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F Galbiati

Publications and source records attributed to F Galbiati.

At least 37 records · Page 2Linked to original sources
PubMed2000

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.

3T3 Cells↗
PubMed2000

The cyclin D1 gene is transcriptionally repressed by caveolin-1.

The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras, neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between -81 to -73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1.

Amino Acid Sequence↗
PubMed2000

IL-12 receptor regulation in IL-12-deficient BALB/c and C57BL/6 mice.

Immunization with protein antigen in complete Freund's adjuvant (CFA) induces Th1 cells in BALB/c and C57BL/6 (B6) mice. Pretreatment with the same protein in soluble form induces Th2 cells in BALB/c but not in B6 mice and inhibits Th1 cell development in both. We have previously shown that inhibition of Th1 in BALB/c mice correlates with the down-regulation of transcripts encoding the IL-12 receptor beta2 (IL-12Rbeta2) chain, which is required for IL-12 signaling and Th1 cell development. We now demonstrate that IL-12-deficient BALB/c mice, when primed with antigen in CFA, mount a Th2 instead of the Th1 response which develops in wild-type mice. Conversely, IL-12-deficient B6 mice fail to develop Th2 cells. Thus, a default Th2 development is induced by antigen priming in IL-12-deficient BALB/c but not B6 mice. IL-12Rbeta2 transcripts are still expressed in antigen-restimulated CD4(+) T cells from IL-12-deficient BALB/c and B6 mice and they are similarly reduced by pretreatment with soluble antigen, suggesting that intrinsic strain differences in IL-12R regulation do not account for the differential polarization to the Th2 pathway. IL-4 is not required for down-regulation of IL-12Rbeta2 transcripts and inhibition of Th1 development in mice pretreated with soluble protein, as shown by their reduction in IL-4-deficient BALB/c mice.

Animals↗
PubMed2000

Inhibition of Th1 development and treatment of chronic-relapsing experimental allergic encephalomyelitis by a non-hypercalcemic analogue of 1,25-dihydroxyvitamin D(3).

1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits production of IL-12, a cytokine involved in the development of Th1 cells and in the pathogenesis of Th1-mediated autoimmune diseases. Here, we show that 1,25(OH)(2)D(3) and a non-hypercalcemic analogue are selective and potent inhibitors of Th1 development in vitro and in vivo without inducing a deviation to the Th2 phenotype. Administration of 1,25(OH)(2)D(3) or its analogue prevents chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide 35 - 55 (MOG(35 - 55)) in Biozzi AB / H mice. The inhibition of EAE induction is associated with a profound reduction of MOG(35 - 55)-specific proliferation and Th1 cell development. Importantly, the non-hypercalcemic analogue also provides long-term protection from EAE relapses induced by immunization with spinal cord homogenate when administered for a short time at symptom onset or even after the first peak of disease. Neuropathological analysis shows a reduction of inflammatory infiltrates, demyelinated areas and axonal loss in brains and spinal cords of treated mice. These resuls indicate that inhibition of IL-12-dependent Th1 cell development is associated with effective treatment of CR-EAE and suggest the feasibility of an approach based on low molecular weight inhibitors of IL-12 production in the treatment of multiple sclerosis.

Animals↗
PubMed2000

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.

3T3 Cells↗
PubMed1999

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.

Caveolin 3↗
PubMed1999

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).

3T3 Cells↗
PubMed1999

Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. Retention of caveolin-2 at the level of the golgi complex.

Caveolins-1 and -2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and -2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.

Biological Transport↗
PubMed1999

Caspase-1 regulates the inflammatory process leading to autoimmune demyelination.

T cell-mediated inflammation is considered to play a key role in the pathogenic mechanisms sustaining multiple sclerosis (MS). Caspase-1, formerly designated IL-1beta-converting enzyme, is crucially involved in immune-mediated inflammation because of its pivotal role in regulating the cellular export of IL-1beta and IL-18. We studied the role of caspase-1 in experimental autoimmune encephalomyelitis (EAE), the animal model for MS. Caspase-1 is transcriptionally induced during EAE, and its levels correlate with the clinical course and transcription rate of proinflammatory cytokines such as TNF-alpha, IL-1beta, IFN-gamma, and IL-6. A reduction of EAE incidence and severity is observed in caspase-1-deficient mice, depending on the immunogenicity and on the amount of the encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide used. In caspase-1-deficient mice, reduced EAE incidence correlates with defective development of anti-MOG IFN-gamma-producing Th1 cells. Finally, pharmacological blockade of caspase-1 in Biozzi AB/H mice, immunized with spinal cord homogenate or MOG35-55 peptide, by the caspase-1-inhibitor Z-Val-Ala-dl -Asp-fluoromethylketone, significantly reduces EAE incidence in a preventive but not in a therapeutic protocol. These results indicate that caspase-1 plays an important role in the early stage of the immune-mediated inflammatory process leading to EAE, thus representing a possible therapeutic target in the acute phase of relapsing remitting MS.

Amino Acid Chloromethyl Ketones↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

3T3 Cells↗
PubMed1999

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.

Acylation↗
PubMed1999

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.

Alginates↗
PubMed1998

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Central nervous system delivery of interleukin 4 by a nonreplicative herpes simplex type 1 viral vector ameliorates autoimmune demyelination.

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.

Animals↗
PubMed1998

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.

3T3 Cells↗
PubMed1998

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.

Amino Acid Sequence↗