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Michael P. Lisanti - One of the best experts on this subject based on the ideXlab platform.
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tyrosine phosphorylation of Caveolin 2 at residue 27 differences in the spatial and temporal behavior of phospho cav 2 py19 and py27
Biochemistry, 2004Co-Authors: Xiao Bo Wang, Shana Marmon, James W Brooks, Roberto Camposgonzalez, Franco Capozza, Federica Sotgia, Michael P. LisantiAbstract:Caveolin-2 is an accessory molecule and the binding partner of Caveolin-1. Previously, we showed that c-Src expression leads to the tyrosine phosphorylation of Cav-2 at position 19. To further investigate the tyrosine phosphorylation of Cav-2, we have now generated a novel phospho-specific antibody directed against phospho-Cav-2 (pY27). Here, we show that Cav-2 is phosphorylated at both tyrosines 19 and 27. We reconstituted this phosphorylation event by recombinantly coexpressing c-Src and Cav-2. We generated a series of Cav-2 constructs harboring the mutation of each tyrosine to alanine, singly or in combination, i.e., Cav-2 Y19A, Y27A, and Y19A/Y27A. Recombinant expression of these mutants in Cos-7 cells demonstrated that neither tyrosine is the unique phosphorylation site, and that double mutation of tyrosines 19 and 27 to alanine abrogates Cav-2 tyrosine phosphorylation. Immunofluorescence analysis of NIH 3T3 cells revealed that the two tyrosine-phosphorylated forms of Cav-2 exhibited some distinct pr...
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The Caveolin proteins
Genome Biology, 2004Co-Authors: Terence M Williams, Michael P. LisantiAbstract:The Caveolin gene family has three members in vertebrates: Caveolin-1, Caveolin-2, and Caveolin-3. So far, most Caveolin-related research has been conducted in mammals, but the proteins have also been found in other animals, including Xenopus laevis, Fugu rubripes , and Caenorhabditis elegans . Caveolins can serve as protein markers of caveolae ('little caves'), invaginations in the plasma membrane 50-100 nanometers in diameter. Caveolins are found predominantly at the plasma membrane but also in the Golgi, the endoplasmic reticulum, in vesicles, and at cytosolic locations. They are expressed ubiquitously in mammals, but their expression levels vary considerably between tissues. The highest levels of Caveolin-1 (also called Caveolin, Cav-1 and VIP2I) are found in terminally-differentiated cell types, such as adipocytes, endothelia, smooth muscle cells, and type I pneumocytes. Caveolin-2 (Cav-2) is colocalized and coexpressed with Cav-1 and requires Cav-1 for proper membrane targeting; the Cav-2 gene also maps to the same chromosomal region as Cav-1 (7q31.1 in humans). Caveolin-3 (Cav-3) has greater protein-sequence similarity to Cav-1 than to Cav-2, but it is expressed mainly in muscle cells, including smooth, skeletal, and cardiac myocytes. Caveolins participate in many important cellular processes, including vesicular transport, cholesterol homeostasis, signal transduction, and tumor suppression.
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Caveolin 2 localizes to the golgi complex but redistributes to plasma membrane caveolae and rafts when co expressed with Caveolin 1
Journal of Biological Chemistry, 1999Co-Authors: Rosalia Mora, Michael P. Lisanti, Philipp E. Scherer, Vera L Bonilha, Alan D Marmorstein, Dennis Brown, Enrique RodriguezboulanAbstract:We have characterized comparatively the subcellular distributions of Caveolins-1 and -2, their interactions and their roles in caveolar formation in polarized epithelial cells. In Fischer rat thyroid (FRT) cells, which express low levels of Caveolin-2 and no Caveolin-1, Caveolin-2 localizes exclusively to the Golgi complex but is partially redistributed to the plasma membrane upon co-expression of Caveolin-1 by transfection or by adenovirus-mediated transduction. In Madin-Darby canine kidney (MDCK) cells, which constitutively express both Caveolin-1 and -2, Caveolin-2 localized to both the Golgi complex and to the plasma membrane, where it co-distributed with Caveolin-1 in flat patches and in caveolae. In FRT cells, endogenous or overexpressed Caveolin-2 did not associate with low density Triton insoluble membranes that floated in sucrose density gradients but was recruited to these membranes when co-expressed together with Caveolin-1. In MDCK cells, both Caveolin-1 and Caveolin-2 associated with low density Triton-insoluble membranes. In FRT cells, transfection of Caveolin-1 promoted the assembly of plasma membrane caveolae that localized preferentially (over 99%) to the basolateral surface, like constitutive caveolae of MDCK cells. In contrast, as expected from its intracellular distribution, endogenous or overexpressed Caveolin-2 did not promote the assembly of caveolae; rather, it appeared to promote the assembly of intracellular vesicles in the peri-Golgi area. The data reported here demonstrate that Caveolin-1 and -2 have different and complementary subcellular localizations and functional properties in polarized epithelial cells and suggest that the two proteins co-operate to carry out specific as yet unknown tasks between the Golgi complex and the cell surface.
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angiogenesis activators and inhibitors differentially regulate Caveolin 1 expression and caveolae formation in vascular endothelial cells angiogenesis inhibitors block vascular endothelial growth factor induced down regulation of Caveolin 1
Journal of Biological Chemistry, 1999Co-Authors: Jun Liu, Babak Razani, Shaoqing Tang, Bruce I Terman, Anthony J Ware, Michael P. LisantiAbstract:Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro andin vivo. Here, we examine the potential role of Caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of Caveolin-1. This down-regulation event was selective for Caveolin-1, as Caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of Caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-β, and thalidomide) effectively blocked VEGF-induced down-regulation of Caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of Caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of Caveolin-1. Furthermore, we show that Caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of Caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.
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sequence and detailed organization of the human Caveolin 1 and 2 genes located near the d7s522 locus 7q31 1 methylation of a cpg island in the 5 promoter region of the Caveolin 1 gene in human breast cancer cell lines
FEBS Letters, 1999Co-Authors: Jeffrey A Engelman, Xiao Lan Zhang, Michael P. LisantiAbstract:The CA microsatellite repeat marker, D7S522, is located at the center of a ∼1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (∼100–200 kb) containing D7S522 and the human genes encoding Caveolins 1 and 2. Here, we present the detailed organization of the Caveolin locus and its relationship to D7S522, as deduced using a shot-gun sequencing approach. We derived two adjacent contigs for a total coverage of ∼250 kb. Analysis of these contigs reveals that D7S522 is located ∼67 kb upstream of the Caveolin-2 gene and that the Caveolin-2 gene is located ∼19 kb upstream of the Caveolin-1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the Caveolin genes; these include the intron-exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the Caveolin genes. The first and second exons of both Caveolin genes are embedded within CpG islands. These results suggest that regulation of Caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5′ promoter region of the Caveolin-1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T-47D) that fail to express the Caveolin-1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non-methylated and these cells express high levels of the Caveolin-1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the Caveolin-1/-2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the Caveolin genes in a variety of human tumors.
Philipp E. Scherer - One of the best experts on this subject based on the ideXlab platform.
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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
Journal of Biological Chemistry, 1999Co-Authors: Isabella Parolini, Philipp E. Scherer, Jeffrey A Engelman, Takashi Okamoto, Tsuneya Ikezu, Massimo Sargiacomo, Giovanni Rizzo, Francesco Grignani, Rosalia MoraAbstract:Abstract 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.
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Caveolin 2 localizes to the golgi complex but redistributes to plasma membrane caveolae and rafts when co expressed with Caveolin 1
Journal of Biological Chemistry, 1999Co-Authors: Rosalia Mora, Michael P. Lisanti, Philipp E. Scherer, Vera L Bonilha, Alan D Marmorstein, Dennis Brown, Enrique RodriguezboulanAbstract:We have characterized comparatively the subcellular distributions of Caveolins-1 and -2, their interactions and their roles in caveolar formation in polarized epithelial cells. In Fischer rat thyroid (FRT) cells, which express low levels of Caveolin-2 and no Caveolin-1, Caveolin-2 localizes exclusively to the Golgi complex but is partially redistributed to the plasma membrane upon co-expression of Caveolin-1 by transfection or by adenovirus-mediated transduction. In Madin-Darby canine kidney (MDCK) cells, which constitutively express both Caveolin-1 and -2, Caveolin-2 localized to both the Golgi complex and to the plasma membrane, where it co-distributed with Caveolin-1 in flat patches and in caveolae. In FRT cells, endogenous or overexpressed Caveolin-2 did not associate with low density Triton insoluble membranes that floated in sucrose density gradients but was recruited to these membranes when co-expressed together with Caveolin-1. In MDCK cells, both Caveolin-1 and Caveolin-2 associated with low density Triton-insoluble membranes. In FRT cells, transfection of Caveolin-1 promoted the assembly of plasma membrane caveolae that localized preferentially (over 99%) to the basolateral surface, like constitutive caveolae of MDCK cells. In contrast, as expected from its intracellular distribution, endogenous or overexpressed Caveolin-2 did not promote the assembly of caveolae; rather, it appeared to promote the assembly of intracellular vesicles in the peri-Golgi area. The data reported here demonstrate that Caveolin-1 and -2 have different and complementary subcellular localizations and functional properties in polarized epithelial cells and suggest that the two proteins co-operate to carry out specific as yet unknown tasks between the Golgi complex and the cell surface.
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expression of Caveolin 1 and 2 in differentiating pc12 cells and dorsal root ganglion neurons Caveolin 2 is up regulated in response to cell injury
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Daniela Volonte, Philipp E. Scherer, Jeffrey A Engelman, Orlando Gil, George Zanazzi, James L Salzer, Massimo Sargiacomo, Amnon Schlegel, Marco ParentiAbstract:Caveolae are cholesterol/sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas Caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no Caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether Caveolins are expressed within neuronal cells. Here we demonstrate the expression of Caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, Caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas Caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, Caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of Caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of Caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express Caveolins.
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cell type and tissue specific expression of Caveolin 2 Caveolins 1 and 2 co localize and form a stable hetero oligomeric complex in vivo
Journal of Biological Chemistry, 1997Co-Authors: Philipp E. Scherer, Daniela Volonte, Jeffrey A Engelman, Stave D Kohtz, Renee Y Lewis, Jacques Couet, Elly Van Donselaar, Peter J Peters, Michael P. LisantiAbstract: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.
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molecular cloning of Caveolin 3 a novel member of the Caveolin gene family expressed predominantly in muscle
Journal of Biological Chemistry, 1996Co-Authors: Zhaolan Tang, Philipp E. Scherer, Kenneth S Song, Takashi Okamoto, Stave D Kohtz, Caryn Chu, Ikuo Nishimoto, Harvey F Lodish, Michael P. LisantiAbstract:Abstract Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second Caveolin gene has been identified and termed Caveolin-2. Here, we report the molecular cloning and expression of a third member of the Caveolin gene family, Caveolin-3. Caveolin-3 is most closely related to Caveolin-1 based on protein sequence homology; Caveolin-1 and Caveolin-3 are 65% identical and 85% similar. A single stretch of eight amino acids (FEDVIAEP) is identical in Caveolin-1, −2, and −3. This conserved region may represent a “Caveolin signature sequence” that is characteristic of members of the Caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, Caveolin-3 is similar to Caveolin-1: (i) Caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) Caveolin-3 colocalizes with Caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a Caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the Caveolin gene family may have implications for understanding the role of Caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown Caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.
Paul A Insel - One of the best experts on this subject based on the ideXlab platform.
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microtubules and actin microfilaments regulate lipid raft caveolae localization of adenylyl cyclase signaling components
Journal of Biological Chemistry, 2006Co-Authors: Brian P Head, Hemal H Patel, David M Roth, Fiona Murray, James S Swaney, Ingrid R Niesman, Marilyn G Farquhar, Paul A InselAbstract:Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of Caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of Caveolin-1, Caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated Caveolin-1 in Caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of Caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of Caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of Caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.
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microtubules and actin microfilaments regulate lipid raft caveolae localization of adenylyl cyclase signaling components
Journal of Biological Chemistry, 2006Co-Authors: Brian P Head, Hemal H Patel, David M Roth, Fiona Murray, James S Swaney, Ingrid R Niesman, Marilyn G Farquhar, Paul A InselAbstract:Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of Caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of Caveolin-1, Caveolin-2, β1-adrenergic receptors (β1-AR), β2-AR, Gαs, and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated Caveolin-1 in Caveolin-1 immunoprecipitates but in parallel increased isoproterenol (β-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of Caveolin-3 and α-tubulin; both Colch and CD decreased co-localization of Caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of Caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/μm of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Gαs from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-Gs-AC in lipid rafts/caveolae.
Jeffrey A Engelman - One of the best experts on this subject based on the ideXlab platform.
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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
Journal of Biological Chemistry, 1999Co-Authors: Isabella Parolini, Philipp E. Scherer, Jeffrey A Engelman, Takashi Okamoto, Tsuneya Ikezu, Massimo Sargiacomo, Giovanni Rizzo, Francesco Grignani, Rosalia MoraAbstract:Abstract 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.
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sequence and detailed organization of the human Caveolin 1 and 2 genes located near the d7s522 locus 7q31 1 methylation of a cpg island in the 5 promoter region of the Caveolin 1 gene in human breast cancer cell lines
FEBS Letters, 1999Co-Authors: Jeffrey A Engelman, Xiao Lan Zhang, Michael P. LisantiAbstract:The CA microsatellite repeat marker, D7S522, is located at the center of a ∼1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (∼100–200 kb) containing D7S522 and the human genes encoding Caveolins 1 and 2. Here, we present the detailed organization of the Caveolin locus and its relationship to D7S522, as deduced using a shot-gun sequencing approach. We derived two adjacent contigs for a total coverage of ∼250 kb. Analysis of these contigs reveals that D7S522 is located ∼67 kb upstream of the Caveolin-2 gene and that the Caveolin-2 gene is located ∼19 kb upstream of the Caveolin-1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the Caveolin genes; these include the intron-exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the Caveolin genes. The first and second exons of both Caveolin genes are embedded within CpG islands. These results suggest that regulation of Caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5′ promoter region of the Caveolin-1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T-47D) that fail to express the Caveolin-1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non-methylated and these cells express high levels of the Caveolin-1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the Caveolin-1/-2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the Caveolin genes in a variety of human tumors.
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targeted downregulation of Caveolin 1 is sufficient to drive cell transformation and hyperactivate the p42 44 map kinase cascade
The EMBO Journal, 1998Co-Authors: Ferruccio Galbiati, Daniela Volonte, Jeffrey A Engelman, Genichi Watanabe, Robert D Burk, Richard G Pestell, Michael P. LisantiAbstract: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.
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expression of Caveolin 1 and 2 in differentiating pc12 cells and dorsal root ganglion neurons Caveolin 2 is up regulated in response to cell injury
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Daniela Volonte, Philipp E. Scherer, Jeffrey A Engelman, Orlando Gil, George Zanazzi, James L Salzer, Massimo Sargiacomo, Amnon Schlegel, Marco ParentiAbstract:Caveolae are cholesterol/sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas Caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no Caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether Caveolins are expressed within neuronal cells. Here we demonstrate the expression of Caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, Caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas Caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, Caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of Caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of Caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express Caveolins.
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chromosomal localization genomic organization and developmental expression of the murine Caveolin gene family cav 1 2 and 3 cav 1 and cav 2 genes map to a known tumor suppressor locus 6 a2 7q31
FEBS Letters, 1998Co-Authors: Jeffrey A Engelman, Xiao Lan Zhang, Michael P. LisantiAbstract:Caveolins (Cav-1, -2, and -3) are a gene family of cytoplasmic membrane-anchored scaffolding proteins that: (i) help to sculpt caveolae membranes from the plasma membrane proper; and (ii) participate in the sequestration of inactive signaling molecules. In the adult, Caveolin-1 and -2 are co-expressed and are most abundant in type I pneumocytes, endothelia, fibroblastic cells and adipocytes, while the expression of Caveolin-3 is restricted to striated muscle cells. However, little is known regarding the genomic organization and developmental expression of the Caveolin gene family. Here, using the mouse as a model system, we examine the chromosomal localization, the detailed intron-exon organization, and developmental expression pattern of the Caveolin gene family. cDNAs encoding Caveolin-1, -2, and -3 were used as probes to isolate murine genomic clones containing these genes. Fluorescence in situ hybridization (FISH) analysis using these genomic clones as probes reveals that all three Caveolin genes are localized to murine chromosome 6. Specifically, Caveolin-1 and -2 co-localize to chromosomal region 6-A2, while Caveolin-3 is located within the chromosomal region 6-E1. Searches of the NCBI Human/Mouse Homology map indicate that murine region 6-A2 corresponds to human chromosome 7q31. As this region (6-A2/7q31) is the site of an as yet unidentified tumor suppressor gene(s), our mapping studies clearly define Caveolin-1 and Caveolin-2 as candidate genes that may be deleted at these loci. All three Caveolin genes show similar intron-exon organization, with the last exon of each gene encoding the bulk of the known Caveolin functional domains. The boundary position of the last exon is essentially identical in all three Caveolin genes, suggesting that they may have arisen through gene duplication events. Developmentally, all three Caveolins were expressed late during mouse embryogenesis as assessed by Northern and Western blot analysis. We examined the localization of the Caveolin proteins in sections of day 16 mouse embryos using a well-characterized panel of antibody probes. Caveolin-1 and -2 were most abundantly expressed in the developing lung parenchyma, while Caveolin-3 was most abundantly expressed in developing tissues that consist primarily of skeletal muscle cells. As the expression of all three Caveolins in the adult is highest in terminally differentiated cell types, this is consistent with the idea that Caveolins may be viewed as late markers of differentiation during embryogenesis.
Marjo Yliperttula - One of the best experts on this subject based on the ideXlab platform.
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distinct prostate cancer related mrna cargo in extracellular vesicle subsets from prostate cell lines
BMC Cancer, 2017Co-Authors: Elisa Lazaroibanez, Taral R Lunavat, Su Chul Jang, Carmen Escobedolucea, Jorge Oliverde La Cruz, Pia Siljander, Jan Lotvall, Marjo YliperttulaAbstract:Multiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed. We isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays. Based on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, Caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP- and PC-3- derived MVs highly correlated with prostate cancer progression. Our study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.