Identification of caveolae and caveolin in C6 glioma cells

Silva, W.I; Maldonado, Héctor; Lisanti, Michael P.; deVellis, Jean; Chompré, Gladys; Mayol, Noel; Ortiz, M; Velázquez, G.; Maldonado, Amanda C; Montalvo, J

doi:10.1016/s0736-5748(99)00040-4

Cited by 37 publications

(44 citation statements)

References 32 publications

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“…(20) contrasting with other studies, including our own, showing that human ASM expresses only caveolin-1 and -2 (41, 46) (with the understanding that caveolin-2 is usually coexpressed with caveolin-1). In airway epithelium, there is currently very little data on the role of caveolins, although both caveolin-1 and -2 have been detected by immunocytochemical and imaging techniques (4,51). These previous data formed the justification for examination of caveolin-1 in this study.…”

Section: Discussionmentioning

confidence: 80%

Caveolin-1 knockout mice exhibit airway hyperreactivity

Aravamudan

VanOosten

Meuchel

et al. 2012

American Journal of Physiology-Lung Cellular and Molecular Phys

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Although caveolins have been found in endothelium and epithelium (where they regulate nitric oxide synthase activity), their role in smooth muscle is still under investigation. We and others have previously shown that caveolae of human airway smooth muscle (ASM), which express caveolin-1, contain Ca 2ϩ and force regulatory proteins and are involved in mediating the effects of inflammatory cytokines such as TNF-␣ on intracellular Ca 2ϩ concentration responses to agonist. Accordingly, we tested the hypothesis that in vivo, absence of caveolin-1 leads to reduced airway hyperresponsiveness, using a knockout (KO) (Cav1 KO) mouse and an ovalbumin-sensitized/challenged (OVA) model of allergic airway hyperresponsiveness. Surprisingly, airway responsiveness to methacholine, tested by use of a FlexiVent system, was increased in Cav1 KO control (CTL) as well as KO OVA mice, which could not be explained by a blunted immune response to OVA. In ASM of wild-type (WT) OVA mice, expression of caveolin-1, the caveolar adapter proteins cavins 1-3, and caveolae-associated Ca 2ϩ and force regulatory proteins such as Orai1 and RhoA were all increased, effects absent in Cav1 KO CTL and OVA mice. However, as with WT OVA, both CTL and OVA Cav1 KO airways showed signs of enhanced remodeling, with high expression of proliferation markers and increased collagen. Separately, epithelial cells from airways of all three groups displayed lower endothelial but higher inducible nitric oxide synthase and arginase expression. Arginase activity was also increased in these three groups, and the inhibitor nor-NOHA (N-omega-nor-L-arginine) enhanced sensitivity of isolated tracheal rings to ACh, especially in Cav1 KO mice. On the basis of these data disproving our original hypothesis, we conclude that caveolin-1 has complex effects on ASM vs. epithelium, resulting in airway hyperreactivity in vivo mediated by altered airway remodeling and bronchodilation. Overall, in vitro data clearly demonstrate an important role for caveolin-1 in enhanced airway contractility relevant to asthma. However, the effect of altered caveolin-1 expression or function in vivo has not been examined and was the focus of this study. Based on previous work, our hypothesis was that reduced caveolin-1 results in airway hyporesponsiveness. To this end we employed the mouse lacking caveolin-1 (Cav1 KO) as a model, with ovalbumin (OVA) sensitization and challenge as a model of allergic airway hyperresponsiveness to test our hypothesis. Cav1 KO mice are known to develop pulmonary fibrosis (9,15,60), and a single study showed that these mice have reduced lung compliance and increased elastance by 3 mo of age (28), with evidence of progressively greater deposition of collagen within airways and parenchyma. Whether such changes are associated with altered airway contractility and the specific role of ASM per se have not been examined. In the present study, we compared airway structure and function in wild-type (WT) vs. Cav1 KO mice that were either unsensitized [control (CTL)] or sensitiz...

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Section: Discussionmentioning

confidence: 80%

Caveolin-1 knockout mice exhibit airway hyperreactivity

Aravamudan

VanOosten

Meuchel

et al. 2012

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Because specialized plasma membrane domains such as lamellipodia and caveolae dictate some of the characteristics of glioma invasion (45,46), the identification and functional characterization of specific molecular players located within these domains should provide further insight into their role in interacting with ECM proteins. For instance, it has been hypothesized that lipids or accessory proteins as- sociated within such lipidic bilayer may significantly modulate the functional activity of CD44 (47,48).…”

Section: Discussionmentioning

confidence: 99%

Hyaluronan Cell Surface Binding Is Induced by Type I Collagen and Regulated by Caveolae in Glioma Cells

Annabi

Thibeault

Moumdjian

et al. 2004

Journal of Biological Chemistry

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Hyaluronan (HA) is a component of the brain extracellular matrix environment that is synthesized and secreted by glioma cells. The primary cell surface receptor for HA is CD44, a membrane glycoprotein that is functionally regulated by a membrane type 1 matrix metalloproteinase (MT1-MMP). Both CD44 and MT1-MMP are partially located in Triton X-100-insoluble domains, but no functional link has yet been established between them. In the present study, we studied the regulation of HA cell surface binding in U-87 glioma cells. We show that an MMP-dependent mechanism regulates the intrinsic cell surface binding of HA as ilomastat, a broad MMP inhibitor, increased HA binding to glioma cells. HA binding was also rapidly and specifically up-regulated by 3-fold by type I collagen in U-87 cells, which also induced a significant morphological reorganization associated with the activation of a latent form of MMP-2 through a MT1-MMP-mediated mechanism. Interestingly, caveolae depletion with a cell surface cholesteroldepleting agent ␤-cyclodextrin triggered an additional increase (9-fold) in the binding of HA, in synergy with type I collagen. On the other hand, HA cell surface binding was diminished by the MEK inhibitor PD98059 and by the overexpression of a recombinant, wild type MT1-MMP, whereas its cytoplasmic-deleted form had no effect. Taken together, our results suggest that MT1-MMP regulates, through its cytoplasmic domain, the cell surface functions of CD44 in a collagen-rich pericellular environment. Additionally, we describe a new molecular mechanism regulating the invasive potential of glioma cells involving a MT1-MMP/CD44/caveolin interaction, which could represent a potential target for anti-cancer therapies.The principal molecules that have been identified in the normal brain extracellular matrix (ECM) 1 are hyaluronan (HA; also known as hyaluronic acid, hyaluronate) and chondroitin sulfate (1, 2). HA is an important glycosaminoglycan constituent believed to be implicated in angiogenesis, the formation of new blood vessels from preexisting vasculature. Although the serum level of HA is already used as an indicator of progressive malignant disease (3), its effects on in vivo angiogenesis and endothelial cell (EC) function are complex and have been reported to depend on HA concentration and molecular size (4, 5). Accordingly, whereas high molecular weight HA was shown to inhibit EC functions (6), low molecular weight HA stimulated EC proliferation, tubulogenesis (6, 7), and neovascularization (8). Moreover, small HA polymers efficiently regulated CD44 cell surface functional expression and promoted tumor cell migration (9). Astrocytic tumors of the central nervous system express CD44 among other cell adhesion receptors of the integrin or immunoglobulin superfamily. Although HA is the principal ligand of CD44, other CD44 ligands include the ECM components collagen, fibronectin, laminin, and chondroitin sulfate, whereas mucosal addressin, serglycin, osteopontin, and the class II invariant chain represent ECM-unrelat...

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“…Lipid rafts were isolated by discontinuous sucrose gradient centrifugation based on the method of Silva et al (1999). In brief, cells from a 100-mm dish were washed twice in ice-cold phosphate-buffered saline, scraped into 1 ml of ice-cold MBS (25 mM MES and 150 mM NaCl) containing 1% Triton X-100, 4 g each leupeptin, antipain, and aprotinin, and 0.5 mM benzamidine, and homogenized with 10 strokes in a hand-held Dounce homogenizer.…”

Section: Biochemistrymentioning

confidence: 99%

Receptor-Mediated Suppression of Potassium Currents Requires Colocalization within Lipid Rafts

Oldfield

Hancock²,

Mason³

et al. 2009

Mol Pharmacol

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Expression of KCNQ2/3 (Kv7.2 and -7.3) heteromers underlies the neuronal M current, a current that is suppressed by activation of a variety of receptors that couple to the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Expression of Kv7.2/ 7.3 channels in human embryonic kidney (HEK) 293 cells produced a noninactivating potassium current characteristic of M current. Muscarinic receptors endogenous to HEK293 cells were identified as being M3 by pharmacology and Western blotting, producing a rise of intracellular calcium ([Ca 2ϩ ] i ) upon activation. Activation of these endogenous muscarinic receptors however, failed to suppress expressed Kv7.2/7.3 current. Current suppression was reconstituted by coexpression of HAtagged muscarinic m1 or m3 receptors. Examination of membrane fractions showed that both expressed receptors and Kv7.2 and -7.3 channel subunits resided within lipid rafts. Disruption of lipid rafts by pretreatment of cells expressing either m1 or m3 muscarinic receptors with methyl-␤-cyclodextrin produced a loss of localization of proteins within lipid raft membrane fractions. This pretreatment also abolished both the increase of [Ca 2ϩ ] i and suppression of expressed Kv7.2/7.3 current evoked by activation of expressed m1 or m3 muscarinic receptors. A similar loss of muscarinic receptor-mediated suppression of M current native to rat dorsal root ganglion neurons was observed after incubating dissociated cells with methyl-␤-cyclodextrin. These data suggested that lipid rafts colocalized both muscarinic receptors and channel subunits to enable receptor-mediated suppression of channel activity, a spatial colocalization that enables specificity of coupling between receptor and ion channel.

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Identification of caveolae and caveolin in C6 glioma cells

Cited by 37 publications

References 32 publications

Caveolin-1 knockout mice exhibit airway hyperreactivity

Caveolin-1 knockout mice exhibit airway hyperreactivity

Hyaluronan Cell Surface Binding Is Induced by Type I Collagen and Regulated by Caveolae in Glioma Cells

Receptor-Mediated Suppression of Potassium Currents Requires Colocalization within Lipid Rafts

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