IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.     

Identification of Reggie-1 and Reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains). © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 502–523, 1998     

Tuberin is a component of lipid rafts and mediates caveolin-1 localization: role of TSC2 in post-Golgi transport

Mutations of the TSC2 gene lead to the development of hamartomas in tuberous sclerosis complex. Their pathology exhibits features indicative of defects in cell growth, proliferation, differentiation, and migration. We have previously shown that tuberin, the TSC2 protein, resides in multiple subcellular compartments and as such may serve multiple functions. To further characterize the microsomal pool of tuberin, we found that it cofractionated with caveolin-1 in a low-density, Triton X-100-resistant fraction (i.e., lipid rafts) and regulated its localization. In cells lacking tuberin, most of the endogenous caveolin-1 was displaced from the plasma membrane to a Brefeldin-A-sensitive, post-Golgi compartment distinct from the endosome and lysosome. Correspondingly, there was a paucity of caveolae at the plasma membrane of Tsc2-/- cells. Reintroduction of TSC2, but not a disease-causing mutant, reversed the caveolin-1 localization to the membrane. Exogenously expressed caveolin-1-GFP and vesicular stomatitis virus G protein, VSVG-GFP in the Tsc2-/- cells failed to be transported to the plasma membrane and were retained in distinct post-Golgi vesicles. Our data suggest a role of tuberin in regulating post-Golgi transport without apparent effects on protein sorting. The presence of mislocalized proteins in Tsc2-/- cells may contribute to the abnormal signaling and cellular phenotype of tuberous sclerosis.     

A new rat type I-like alveolar epithelial cell line R3/1: bleomycin effects on caveolin expression

The study of function and regulation of the phenotype of alveolar type I (AT I) epithelial cells is limited by the rareness of suitable cell lines or primary cultures of this cell type. We describe in the present study the type I-like rat epithelial cell line R3/1. This cell line displays in vitro a phenotype with several characteristic features of AT I cells. R3/1 cells were analysed for mRNA and protein content of markers related to the AT I cell type (T1, ICAM-1, connexin-43, caveolins-1 and -2) and AT II phenotypes [surfactant proteins (SPs) A, B, C and D]. The mRNAs for SPs were found to be at a low level. Moderate protein levels for SP-A and SP-B were found, and SP-C and SP-D proteins were not detectable. R3/1 cells are positive for CD44s, E-cadherin, cytokeratin, vimentin and RAGE, and bind the lectins BPA and SBA. For demonstration of the suitability of R3/1 cells for in vitro studies on epithelial injury, the cells were treated with bleomycin. As shown by real-time RT-PCR and immunoblotting, bleomycin-treatment of R3/1 cells resulted in a decrease in mRNA and protein for both caveolin-1 and caveolin-2 in comparison with controls. The AT I-like cell line R3/1 may serve as a promising tool for the study of lung cell biology.Roland Koslowski and Kathrin Barth contributed equally to the study     

Microdomain-forming proteins and the role of the reggies/flotillins during axon regeneration in zebrafish

The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin—which, however, are responsible for their own microdomains and functions—are introduced. Moreover, the function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown. Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5′-triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo—a process which is required for process extension and growth. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Linking membrane microdomains to the cytoskeleton: regulation of the lateral mobility of reggie-1/flotillin-2 by interaction with actin

The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie-1/flotillin-2 microdomains are organized along cortical F-actin in several cell types. Interaction with F-actin is mediated by the SPFH domain as shown by in vivo co-localization and in vitro binding experiments. Reggie-1/flotillin-2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie-1/flotillin-2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie-1/flotillin-2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.     

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.     

Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells

Background  

Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells.     

Cutting edge: differential expression of chemokines in Th1 and Th2 cells is dependent on Stat6 but not Stat4

The in vivo function of Th cell subsets is largely dependent on the ability of differentiated CD4+ T cells to be recruited to specific sites and secrete restricted sets of cytokines. In this paper we demonstrate that Th1 and Th2 cells secrete discrete patterns of chemokines, small m.w. cytokines that function as chemoattractants in inflammatory reactions. Th2 cells secrete macrophage-derived chemokine and T cell activation gene 3, and acquisition of this pattern of expression is dependent on Stat6. In contrast, Th1 cells secrete lymphotactin and RANTES, though unlike IFN-gamma, expression of these chemokines is independent of Stat4. We further show that supernatants from activated Th2 cells preferentially induce the chemotaxis of Th2 over Th1 cells, corresponding with Stat6-dependent expression of CCR4 and CCR8 in Th2 cells. These data provide the basis for restricted and direct T cell-mediated cellular recruitment to sites of inflammation.     

Sorting and function of the human folate receptor is independent of the caveolin expression in Fisher rat thyroid epithelial cells

Caveolae are small, flask-shaped, non-clathrin coated invaginations of the plasma membrane of many mammalian cells. Caveolae have a coat that includes caveolin. They have been implicated in numerous cellular processes, including potocytosis. Since the human folate receptor (hFR) and other glycosyl-phosphatidylinositol GPI)-tailed proteins have been co-localized to caveolae, we studied the caveolin role in the hFR function by transfecting hFR and/or caveolin cDNA into Fisher rat thyroid epithelial (FRT) cells that normally do not express detectable levels of either protein. We isolated and characterized stable clones as follows: they express (1) high levels of caveolin alone, (2) hFR and caveolin, or (3) hFR alone. We discovered that hFR is correctly processed, sorted, and anchored by a GPI tail to the plasma membrane in FRT cells. No difference in the total folic acid binding or cell surface folic acid binding activity were found between the FRT cells that were transfected with hFR, or cells that were transfected with hFR and caveolin. The hFR that was expressed on the cell surface of clones that were transfected with hFR was also sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) release, and incorporated radiolabeled ethanolamine that supports the attachment of a GPI-tail on hFR. We conclude that the processing, sorting, and function of hFR is independent on the caveolin expression in FRT cells.     

Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells

In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.     

Fas ligand is enriched in the caveolae membrane domains of thymic epithelial cells

Both Fas and Fas ligand (FasL) are expressed in the thymus. Although reports suggest that they are important throughout the thymocyte maturation process their precise role remains elusive. The present paper characterizes the expression of FasL in the thymus and in the TEA3A1 and BT1B functional thymic epithelial cell (TEC) lines. FasL expression by thymus fractions, TEA3A1, and BT1B cells was detected by Northern blot analysis. In TEA3A1 cells, we discovered that FasL protein expression was localized to caveolae membrane domains. This restricted subcellular localization of FasL, together with reports describing the localization of the major histocompatibility complex proteins, the T cell receptor and Fas to caveolae membrane domains, may provide a mechanism for the deletion of thymocytes during negative selection. Finally, using semi-quantitative RT-PCR we found that FasL expression by TECs is regulated by glucocorticoids.     

Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells

Acute H(2)O(2) exposure to placental artery endothelial cells induced an array of tyrosine-phosphorylated proteins, including caveolin 1 (CAV1) rapid and transient tyr(14) phosphorylated in a time- and concentration-dependent manner. Basal tyr(14) phosphorylated CAV1 was primarily located at the edges of cells and associated with actin filaments. Phosphorylated CAV1 was markedly increased and diffused with the disorganization of actin filaments at 20 min, disappeared at 120 min treatment with 0.2 mM H(2)O(2). Treatment with H(2)O(2) also disorganized actin filaments and changed cell shape in a time-dependent manner. Pretreatment with antioxidants catalase completely, whereas the other tested superoxide dismutase, N-acetyl-l-cysteine and sodium formate partially attenuated H(2)O(2)-induced CAV1 phosphorylation in a concentration-dependent manner. Acute treatment with H(2)O(2) activated multiple signaling pathways, including the mitogen-activated protein kinases (MAPK) members (MAPK3/1-ERK2/1, MAPK8/9-JNK1/2, and MAPK11-p38(mapk)) and the c-src tyrosine kinase (CSK). Pharmacological studies demonstrated that, among these pathways, only the blockade of CSK activation abolished H(2)O(2)-induced CAV1 phosphorylation. Additionally, H(2)O(2)-induced CAV1 phosphorylation was reversible rapidly (<10 min) upon H(2)O(2) withdrawal. Because maternal and fetal endothelia must make dynamic adaptations to oxidative stress resulting from enhanced pregnancy-specific oxygen metabolism favoring prooxidant production, which is emerging as one of the leading causes of the dysfunctional activated endothelium during pregnancy, these unique features of CAV1 phosphorylation on oxidative stress observed implicate an important role of CAV1 in placental endothelial cell biology during pregnancy.     

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.     

Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells

The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin-1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction.     

Induction or suppression of expression of cytochrome C oxidase subunit II by heregulin beta 1 in human mammary epithelial cells is dependent on the levels of ErbB2 expression

The ErbB family of receptor kinases is composed of four members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Amplification of the ErbB2/neu is found in about 30% of breast cancer patients and is associated with a poor prognosis. Heregulin (HRG) activates the ErbB2 via induction of heterodimerization with ErbB3 and ErbB4 receptors. With suppression subtractive hybridization, we demonstrated that the expression of cytochrome c oxidase subunit II (COXII) is HRG-responsive. Two nontransformed human mammary epithelial cell lines, the HB2 and the HB2(ErbB2) (the HB2 engineered to overexpress ErbB2), displayed an opposite response to HRG-mediated regulation. HRG upregulated mRNA expression of COXII in the HB2 cells, but suppressed COXII expression in the HB2(ErbB2) cells. A human breast cancer cell line (T47D), which expresses ErbB2 at a level similar to that of the HB2 cells, also responded to HRG by increasing COXII mRNA levels. Therefore, HRG regulation of COXII expression depends on the levels of ErbB2 expression. Furthermore, the expression of COXII was inversely correlated to the levels of ErbB2, i.e., the cells overexpressing ErbB2 exhibited lower COXII levels. HRG-evoked signal transduction differed between the cells with normal ErbB expression and the cells overexpressing ErbB2. The activation of both ERK and PI3-K was essential for HRG regulation of COXII, i.e., blockage of either pathway eliminated HRG-mediated alteration. This is the first report demonstrating that the expression of mitochondria-encoded COXII is HRG-responsive. The levels of ErbB2 expression are decisive for the diverse biological activities of HRG.     

Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells

Gamma₂-melanocyte-stimulating hormone (γ₂MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle³, D-Phe⁶]-γ₂MSH (NDP-γ₂MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ₂MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400 mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1 nM NDP-γ₂MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1 h with 1 nM NDP-γ₂MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ₂MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.