ABSTRACT
Background Microalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. Methods We established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. Results In vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner (AU)
Antecedentes La microalbuminuria es un síntoma clínico común que se manifiesta en las fases tempranas de la enfermedad renal diabética (ERD), y también es característica del daño de las células endoteliales glomerulares (GEC). Existe evidencia creciente en cuanto a que la transcitosis de la albúmina a través de las GEC está estrechamente relacionada con la formación de albuminuria. Nuestros estudios previos reflejaron que angiopoyetina 2 (ANGPT2) puede inhibir la transcitosis de la albúmina a través de las células epiteliales tubulares renales activando la fosforilación de caveolina 1 (CAV1) durante la exposición a hiperglucemia (HG). El rol de ANGPT2 en la transcitosis de la albúmina a través de las GEC resulta incierto. Losartan reduce considerablemente la albuminuria, aunque no se ha esclarecido el mecanismo. Métodos Establecimos un modelo in vitro de transcitosis de la albúmina para investigar el cambio de dicho mecanismo a través de las células endoteliales glomerulares renales humanas (hrGEC) en condiciones de glucosa normal (GN), hiperglucemia (HG) e intervención de losartan. Realizamos breakdown de ANGPT2 y CAV1 para evaluar sus roles en la transcitosis de la albúmina a través de las hrGEC, y verificamos la relación entre ellas. Se establecieron modelos in vivo de ratones con ERD, tratados con diferentes dosis de losartan. Se utilizaron pruebas de inmunohistoquímica e inmunotransferencia para detectar la expresión de ANGPT2 y CAV1. Resultados In vitro, la transcitosis de la albúmina a través de hrGEC se incrementó considerablemente en condiciones de estimulación de la hiperglucemia, inhibiendo losartan este proceso. La expresión de ANGPT2 y CAV1 se incrementó en las hrGEC en condiciones de HG, reduciendo la intervención de losartan la expresión de ambas (AU)
Subject(s)
Animals , Male , Mice , Diabetes Mellitus, Experimental/metabolism , Kidney Glomerulus/metabolism , Albumins/metabolism , Transcytosis , Angiopoietins/metabolism , Mice, Inbred C57BL , Caveolins/pharmacology , Losartan/pharmacology , Models, AnimalABSTRACT
Caveolae have impressive morphological highlights of the cytomembrane of mammalian cells which involve in wide diversity of cellular functions involving signaling pathways and cholesterol hastening. Caveolin proteins possess a 'scaffolding' domain which for caveolin-1 and caveolin-3 appear to act a dominant role in signal regulation through caveolae. Caveolin-1 is treated to be protein in the cytomembrane entrapped with caveolae in endothelial cells and vascular smooth muscle cells which diminish nitric oxide (NO) by fill up the calcium/calmodulin (Ca2+/CaM) confining point of endothelial nitric oxide synthase (eNOS), decrease NO generation produce endothelial dysfunction and atherosclerotic injury development. It is a cholesterol-binding layer protein associated with cell cholesterol transport and also shows cardioprotective action through ischemic preconditioning (IPC) in diabetic and postmenopausal rat heart. Additionally it is ensnared in the procedures of tumorigenesis, prostate disease, and inflammation. The present study in the paper is to explore the structural functionalities of caveolins and their contributory role in CVS disorders and various other diseases.
Subject(s)
Caveolins/physiology , Adipocytes/chemistry , Adipocytes/ultrastructure , Alzheimer Disease/etiology , Animals , Cardiovascular Diseases/etiology , Caveolae/chemistry , Caveolins/pharmacology , Caveolins/therapeutic use , Cholesterol/physiology , Diabetes Mellitus, Type 2/etiology , Inflammation/etiology , Insulin/physiology , Ischemic Preconditioning , Kidney/physiology , Kidney/physiopathology , Muscular Diseases/etiology , Neoplasms/etiology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/physiology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Respiratory System/cytology , Signal Transduction , Testosterone/deficiency , Testosterone/physiology , Vertebrates/anatomy & histologyABSTRACT
Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because ß-adrenergic receptor (ßAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to ßAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with ß(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the ß(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.
Subject(s)
Adenylyl Cyclases/metabolism , Bronchi/metabolism , Muscle, Smooth/metabolism , Myocytes, Smooth Muscle/metabolism , Receptors, Prostaglandin E, EP2 Subtype/metabolism , Alprostadil/analogs & derivatives , Alprostadil/pharmacology , Animals , Bronchi/drug effects , Caveolins/pharmacology , Cell Line, Transformed , Colforsin/pharmacology , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , HEK293 Cells , Humans , Isoproterenol/pharmacology , Male , Mice , Muscle, Smooth/drug effects , Muscle, Smooth/enzymology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/enzymology , Receptors, Adrenergic, beta-2/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effectsABSTRACT
Peroxisome proliferator-activated receptor gamma (PPARgamma) activation results in an increased rate of amyloid-beta (Abeta) clearance from the media of diverse cells in culture, including primary neurons and glial cells. Here, we further investigate the mechanism for Abeta clearance and found that PPARgamma activation modulates a cell surface metalloprotease that can be inhibited by metalloprotease inhibitors, like EDTA and phenanthroline, and also by the peptide hormones insulin and glucagon. The metalloprotease profile of the Abeta-degrading mechanism is surprisingly similar to insulin-degrading enzyme (IDE). This mechanism is maintained in hippocampal and glia primary cultures from IDE loss-of-function mice. We conclude that PPARgamma activates an IDE-like Abeta degrading activity. Our work suggests a drugable pathway that can clear Abeta peptide from the brain.
Subject(s)
Amyloid beta-Peptides/metabolism , Insulysin/metabolism , PPAR gamma/pharmacology , Animals , Biotinylation , Caveolins/pharmacology , Cells, Cultured , Clathrin/pharmacology , Down-Regulation/drug effects , Electrophoresis, Polyacrylamide Gel , Endocytosis/drug effects , Epitopes , Female , Glucagon/pharmacology , Insulysin/genetics , Membrane Proteins/metabolism , Metalloproteases/metabolism , Mice , Mice, Knockout , Neprilysin/genetics , Neprilysin/metabolism , Neuroglia/drug effects , Neuroglia/metabolism , Neurons/drug effects , Neurons/metabolism , Phenanthrolines/pharmacology , Plasmids/genetics , Pregnancy , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Las caveolas participan en múltiples procesos celulares tales como el transportevesicular, homeostasis del colesterol, regulación de la señalización intracelular,por integrinas y proliferación celular. Sin embargo, su función en el hígado no estábien establecida. La expresión de caveolina 1 (Cav), la proteína más abundante enlas caveolas, está bien descrita en el hígado y en varias líneas de hepatocitos y enhígado cirrótico humano y en carcinoma hepatocelular. Sin embargo, el papel deCav-1 en la fisiopatología hepática es controvertido, ya que se ha propuesto un papel crítico en el proceso de regeneración tras hepatectomía parcial (HP). Contrariamentea esta observación, nuestros datos sugieren que Cav-1 aumenta en elhígado regenerante, con una re-distribución de la proteína desde las caveolas haciadominios no caveolares. Además, la Cav-1 localizada en estas fracciones está fosforiladaen la tirosina 14. A pesar de ello, el gen de la Cav-1 es dispensable parala regeneración hepática tras HP, tal como se deduce de animales que carecen deeste gen. En conjunto, estos datos muestran un papel dinámico de la Cav-1 en laproliferación hepática tras HP y en líneas hepáticas en cultivo, pero con mínimasimplicaciones en el proceso regenerativo
Although caveolae participate in many cellular processes such as vesicular transport,cholesterol homeostasis, regulation of signal transduction, integrin signalingand cell growth, their role in liver remains elusive. Expression of caveolin 1 (Cav),the most abundant protein of caveolae, has been reported in liver and in differenthepatocyte cell lines, in human cirrhotic liver and in hepatocellular carcinomas.However, the role of Cav-1 in liver pathophysiology remains controversial and acritical role in regeneration after partial hepatectomy (PH) has been reported.Opposite to this observation, our data support the view that Cav-1 increases inliver after PH with a redistribution of the protein from the caveolae enricheddomain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolarfraction is phosphorylated in tyrosine 14 (Tyr14). Even though, the Cav-1 geneis dispensable for liver regeneration after PH as deduced from data obtained withcommercially available animals lacking this gene. Taken together these resultssupport a dynamic role for Cav-1 in liver proliferation both in vivo after PH, andin vitro in cultured hepatic cell lines, but with minimal implications in the liverregeneration process
Subject(s)
Caveolins/chemistry , Caveolins/pharmacology , Liver Regeneration , Liver/chemistry , Hepatectomy/methods , Hepatectomy/rehabilitation , Caveolins/analysis , Caveolins/chemical synthesis , Caveolins/pharmacokinetics , Liver Regeneration/immunology , Liver Regeneration/physiology , Caveolae/chemistry , Caveolae , Liver , Hepatocyte Growth Factor/chemical synthesis , Hepatocyte Growth Factor/pharmacologyABSTRACT
This study investigates the role of lipid rafts and caveolae, a subclass of lipid raft microdomains, in the binding and uptake of long-chain fatty acids (LCFA) by 3T3-L1 cells during differentiation. Disruption of lipid rafts by beta-cyclodextrin (betaCD) or selective inhibition of caveolae by overexpression of a dominant-negative mutant of caveolin-3 (Cav(DGV)) resulted in disassembly of caveolae structures at the cell surface, as assessed by electron microscopy. While in 3T3-L1 fibroblasts, which express few caveolae, Cav(DGV) or betaCD had no effect on LCFA uptake, in 3T3-L1 adipocytes the same treatments decreased the level of [(3)H]oleic acid uptake by up to 55 +/- 8 and 49 +/- 7%, respectively. In contrast, cholesterol loading of 3T3-L1 adipocytes resulted in a 4-fold increase in the extent of caveolin-1 expression and a 1.7-fold increase in the level of LCFA uptake. Both the inhibitory and enhancing effects of these treatments were constantly increasing with the [(3)H]oleic acid incubation time up to 5 min. Incubation of 3T3-L1 adipocytes with [(3)H]stearate followed by isolation of a caveolin-1 positive detergent-resistant membrane (DRM) fraction revealed that [(3)H]stearate binds to caveolae. Fatty acid translocase (FAT/CD36) was found to be present in this DRM fraction as well. Our data thus strongly indicate a critical involvement of lipid rafts in the binding and uptake of LCFA into 3T3-L1 adipocytes. Furthermore, our findings suggest that caveolae play a pivotal role in lipid raft-dependent LCFA uptake. This transport mechanism is induced in conjunction with cell differentiation and might be mediated by FAT/CD36.
Subject(s)
Adipocytes/metabolism , Fatty Acids/metabolism , Membrane Microdomains/physiology , beta-Cyclodextrins , 3T3-L1 Cells , Animals , Binding Sites , CD36 Antigens/metabolism , Caprylates/metabolism , Caveolae/drug effects , Caveolae/metabolism , Caveolin 3 , Caveolins/biosynthesis , Caveolins/genetics , Caveolins/pharmacology , Cholesterol/metabolism , Cholesterol/pharmacology , Cholic Acids/pharmacology , Cyclodextrins/pharmacology , Detergents/pharmacology , Membrane Microdomains/drug effects , Membrane Microdomains/metabolism , Mice , Oleic Acid/metabolism , Stearic Acids/metabolismABSTRACT
Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.
Subject(s)
Caveolins/physiology , Muscle, Smooth, Vascular/physiology , Vasoconstriction/physiology , Animals , Anticholesteremic Agents/pharmacology , Aorta/metabolism , Calmodulin-Binding Proteins/metabolism , Calmodulin-Binding Proteins/pharmacology , Caveolin 1 , Caveolins/pharmacology , Ferrets , Mitogen-Activated Protein Kinases/metabolism , Peptide Fragments/pharmacology , Phorbol Esters/pharmacology , Phosphorylation , Protein Kinase C/metabolism , Vasoconstriction/drug effectsABSTRACT
Activation of the enzyme phospholipase (PLA 2) has been proposed to be part of the molecular mechanism involved in the alteration of 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor responsiveness during long term changes in synaptic plasticity (long term potentiation). This study assesses the effect of the caveolin-1 scaffolding domain (CSD) on the activity of the regulatory enzyme PLA2. Caveolin-1 is a 22-kDa cholesterol-binding membrane protein known to inhibit the activity of most of its interacting partners. Our results show that the calcium-dependent cytosolic form of PLA2 (cPLA2) and caveolin-1 co-localized in mouse primary hippocampal neuron cultures and that they were co-immunoprecipitated from mouse hippocampal homogenates. A peptide corresponding to the scaffolding domain of caveolin-1 (Cav-(82-101)) dramatically inhibited cPLA2 activity in purified hippocampal synaptoneurosomes. Activation of endogenous PLA2 activity with KCl or melittin increased the binding of [3H]AMPA to its receptor. This effect was almost completely abolished by the addition of the CSD peptide to these preparations. Moreover, we demonstrated that the inhibitory action of the CSD peptide on AMPA receptor binding properties is specific (because a scrambled version of this peptide failed to have any effect) and that it is mediated by an inhibition of PLA2 enzymatic activity (because the CSD peptide failed to have an effect in membrane preparations lacking endogenous PLA2 activity). These results raised the possibility that caveolin-1, via the inhibition of cPLA2 enzymatic activity, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.
Subject(s)
Caveolins/metabolism , Phospholipases A/metabolism , Receptors, AMPA/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/metabolism , Animals , Arachidonic Acid/metabolism , Binding Sites , Caveolin 1 , Caveolins/chemistry , Caveolins/isolation & purification , Caveolins/pharmacology , Cytosol/enzymology , Hippocampus/chemistry , Kinetics , Mice , Neurons/physiology , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Receptors, AMPA/chemistry , Synaptosomes/physiologyABSTRACT
1. The formation of NO from endothelial nitric oxide synthase (eNOS) in rat superior mesenteric artery rings was dependent on extracellular L-arginine, and was optimal at a concentration of L-arginine close to the plasma level (carbachol-stimulated NO: control 15.7+/-0.9, L-arginine 100 micro M 22.8+/-1.3 nM). 2. Enhancement of NO output by L-arginine was stereospecific, required the cationic amino-acid transporter and was dependent on caveolin. 3. Induction of inducible nitric oxide synthase (iNOS) impaired the stimulated NO synthesis from eNOS (100 nM carbachol-stimulated NO: control 5.7+/-0.6, iNOS 0.3+/-0.3 nM). 4. The interaction between iNOS and eNOS was reversed by the superoxide scavenger MnTMPyP. Impairment of eNOS by iNOS was also prevented by L-arginine 100 micro M administered simultaneously with carbachol, but not by L-arginine administered during incubation with lipopolysaccharide. 5. These data provide functional evidence that supplementing L-arginine from the extracellular medium optimises the formation of NO from eNOS and suggests that the impairment of eNOS by iNOS is caused by excess formation of superoxide by NO synthase, which can be prevented by L-arginine. These results provide an explanation for the observations that extracellular L-arginine can enhance endothelium function only when the endothelium is impaired or when iNOS has been induced.
Subject(s)
Arginine/pharmacology , Mesenteric Arteries/metabolism , Nitric Oxide Synthase/biosynthesis , Nitric Oxide/biosynthesis , Animals , Carbachol/pharmacology , Caveolin 1 , Caveolins/pharmacology , Enzyme Induction , In Vitro Techniques , Lipopolysaccharides/pharmacology , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/enzymology , Metalloporphyrins/pharmacology , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolismABSTRACT
Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.
Subject(s)
Endocytosis/physiology , Endothelium, Vascular/metabolism , Intercellular Adhesion Molecule-1/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Amides/pharmacology , Amiloride/pharmacology , Antibodies, Monoclonal/metabolism , Caveolin 1 , Caveolins/pharmacology , Cell Line, Tumor , Cells, Cultured , Clathrin/pharmacology , Dynamins/genetics , Dynamins/physiology , Endocytosis/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Humans , Intercellular Adhesion Molecule-1/immunology , Lactones/pharmacology , Macrolides , Models, Biological , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Protein Binding , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Pyridines/pharmacology , Thiazoles/pharmacology , Time FactorsABSTRACT
Cell swelling triggers in most cell types an outwardly rectifying anion current, I(Cl,swell), via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I(Cl,swell). However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of Galpha (a Rho activating heterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway.
Subject(s)
Anions/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Ion Channels/metabolism , rhoA GTP-Binding Protein/physiology , Animals , Caco-2 Cells , Cattle , Caveolin 1 , Caveolins/pharmacology , Cells, Cultured , Chloride Channels/physiology , DNA-Binding Proteins/physiology , Endothelium, Vascular/drug effects , GTP-Binding Protein alpha Subunits, G12-G13 , Humans , Hypotonic Solutions/pharmacology , Intracellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases/physiology , rho GTP-Binding Proteins/physiology , rho-Associated KinasesABSTRACT
Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.
Subject(s)
Caveolins/genetics , Caveolins/pharmacology , Cholesterol/pharmacology , Membrane Microdomains/drug effects , Muscular Dystrophies/metabolism , Signal Transduction/drug effects , Animals , Caveolin 3 , Cell Line , Cricetinae , Genes, ras , Golgi Apparatus/metabolism , Muscles/metabolism , Muscular Dystrophies/genetics , PC12 Cells , Point Mutation , RatsABSTRACT
Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.
Subject(s)
Caveolae/physiology , Caveolins/pharmacology , Endocytosis/physiology , Endoplasmic Reticulum/physiology , 3T3 Cells , Amino Acid Substitution , Animals , Caveolae/drug effects , Caveolin 1 , Cell Transformation, Neoplastic , Dynamins , Endoplasmic Reticulum/drug effects , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Genes, abl , Genes, ras , Homeostasis , Kinetics , Mice , Mutagenesis, Site-Directed , Receptors, Autocrine Motility Factor , Receptors, Cytokine/physiology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Ubiquitin-Protein LigasesABSTRACT
Caveolin-1 traffics cholesterol between the endoplasmic reticulum and cell surface caveolae in a non-vesicle chaperone complex which contains heat shock protein 56, cyclophilin 40, and cyclophilin A. Recent studies demonstrate that endothelial nitric oxide synthase (eNOS), caveolin, hetero-trimeric G-protein coupled receptors, and a calcium channel form an activation complex that is associated with cholesterol-rich caveolae. Oxidized LDL depletes caveolae of cholesterol and prevents agonist stimulation of eNOS by disrupting the activation complex. HDL antagonizes the effects of oxLDL by donating cholesterol to caveolae, thereby preserving the structure and function of caveolae. These findings and others provide a possible mechanistic basis for some of the molecular changes observed in vascular disease.
Subject(s)
Caveolins/pharmacology , Cholesterol/pharmacology , Lipoproteins/pharmacology , Nitric Oxide Synthase/metabolism , Vascular Diseases/metabolism , Caveolin 1 , Humans , Nitric Oxide Synthase Type III , Receptors, Lipoprotein/metabolismABSTRACT
Endothelial nitric oxide synthase (eNOS) is regulated both by caveolin-1 and 17beta-estradiol (E(2)). Temporal relationships between effects of estrogen on caveolin-1 and nitric oxide (NO) are not known. Therefore, this study was designed to determine whether estrogen regulates caveolin-1 and, if so, whether such regulation corresponds to changes in nitrite/nitrate (NO(x)) production. Bovine aortic endothelial cells (BAECs) were cultured in the absence and presence of 17beta-estradiol or 17alpha-estradiol (10(-8) and 10(-10) M) for 12, 24, and 48 h. eNOS protein expression and NO(x) production increased significantly after 24 h but not after 12-h treatment with 17beta- and not 17alpha-estradiol. Both mRNA and protein for caveolin-1 were increased significantly only after 48-h treatment with E(2), but eNOS protein and NO(x) production were decreased compared with cells treated for 24 h. These increases in caveolin-1 were inhibited by the estrogen receptor antagonist ICI-182,780 (10(-6) M). Results of this study suggest that E(2) stimulates caveolin-1 transcription and translation through estrogen receptor-mediated mechanisms. The results further suggest that estrogen may indirectly regulate NO(x) through caveolin-1 expression, which inhibits eNOS catalytic activity.
Subject(s)
Caveolins/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Estradiol/pharmacology , RNA, Messenger/metabolism , Animals , Aorta , Cattle , Caveolin 1 , Caveolins/genetics , Caveolins/pharmacology , Cells, Cultured , Enzyme Activation/drug effects , Estradiol/analogs & derivatives , Estrogen Antagonists/pharmacology , Female , Fulvestrant , Gene Expression/drug effects , Male , Nitrates/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type III , Nitrites/metabolism , Stereoisomerism , Time FactorsABSTRACT
Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the control of the caveolar system. When calf pulmonary artery endothelial (CPAE) cells were transfected with caveolin-1 Delta1-81 (deletion of amino acids 1 to 81), activation of VRAC by hypotonic cell swelling was strongly impaired. Concomitantly, caveolin-1 Delta1-81 disturbed the formation of caveolin-1 containing lipid rafts as evidenced by sucrose density gradient centrifugation. In nontransfected cells, endogenous caveolin-1 typically associated with low-density, detergent-resistant lipid rafts. However, transient expression of caveolin-1 Delta1-81 caused a redistribution of endogenous caveolin-1 to high-density, detergent-soluble membrane fractions. We therefore conclude that the interaction between caveolin-1 and detergent-resistant lipid rafts is an important prerequisite for endothelial VRAC activity.
Subject(s)
Anions/metabolism , Caveolae/metabolism , Caveolins/metabolism , Ion Channels/metabolism , Animals , Caco-2 Cells , Cattle , Caveolin 1 , Caveolins/genetics , Caveolins/pharmacology , Cell Line , Detergents/chemistry , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression , Genes, Dominant , Humans , Hypotonic Solutions/pharmacology , Ion Channels/antagonists & inhibitors , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Mutation , Pulmonary Artery , Rats , Sequence Deletion , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism , TransfectionABSTRACT
Sphingomyelinases hydrolyse sphingomyelin to ceramide, a process involved in signal-transduction routes leading to apoptosis and various other cellular responses. In the present study, we investigated the sphingomyelinase content of caveolae, invaginated plasma-membrane microdomains that contain a variety of signalling molecules. These structures are highly enriched in sphingomyelin as well as in ceramide, which suggests that metabolism of these lipids might, to some extent, occur locally. By cell fractionation, we demonstrate that, in addition to a previously reported minute amount of acidic sphingomyelinase activity, a substantial amount of neutral sphingomyelinase activity resides in caveolae of human skin fibroblasts. This caveolar neutral sphingomyelinase activity was also detected in Niemann-Pick disease type A fibroblasts, which are completely devoid of functional acidic sphingomyelinase. Neutral (but not acidic) sphingomyelinase activity was specifically inhibited by a peptide that corresponds to the scaffolding domain of caveolin, which suggests a direct molecular interaction between the two proteins. In addition, this finding implies a cytosolic orientation of the caveolar neutral sphingomyelinase. Interestingly, stimulation of fibroblasts with tumour necrosis factor alpha (TNFalpha) resulted in a partial shift of its p55 receptor to caveolin-enriched membrane fractions and the appearance of caveolin-sensitive neutral sphingomyelinase activity in the non-caveolar fractions. These results suggest that (part of) the presently identified caveolar neutral sphingomyelinase activity is involved in TNFalpha signalling.
Subject(s)
Caveolins/pharmacology , Signal Transduction/physiology , Sphingomyelin Phosphodiesterase/metabolism , Tumor Necrosis Factor-alpha/metabolism , Caveolae/metabolism , Caveolins/chemistry , Cells, Cultured , Cholesterol/deficiency , Cholesterol/metabolism , Enzyme Activation/drug effects , Humans , Niemann-Pick Diseases/pathology , Protein Structure, Tertiary , Sphingolipids/metabolism , Sphingomyelin Phosphodiesterase/antagonists & inhibitorsABSTRACT
Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its "scaffolding domain" to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82-101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.