Caveolae In Cells Research Articles

Abstract 19749: Cardiac-specific Deletion of Caveolin-3 Delays Repolarization and Increases Susceptibility to Ventricular Arrhythmia

Caveolin-3 (Cav-3) is an essential scaffolding protein for formation of caveolae in muscle cells. Cav-3 is part of a macromolecular complex including several ion channels. Mutations in Cav-3 have been associated with the inherited long QT syndrome as well as a variety of skeletal myopathies. To investigate the role of Cav-3 in heart and whether loss of function of Cav-3 explains the long QT phenotype, we generated cardiac-specific, inducible Cre-lox Cav-3 knockout mice. 8 week old mice were treated with tamoxifen in the chow to induce cardiac-specific recombination. Western blot analysis and transmitted electron microscopy demonstrated a graded loss of Cav-3 and caveolae in Cav-3 KO heterozygous mice (Cav-3-/+), Cav-3 KO homozygous mice (Cav-3-/-) relative to the littermate controls mice (WT). Echocardiography revealed no significant difference in %EF, %FS, LV chamber dimensions, and LV wall thickness between the different genotypes. Histopathological examination demonstrated no significant difference in HW/BW ratio, cardiac structure or fibrosis comparing Cav-3-/- and WT mice. Telemetry ECG recordings revealed a significant increase in QTc interval Cav-3-/- (68.5±7 ms) compared to WT (54.83±6 ms). Whole cell patch clamp analysis from isolated ventricular myocytes indicated a progressive increase in action potential duration (APD) with loss of Cav-3: WT (APD50: 4.7 ± 1ms; APD90: 28.0±3 ms; n=9); Cav-3-/+(APD50: 10.3±2 ms; APD90: 42.4±3 ms; n=13), Cav-3-/- (APD50: 32.4±6ms; APD90: 97.4±7ms; n=12). Whole cell voltage clamp measurements from Cav-3-/- revealed increased late INa, decrease in ICa,L, Ito,Iss current density without altering peak INa compared to WT cells, and these current changes were adequate to explain the increased APD based on computational representation using the Morotti et al. mouse ventricular cell model. Intracardiac programmed electrical stimulation (ventricular burst pacing) induced VT/Vfib in 8 out of 9 Cav3-/- but none of WT mice (0/5). Our results demonstrate that loss of Cav-3 and caveolae in adult mice does not alter cardiac structure or contractile function but leads to prolonged APD, an increased in QTc, and increased susceptibility to ventricular arrhythmias.

Caveolin-1 in Cardiovascular Disease: A Double-Edged Sword.

Endothelial dysfunction, as manifested by an attenuation of nitric oxide (NO)–mediated vasodilation, is recognized to be a fundamental abnormality in the genesis of hypertension, atherosclerosis, and coronary artery disease (1). Metabolic risk factors, such as obesity, insulin resistance, and type 2 diabetes (T2D), can initiate and accelerate endothelial dysfunction leading to cardiovascular disease (CVD) (1). Endothelial dysfunction in association with metabolic abnormalities is typically caused by a combination of reduced production and increased destruction of NO leading to a decrease in NO bioavailability (1,2). NO production in response to various factors, such as increased shear stress, is mediated by endothelial nitric oxide synthase (eNOS), which is constitutively expressed in endothelial cells (ECs) and is tightly controlled by various membrane-bound receptors and regulatory proteins under physiological conditions (3). Caveolin-1 (Cav-1), an anchoring protein in the plasma membrane caveolae in ECs and vascular smooth muscle cells (VSMCs), attenuates endothelial NO production by occupying the calcium/calmodulin (Ca2+/CaM) binding site of eNOS (4) (Fig. 1). Increases in caveolin and eNOS interaction, as may occur with hyperlipidemia, reduce NO production and promote endothelial dysfunction and atherosclerotic lesion formation. This process is mediated by increased lipoprotein trafficking across the vascular endothelium (5,6). Therefore, treating hyperlipidemia as an early intervention to help …

Caveolae regulate vasoconstriction of conduit arteries to angiotensin II in hindlimb unweighted rats.

Weightlessness induces the functional remodelling of arteries, but the changes to angiotensin II (Ang II)-elicited vasoconstriction and the underlying mechanism have never been reported. Caveolae are invaginations of the cell membrane crucial for the contraction of vascular smooth muscle cells, so we investigated the adaptation of Ang II-elicited vasoconstriction to simulated weightlessness and the role of caveolae in it. The 4 week hindlimb unweighted (HU) rat was used to simulate the effects of weightlessness. Ang II-elicited vasoconstriction was measured by isometric force recording. The morphology of caveolae was examined by transmission electron microscope. The binding of the angiotensin II type 1 receptor (AT1 ) and caveolin-1 (cav-1) was examined by coimmunoprecipitation and Western blot. We found that the maximal developing force (E(max)) of Ang II-elicited vasoconstriction was decreased in abdominal aorta by 30.6%, unchanged in thoracic aorta and increased in carotid artery by 17.9% after HU, while EC50 of the response was increased in all three arteries (P < 0.05). AT1 desensitization upon activation was significantly reduced by HU in all three arteries, as was the number of caveolae (P < 0.05). Furthermore, Ang II promoted the binding of AT1 and cav-1 significantly in control but not HU arteries. Both the number of caveolae and the binding of AT1 and cav-1 in HU arteries were restored by cholesterol pretreatment which also reinstated the change in EC50 as well as the level of AT1 desensitization. These results indicate that modified caveolae in vascular smooth muscle cells could interfere with the binding of AT1 and cav-1 mediating the adaptation of Ang II-elicited vasoconstriction to HU.

Cavin 1 function does not follow caveolar morphology.

The function of caveolae, small invaginations of the plasma membrane, remains a matter of debate. We discuss endocytosis and compartmentalization of metabolic and signaling pathways. Caveolin 3 (CAV3) and polymerase I and transcript release factor (PTRF) are important proteins that ensure shaping of caveolae in muscle cells. We investigated caveolae morphologically by electron microscopy in myotubes obtained from patients with CAV3 mutations and performed functional analyses in fibroblasts from a patient with a mutation in PTRF. Despite the complete clinical picture of a caveolinopathy, we found that caveolae in the CAV3-deficient myotubes were normal in shape and number. Furthermore, we found a difference in uptake of cholera toxin B between PTRF-deficient fibroblasts devoid of caveolae and normal fibroblasts. However, after caveolae were rescued by transfection of PTRF, cholera toxin B uptake did not normalize. We conclude that the presence of caveolae as an anatomic structure is not sufficient to ensure their proper function. Alternatively, the functional properties assigned to caveolae might be mediated by different mechanisms that have yet to be resolved.

Eisosomes provide membrane reservoirs for rapid expansion of the yeast plasma membrane.

Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires 'membrane reservoirs' that provide surface area and buffer membrane tension, but the sources of this membrane remain poorly understood. In principle, the flattening of invaginations and buds within the plasma membrane could provide this additional surface area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study the rapid expansion of the yeast plasma membrane in protoplasts, which lack the rigid cell wall. To survive hypoosmotic stress, yeast cell protoplasts required eisosomes, protein-based structures that generate long invaginations at the plasma membrane. Both budding yeast and fission yeast protoplasts lacking eisosomes were unable to expand like wild-type protoplasts during hypoosmotic stress, and subsequently lysed. By performing quantitative fluorescence microscopy on single protoplasts, we also found that eisosomes disassembled as surface area increased. During this process, invaginations generated by eisosomes at the plasma membrane became flattened, as visualized by scanning electron microscopy. We propose that eisosomes serve as tension-dependent membrane reservoirs for expansion of yeast cells in an analogous manner to caveolae in animal cells.

Caveolin 1 is critical for abdominal aortic aneurysm formation induced by angiotensin II and inhibition of lysyl oxidase

Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1-/- mice and the control wild-type mice were co-infused with AngII and β-aminopropionitrile to induce AAA. We found that Cav1-/- mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1-/- aortas with the co-infusion. Furthermore, aortas from Cav1-/- mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.

How antepartum and postpartum acute urinary retention affects the function and structure of the rat bladder

To examine the effect of acute urinary retention (AUR) on the urodynamic function and molecular structure of rat bladders in pregnancy and after parturition. Forty-eight nulliparous rats were distributed into AUR and non-AUR groups. AUR was induced by clamping the distal urethra of each rat after infusing 3 ml of saline for 60 min. Plasma progesterone levels and cystometric data were evaluated on the 14th day of gestation, 3 and 10 days postpartum, and in virgin rats. The immunoreactivity of caveolins and nerve growth factor (NGF) was analyzed. The number of caveolae in bladder muscle cells was evaluated by electron microscopy. Progesterone levels significantly increased during pregnancy and 3 days postpartum. In cystometric results, the AUR group has significantly shorter intercontraction interval, lower void volume and greater residual volume compared with the non-AUR. AUR rats exhibited higher NGF immunoreactivity, lower caveolin-1 immunoreactivity, and less caveolae in the bladder compared with the non-AUR. The caveolin-1 and NGF immunoreactivity and the number of caveolae in the bladder decreased during pregnancy and 3 days postpartum compared with virgin rats. By using Pearson correlations, we found significant correlations between urodynamic variables (residual volumes and intercontraction intervals) and the expressions of caveolin-1, caveolae and NGF in the AUR rats on the 14th day of gestation and 3 days postpartum. Bladder dysfunction in pregnancy and immediately postpartum in a rat model caused by AUR is associated with the plasma progesterone level change and the expressions of caveolin, caveolae, and NGF in bladder muscle cells.

Extrusion of Amyloid Fibrils to the Extracellular Space in Experimental Mesangial AL-Amyloidosis: Transmission and Scanning Electron Microscopy Studies and Correlation with Renal Biopsy Observations

In vitro studies have provided much information regarding the process of glomerular AL-amyloidogenesis. Research efforts have been successful in deciphering how glomerulopathic light chains interact with mesangial cells. The sequential steps involved in the genesis of amyloid fibrils include interactions with surface caveolae in mesangial cells and internalization of the monoclonal light chains through a clathrin-mediated process followed by trafficking in the mesangial cells to the mature lysosomal compartment where fibrils are formed. This manuscript focuses on how mesangial cells, once amyloid has been formed, deliver the fibrils to the extracellular matrix. The delivery of amyloid fibrils to the outside of the cells is carried out by lysosomes, which abut the mesangial cell membranes and extrude their contents into the extracellular space. This final step responsible for the fibrils to be present predominantly in the extracellular space is well demonstrated with scanning electron microscopy.

Striatin Is a Novel Regulator Of Aldosterone-Mediated Endothelial Cell Activation

Activation of the minerolocorticoid receptor (MR) by aldosterone (ALDO) has been shown to play an important role in inflammatory vascular responses in addition to its well described effects on sodium homeostasis. Steroid responses are mediated by well-known genomic and less known rapid/nongenomic responses. However, characterization of the mechanisms underlying ALDO's rapid/nongenomic actions have been difficult to study and are not clearly understood. We recently reported that in vivo and in vitro activation of MR leads to increases of striatin levels in endothelial cells, aortas and heart tissue (Pojoga, Amer J Hypertens, 2012) and that MR forms a complex with caveolin-1 and striatin within caveolae in endothelial cells. We hypothesized that striatin is a critical intermediary of the rapid effects of ALDO and that striatin serves as a novel link for MR regulation in endothelial cells activation. Endothelial cell activation promotes, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI), a redox modifying enzyme that catalyze disulfide interchange reactions. We studied EA.hy926 cells (EA), a human endothelial cell line that expresses MR, striatin and maintains its caveolae while in culture. We incubated EA cells with ALDO (10–9–10–7M) for 60 min and observed a dose-dependent rise in ROS production (P<0.001, n=4) using the oxidative fluorescent indicator dye 5-6-chloromethyl-2ʼ,7ʼ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) that peaked at around 10-8M ALDO, an event that was blocked by pre-incubation of EA cells for 30 mins with 1μM canrenoic acid (CA), an MR antagonist (P<0.03, n=3). Time course analyses showed ALDO stimulated ROS responses that increased for up to 3 hours following the addition of ALDO. As there are no known inhibitors for striatin we then used siRNA technology to down regulate striatin in these cells. EA cells were transfected with striatin siRNA and subsequently stimulated with ALDO and ROS production measured. The transfection process itself did not modify baseline levels of ROS significantly, as assessed in cells transfected with scrambled siRNA and non-transfected cells, which had nearly identical ROS levels, basally and in response to ALDO. In addition, in the presence of lower levels of striatin protein the ALDO-stimulated ROS response was abrogated, supporting the concept that striatin is necessary for the rapid effects of ALDO. We also measured phosphorylated ERK-1/2 (pERK) levels that peaked within 10 minutes in EA as estimated by western blot analyses. Consistent with these observations pre-incubation of EA cells with 10-6M PD0325901, a selective MEK-1/2 inhibitor was associated with greater than 90% reduction of the ALDO-stimulated ROS responses (7244.3±497 vs 4386.6±586 RFU, P<0.02, n=3). Qualitatively similar responses were observed using another MEK inhibitor, U0126 [10-5M] (P<0.01, n=3). We then tested the effects of ALDO on PDI secretion. Incubation of EA cells with ALDO (10-7M) led to PDI increases when compared to vehicle treated cells (P<0.01, n=3). We also tested the effects of low levels of striatin using siRNA on PDI activity in EA cells. We found that PDI secretion was reduced by 62% in striatin knockdown conditions. We then tested the effects of Methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted 10nM ALDO–stimulated PDI response (530±117 to 215±99 RFU/mg protein, n=3, P<0.01). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from endothelial-specific caveolin-1 knockout mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from caveolin-1 knockout mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). These results suggest that striatin is a novel mediator for ALDO's rapid effects on PDI and ROS, thereby suggesting a unique level of interaction between the MR and striatin in endothelial cell activation. Supported by NIH R01HL090632 (AR), R01HL104032 (LHP) and R01HL096518 (JRR). Disclosures:No relevant conflicts of interest to declare.

The Long Coiled-Coil Protein NECC2 Is Associated to Caveolae and MODULATES NGF/TrkA Signaling IN PC12 CELLS

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.

The less-often-traveled surface of stem cells: caveolin-1 and caveolae in stem cells, tissue repair and regeneration

Stem cells are an important resource for tissue repair and regeneration. While a great deal of attention has focused on derivation and molecular regulation of stem cells, relatively little research has focused on how the subcellular structure and composition of the cell membrane influences stem cell activities such as proliferation, differentiation and homing. Caveolae are specialized membrane lipid rafts coated with caveolin scaffolding proteins, which can regulate cholesterol transport and the activity of cell signaling receptors and their downstream effectors. Caveolin-1 is involved in the regulation of many cellular processes, including growth, control of mitochondrial antioxidant levels, migration and senescence. These activities are of relevance to stem cell biology, and in this review evidence for caveolin-1 involvement in stem cell biology is summarized. Altered stem and progenitor cell populations in caveolin-1 null mice suggest that caveolin-1 can regulate stem cell proliferation, and in vitro studies with isolated stem cells suggest that caveolin-1 regulates stem cell differentiation. The available evidence leads us to hypothesize that caveolin-1 expression may stabilize the differentiated and undifferentiated stem cell phenotype, and transient downregulation of caveolin-1 expression may be required for transition between the two. Such regulation would probably be critical in regenerative applications of adult stem cells and during tissue regeneration. We also review here the temporal changes in caveolin-1 expression reported during tissue repair. Delayed muscle regeneration in transgenic mice overexpressing caveolin-1 as well as compromised cardiac, brain and liver tissue repair and delayed wound healing in caveolin-1 null mice suggest that caveolin-1 plays an important role in tissue repair, but that this role may be negative or positive depending on the tissue type and the nature of the repair process. Finally, we also discuss how caveolin-1 quiescence-inducing activities and effects on mitochondrial antioxidant levels may influence stem cell aging.

Caveolae-dependent internalization and homologous desensitization of VIP/PACAP receptor, VPAC2, in gastrointestinal smooth muscle

The main membrane proteins of caveolae (caveolin-1, -2 and -3) oligomerize within lipid rich domains to form regular invaginations of smooth muscle plasma membrane and participate in receptor internalization and desensitization independent of clathrin-coated vesicle endocytosis. We have previously shown that Gs-coupled VIP/PACAP receptors, VPAC2, predominantly expressed in smooth muscle cells of the gut, are exclusively phosphorylated by GRK2 leading to receptor internalization and desensitization. Herein, we characterized the role of caveolin-1 in VPAC2 receptor internalization and desensitization in gastric smooth muscle using three approaches: (i) methyl β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae in dispersed muscle cells, (ii) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and (iii) caveolin-1 knockout mice (caveolin-1−/−). Pretreatment of gastric muscle cells with VIP stimulated tyrosine phosphorylation of caveolin-1, and induced VPAC2 receptor internalization (measured as decrease in 125I-VIP binding after pretreatment) and desensitization (measured as decrease in VIP-induced cAMP formation after pretreatment). Caveolin-1 phosphorylation, and VPAC2 receptor internalization and desensitization were blocked by disruption of caveolae with MβCD, suppression of caveolin-1 with caveolin-1 siRNA or inhibition of Src kinase activity by PP2. Pretreatment with VIP significantly inhibited adenylyl cyclase activity and muscle relaxation in response to subsequent addition of VIP in freshly dispersed muscle cells and in muscle strips isolated from wild type and caveolin-1−/− mice; however, the inhibition was significantly attenuated in caveolin-1−/− mice. These results suggest that caveolin-1 plays an important role in VPAC2 receptor internalization and desensitization.

Abstract 19025: A Signaling Complex of HCN2/HCN4/Cav3 Resides in Caveolae of Cardiac Cells

Macromolecular signaling complexes including ion channels present in the caveolar micro-domains can provide highly specific and localized regulation of cell function. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key constituents of the cardiac pacemaker, regulating the rate of diastolic depolarization and cardiac rhythm. HCN4 and HCN2 are the major HCN isoforms found in cardiac tissues. HCN4 is physically associated with Caveolin-3 (Cav3), the “coat protein” for caveolae in cardiac cells. In this report, we dissected the regulation of Cav3 on HCN2. Cardiac HCN2 (60-kDa, HCN2s) or HCN4 was co-localized with Cav3 in mouse LV membrane fractions. A dominant-negative P104L-Cav3 mutation was used to “probe” the association of HCN2/Cav3. HCN2s itself does not encode currents but HCN2s/HCN4 channels generated currents resembled to those recorded from native cells. HEK293 cells expressing full-length-HCN2 (HCN2f)/WTCav3 showed comparable HCN2 density and channel activity to those cells expressing HCN2f/P104L or HCN2 alone. Bioluminescence Resonance Energy Transfer (BRET) assays in conjunction with stable HCN cell lines and lipid raft inhibitors were used to quantify the intra-molecular interactions of each HCN form and Cav3. WTCav3 or P104L was fused to a Renilla Luciferase (Rluc) as the “donor” while each HCN form was fused to YFP as the “acceptor”. All HCN/Cav3 pairs displayed positive BRET ratios, suggesting that HCN and Cav3 were within a 10-100 Å distance to permit energy transfer from RLuc (470-nm) to YFP (530-nm) in the presence of coelenterazine-h. We did not record positive BRET ratios from samples containing no assay substrate. Average BRET ratio data (n=6, p&lt;0.05) suggested that the association for HCN2s/Cav3 is the strongest. The P104L study showed that aa104 is involved in the interactions of HCN2s/Cav3 or HCN4/Cav3 but not HCN2f/Cav3. Our results indicate that Cav3 interacts with HCN4 via HCN2s to form a HCN2s/HCN4/Cav3/caveolae complex, and it may play an important role in cardiac signaling.

Caveolin-1, caveolae, and glioblastoma

Glioblastoma multiforme (GBM) is the most common malignant brain tumor and is characterized by high invasiveness, poor prognosis, and limited therapeutic options. Biochemical and morphological experiments have shown the presence of caveolae in glioblastoma cells. Caveolae are flask-shaped plasma membrane subdomains that play trafficking, mechanosensing, and signaling roles. Caveolin-1 is a membrane protein that participates in the formation of caveolae and binds a multitude of signaling proteins, compartmentalizing them in caveolae and often directly regulating their activity via binding to its scaffolding domain. Caveolin-1 has been proposed to behave either as a tumor suppressor or as an ongogene depending on the tumor type and progress. This review discusses the existing information on the expression and function of caveolin-1 and caveolae in GBM and the role of this organelle and its defining protein on cellular signaling, growth, and invasiveness of GBM. We further analyze the available data suggesting caveolin-1 could be a target in GBM therapy.

Deletion of myosin light chain kinase in endothelial cells has a minor effect on the lipopolysaccharide‐induced increase in microvascular endothelium permeability in mice

There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)-mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS-induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild-type mice. However, the LPS-induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS-mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.

Phospholipase A2 activating protein is required for 1α,25-dihydroxyvitamin D3 dependent rapid activation of protein kinase C via Pdia3

1α,25-Dihydroxyvitamin D3 (1,25D3) regulates musculoskeletal cells via two different mechanisms: vitamin D receptor (VDR)-dependent gene transcription and rapid membrane-signaling via VDR as well as protein disulfide isomerase, family A, member 3 (Pdia3). In chondrocytes from the costochondral cartilage growth zone (GC), ligand binding to Pdia3 causes a rapid increase in phospholipase A2 (PLA2) activity leading to release of arachidonic acid and formation of lysophospholipid (LPL). LPL activates phospholipase C (PLC), and resulting inositol trisphosphate (IP3) and diacylglycerol contribute to PKCα activation and downstream activation of ERK1/2. PLA2 activating protein (PLAA) is increased in the growth zone of rat growth plates suggesting that it mediates the 1,25D3-dependent pathway. This study examined the role of PLAA in mediating 1,25D3-dependent PKC activation using GC cells and MC3T3-E1 wild-type and PLAA-silenced osteoblasts as models. PLAA, Pdia3, and caveolin-1 (Cav-1) were detected in plasma membranes and caveolae of GC and MC3T3-E1 cells. Pdia3-immunoprecipitated samples were positive for PLAA only after 1,25D3 treatment. Cav-1 was detected when immunoprecipitated with anti-Pdia3 and anti-PLAA in both vehicle and 1,25D3 treated cells. These observations were confirmed by immunohistochemistry. 1,25D3 failed to activate PLA2 and PKC or cause PGE2 release in PLAA-silenced cells. PLAA-antibody successfully blocked the PLAA protein and consequently suppressed PKC activity in GC and MC3T3-E1 cells. Crosslinking studies confirmed the localization of PLAA on the extracellular face on the plasma membrane in untreated MC3T3-E1 cells. Taken together, our results suggest that PLAA is an important mediator of 1α,25(OH)2D3 rapid membrane mediated signaling. 1α,25(OH)2D3 likely causes conformational changes bringing Pdia3 into proximity with PLAA, and aiding in transducing the signal from caveolae to the plasma membrane.

Role of protein kinase C in phospholemman mediated regulation of α2β1 isozyme of Na+/K+-ATPase in caveolae of pulmonary artery smooth muscle cells

We have recently reported that α2β1 and α1β1 isozymes of Na+/K+-ATPase (NKA) are localized in the caveolae whereas only the α1β1 isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α2 isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α1 isoform of NKA. To investigate the mechanism of regulation of α2 isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na+; and (iii) even after phosphorylation by PKC, PLM still remains associated with the α2 isoform of NKA.

Genetic variation in the mouse model of Niemann Pick C1 affects female, as well as male, adiposity, and hepatic bile transporters but has indeterminate effects on caveolae

We have previously shown that male Npc1 heterozygous mice ( Npc1 +/− ), as compared to homozygous wild-type mice ( Npc1 +/+ ), both maintained on the “lean” BALB/cJ genetic background, become obese on a high fat but not on a low fat diet. We have now extended this result for female heterozygous mice. When fed high-fat diet, the Npc1 +/− white adipose weight is also increased in females, therefore following the same trend as males. Bile transporters which had previously been found to be altered in Npc1 −/− mice on a high fat diet, showed related, but small, changes in mRNA levels but large changes in protein expression. We have addressed the possible role of caveolae in these differences. It has long been known that caveolin 1 is increased in the liver (sex not specified) of Npc1 +/− (compared to Npc1 +/+ and Npc1 −/− ) mice and in heterozygous cultured skin fibroblasts of NPC1 carriers. We now find that caveolin 1 is increased in male, but not female liver and female, but not male adipose tissue. The caveolin 1 increase was not accompanied by changes in another caveolar protein, polymerase1 and transcript release factor (Ptrf). The numbers of caveolae in female adipose cells could not be correlated with levels of caveolae. Thus, we conclude that Npc1 affects female as well as male obesity and bile transporters but that effects on caveolin 1 are not discernible.

Caveolar vesicles generate DNA damage and perpetuate cellular aging

Caveolar vesicles generate DNA damage and perpetuate cellular aging

Changes in caveolae, caveolin, and polymerase 1 and transcript release factor (PTRF) expression in prostate cancer progression

Caveolae are specialized invaginations in the cell membrane involved in the regulation of cell transport and signal transduction. The aims of this study were to investigate the number of caveolae and expression of caveolae-associated proteins, caveolin-1 and -2, and polymerase 1 and transcript release factor (PTRF) with development of prostate cancer. Transmission electron microscopy was used to investigate the number of caveolae in normal human prostate stromal, epithelial cells, and androgen-dependent (LNCaP) and androgen-independent (PC3) cancer cell lines. Surgical tissue was obtained from patients with benign prostatic hyperplasia (BPH), well-differentiated and poorly differentiated prostate cancer. Caveolin-1, caveolin-2, and PTRF expression was identified by immunohistochemistry in tissue samples and quantified by Western blot analysis in cell lines. Caveolae were identified in normal epithelial and stromal prostate cells. The number of caveolae was significantly reduced in LNCaP and PC3 cells (P < 0.0001). PTRF was localized to stromal and epithelial cells in tissue from patients with BPH and in normal stromal and epithelial cells in vitro. PTRF expression was significantly decreased in LNCaP and PC3 cells and also in cancer tissue. In prostate tissue, caveolin-1 and -2 expression appeared to increase in prostate cancer. Caveolin-1 and -2 expression was decreased in LNCaP cells but caveolin-2 expression was significantly increased in PC3 cells compared to normal epithelial cells. This study demonstrates that changes in the cell membrane involving loss of caveolae and PTRF expression occur with the development of prostate cancer. These changes are accompanied by an up-regulation of caveolin-2.