The present study has tested the hypothesis that leptin receptors are localized in caveolae and that caveolae are involved in the leptin-induced stimulation of lipolysis in 3T3-L1 adipocytes. Leptin, a peptide hormone, is secreted primarily by adipocytes and has been postulated to regulate food intake and energy expenditure via hypothalamic-mediated effects. Exposure to leptin increases the lipolytic activity in 3T3-L1 adipocytes. We isolated caveolae from 3T3-L1 adipocytes using a detergent free sucrose gradient centrifugation method. Leptin receptors were localized in the same gradient fraction as caveolin-1. Confocal microscopic studies demonstrated the colocalization of leptin receptors with caveolin-1 in the plasma membrane, indicating distribution of leptin receptors in the caveolae. We disrupted caveolae by treating cells with methyl--cyclodextrin and found that leptin induced lipolytic activity was reduced after caveolae disruption, indicating an important role of caveolae in the signaling mechanism of leptin.

Allergic asthma is a syndrome characterized by respiratory distress in response to environmental triggers. This atypical response to an allergen is an over reaction of the immune system causing an influx of inflammatory cells into the airway and concomitant airway smooth muscle constriction. Firstly, we demonstrate using whole house dust mite (HDM) extract as a sensitizing allergen produces an equivalent or more robust hyperresponsive and inflammatory reaction than can be achieved with the widely used ovalbumin (OVA) sensitization / challenge protocol. Secondly, we investigated the role of caveolin-1 in the pathophysiology of allergic asthma . Our data suggest an important role for cav-1 in down regulating allergic airway inflammation, leading to reduced airways hyperresponsiveness and mucus overproduction.

Background: Caveolin-1 (Cav-1), the major component of caveolae, is a multifunctional scaffolding protein that serves as a platform for the cell’s signal-transduction and plays a role in inflammation. However, its role in inflammatory bowel disease (IBD), a chronic inflammatory condition in the gastrointestinal tract, is not clear. A recent study shows that Cav-1 mediates angiogenesis in dextran sodium sulphate (DSS)-induced colitis. These results contradict our data, in which Cav-1 levels decreased significantly in 2,4,6-trinitrobenzene sulphonic acid (TNBS)–induced colitis. Methods: To test whether Cav-1 is involved in IBD pathogenesis, various models representing different dominant Th subtype responses and mimicking the immune pathologic mechanisms of different clinical IBD setting were employed: acute colitis was induced by intra-rectal administration of a single dose of TNBS in BALB/c and C57BL/6J mice, or by drinking 3% DSS water for 6 days in C57BL/6J mice. Chronic colitis was induced by administration of TNBS once a week for 7 weeks in BALB/c mice. To assess the effects of complete loss of Cav-1, Cav-1 knock-out (Cav-1-/-) and control wild-type C57BL/6J mice received a single TNBS administration. To further test the possible role of Cav-1, one of two peptides (that either mimicked (Caveolin scaffolding domain; CSD) or antagonized (Caveolin-1 binding domain; CBD1) Cav-1)) was administered intraperitoneally to mice receiving TNBS. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified by ELISA. Inflammation was evaluated through histological analysis. Results: Cav-1 levels in mouse colon tissue were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation and cytokine levels. Furthermore, a loss of Cav-1 (Cav-1-/-) showed increased clinical and inflammatory scores and increased body weight loss. Mice receiving peptides to alter Cav-1 levels, showed surprising effects. The mimicking peptide (CSD) showed decreased Cav-1 levels, while the antagonizing peptide (CBD1) showed increased Cav-l levels. These changes in levels were associated with clinical and inflammatory scores and body weight loss that supported the TNBS-induced data. DSS-induced colitis mice showed increased disease activity index, however no significant difference in Cav-1 levels was found between colitis and normal mice. Conclusions: Cav-1 plays an important role in the protection of TNBS-induced colitis, but not in DSS-induced colitis, an entirely different result from a previous report, suggesting that enhancement of Cav-1 expression and functions may be beneficial to IBD treatment in some specific clinical settings. Further studies are warranted.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

La cavéoline-1, une petite protéine membranaire de 21 kDa, est la protéinemembranaire majoritaire d'invaginations de la membrane cytoplasmique appeléescavéoles. Enrichis en cholestérol et sphingolipides, ces domaines ont un rôleimportant dans de nombreux aspects de la vie cellulaire et constituent une véritableplateforme d'interactions protéiques et lipidiques. Ces dernières années, malgré lenombre important de travaux concluant à l'implication de la cavéoline-1, ou descavéoles, dans de nombreux processus cellulaires ou pathologiques, les donnéesquant à l'organisation de cette protéine au sein de la membrane plasmique restent trèséparses. Aussi, l'objectif principal de ce travail est de contribuer à l'acquisition dedonnées structurales sur cette protéine. Ce travail se base sur les expressionshétérologues d'une isoforme de la cavéoline-1 canineou de l'un de ses fragments, dans un hôte bactérien, sous la forme de protéines defusion associées à la Maltose Binding Protein. Ces expressions induisent laformation de vésicules intracytosoliques composées majoritairement de la protéineexprimée. Aussi, la première partie du travail est consacré à la mise en place d'unprotocole de purification de ces vésicules dans des conditions natives, répondant aucritère de réaliser une étude structurale de cette protéine n'impliquant pas l'usage dedétergent. La deuxième partie porte sur une application potentielle de ces vésicules, eten particulier pour des essais de caractérisation d'une enzyme membranaire,la glutathion-S-transférase microsomale de rat. Enfin une troisième partie est dédiée àl'analyse de simulations de dynamique moléculaire dans le cadre d'étudesd'interactions au sein de systèmes membranaires. / Caveolin-1, a 21 kDa membrane protein, is the principal membrane protein ofcytoplasmic membrane domains named caveolae. Specifically enriched incholesterol and sphingolipids those domains are important in many aspect of the cell's life and make up a proteins and lipids interaction platform. In the past years, despite a large number of publications stating the implication of caveolin-1 or caveolae in many cell processes and pathologies, very few is known about theway this protein is organized at the cell membrane. Hence, the main purpose of thiswork is to contribute to the acquisition of structural data on this protein. At the base of this work is the heterologous expression within a bacterial host, and as a fusion protein, of the canin beta isoform of cavéolin-1 or one of its fragment. These expressions lead to the formation of cytoplasmic vesicles composed mainly ofthe expressed protein. Thence, the first part of this work focus on developping a method to purify those vesicles that does not rely on using any kind of detergent which could enable structural studies in a native environnment. The second part present a potentiel application of those vesicles and inparticular the use of those vesicles to characterize a membrane enzyme, namelythe murin microsomal glutathion-Stransferase. The last part will be a contribution to data analysis within the context of molecular dynamics simulationof membraneous systems.

Cardiovascular disease (CVD) is the leading global cause of death. Coronary Artery Disease (CAD) is a grouping of the most common cardiovascular diseases and is the current leading cause of death in developed countries. Treatments for CAD include pharmaceuticals as well as surgical interventions such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting. However, these treatments do not completely remove the risk of adverse outcomes. Endothelial dysfunction is the underlying cause of CAD and is initiated by the chronic inflammation of the vasculature due to increased oxidative stress and production of reactive oxygen species (ROS). Previous studies have shown that the deletion of caveolin, a signaling molecules abundant within endothelial cells, can enhance inflammatory responses and lead to increased oxidative stress and ROS production. Mitochondrial ROS created from dysfunctional mitochondrial dynamics has also been shown to contribute to the inflammation of the endothelium. We hypothesize that due to the link between caveolin and endothelial dysfunction, and the link between mitochondria and endothelial dysfunction, caveolin has an important function in mitochondrial dynamics and that the loss of caveolin increases the mitochondrial fission via a Drp1-dependent pathway. Our data shows that adenoviral silencing of caveolin-1 in rat aortic endothelial cells increases Drp1 expression but does not significantly alter mitochondrial morphology. Overexpression of caveolin-1 via an adenoviral construct in these cells produces a decrease in Drp1 expression without altering mitochondrial morphology. This data provides insight into the pathophysiology of CAD and could provide us with new therapeutic targets in the future. / Biomedical Sciences

Reactive oxygen species (ROS) generated during ischemia-reperfusion (I/R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion [ischemic preconditioning (IP)] induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of GLUT-4 from the intracellular vesicles to the sarcolemma. In the present study we demonstrated involvement of ROS in IP-mediated GLUT-4 translocation along with increased expression of caveolin (Cav)-3, phospho (p)-endothelial nitric oxide synthase (eNOS), p-Akt, and decreased expression of Cav-1. The rats were divided into the following groups: 1) control sham, 2) N-acetyl-L-cysteine (NAC, free radical scavenger) sham (NS), 3) I/R, 4) IP + I/R (IP), and 5) NAC + IP (IPN). IP was performed by four cycles of 4 min of ischemia and 4 min of reperfusion followed by 30 min of ischemia and 3, 24, 48 h of reperfusion, depending on the protocol. Increased mRNA expression of GLUT-4 and Cav-3 was observed after 3 h of reperfusion in the IP group compared with other groups. IP increased expression of GLUT-4, Cav-3, and p-AKT and p-eNOS compared with I/R. Coimmunoprecipitation demonstrated decreased association of Cav-1/eNOS in the IP group compared with the I/R group. Significant GLUT-4 and Cav-3 association was also observed in the IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents a significant role of ROS signaling in Akt/eNOS/Cav-3-mediated GLUT-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP-induced eNOS and GLUT-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia, leading to myocardial protection in a clinically relevant rat ischemic model.

Caveolae are integral part of the smooth muscle membrane and caveolins, the defining proteins of caveolae, act as scaffolding proteins for several G protein-coupled receptor signaling molecules and regulate cellular signaling through direct and indirect interactions with signaling proteins. Caveolin-1 is the predominant isoform in the smooth muscle and drives the formation of caveolae. However, little is known about the role of caveolin-1 in the regulation of excitation-contraction and excitation-transcription coupling in gastrointestinal smooth muscle. In the present study we have characterized muscarinic m2 and m3 receptor signaling in gastric smooth muscle and tested the hypothesis that caveolin-1 positively regulates muscarinic receptor signaling and contractile protein expression in smooth muscle. The role of caveolae/caveolin-1 in the regulation of muscarinic signaling was examined using complementary approaches: a) methyl b-cyclodextrin (MbCD) to deplete cholesterol in dispersed muscle cells, b) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and c) caveolin-1 knockout (KO) mice. RT-PCR, western blot and radioligand binding studies demonstrated the selective expression of m2 and m3 receptor in gastric smooth muscle cells. Carbachol (CCh), acting via m3 receptors caused stimulation of phosphoinositide (PI) hydrolysis, Rho kinase and ZIP kinase activity, and induced phosphorylation of MYPT1 (at Thr696) and MLC20 (at Ser19), and muscle contraction, and acting via m2 receptors caused inhibition of forskolin stimulated cAMP formation. Stimulation of PI hydrolysis, Rho kinase and ZIP kinase activities, phosphorylation of MYPT1 and MLC20 phosphorylation and muscle contraction in response to CCh was attenuated in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. Similar inhibition of all responses was obtained in gastric muscle cells from caveolin-1 KO mice compared to gastric muscle cells to WT mice. Although, caveolin-1 had no effect on m2 receptor signaling, agonist-induced internalization of m2, but not m3 receptors was blocked in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. These results suggest that caveolin-1 selectively and positively regulates Gq/13-coupled m3 receptor signaling, Gi-coupled m2 receptor internalization. The expression of contractile proteins, g-actin and caldesmone and the transcription factors SRF and myocardin that regulate the expression of contractile proteins are down regulated, whereas EGF-stimulated EGF receptor phosphorylation and ERK1/2 activity are up-regulated in cells transfected with caveolin-1 siRNA. These results suggest using pharmacological, molecular and genetic approaches provide conclusive evidence that caveolae and caveolin-1 play an important role in orchestrating G protein coupled receptor signaling to have dual pro- excitation-contraction and excitation-transcription coupling, and anti-proliferative role in gastric smooth muscle.

Matrix metalloproteinase2 (MMP2) was recently revealed to have targets and actions within the cardiac myocyte. In ischemia/reperfusion (I/R) injury, MMP2 is activated and degrades troponin I (TnI) and actinin. The regulation of intracellular MMP2 activity is relatively unknown and is thus the subject of this thesis. The localization of MMP2 in caveolae of endothelial cells suggests that caveolin1 (Cav1) may play a role in regulating MMP2. Whether Cav1 is responsible for regulating MMP2 in the heart is unknown. A Cav1 knockout mouse model was used to explore the role Cav1 may play in the regulation of MMP2 activity. The initial studies found that MMP2 and Cav1 were colocalized in cardiomyocytes and that MMP2 activity in Cav1/ hearts was markedly enhanced. Additionally, the caveolin scaffolding domain inhibited MMP2 activity in a concentrationdependent manner. To explore whether increased MMP2 in Cav1/ hearts translates to impaired cardiac function, Cav1+/+ and Cav1/ isolated working hearts were physiologically challenged with increasing increments of left atrial preload followed by increasing concentrations of isoproterenol. Cav1/ hearts show similar or better cardiac function compared to Cav1+/+ hearts following preload challenge or adrenergic stimulation in vitro, and this appears unrelated to changes in MMP2. Though the function of Cav1/ hearts appears similar to that of Cav1+/+ hearts during physiological situations, whether this is the case during I/R injury is not known. Cav1+/+ and Cav1/ isolated working mouse hearts exposed to global, noflow ischemia showed no functional differences. However, Cav1/ hearts had significantly higher levels of both TnI and actinin following I/R than Cav1+/+ hearts. Posttranslational modifications of the intracellular MMP2 substrates could alter susceptibility to MMP2 proteolysis. Isolated working mouse hearts were exposed to isoproterenol and/or I/R injury to examine the phosphorylation status of TnI. Isoproterenol and I/R both result in the phosphorylation of TnI, however, isoproterenol lead to a more highly phosphorylated form of TnI than that observed in hearts exposed I/R alone. These and subsequent studies will further reveal the molecular mechanisms that underlie the complex interactions between Cav1 and MMP2. This may eventually lead to a novel avenue of therapeutic intervention for heart diseases.