Caveolae Cholesterol Research Articles

Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder.

Cholesterol rich membrane invaginations, caveolae, have important roles in various cellular activities, one of them being signal transduction. This signaling pathway seems to be affected during various bladder disorders and the current study aimed to elucidate the plausible involvement of caveolae mediated signal transduction during cyclophosphamide induced cystitis. Furthermore, the urothelial cholinergic part of ATP-evoked contractions and its possible link to caveolae were investigated. Cholinergic, as well as purinergic, contractile responses in rat urinary bladders were examined using a classic organ bath set-up with full-thickness strip preparations or a whole bladder model that enabled luminal administration of substances. Furthermore, sub groups with and without urothelium were examined. The expression of caveolin-1 was also tested using western blot and immunofluorescence. Caveolae cholesterol depletion by methyl-β-cyclodextrin entailed a significant decrease of ATP-evoked bladder contractility. Interestingly, after muscarinic blockade the ATP induced contractions were significantly reduced in the same manner. Furthermore, this atropine-sensitive part of ATP-evoked responses was absent in denuded as well as inflamed bladders. A tendency towards a reduced expression of caveolin-1 was observed in rats with experimental cystitis. The cholinergic part of ATP-induced contractile responses seemed to be affected by urothelium denudation as well as caveolae depletion. Removing one of these structures nullifies the effect of the other, suggesting an important interaction between the urothelium and the caveolar structures. These effects are absent in inflamed animals and might be one pathophysiological aspect behind BPS/IC.

Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility.

Abstract 237: Acute Activation of eNOS by Lovastatin Involves Scavenger Receptor-B1

Statins (HMG CoA reductase inhibitors) have beneficial vascular effects independent of their ability to reduce cholesterol synthesis. We and others have previously shown that statins acutely activate endothelial cell NOS (eNOS). Our present study characterized signaling pathways by which statins activate NOS using cultured bovine aortic endothelial cells (BAEC) and COS 7 cells. It also examined involvement of scavenger receptor - B1 (SR-B1), which is expressed in endothelial cells and maintains caveolae cholesterol concentration and eNOS localization. BAEC were exposed to lovastatin (LOV, 10 −6 M) for 2–5 min after pretreatment with the end product of HMG CoA reductase (mevalonic acid, 5×10 −4 M), a Gi protein inhibitor (pertussis toxin, 10 −4 M), a PLC inhibitor (U-73122, 10 −5 M), a receptor tyrosine kinase (RTK) inhibitor (tyrphostine 51A, 2×10 −6 M) or intracellular and extracellular calcium chelators -BAPTA-AM (10 −5 M) and EGTA (10 −4 M), respectively, and levels of NO production were determined. LOV alone acutely increased NO production by 67%. NO production in response to LOV was not blocked by mevalonic acid indicating that it occurs independent of HMG CoA reductase inhibition. Inhibition of Gi protein, PLC, and RTK almost completely blocked the LOV-induced NO generation. Removal of extracellular Ca ++ prevented statin-induced NO production by 90%, while intracellular Ca ++ chelation with BAPTA-AM reduced NO production by 45%. COS 7 cells transfected with eNOS did not show increased NO production to LOV or HDL, indicating NOS activation occurs through an endothelial cell receptor. In COS 7 cells, co-transfected with eNOS and the endothelial SR-B1, LOV or HDL (10 −5 M), a known agonist of SR-B1, increased NO production by over 200%. In COS 7 cells transfected with eNOS alone or co-transfected with the bradykinin B2 receptor, neither LOV nor HDL increased NO production, indicating effects specific for the SR-B1 receptor. In conclusion, our study shows LOV acutely activates eNOS independently of HMG CoA reductase inhibition. eNOS activation appears to involve an endothelial Gi protein receptor and subsequent activation of PLC and RTK pathways, leading to entry of extracellular calcium. Furthermore, LOV can activate eNOS via the SR-B1 receptor.

Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

Regulation of Growth Plate Chondrocytes by 1,25-Dihydroxyvitamin D3 Requires Caveolae and Caveolin-1

We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice. 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology. Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons. Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis. The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology.

Cholesterol loading increases the translocation of ATP synthase β chain into membrane caveolae in vascular endothelial cells

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs) and have been suggested to contribute to cell signaling and cholesterol trafficking. This study investigated the effect of cholesterol on the movement of caveolae-related proteins in human umbilical vein ECs with use of caveolae functional proteomics. After cholesterol exposure to ECs for 2 to 4 h, caveolae were isolated and separated on 2-D protein gels. Among 40 protein spots revealed in caveolae fractions, the ATP synthase β subunit (ATPS-β), one of the 3 proteins enriched by cholesterol in caveolae, was confirmed by western blotting and confocal microscopy. Further, cholesterol exposure increased the level of ATPS-β, along with Cav-1 and cholesterol in caveolae. These effects could be blocked by cytochalasin B, an actin cytoskeleton disruptor. ATPS-β was physically associated with Cav-1, as demonstrated by co-immunoprecipitation and GST-Cav-1 fusion protein pull-down assay. Cholesterol increased the extracellular ATP release mediated by ATPS-β, since this action could be blocked by piceatannol or oligomycin, ATPS inhibitors. Thus, the ectopic localization of ATPS-β may participate in the energy balance of cells in response to the change in intracellular cholesterol levels.

Endothelial nitric oxide synthase, caveolae and the development of atherosclerosis.

Early hypercholesterolaemia-induced vascular disease is characterized by an attenuated capacity for endothelial production of the antiatherogenic molecule nitric oxide (NO), which is generated by endothelial NO synthase (eNOS). In recent studies we have determined the impact of lipoproteins on eNOS subcellular localization and action, thereby providing a causal link between cholesterol status and initial abnormalities in endothelial function. We have demonstrated that eNOS is normally targeted to cholesterol-enriched caveolae where it resides in a signalling module. Oxidized low density lipoprotein (LDL; oxLDL) causes displacement of eNOS from caveolae by binding to endothelial cell CD36 receptors and by depleting caveolae cholesterol content, resulting in the disruption of eNOS activation. The adverse effects of oxLDL are fully prevented by high density lipoprotein (HDL) via binding to scavenger receptor BI (SR-BI), which is colocalized with eNOS in endothelial caveolae. This occurs through the maintenance of caveolae cholesterol content by cholesterol ester uptake from HDL. As importantly, HDL binding to SR-BI causes robust stimulation of eNOS activity in endothelial cells, and this process is further demonstrable in isolated endothelial cell caveolae. HDL also enhances endothelium- and NO-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, lipoproteins have potent effects on eNOS function in caveolae via actions on both membrane cholesterol homeostasis and the level of activation of the enzyme. These processes may be critically involved in the earliest phases of atherogenesis, which recent studies suggest may occur during fetal life.

Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae).

We have studied the effect of lipid-free human plasma apolipoprotein A-I (apoA-I) on the transport of newly synthesized cholesterol to cell-surface cholesterol-rich domains, which in human skin fibroblasts are mainly represented by caveolae. Changes in transport of newly synthesized cholesterol were assessed after labelling cells with [(14)C]acetate at 15 degrees C and warming cells to permit the transfer of cholesterol, followed by the selective oxidation of cholesterol in cholesterol-rich domains (caveolae) in the plasma membrane before their partial purification. ApoA-I, but not BSA added in an equimolar concentration, enhanced the transport of cholesterol to the caveolae up to 5-fold in a dose- and time-dependent manner. The effect of apoA-I on cholesterol transport exceeded its effect on cholesterol efflux, resulting in an accumulation of intracellular cholesterol in caveolae. Methyl-beta-cyclodextrin, added at a concentration promoting cholesterol efflux to the same extent as apoA-I, also stimulated cholesterol trafficking, but was 3-fold less effective than apoA-I. Progesterone inhibited the transport of newly synthesized cholesterol to the caveolae. Treatment of cells with apoA-I stimulated the expression of caveolin, increasing the amount of caveolin protein and mRNA by approx. 2-fold. We conclude that apoA-I induces the transport of intracellular cholesterol to cell-surface caveolae, possibly in part through the stimulation of caveolin expression.

Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae)

We have studied the effect of lipid-free human plasma apolipoprotein A-I (apoA-I) on the transport of newly synthesized cholesterol to cell-surface cholesterol-rich domains, which in human skin fibroblasts are mainly represented by caveolae. Changes in transport of newly synthesized cholesterol were assessed after labelling cells with [14C]acetate at 15°C and warming cells to permit the transfer of cholesterol, followed by the selective oxidation of cholesterol in cholesterol-rich domains (caveolae) in the plasma membrane before their partial purification. ApoA-I, but not BSA added in an equimolar concentration, enhanced the transport of cholesterol to the caveolae up to 5-fold in a dose- and time-dependent manner. The effect of apoA-I on cholesterol transport exceeded its effect on cholesterol efflux, resulting in an accumulation of intracellular cholesterol in caveolae. Methyl-β-cyclodextrin, added at a concentration promoting cholesterol efflux to the same extent as apoA-I, also stimulated cholesterol trafficking, but was 3-fold less effective than apoA-I. Progesterone inhibited the transport of newly synthesized cholesterol to the caveolae. Treatment of cells with apoA-I stimulated the expression of caveolin, increasing the amount of caveolin protein and mRNA by approx. 2-fold. We conclude that apoA-I induces the transport of intracellular cholesterol to cell-surface caveolae, possibly in part through the stimulation of caveolin expression.

Site-specific Phosphorylation of Tau Accompanied by Activation of Mitogen-activated Protein Kinase (MAPK) in Brains of Niemann-Pick Type C Mice

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.