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Department of Molecular Pharmacology and the Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York
ABSTRACT I. PERSPECTIVE II. CAVEOLAE A. Discovery and Morphology B. Tissue Distribution C. Biochemical Properties III. THE CAVEOLINS A. Initial Discovery B. The Caveolin Gene Family C. Caveolin Protein Characterization 1. Membrane topology and attachment 2. Oligomerization domain 3. The caveolin scaffolding domain IV. FUNCTIONAL ROLES OF CAVEOLAE/CAVEOLINS A. Vesicular Transport 1. Transcytosis 2. Endocytosis B. Cholesterol Homeostasis C. Signal Transduction D. Proteomics V. CAVEOLINS AND HUMAN DISEASE: KNOCKOUT MOUSE MODELS A. Caveolin-1 1. Cellular transformation and tumorigenesis A) BREAST CANCER. B) GASTROINTESTINAL CANCER. C) PROSTATE CANCER. D) MULTIDRUG RESISTANT CANCERS. 2. Glucose metabolism, insulin signaling, and diabetes 3. Modulation of lipid storage and breakdown 4. Vascular abnormalities: hypertrophic cardiomyopathy 5. Vascular abnormalities: increased nitric oxide production 6. Vascular abnormalities: impaired angiogenesis 7. Vascular abnormalities: atheroprotection 8. Abnormalities of the urogenital system 9. Ocular disease 10. Reductions in life span B. Caveolin-2 1. Interstitial lung disease C. Caveolin-3 1. Specialized function: a muscle-specific isoform 2. Pathogenesis of muscular dystrophy 3. Caveolin-3 null mice 4. Cardiomyopathy D. Caveolin-1/3 VI. CONCLUSIONS AND FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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I. PERSPECTIVE |
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Indeed, evidence is accumulating that implicates the caveolin gene family in the pathogenesis of numerous human diseases, including cancer, muscular dystrophy, and type II diabetes. This review describes the 1) biochemical properties of the caveolin protein products, 2) the physiological consequences of caveolin gene ablation in mice, and 3) the relationship between caveolin expression and the development/progression of certain human diseases.
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II. CAVEOLAE |
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Caveolae are morphologically identifiable plasma membrane invaginations that are distinct from the larger electron-dense clathrin-coated pits. Originally identified in the 1950s by electron microscopists investigating cellular ultrastructure, caveolae appear as "smooth" uncoated pits or vesicles at the plasma membrane, typically observed using conventional resin-embedded techniques. In general, caveolae are 50- to 100-nm flask-shaped invaginations of the plasma membrane that can be singular or found in detached grapelike clusters (rosette formation) and long tubular structures thought to evolve from the fusion of individual caveolae (Fig. 1). Isolated caveolae appear as vesicular or curved U-shaped structures possessing a distinctive granular appearance using techniques such as transmission electron microscopy and low-angle platinum shadowing (Fig. 2). While the overall function of the prototypical caveolae organelle has been an area of intense exploration, little attention has focused on the specialized function, if any, of the various morphological subsets of caveolae. Consequently, the functional significance of these caveolae-related organelles remains unknown.
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Caveolae were simultaneously identified in capillary endothelial cells and epithelial cells from the mouse gall bladder (181, 285). Since then, caveolae have been identified in a wide variety of tissues and cell types. While no all-encompassing ultrastructural study has been undertaken, a review of published literature reveals that caveolae are present to some degree in most differentiated cell types. Particularly, caveolae have been well-described in adipocytes, where they are extremely abundant, endothelial cells, type I pneumocytes of the lung, and striated and smooth muscle cells. Because of their relative abundance in endothelial cells and type I pneumocytes, the two major constituents of lung alveoli, the lung stands out as one of the most abundant sources of identifiable caveolae, second only to adipocytes. Ultrastructural analysis of adipocytes has shown that as much as 20% of the total plasma membrane is occupied by caveolae (53). Thus caveolae can greatly increase the surface area of numerous cell types, an observation that lends credence to the original speculation that caveolae are involved in macromolecular transport and mechanotransduction events.
Unlike earlier views of the plasma membrane as a "fluid mosaic" (235), where integral membrane proteins were thought to float and diffuse freely through a sea of homogeneous lipids, a more contemporary view of the plasma membrane is that proteins are much more heterogeneously distributed and can be found clustered within specialized microdomains, termed lipid rafts. These lipid rafts are thought to form via the aggregation of glycosphingolipids and sphingomyelin in the Golgi apparatus (held together by transient and weak molecular interactions) and are then delivered to the plasma membrane as concentrated units (263, 264). These lipid rafts are also enriched in cholesterol and several resident proteins, including glycophosphatidylinositol (GPI)-linked proteins. Relative to the plasma membrane proper, which contains an abundance of cis-unsaturated phospholipids, the sphingolipids in lipid rafts contain primarily saturated fatty acyl chains, allowing tighter molecular packing that results in a higher melting temperature (Tm 
41°C vs. Tm <0°C for phospholipids) (223). The high cholesterol and sphingolipid content of lipid rafts imparts a resistance to extraction in nonionic detergents such as Triton X-100 at 4°C and a light buoyant-density in sucrose gradients, properties instrumental for their purification and biochemical characterization. It should be mentioned that the exact nature and defining characteristics of lipid rafts, as well as the techniques involved in isolating them, are now quite well-developed (for recent reviews of this subject, see Refs. 90, 165, 187).
Caveolae represent a morphologically identifiable subset of lipid rafts. They contain the coat protein caveolin, which is essential for the invagination of the plasma membrane through a largely unknown process, giving them their characteristic flasklike appearance. While the overall biochemical composition of lipid rafts and caveolae is thought to overlap, these microdomains are not completely equivalent. In addition to the caveolins, several proteins have been shown to preferentially localize to either caveolae or lipid rafts, respectively (134).
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III. THE CAVEOLINS |
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In the late 1980s, investigators attempting to identify possible targets for phosphorylation-mediated cellular transformation used a then recently produced antiphosphotyrosine antibody (PY-20) to affinity purify several phosphotyrosine-containing proteins from Rous sarcoma-transformed chicken embryonic fibroblast (77). One of the four major proteins identified was found to be resistant to extraction with nonionic detergents and demonstrated a staining pattern that was simultaneously punctate, concentrated at cell margins, and focused in parallel arrays along actin stress fibers. The localization of this 22-kDa protein was also shown to change after cellular transformation; indeed, the tyrosine phosphorylation of this protein was dependent on transformation with v-Src, suggesting a role for this protein in oncogenesis (76). This 22-kDa protein was localized to caveolae by immunoelectron microscopy, and ultrastructural analysis revealed that numerous striated filaments coat the cytoplasmic surface of these organelles. In searching for the protein components of these striations, it was found that antibodies generated against the 22-kDa protein stained the striated filaments, and thus the 22-kDa protein was termed caveolin (now called caveolin-1). Furthermore, the caveolin filaments were found to be resistant to extraction with high salt, and treatment of cells with cholesterol-chelating agents resulted in the flattening of membrane caveolae, with disassociation of the striated coat (203). Later, using oligonucleotides predicted from its peptide sequence, the caveolin gene was cloned. Overexpression of the caveolin-1 cDNA in fibroblasts resulted in caveolin-1 localization to caveolae, confirming that it is the major resident protein of this organelle (75).
Working on a completely different aspect of cell biology, Kurzchalia et al. (115) identified a set of CHAPS-insoluble proteins, one of which they termed VIP21 (vesicular integral-membrane protein of 21 kDa). The cDNA for this protein was cloned and transiently expressed in mammalian cells. By immunofluorescence microscopy, VIP21 was found to localize to the Golgi apparatus, the plasma membrane, and membrane-bound vesicles (115). In later work, Glenney (75) showed that the sequences for VIP21 and caveolin were identical and thus these two research groups had independently identified the same protein by distinct biochemical methods.
To date, three members of the caveolin (CAV) gene family have been identified (Fig. 3). Caveolin (later termed caveolin-1; Ref. 216) was the first gene discovered and is composed of three exons that are highly conserved in sequence and structure across species. Caveolin-2 was discovered when micro-sequencing of purified adipocyte caveolae membrane domains revealed a strikingly similar peptide sequence to that of caveolin-1, differing in several key conserved caveolin-1 residues. Cross-referencing this new peptide sequence with known expressed sequence tags (EST) revealed an EST encoding a caveolin-1-like protein of 162 amino acids, subsequently termed caveolin-2 (216). Caveolin-3 was cloned from a cDNA library using a caveolin-1-related sequence found immediately downstream of the rat oxytocin receptor (256).
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, with the -isoform consisting of residues 1–178 and the -isoform containing residues 32–178, resulting in a protein 3 kDa smaller in size (128, 217). The -isoform is thought to derive from an alternate translation initiation site occurring at a methionine in position 32. When expressed in Sf21 insect cells, which lack endogenous caveolae, both isoforms of caveolin-1 (Cav-1 and Cav-1) are capable of driving caveolae formation (128). In addition, analysis of the size distribution of caveolae in this setting revealed a mean of 80 ± 14.8 nm with 95% of caveolae being between 50–105 nm (Fig. 4). Furthermore, whole-mount electron microscopic evaluation of isolated recombinant caveolae revealed that both the - and -isoforms of caveolin-1 generate morphologically similar cup-shaped caveolae (Fig. 4). While the exact functional significance of these distinct isoforms remains unclear, studies have suggested that the caveolin-1 isoform is localized predominantly to deeply invaginated caveolae and can more efficiently drive the formation of caveolae than the -isoform (65, 217). Caveolin-2 has three identified isoforms, the full-length caveolin-2, and two truncated variants, termed caveolin-2 and -2
. The -isoform is thought to be an alternate splice variant, with a distinct subcellular distribution from full-length caveolin-2 (113). Little is known about the functional significance, if any, of the Cav-2 and -2 isoforms.
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38% identical and 58% similar to human caveolin-1, while caveolin-3 is 65% identical and 85% similar to caveolin-1. In addition, the caveolin proteins are highly conserved throughout evolution (Fig. 5). Moreover, a short stretch of eight amino acids has been identified (FEDVIAEP) that constitutes the "caveolin signature sequence," a motif that is identical between all three caveolin proteins.
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C. Caveolin Protein Characterization
As the prototypical caveolin family member, caveolin-1 has been the primary focus of many biochemical studies and provides the basis of our knowledge regarding the interaction of these proteins with their subcellular environment. Therefore, the attributes of caveolin-1 are considered here; however, it can be inferred that the majority of these characteristic are valid for the other caveolin family members. Caveolin-1 is a 22-kDa protein of 178 amino acids with adipocytes, endothelial cells, fibroblasts, and type I pneumocytes having the highest levels of expression. It is now known that caveolin-1 expression is sufficient and necessary to drive the formation of morphologically identifiable caveolae (128, 197). In addition, caveolin-1 expression is necessary for the stable expression and membrane localization of caveolin-2. Indeed, caveolin-2 alone is insufficient to induce caveolae biogenesis (199). The Cav-3 protein is muscle specific and, like Cav-1, is sufficient to drive the formation of caveolae (67).
1. Membrane topology and attachment
Initial studies into the subcellular localization of caveolin-1 revealed that it is an integral membrane protein, as it was found to be resistant to extraction with sodium carbonate and high salt concentrations (115, 203, 212). In addition, several lines of evidence have contributed to the generally accepted view that both the NH2 and COOH termini of caveolin-1 face the cytoplasm, with an intervening hydrophobic domain inserted into the membrane (Fig. 6). Preliminary findings indicative of this unusual topology were suggested by Dupree et al. (42) after antibodies directed against either the NH2- or COOH-terminal domains of caveolin-1 were unable to label the protein in the absence of cellular permeablization. Additional studies demonstrated that the COOH terminus of caveolin-1 is palmitoylated and the NH2 terminus is tyrosine phosphorylated, two posttranslational modifications that require both ends of the protein to remain cytoplasmic (39, 127). Furthermore, surface biotinylation studies failed to label caveolin-1, further supporting the idea that no portion of the caveolin-1 protein is extracellular (211). The membrane insertion of caveolin-1 occurs via the classical endoplasmic reticulum (ER) machinery, resulting in an unusually short transmembrane domain of 32 hydrophobic amino acids (residues 102–134) thought to form a unique hair-pin loop configuration that prevents caveolin-1 from completely spanning the plasma membrane in a traditional double-pass fashion (Fig. 6) (159).
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Caveolin-1 isoforms form high-molecular-mass oligomers of 400 kDa, as demonstrated using velocity gradient centrifugation (159, 211). Interestingly, deletion mutagenesis studies showed that caveolin-1 contains an oligomerization domain mapping to residues 61–101, which mediates the homo-oligomerization of 14–16 individual caveolin-1 molecules (Fig. 6) (211). These caveolin-1 oligomers are thought to undergo a second stage of oligomerization during transport from the trans-Golgi to plasma membrane caveolae, whereby several oligomers self-associate via COOH-terminal interactions, forming a large network of caveolin (243). Sargiacomo et al. (211) examined the ultrastructure of purified caveolin-1 homo-oligomers derived from rat lung tissue and found that these individual homo-oligomers appear as globular particles of 4–6 nm in the presence of the nonionic detergent octyl glucoside (Fig. 7). After removal of this detergent, caveolin-1 homo-oligomers associated into larger, nonlinear polymers of 25 nm in an apparent side-by-side fashion, further supporting the notion that this may be the mechanism by which individual homo-oligomers associate into larger multimeric caveolar complexes in vivo.
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Caveolin-3 is known to form high-molecular-mass homo-oligomeric complexes in situ, in a fashion similar to that of caveolin-1 (239, 256). In addition, while it has long been thought that caveolin-3 does not bind to caveolin-2, recent findings in isolated neonatal cardiac myocytes and smooth muscle cells suggest otherwise (206, 278). In these systems, it was found that caveolin-2 coimmunoprecipitates with caveolin-3 and cofractionates with caveolin-3 in sucrose density gradient centrifugation (206, 278).
3. The caveolin scaffolding domain
The caveolin-1 scaffolding domain (CSD) was originally defined by a series of experiments, including deletion mutagenensis of Cav-1-GST fusion proteins, which identified a region within caveolin-1 (residues 82–101) capable of mediating protein-protein interactions (33–35, 104, 124, 243). Using a GST-fusion protein containing the caveolin-1 scaffolding domain as a receptor to select peptide ligands from a bacteriophage display library, two related but distinct caveolin binding motifs (CBM) were identified in most proteins shown to interact with caveolin-1 (CBM; 
XXXXXX and XXXXX, where represents an aromatic amino acid) (33). The scaffolding domain has since been shown to serve a dual role, acting both as an anchor holding various proteins within caveolae as well as a regulatory element capable of either inhibiting or enhancing a given protein's signaling activity.
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IV. FUNCTIONAL ROLES OF CAVEOLAE/CAVEOLINS |
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Based purely on their ultrastructural appearance as plasma membrane invaginations, caveolae were originally thought to function as macromolecular transport vesicles. Initially, their proposed function was limited to the process of pinocytosis or "cellular drinking." However, with the advent of new tools to investigate their function, their role as vesicular transporters has expanded to include transcytosis and endocytosis.
The term transcytosis was first used by Simionescu et al. (233) to describe the movement of macromolecules from the luminal side of capillary endothelial cells to the interstitial space via membrane-bound vesicles. This early research involved ultrastructural quantification of probes, such as gold-labeled albumin and peroxidase-labeled small heme derivatives, in what appeared to be transendothelial channels formed by the coalescence of caveolae (74, 233). While these reports demonstrate an accumulation of labeled probes in both abluminal caveolae and the interstitial space, indicative of transcytosis, the possibility remained that these tracers were subject to paracellular transport (between cells). Furthermore, their presence in abluminal caveolae could also have been attributed to retrograde filling, i.e., paracellular transport followed by entry into caveolae.
It was not until two major advancements in the field, 1) the introduction of novel caveolae-specific tracer molecules and 2) the development of caveolin knockout mice, that the role of caveolae in transcytosis could be resolved. Recently, Schnitzer and co-workers (151) used a novel approach that employed antibodies generated against antigens present only on the luminal surface of caveolae. Using these antibodies, this group demonstrated that caveolae are capable of macromolecular transcytosis, as they found an 50-fold increase in the interstitial accumulation of caveolae-specific antibodies, compared with control antibodies (151). Because their specific probe accumulates in tissue, while a control probe of similar structure does not, these authors conclude that nonspecific pathways, such as paracellular transport, cannot account for the interstitial accumulation.
Measurements of transcytosis in caveolin-1 knockout mice, which lack detectable caveolae in cell types such as endothelial and epithelial cells, show no interstitial accumulation of gold-labeled albumin in knockout, but significant accumulation in wild-type mice (224, 225). Similarly, when incubated with radio-iodinated albumin, isolated aortic rings from caveolin knockout mice showed no uptake of this tracer, while aortic rings from wild-type animals showed temperature- and time-dependent albumin uptake (225). In contrast to these findings however, Drab et al. (40) found no change in the apparent transcytosis of albumin into the cerebrospinal fluid (CSF) of their independently generated Cav-1 null mice. Here, these authors assessed albumin transcytosis across endothelial cells by measuring the concentration of albumin in the CSF of wild-type and Cav-1 null mice using SDS-PAGE. These authors conclude that either 1) caveolae are not involved in endothelial cell transcytosis or 2) an unidentified compensatory mechanism exists. To further clarify this issue, Schubert et al. (225) examined the microvascular permeability of wild-type and Cav-1 null mice, finding that indeed Cav-1 null mice demonstrate significant increases in the extravascular deposition of radioiodinated albumin. Additional experiments led us to conclude that defective transcytosis of albumin is compensated for by increased paracellular transport between endothelial cells, a process mediated by increases in plasma nitric oxide levels. Thus with these studies it now seems clear that caveolae do indeed mediate transcytosis (i.e., transcellular transport) of specific macromolecules in endothelial cells.
While clathrin-mediated endocytosis stands as the prototypical pathway for the internalization of many extracellular substances, alternative mechanisms also exist, including those mediated by caveolae. Caveolar transport may overlap with those events mediated by coated pits, but caveolae may serve selective transport functions as well.
This alternative caveolar pathway was first recognized by the observation that cholera toxin was preferentially bound and internalized via caveolae (177). Additional observations suggesting a role for caveolae in the process of endocytosis include 1) the identification of several molecules involved in vesicle docking and fusion (i.e., SNARE proteins) shared by clathrin-coated pits (CCPs) and 2) the detection of the small GTPase dynamin, which is known to be involved in the internalization of CCPs, in the necks of caveolae following specific stimuli (91, 173, 222). Taken together, these data have helped solidify the idea that caveolae are indeed functional endocytic vesicles. Dynamin seems to play a homologous role in the internalization of both CCPs and caveolae, as overexpression of a dominant negative dynamin mutant inhibits endocytosis by both pathways (173). Although the exact process mediating caveolar endocytosis remains unknown, several recent findings suggest that the activation of tyrosine kinase-dependent signaling is an important step (190). Indeed, treating cells with phosphatase inhibitors increases endocytosis via caveolae. Similarly, ligands that are known to be internalized via caveolae, including several pathogenic agents, activate tyrosine kinase signaling pathways upon binding to their cellular receptors (21, 170, 171, 188, 192).
Perhaps the most well-documented infectious agent that selectively uses caveolae to enter cells is simian virus 40 (SV40). It has been shown that SV40 is internalized specifically by caveolae by several means, including that the overexpression of a dominant negative epidermal growth factor (EGF) receptor pathway mutant (Eps15) will selectively inhibit clathrin-mediated endocytosis without effects on SV40 internalization (192). Additionally, the overexpression of a dominant negative caveolin mutant, which inhibits caveolar-mediated endocytosis, resulted in little or no SV40 infection (191, 204). Following its initial binding, SV40 accumulates in what has now been termed a "caveosome," an endocytic vesicle containing caveolin-1 and devoid of markers of clathrin-mediated endocytosis (191). After accumulation, SV40 is delivered to the ER, completing a mechanism that bypasses the lysosomal compartment thereby preventing SV40 inactivation (171, 191). Although SV40 is the only virus shown unequivocally to enter cells via caveolae en route to the ER, this may prove prototypical for many viruses (170). It is unclear if SV40 is an infectious agent to humans or a mediator of disease, but its exploitation of caveolae to gain entry into cells and avoid degradation may prove a robust strategy co-opted by many pathogens. It should also be noted that viral endocytosis via caveolae may be a relatively slow process and that caveolae, on the whole, are thought to be relatively stable structures (190, 260); however, this remains a hotly debated topic (164).
A growing list of pathogens, including viruses, bacteria and their associated toxins, fungi, and even prions, can interact with caveolae membrane domains (Table 1). The intracellular trafficking of these agents via caveolae differs dramatically from the usual route of ligands internalized by clathrin-medited endocytosis. The use of caveolae for cellular entry allows the pathogen to avoid classical endosome-lysosome trafficking and, consequently, avoid such degradative compartments in the cell. The mechanism of pathogen binding usually involves the utilization of one or more of the glycolipid or GPI-anchored moieties that cluster within the caveolae of host cells. It appears that disparate pathogens can utilize common binding sites localized within caveolae (170, 231, 232). The numbers of currently known pathogens that can use caveolae for cellular invasion probably represents a small fraction that will ultimately be shown to use caveolae-mediated cell entry (Table 1). Interestingly, the Marburg and Ebola viruses, which result in lethal hemorrhagic fever, have been shown to utilize the folate receptor as a cofactor to gain entry into cells (20). Consequently, these pathogens may exploit caveolar potocytosis as a mechanism for cellular invasion. In the same auspice, strategies aimed to exploit the folate receptor for therapeutic gene transfer may prove useful for the treatment of human disease (37, 236). Importantly, as pathogens exploit caveolae as a common mechanism to gain entry into cells and avoid degradative membrane compartments, this may benefit pharmaceutical strategies aimed at reengineering common pathogen moieties to facilitate entry, translocation, and delivery of therapeutic agents.
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The interrelationship between cholesterol and caveolin has a wide and varied history. Rothberg et al. (203) first recognized the importance of cellular cholesterol balance on caveolar structure when they found that treating cells with cholesterol binding agents, such as filipin and nystatin, resulted in the flattening of morphologically identifiable caveolae and the disruption of the striated caveolin protein coat. Additionally, cholesterol regulates caveolin-1 expression at the level of transcription through two steroid regulatory binding elements in the caveolin-1 promoter. This results in decreased caveolin-1 mRNA levels during periods of cholesterol depletion, while cholesterol loading has the opposite effect (8, 58). Additionally, the oxidation of cholesterol into cholesterone by cholesterol oxidase functionally disrupts cellular cholesterol balance and results in the internalization of caveolin-1 from the plasma membrane with its translocation to the ER and the Golgi apparatus (237). Thus caveolin-1 expression and its intracellular distribution are clearly dependent upon cellular cholesterol levels.
In an attempt to determine the interrelationship between caveolin-1 and the cellular cholesterol transport machinery, Frank et al. (63) identified a direct role for cholesterol in the stabilization of caveolin-1. These authors found that the expression of endogenous caveolin-1 increased 18-fold when human embryonic kidney (HEK) 293 cells were transfected with the scavenger receptor B1 (SR-B1) cDNA, but not with a cDNA encoding CD36 (63). Because SR-B1, but not CD36, results in the accumulation of intracellular cholesterol, these authors postulated that cholesterol, not SR-B1 itself, was responsible for the increase in caveolin-1 expression. Indeed, when HEK 293 cells were loaded with cholesterol, caveolin-1 protein expression increased dramatically by Western blot analysis. In addition, the distribution of caveolin-1 expression was also affected, with the intracellular clustering of caveolin-1 in cholesterol-loaded COS-7 cells. Thus, in addition to positive transcriptional regulation, cholesterol also directly modulates caveolin-1 expression via stabilization of the protein itself.
This interrelationship between caveolin-1 and cholesterol is further complicated by recent evidence suggesting that caveolin-1 can regulate cholesterol levels by modulating its cellular influx and efflux. Caveolin-1 has been shown to transport newly synthesized cholesterol from the ER to membrane caveolae, where it is delivered to plasma high-density lipoproteins (HDL). Furthermore, while extracellular cholesterol primarily enters cells via clathrin-mediated endocytosis of low-density lipoproteins (LDL), a secondary pathway involving caveolae may mediate the influx of cholesterol from HDL particles by way of the scavenger receptor B1 (SR-B1), which has recently been shown to colocalize with caveolin-1 in caveolae (81). Therefore, caveolae may be the principal location whereby plasma membrane cholesterol is exchanged between HDL and the cell membrane.
Perhaps one of the most important realizations concerning caveolae and caveolins was that these elements play an important functional role in the modulation of cell signal transduction. For a more in-depth review of caveolae and caveolins in signal transduction, the reader is referred to References 117, 200, 219, 234. Lisanti and colleagues (131, 212) were the first to recognize this property when they made the surprising discovery that biochemically purified caveolae microdomains contained an abundance of signaling molecules, such as Src-like tyrosine kinases and heterotrimeric G proteins (131, 212). Such initial discoveries led these authors to propose that caveolin-1 may serve to compartmentalize certain signaling molecules within caveolae, thereby concentrating and localizing these elements within the cell with the prospect of rapidly and selectively modulating cell signaling events (130). Thus caveolae may serve as docking points for numerous cell surface receptors, which when activated by ligand binding are recruited to caveolae (Fig. 8). Here, activated receptors interact with their respective caveolae-associated signaling components, leading to their rapid and selective activation in the confines of a specific subcellular environment. This hypothesis is strengthened by studies showing that the GTPase activity of G protein -subunits could be suppressed by a peptide derived from the NH2 terminus of caveolin-1, the caveolin scaffolding domain, demonstrating the interdependence of these proteins for functional activity (126).
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Proteomic analysis of purified caveolae has identified a wide assortment of proteins that are localized to these structures. The first detailed proteomic analysis of caveolae was carried out in 1994. Based on their buoyancy and resistance to detergent solubilization, Lisanti et al. (131) used sucrose density ultracentrifugation to purify caveolae-rich membrane domains from murine lung tissue. This procedure allowed for the exclusion of 
98% of an integral plasma membrane protein marker, while retaining 85% of total caveolin and 55% of GPI-linked marker proteins. When these purified caveolae were analyzed by protein microsequencing and Western blotting, these authors provided the first glimpse into resident caveolar protein components, identifying a preponderance of cytoplasmically oriented signaling molecules (Src-like kinases and heterotrimeric G protein subunits), including the small GTPases Rap 1, Rap 2, and TC 21, and cytoskeletal elements, such as monomeric G-actin and myosin regulatory light chain (Fig. 9A). In addition, several transmembrane proteins were identified, including CD36 and RAGE, as well as an abundance of GPI-linked proteins (131). This initial proteomic analysis of caveolae allowed for the first large-scale characterization of caveolae-enriched protein constituents and provided the basis for many follow-up investigations into the functional significance that caveolar localization may confer upon these proteins. Importantly, extremely similar results were obtained during the proteomic analysis of purified adipocyte caveolae (Table 2).
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), and two novel 45-kDa proteins (now known as flotillin-1/2) (217). More recently, Sprenger et al. (247) explored the proteomic composition of endothelial cell caveolae isolated by two distinct methodologies. In their first approach, these authors coated the outer plasma membrane of cultured human endothelial cells with positively charged silica, allowing subfractionation of luminal membrane components. Homogenization of the silica-bound membranes in cold Triton X-100 allows for the release of caveolar vesicles, which can be subsequently purified by sucrose density gradient centrifugation. However, when the resultant fractions were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization (MALDI), it was found that this methodology fails to purify caveolae, as almost no known caveolar resident proteins were identified. Furthermore, it seems that this methodology provides for an enrichment of ER proteins, as these were the main resident proteins identified (247).
When these authors next used a procedure to isolate caveolae based on their resistance to extraction in cold Triton X-100 and their propensity to float in a sucrose density gradient, they were able to confirm the validity of this methodology, showing an abundance of known caveolar resident proteins, such as caveolin-1 and flotillin-1. Subsequent two-dimensional gel and MALDI analysis of these isolated microdomains provided insight into their protein components, identifying a host of proteins similar to those described above. In addition, these authors recognized, for the first time, the existence and caveolar localization of several proteins whose identity had only been postulated based on analysis of the human genome (247). These proteins include a stomatin-like protein (SLP-2), a glucose-regulated-like protein (SGRP58), as well as the X-linked retinitis pigmentosa 2 protein (XRP-2). Because at least one of these genes has been identified as causing human disease (XRP-2), its localization to caveolae provides a new avenue by which its molecular function may be addressed. Taken together, proteomic studies such as those described above provide powerful tools by which new caveolar protein components can be identified and may present a basis for the further implication of these structures in human disease.
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V. CAVEOLINS AND HUMAN DISEASE: KNOCKOUT MOUSE MODELS |
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Cav-1 null mice were generated and initially characterized by two independent groups simultaneously (40, 198). As reported by both groups, these mice have a complete absence of morphologically identifiable caveolae in all tissues and cell types that normally express caveolin-1 (i.e., endothelia and adipocytes), while retaining identifiable caveolae in tissues that normally express caveolin-3 (i.e., skeletal and cardiac myocytes). Indeed, the Cav-1 null mouse unequivocally demonstrates the essential role of caveolin-1 in caveolar biogenesis in nonmuscle cells. An interesting caveat of caveolin-1 ablation is a consequential loss of the caveolin-2 protein by 90%, in all caveolin-1-expressing tissues. This finding was not completely unexpected considering the known role of caveolin-1 in its hetero-oligomerization with caveolin-2, and the ability of caveolin-1 to recruit caveolin-2 from the Golgi to the plasma membrane (125, 160, 185). This reduction in caveolin-2 protein levels is the result of caveolin-2 destabilization and subsequent proteasomal degradation, rather than altered transcriptional regulation. Indeed, the treatment of mouse embryonic fibroblasts (MEFs) derived from Cav-1 null animals with the proteasomal inhibitor MG-132 is sufficient to rescue the caveolin-2 protein expression (197). These data were supported by the observation that, in untreated Cav-1 null MEFs, caveolin-2 remained trapped in the Golgi apparatus, apparently unable to transit to plasmalemmal caveolae in the absence of its molecular chaperone, caveolin-1. In MEFs treated with MG-132, caveolin-2 expression increased to levels near those in wild-type cells; however, the protein remained confined to the Golgi (197). Thus Cav-1 null mice are essentially caveolin-2 deficient as well, and this must be considered in the phenotypic characterization of Cav-1-deficient mice. In support of this notion, our group has shown that the restrictive lung phenotype initially described in Cav-1 null animals is actually due to a selective loss of caveolin-2, as determined through the generation and characterization of Cav-2 null mice (199).
The sections below detail the various phenotypes identified to date in Cav-1 null mice and their relation to human disease.
1. Cellular transformation and tumorigenesis
Caveolin-1 was initially identified as a major substrate for tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryonic fibroblasts, suggesting that it may be a target for inactivation during oncogenesis (76). Thus it has been proposed that caveolin-1 acts as a tumor suppressor protein, inhibiting the functional signaling activity of several protooncogenes and consequently disrupting the process of cellular transformation (48–51, 66, 70, 114, 123, 209).
Numerous follow-up studies designed to test this hypothesis have contributed a myriad of evidence suggesting that caveolin-1 may indeed possess tumor suppressor capabilities. For instance, caveolin-1 mRNA and protein expression are downregulated in NIH-3T3 cells transformed with several activated oncogenes, such as v-Abl, Bcr-Abl, and H-Ras (G12V) (48, 114). The ability of these transformed cells to grow in soft agar, a hallmark of cellular transformation, was found to inversely correlate with caveolin-1 protein levels. The reintroduction of caveolin-1 under the control of an inducible promoter was sufficient to inhibit the anchorage-independent growth of these cells, thus reverting their transformed phenotype (48). In addition, some studies have identified similar reductions in caveolin-1 protein and mRNA levels in breast cancer cell lines (47, 51, 60, 94, 123, 292).
In further support of a role of caveolin-1 as a tumor suppressor, it has been shown that targeted downregulation of caveolin-1 using a vector-based anti-sense approach resulted in the transformation of NIH-3T3 cells, enhanced their anchorage independent growth, and hyperactivated the Ras-p42/44 mitogen-activated protein (MAP) kinase cascade (70). When injected into nude mice, these NIH-3T3 cells expressing the caveolin-1 anti-sense construct were capable of forming large tumors, compared with matched NIH-3T3 cells lacking the caveolin-1 anti-sense vector.
Importantly, these results were reversible, as loss of the caveolin-1 anti-sense vector, and thus reexpression of caveolin-1, reverted the transformed phenotype. Although it is generally accepted that caveolin-1 is indeed targeted for downregulation during oncogenesis, the mechanisms required for this reduction in mRNA levels have, for the most part, remained enigmatic (52). The identification of c-Myc-repressive and p53-responsive elements in the caveolin-1 promoter may provide common targets by which various oncogenes regulate caveolin-1 gene expression (183, 196).
Genetic evidence supporting the role of caveolin-1 as a tumor suppressor has emerged from gene mapping studies, which revealed that the human CAV-1 gene maps to the long arm of human chromosome 7 (7q31.1). This region, the D7S522 locus, encompasses a known fragile site (FRA7G) and is often associated with loss of heterozygosity (LOH) in various cancers, including breast, prostate, ovarian, and renal carcinomas (105, 110, 289–291). The deletion of this region and its association with the pathogenesis of several different types of cancers lends credible support to the presence of a tumor suppressor gene within this genetic locus. While no genes have been directly mapped to the D7S522 locus, the closest genes to this region encode caveolin-2 and caveolin-1 (Fig. 10) (50, 51). The CAV-2 gene is found 67 kb downstream of the microsatellite repeat marker D7S522, while the CAV-1 gene is located 86 kb downstream. Furthermore, the CAV-1 promoter has been reported to be hypermethylated in several cancer cell lines, including those derived from breast and prostate, suggesting that transcriptional silencing may abrogate caveolin-1 expression in human cancers (36, 51). As others have not shown significant changes in the methylation status of the caveolin-1 gene in human tumor cell lines, a result which may reflect technical differences, the transcriptional downregulation of caveolin-1 by promoter silencing will require further clarification (99).
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With so much data indicating that caveolin-1 possesses important tumor suppressor properties, it was somewhat surprising that Cav-1 null mice do not form spontaneous tumors. However, the characterization of the proliferative capacity of cells derived from these mice did reveal their hyperproliferation. For instance, cultured primary mouse embryonic fibroblasts (MEFs) derived from Cav-1 null mice show a marked increase in growth rate, compared with matched control wild-type MEFs (197). In addition, the lungs of Cav-1 null animals were found to be hypercellular, indicative of altered cell cycle regulation. Although Cav-1 null mice do not show any increased incidence of spontaneous mammary tumor formation, they develop significant mammary epithelial cell hyperplasia as early as 6 wk of age, in the nulliparous state. In 9-mo-old Cav-1 null mice, this mammary epithelial cell hyperplasia is also accompanied by an increase in lobular development, with acini formation and fibrosis (121).
Because these results indicate that loss of caveolin-1 may be a contributing factor in oncogenesis, Capozza et al. (18) subjected the skin of Cav-1 null mice to the carcinogen 7,12-dimethylbenzanthracene (DMBA). Here, it was shown that repeated applications of this compound resulted in a 10-fold increase in tumor incidence, a 15-fold increase in tumor multiplicity, and a 35-fold increase in tumor area per mouse, compared with wild-type mice treated identically (18). In addition, before the formation of overt skin tumors, Cav-1 null mice showed severe epidermal hyperplasia, accompanied by increases in cyclin D1 expression and the hyperactivation of the p42/44 MAP kinase cascade. These findings suggest that loss of caveolin-1 sensitizes mouse keratinocytes to oncogenic transformation and that caveolin-1 does indeed function as a tumor suppressor gene.
Following a similar rationale, Cav-1 null mice were interbred with a tumor-prone transgenic model of breast cancer (MMTV-PyMT). MMTV-PyMT mice develop spontaneous mammary tumors by 8 wk of age involving the entire mammary (146). When crossed with MMTV-PyMT mice, Cav-1 null mice develop multifocal dysplastic mammary lesions at a much earlier age (3 wk) than MMTV-PyMT mice alone (274). At this early age, there is an approximately twofold increase in the number of lesions per mouse, as well as an approximately sixfold increase in the area occupied by these lesions in PyMT/Cav-1 (–/–) mice compared with PyMT/Cav-1 (+/+) mice. Furthermore, PyMT/Cav-1 (–/–) lesions were of a higher histological grade and contained many more papillary projections than those of PyMT/Cav-1 (+/+) mice. Western blot analysis of these dysplatic lesions revealed a dramatic elevation in cyclin D1 protein expression, thus providing a potential mechanism by which loss of caveolin-1 accelerates the appearance of these lesions.
Thus the loss of caveolin-1 alone appears insufficient to induce cell transformation in vivo, but loss of caveolin-1 potentiates this process when combined with a transforming agent (a carcinogen or tumor-prone genetic background) (18, 274). This is not surprising, as the process of cell transformation and the development of cancer in vivo is a multistep process that involves the selective and progressive loss of several tumor suppressors (such as p53, INK4a, and Rb), as well as the mutational activation or upregulation of certain key protooncogenes [Ras(G12V), c-Myc, and c-Neu/ErbB2]. Indeed, the etiology of most cancers does not reflect alterations in a single gene, but rather the functional loss or induction of a series of key regulatory proteins that, in combination, disrupts the normal regulation of the cell cycle and subsequently leads to uncontrolled cell growth (140).
A) BREAST CANCER. Fueled by evidence suggesting a role for caveolin-1 in tumorigenesis, many clinically oriented studies have been undertaken to examine the regulation of caveolin-1 in a variety of human cancers. Analysis of human breast cancer samples revealed that up to 16% of these cancers have a CAV-1 gene point mutation (P132L), with the majority of the mutations being found in invasive carcinomas (88). Following up on this finding, Lee et al. (121) examined the behavior of the Cav-1 (P132L) mutant in a mammary cell culture model and found that it is mislocalized, being retained in the Golgi apparatus. Furthermore, coexpression of the P132L mutant along with the wild-type caveolin-1 cDNA resulted in the retention of the wild-type caveolin-1 protein in the Golgi, indicating that the P132L mutant acts in a dominant negati
