Decreased expression of caveolin 1 in patients with systemic sclerosis: crucial role in the pathogenesis of tissue fibrosis.

OBJECTIVE: Recent studies have implicated caveolin 1 in the regulation of transforming growth factor beta (TGFbeta) downstream signaling. Given the crucial role of TGFbeta in the pathogenesis of systemic sclerosis (SSc), we sought to determine whether caveolin 1 is also involved in the pathogenesis of tissue fibrosis in SSc. We analyzed the expression of CAV1 in affected SSc tissues, studied the effects of lack of expression of CAV1 in vitro and in vivo, and analyzed the effects of restoration of caveolin 1 function on the fibrotic phenotype of SSc fibroblasts in vitro. METHODS: CAV1 expression in tissues was analyzed by immunofluorescence and confocal microscopy. The extent of tissue fibrosis in Cav1-knockout mice was assessed by histologic/histochemical analyses and quantified by hydroxyproline assays. Cav1-null and SSc fibroblast phenotypes and protein production were analyzed by real-time polymerase chain reaction, immunofluorescence, Western blot, and multiplexed enzyme-linked immunosorbent assay techniques. The effects of restoration of caveolin 1 function in SSc fibroblasts in vitro were also examined using a cell-permeable recombinant CAV1 peptide. RESULTS: CAV1 was markedly decreased in the affected lungs and skin of SSc patients. Cav1-knockout mice developed pulmonary and skin fibrosis. Down-regulation of caveolin 1 was maintained in cultured SSc fibroblasts, and restoration of caveolin 1 function in vitro normalized their phenotype and abrogated TGFbeta stimulation through inhibition of Smad3 activation. CONCLUSION: Caveolin 1 appears to participate in the pathogenesis of tissue fibrosis in SSc. Restoration of caveolin 1 function by treatment with a cell-permeable peptide corresponding to the CAV1 scaffolding domain may be a novel therapeutic approach in SSc

The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells

Background: Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCα) with caveolin in CHO cells. PKCα is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult. Results: Fluorescence energy transfer (FRET), between GFP-PKCα and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from ~2.2 ns to ~1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCα that was not associated with caveolin in the nucleus and cytoplasm, PKCα associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin. Conclusion: Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCα and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell

Plasma from Volunteers Breathing Helium Reduces Hypoxia-Induced Cell Damage in Human Endothelial Cells-Mechanisms of Remote Protection Against Hypoxia by Helium.

PurposeRemote ischemic preconditioning protects peripheral organs against prolonged ischemia/reperfusion injury via circulating protective factors. Preconditioning with helium protected healthy volunteers against postischemic endothelial dysfunction. We investigated whether plasma from helium-treated volunteers can protect human umbilical vein endothelial cells (HUVECs) against hypoxia in vitro through release of circulating of factors.MethodsHealthy male volunteers inhaled heliox (79% helium, 21% oxygen) or air for 30 min. Plasma was collected at baseline, directly after inhalation, 6 h and 24 h after start of the experiment. HUVECs were incubated with either 5% or 10% of the plasma for 1 or 2 h and subjected to enzymatically induced hypoxia. Cell damage was measured by LDH content. Furthermore, caveolin 1 (Cav-1), hypoxia-inducible factor (HIF1α), extracellular signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription (STAT3) and endothelial nitric oxide synthase (eNOS) were determined.ResultsPrehypoxic exposure to 10% plasma obtained 6 h after helium inhalation decreased hypoxia-induced cell damage in HUVEC. Cav-1 knockdown in HUVEC abolished this effect.ConclusionsPlasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1

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

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

Pathophysiological Role of Caveolae in Hypertension

Caveolae, flask-shaped cholesterol-, and glycosphingolipid-rich membrane microdomains, contain caveolin 1, 2, 3 and several structural proteins, in particular Cavin 1-4, EHD2, pacsin2, and dynamin 2. Caveolae participate in several physiological processes like lipid uptake, mechanosensitivity, or signaling events and are involved in pathophysiological changes in the cardiovascular system. They serve as a specific membrane platform for a diverse set of signaling molecules like endothelial nitric oxide synthase (eNOS), and further maintain vascular homeostasis. Lack of caveolins causes the complete loss of caveolae; induces vascular disorders, endothelial dysfunction, and impaired myogenic tone; and alters numerous cellular processes, which all contribute to an increased risk for hypertension. This brief review describes our current knowledge on caveolae in vasculature, with special focus on their pathophysiological role in hypertension

Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation

Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells

Canine respiratory coronavirus (CRCoV), identified in 2003, is a member of the Coronaviridae family. The virus is a betacoronavirus and a close relative of human coronavirus OC43 and bovine coronavirus. Here, we examined entry of CRCoV into human rectal tumor cells (HRT-18G cell line) by analyzing co-localization of single virus particles with cellular markers in the presence or absence of chemical inhibitors of pathways potentially involved in virus entry. We also targeted these pathways using siRNA. The results show that the virus hijacks caveolin-dependent endocytosis to enter cells via endocytic internalization

Caveolin-1 Phosphorylation Is Essential for Axonal Growth of Human Neurons Derived From iPSCs.

Proper axonal growth and guidance is essential for neuron differentiation and development. Abnormal neuronal development due to genetic or epigenetic influences can contribute to neurological and mental disorders such as Down syndrome, Rett syndrome, and autism. Identification of the molecular targets that promote proper neuronal growth and differentiation may restore structural and functional neuroplasticity, thus improving functional performance in neurodevelopmental disorders. Using differentiated human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs), the present study demonstrates that during early stage differentiation of human NPCs, neuron-targeted overexpression constitutively active Rac1 (Rac1CA) and constitutively active Cdc42 (Cdc42CA) enhance expression of P-Cav-1, T-Cav-1, and P-cofilin and increases axonal growth. Similarly, neuron-targeted over-expression of Cav-1 (termed SynCav1) increases axonal development by increasing both axon length and volume. Moreover, inhibition of Cav-1(Y14A) phosphorylation blunts Rac1/Cdc42-mediated both axonal growth and differentiation of human NPCs and SynCav1(Y14A)-treated NPCs exhibited blunted axonal growth. These results suggest that: (1) SynCav1-mediated dendritic and axonal growth in human NPCs is dependent upon P-Cav-1, (2) P-Cav-1 is necessary for proper axonal growth during early stages of neuronal differentiation, and (3) Rac1/Cdc42CA-mediated neuronal growth is in part dependent upon P-Cav-1. In conclusion, Cav-1 phosphorylation is essential for human neuronal axonal growth during early stages of neuronal differentiation