRC-RF) both block the stress-induced tyrosine phosphorylation of caveolin-1 (Tyr(P)(14)). In contrast, inhibition of the p42/44 MAP kinase cascade did not affect the tyrosine phosphorylation of caveolin-1. These results indicate that extracellular stressors can induce caveolin-1 tyrosine phosphorylation through the activation of well established upstream elements, such as p38 MAP kinase and c-Src kinase. However, heat shock did not promote the tyrosine phosphorylation of caveolin-1 and did not activate p38 MAP kinase. Finally, we show that after hyperosmotic shock, tyrosine-phosphorylated caveolin-1 is localized near focal adhesions, the major sites of tyrosine kinase signaling. In accordance with this localization, disruption of the actin cytoskeleton dramatically potentiates the tyrosine phosphorylation of caveolin-1. Taken together, our results clearly define a novel signaling pathway, involving p38 MAP kinase activation and caveolin-1 (Tyr(P)(14)). Thus, tyrosine phosphorylation of caveolin-1 may represent an important downstream element in the signal transduction cascades activated by cellular stress.

Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette.

Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3.

Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C.

Transgenic overexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype.

It recently was reported that Duchenne muscular dystrophy (DMD) patients and mdx mice have elevated levels of caveolin-3 expression in their skeletal muscle. However, it remains unknown whether increased caveolin-3 levels in DMD patients contribute to the pathogenesis of DMD. Here, using a genetic approach, we test this hypothesis directly by overexpressing wild-type caveolin-3 as a transgene in mice. Analysis of skeletal muscle tissue from caveolin-3- overexpressing transgenic mice reveals: (i) a dramatic increase in the number of sarcolemmal muscle cell caveolae; (ii) a preponderance of hypertrophic, necrotic, and immature/regenerating skeletal muscle fibers with characteristic central nuclei; and (iii) down-regulation of dystrophin and beta-dystroglycan protein expression. In addition, these mice show elevated serum creatine kinase levels, consistent with the myo-necrosis observed morphologically. The Duchenne-like phenotype of caveolin-3 transgenic mice will provide an important mouse model for understanding the pathogenesis of DMD in humans.

Caveolin-1 expression inhibits Wnt/beta-catenin/Lef-1 signaling by recruiting beta-catenin to caveolae membrane domains.

Caveolin-1 is a principal component of caveolae membranes. In NIH 3T3 cells, caveolin-1 expression is dramatically up-regulated in confluent cells and localizes at areas of cell-cell contact. However, it remains unknown whether caveolin-1 is involved in cell-cell signaling. Here, we examine the potential role of caveolin-1 in regulating beta-catenin signaling. beta-Catenin plays a dual role in the cell, linking E-cadherin to the actin cytoskeleton and in Wnt signaling by forming a complex with members of the lymphoid enhancing factor (Lef-1) family of transcription factors. We show that E-cadherin, beta-catenin, and gamma-catenin (plakoglobin) are all concentrated in caveolae membranes. Moreover, we demonstrate that activation of beta-catenin/Lef-1 signaling by Wnt-1 or by overexpression of beta-catenin itself is inhibited by caveolin-1 expression. We also show that recombinant expression of caveolin-1 in caveolin-1 negative cells is sufficient to recruit beta-catenin to caveolae membranes, thereby blocking beta-catenin-mediated transactivation. These results suggest that caveolin-1 expression can modulate Wnt/beta-catenin/Lef-1 signaling by regulating the intracellular localization of beta-catenin.

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.

Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia.

This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats. All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining. In newborn rats, only caveolin-1 was found in the hyaline cartilage. Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures. Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes. These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes. There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation.

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).

Flotillins/cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. Characterization and epitope-mapping of a novel flotillin-1 monoclonal antibody probe.

Caveolae are vesicular organelles that represent a subcompartment of the plasma membrane. Caveolins and flotillins are two families of mammalian caveolae-associated integral membrane proteins. However, it remains unknown whether flotillins interact with caveolin proteins to form a stable caveolar complex or if expression of flotillins can drive vesicle formation. Here, we examine the cell type and tissue-specific expression of the flotillin gene family. For this purpose, we generated a novel monoclonal antibody probe that recognizes only flotillin-1. A survey of cell and tissue types demonstrates that flotillins 1 and 2 have a complementary tissue distribution. At the cellular level, flotillin-2 was ubiquitously expressed, whereas flotillin-1 was most abundant in A498 kidney cells, muscle cell lines, and fibroblasts. Using three different models of cellular differentiation, we next examined the expression of flotillins 1 and 2. Taken together, our data suggest that the expression levels of flotillins 1 and 2 are independently regulated and does not strictly correlate with known expression patterns of caveolin family members. However, when caveolins and flotillins are co-expressed within the same cell, as in A498 cells, they form a stable hetero-oligomeric "caveolar complex." In support of these observations, we show that heterologous expression of murine flotillin-1 in Sf21 insect cells using baculovirus-based vectors is sufficient to drive the formation of caveolae-like vesicles. These results suggest that flotillins may participate functionally in the formation of caveolae or caveolae-like vesicles in vivo. Thus, flotillin-1 represents a new integral membrane protein marker for the slightly larger caveolae-related domains (50-200 nm) that are observed in cell types that fail to express caveolin-1. As a consequence of these findings, we propose the term "cavatellins" be used (instead of flotillins) to describe this gene family.

Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras. The subcellular distribution of caveolin-1 is modulated by cell-cell contact.

Caveolin-1, a suspected tumor suppressor, is a principal protein component of caveolae in vivo. Recently, we have shown that NIH 3T3 cells harboring anti-sense caveolin-1 exhibit a loss of contact inhibition and anchorage-independent growth. These observations may be related to the ability of caveolin-1 expression to positively regulate contact inhibition. In order to understand the postulated role of caveolin-1 in contact inhibition, it will be necessary to follow the distribution of caveolins in living cells in response to a variety of stimuli, such as cell density. Here, we visualize the distribution of caveolin-1 in living normal NIH 3T3 cells by creating GFP-fusion proteins. In many respects, the behavior of these GFP-caveolin-1 fusion proteins is indistinguishable from endogenous caveolin-1. These GFP-caveolin-1 fusion proteins co-fractionated with endogenous caveolin-1 using an established protocol that separates caveolae-derived membranes from the bulk of cellular membranes and cytosolic proteins, and co-localized with endogenous caveolin-2 in vivo as seen by immunofluorescence microscopy. We show here that as NIH 3T3 cells become confluent, the distribution of GFP-caveolin-1 and endogenous caveolin-1 shifts to areas of cell-cell contact, coincident with contact inhibition. However, unlike endogenous caveolin-1, the levels of GFP-caveolin-1 expression are unaffected by changes in cell density, serum starvation, or growth factor stimulation. These results are consistent with the idea that the levels of endogenous caveolin-1 are modulated by either transcriptional or translational control, and that this modulation is separable from density-dependent regulation of the distribution of caveolin-1. These studies provide a new living-model system for elucidating the dynamic mechanisms underlying the density-dependent regulation of the distribution of caveolin-1 and how this relates to contact inhibition.

The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo.

Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.

Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types.

Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.

Upregulation of caveolin-1 and caveolae organelles in Taxol-resistant A549 cells.

Caveolin is a principal component of caveolae membranes. It has been demonstrated that the interaction of the caveolin scaffolding domain with signaling molecules can functionally inhibit the activity of these molecules. Taxol is an antitumor agent that suppresses microtubule dynamics and binds to microtubules thereby stabilizing them against depolymerization. The drug also has been implicated in the induction of apoptosis through activation of components in signal transduction cascades. Here we have investigated the role of caveolin in the development of drug resistance by examining the expression of caveolins in low- and high-level drug-resistant cell lines. Caveolin-1, but not caveolin-2, was upregulated in highly multidrug resistant SKVLBI cells that express high levels of P-glycoprotein, and in low-level Taxol-resistant A549 cell lines that express low amounts of P-glycoprotein. Two drug-resistant A549 cell lines (one 9-fold resistant to Taxol and the other 1.5-fold resistant to epothilone B), both of which express no P-glycoprotein, demonstrate a significant increase in the expression of caveolin-1. These results indicate that in low-level epothilone B- or Taxol-resistant A549 cells, increased caveolin-1 expression occurs independently of P-glycoprotein expression. Electron microscopic studies clearly demonstrate the upregulation of caveolae organelles in Taxol-resistant A549 cells. Upregulation of caveolin-1 expression in drug-sensitive A549 cells was observed acutely beginning 48 h after incubation with 10 nM Taxol. Thus, caveolin-1 may play a role in the development of Taxol resistance in A549 cells.

Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin-1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1 but contain normal amounts of caveolin-2. NIH 3T3 cells harboring antisense caveolin-1 exhibit anchorage-independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin-1 downregulation is reversed when caveolin-1 protein levels are restored to normal by loss of the caveolin-1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin-1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin-1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.

Crowded little caves: structure and function of caveolae.

Caveolae are small vesicular invaginations of the cell membrane. It is within this organelle that cells perform transcytosis, potocytosis and signal transduction. These "little caves" are composed of a mixture of lipids and proteins unlike those found in the plasma membrane proper. The chief structural proteins of caveolae are caveolins. To date, three caveolins (Cav-1, -2 and -3) with unique tissue distributions have been identified. Caveolins form a scaffold onto which many signalling molecules can assemble, to generate pre-assembled signalling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signalling molecules.