Role Of Caveolin-1 Research Articles

A Possible Role for Caveolin as a Signaling Organizer in Olfactory Sensory Membranes

Fast kinetics and sensitivity of olfactory signaling raise the question of whether the participating proteins may be associated in supramolecular transduction complexes. We found evidence that caveolin proteins could play an important role in organizing signaling elements in olfactory sensory neurons. Western blot analysis indicated that caveolins are highly enriched in olfactory sensory membranes, where they co-localize in detergent-insoluble complexes with key components of the signaling pathways. Furthermore, the results of immunoprecipitation experiments suggest that G proteins and effector enzyme form preassembled subcellular complexes with caveolins. Since anti-caveolin antibodies and synthetic peptides derived from the scaffolding domains of caveolin-1 and caveolin-2 effectively attenuated second messenger responses in sensory cilia preparations in a characteristic manner, the data led to the suggestion that caveolins could mediate the assembly of signaling complexes within specialized membrane microdomains of olfactory sensory neurons.

Caveolin-1 Inhibits Epidermal Growth Factor-stimulated Lamellipod Extension and Cell Migration in Metastatic Mammary Adenocarcinoma Cells (MTLn3): TRANSFORMATION SUPPRESSOR EFFECTS OF ADENOVIRUS-MEDIATED GENE DELIVERY OF CAVEOLIN-1

Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.

Role of caveolin in hemodynamic force-mediated endothelial changes.

Caveolin has been shown to play an important role in signal transduction and nitric oxide synthase production. The purpose of this study was to investigate whether caveolin was tyrosine phosphorylated or activated by shear stress or cyclic strain in bovine aortic endothelial cells (BAECs). BAECs were subjected to an average of 10% strain at a rate of 60 cycles/min or a laminar shear stress of 10 dyn/cm(2) for up to 4 h. Immunoblotting with anticaveolin antibody was performed to assess activation of caveolin. Coimmunoprecipitation of anticaveolin antibody with anti-tyrosine phosphorylation antibody was performed to detect the tyrosine phosphorylation of caveolin. Neither cyclic strain nor shear stress at physiologic levels altered the level of caveolin protein. Tyrosine phosphorylation of caveolin could not be observed at any time under either cyclic strain or shear stress condition. Although hemodynamic forces alter nitric oxide synthase production and activate signal transduction, caveolin levels or activity is not altered in endothelial cells exposed to shear stress or cyclic strain.

The role of caveolin in class B scavenger receptor-BI mediated cholesterol uptake in the intestine

The role of caveolin in class B scavenger receptor-BI mediated cholesterol uptake in the intestine

Concentration of Caveolin-1 in the Cleavage Furrow as Revealed by Time-Lapse Analysis

Caveolin-1 is a major component of caveolae. Recent studies have suggested a possible role of caveolin-1 in cell transformation and normal cell proliferation. To observe the behavior of caveolin-1 in living mitotic cells, we prepared cDNA constructs encoding the chimeric protein of α- or β-caveolin-1 and green fluorescent protein (GFP) and transfected culture cells with them. Correct targeting of the chimera to the caveolae was confirmed by colocalization with the caveolar inositol 1,4,5-trisphosphate receptor-like protein. By time-lapse observation of mitotic MDCKII cells, the GFP–caveolin-1 chimeras were seen throughout the plasma membrane before cell division, but became markedly concentrated at the cleavage furrow during cytokinesis. Accumulation around the spindle poles was also observed at late telophase. The result showed that caveolin-1 undergoes a drastic distributional change during cell division and suggested that the protein may be involved in the cytokinetic process.

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling.

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.

Intracellular cholesterol transport in synchronized human skin fibroblasts.

Normal human skin fibroblasts maintained in serum-containing medium were synchronized with aphidicolin. After removal of inhibitor, free cholesterol (FC) homeostasis was determined at intervals during the following cell cycle. FC mass per cell doubled following S-phase, and reached its maximum well before mitosis. This increase was mainly the result of stimulation of the rate of selective uptake of FC from medium lipoproteins, and reduction of FC efflux. Rates of cholesterol synthesis, endocytosis of intact low-density lipoprotein, and HDL receptor (CLA-1) activity were relatively low and little changed during the cell cycle. The expression of caveolin (structural protein of cell surface caveolae) and caveolar FC were decreased along with FC efflux. To test the hypothesis that regulation of caveolin expression could contribute to changes in FC efflux during cell division, cells were transfected with human caveolin cDNA, synchronized with aphidicolin, and then allowed to divide. In the transfected cells, caveolar FC and FC efflux were both increased. FC accumulation and entry into mitosis were markedly inhibited compared to controls. The contribution of transcriptional regulation to caveolin mRNA levels was determined with a 705 bp caveolin 5'-flanking sequence ligated to the pGL3 luciferase expression vector. Expression of the reporter gene was downregulated at S-phase of synchronized cells. Deletion of a hybrid E2F/ Sp1-like site between -139 and -150 bp abolished this downregulation. These data are consistent with a role for caveolin in cell cycle kinetics, which may be mediated, at least in part, at the transcriptional level.

Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer.

Metastasis represents the ultimate target in cancer therapy as this complex biological process is the direct cause of mortality for a variety of human malignancies. The current high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. Currently, there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. In using this system a number of metastasis-related sequences were identified including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin-1, androgen-insensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression.

Caveolin Is an Activator of Insulin Receptor Signaling

Recent data have demonstrated that caveolin, a major structural protein of caveolae, negatively regulates signaling molecules localized to caveolae. The interaction of caveolin with several caveolae-associated signaling proteins is mediated by the binding of the scaffolding region of caveolin to a hydrophobic amino acid-containing region within the regulated proteins. The presence of a similar motif within the insulin receptor kinase prompted us to investigate the caveolar localization and regulation of the insulin receptor by caveolin. We found that overexpression of caveolin-3 augmented insulin-stimulated phosphorylation of insulin receptor substrate-1 in 293T cells but not the phosphorylation of insulin receptor. Peptides corresponding to the scaffolding domain of caveolin potently stimulated insulin receptor kinase activity toward insulin receptor substrate-1 or a Src-derived peptide in vitro and in a caveolin subtype-dependent fashion. Peptides from caveolin-2 exhibited no effect, whereas caveolin-1 and -3 stimulated activity 10- and 17-fold, respectively. Peptides which increased insulin receptor kinase activity did so without affecting insulin receptor auto-phosphorylation. Furthermore, the insulin receptor bound to immobilized caveolin peptides, and this binding was inhibited in the presence of free caveolin-3 peptides. Thus, we have identified a novel mechanism by which the insulin receptor is bound and activated by specific caveolin subtypes. Furthermore, these data define a new role for caveolin as an activator of signaling.

Specificity of Receptor–G Protein Coupling: Protein Structure and Cellular Determinants

G protein-coupled receptors are involved in a tremendous range of signaling processes in the nervous system. The outlines of a molecular basis for specificity of receptor–G protein coupling has been established, but important details remain unclear. The second and third intracellular loops of most G protein-coupled receptors and the carboxyl terminus of the G protein α subunit have the most clearly established roles in specificity of coupling. The role of other regions of receptor and G protein, especially the Gβγ subunit, is an area needing more investigation. A discrepancy between specificity observedin vitroand in intact cells suggests a role for targeting and compartmentation of signaling components to account for the striking specificity observed in intact cells. The role of caveolin and PDZ domain-containing proteins will be of significant interest.

Metastasis-related genes in prostate cancer: the role of caveolin-1.

Prostate cancer continues to be a significant cause of death in U.S. men despite screening of asymptomatic men and extensive treatment with potentially curative therapies, predominantly radical prostatectomy and irradiation therapy. The reason for the persistent mortality resides in part in the presence of occult metastases at the time of treatment. Currently there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to compare and isolate mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. This strategy has proven to be successful and multiple gene sequences associated with metastasis in this model are being investigated. One of the genes isolated by this method was caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also human metastatic disease. Recent studies have indicated that suppression of caveolin expression induces androgen sensitivity in high-caveolin, androgen-insensitive mouse prostate cancer cells derived from metastases. Overexpression of caveolin leads to androgen insensitivity in low-caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is a metastasis-related gene and a candidate gene for hormone-resistant prostate cancer in man.

A Role for Caveolin in Transport of Cholesterol from Endoplasmic Reticulum to Plasma Membrane

Caveolin is a 22-kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half-time of approximately 10 min. The cholesterol then rapidly flowed from caveolae to non-caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 microg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting cholesterol transport is linked to caveolin movement. Caveolae fractions from cells expressing caveolin were enriched in cholesterol 3-4-fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.