Daniela Volonté

Cell-type and Tissue-specific Expression of Caveolin-2 CAVEOLINS 1 AND 2 CO-LOCALIZE AND FORM A STABLE HETERO-OLIGOMERIC COMPLEX IN VIVO

Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21–24-kDa integral membrane protein, is a principal structural component of caveolae membranein vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been re-termed caveolin-1. Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.

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.

Cellular Stress Induces the Tyrosine Phosphorylation of Caveolin-1 (Tyr14) 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.

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.

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.

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.

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 (ΔTFT) 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 (ΔTFT 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 ∼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).

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 SKVLB1 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.

Mutational analysis of caveolin-induced vesicle formation Expression of caveolin-1 recruits caveolin-2 to caveolae membranes

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50–100 nm. Although recombinant expression of caveolin‐1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin‐caveolin interactions regulate the size of caveolae, as caveolin‐1 undergoes two stages of self‐oligomerization. To test this hypothesis directly, we have created two caveolin‐1 deletion mutants that lack regions of caveolin‐1 that are involved in directing the self‐assembly of caveolin‐1 oligomers. More specifically, Cav‐1 Δ61–100 lacks a region of the N‐terminal domain that directs the formation of high molecular mass caveolin‐1 homo‐oligomers, while Cav‐1 ΔC lacks a complete C‐terminal domain that is required to allow caveolin homo‐oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav‐1 Δ61–100 and Cav‐1 ΔC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500–1000 nm. In addition, caveolin‐induced vesicle formation appears to be isoform‐specific. Recombinant expression of caveolin‐2 under the same conditions failed to drive the formation of vesicles, while caveolin‐3 expression yielded caveolae‐sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin‐1 expression, but continue to express caveolin‐2, no morphologically distinguishable caveolae are observed. In addition, as caveolin‐2 alone exists mainly as a monomer or homo‐dimer, while caveolins 1 and 3 exist as high molecular mass homo‐oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae‐sized vesicles. In direct support of this notion, regulated induction of caveolin‐1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin‐2 to caveolae membranes. The ability of caveolin‐1 to recruit caveolin‐2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co‐expressed and interact with each other to form high molecular mass hetero‐oligomers containing both caveolins 1 and 2.

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.