Caveolin-1 Phosphorylation Research Articles

Src-mediated phosphorylation, ubiquitination and degradation of Caveolin-1 promotes breast cancer cell stemness

Cancer stem cells (CSCs) are responsible for tumor initiation, metastasis and recurrence. Caveolin-1 (Cav-1) is a major protein of caveolae, which participates in various cellular functions, such as vesicle trafficking, cholesterol homeostasis, tumor progression, etc. In the present study, we investigated a role for Cav-1 in regulating the stemness of human breast cancer (MDA-MB-231) cells. Cav-1 expression was significantly lower in tumorspheres than in adherent cells. The silencing of Cav-1 enhanced stemness of MDA-MB-231 cells. Mechanistically, Cav-1 silencing was accompanied by enhanced expression of Bmi-1, which is a representative self-renewal regulator, and promoted epithelial-mesenchymal transition. In a CSC-like state, reduced Cav-1 depends on its destabilization through ubiquitin-proteasome degradation. We further found that Src-mediated phosphorylation of Cav-1 at the Tyr 14 residue is essential for its degradation. Taken together, these findings suggest that Cav-1 destabilization by Src may play a pivotal role in manifestation and maintenance of stemness in breast cancer cells.

Identification of specific integrin cross-talk for dermal fibroblast cell adhesion using a mixed peptide-chitosan matrix.

Extracellular matrix molecules are recognized by several integrin subtypes, making identification of cross-talk among different integrin subtypes difficult. Here, we evaluated the cross-talk of integrin subtypes using four different integrin-binding peptides (FIB1; integrin αvβ3/α5β1, A2G10; integrin α6β1, EF1zz; integrin α2β1, or 531; integrin α3β1) derived from extracellular matrix molecules. Various combinations of two different integrin-binding peptides were mixed and conjugated on a chitosan matrix at various molar ratios and were evaluated for cell attachment activity. FIB1/A2G10 (molar ratio 5:5; total 10 nmol/well)-chitosan matrix significantly enhanced cell attachment activity compared with sum of the cell attachment activity on FIB1 (5 nmol/well)-chitosan matrices and A2G10 (5 nmol/well)-chitosan matrices, respectively. However, none of the other peptides showed a significant activity change when they were mixed and conjugated on a chitosan matrix. We investigated the mechanisms of this enhancement. FIB1/A2G10 (8:2 or 6:4)-chitosan matrix increased the cell spreading, phosphorylation of focal adhesion kinase at Y397, and slightly decreased phosphorylation of caveolin-1 at Y14 in fibroblasts compared with FIB1-chitosan and A2G10-chitosan matrices. These results indicate that FIB1/A2G10 (8:2 or 6:4)-chitosan matrix synergistically enhances cell attachment, suggesting that integrins αvβ3/α5β1 and α6β1 are involved in a cross-talk and synergistically enhance cell attachment. These findings also suggest that the mixed peptide-chitosan matrix system can regulate the ratio of two different peptides and is useful for evaluating cellular functions through receptor-specific cross-talk. Further, FIB1/A2G10 (8:2 or 6:4)-chitosan matrix could be a useful material for tissue engineering.

Oxidative stress-induced activation of Abl and Src kinases rapidly induces P-glycoprotein internalization via phosphorylation of caveolin-1 on tyrosine-14, decreasing cortisol efflux at the blood–brain barrier

Exposure of the brain to high levels of glucocorticoids during ischemia–reperfusion induces neuronal cell death. Oxidative stress alters blood–brain barrier (BBB) function during ischemia–reperfusion, and so we hypothesized that it might impair P-glycoprotein (P-gp)-mediated efflux transport of glucocorticoids at the BBB. Therefore, the purpose of this study was to clarify the molecular mechanism of this putative decrease of P-gp-mediated efflux function. First, we established that H2O2 treatment of a human in vitro BBB model (hCMEC/D3) reduced both P-gp efflux transport activity and protein expression on the plasma membrane within 20 min. These results suggested that the rapid decrease of efflux function might be due to internalization of P-gp. Furthermore, H2O2 treatment markedly increased tyrosine-14-phosphorylated caveolin-1, which is involved in P-gp internalization. A brain perfusion study in rats showed that cortisol efflux at the BBB was markedly decreased by H2O2 administration, and inhibitors of Abl kinase and Src kinase, which phosphorylate tyrosine-14 in caveolin-1, suppressed this decrease. Overall, these findings support the idea that oxidative stress-induced activation of Abl kinase and Src kinase induces internalization of P-gp via the phosphorylation of tyrosine-14 in caveolin-1, leading to a rapid decrease of P-gp-mediated cortisol efflux at the BBB.

MiR‐132‐3p boosts caveolae‐mediated transcellular transport in glioma endothelial cells by targeting PTEN/PI3K/PKB/Src/Cav‐1 signaling pathway

Blood-brain tumor barrier (BTB) impedes the transportation of antitumor therapeutic drugs into brain tumors. Its mechanism is still unknown, but learning how to improve the BTB permeability is critical for drug intervention. Recently, microRNAs (miRNAs) have appeared as regulation factors of numerous biologic processes and therapeutic targets of diverse diseases. In this study, we have identified that miR-132-3p is an essential miRNA by increasing the transcellular transport through the BTB. We found that miR-132-3p expression was significantly up-regulated in glioma endothelial cells (GECs). Furthermore we showed that miR132-3p+ greatly induced the endocytosis of cholera toxin subunit B and FITC-bovine serum albumin and up-regulated the expression of p-PKB, p-Src and Tyr14 phosphorylation of caveolin-1 (p-Cav-1), while phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression was markedly down-regulated in GECs. Our results identify PTEN as a direct and functional downstream target of miR-132-3p, which is involved in the regulation of p-PKB, p-Src, and p-Cav-1. The inhibitors for PI3K and Src significantly reversed the increase of p-Cav-1 induced by miR-132-3p. Moreover, overexpression of PTEN greatly reduced the endocytosis of cholera toxin subunit B and the up-regulation of p-Cav-1 induced by agomiR132-3p, suggesting that miR132-3p+ increases the endothelial permeability by inhibition of PTEN expression. In addition, miR132-3p+ significantly increased the delivery of doxorubicin across the BTB in vitro and contributed to the accumulation of doxorubicin within the brain tumor tissue. Our results show that miR-132-3p contributes to the increased permeability of BTB by targeting PTEN/PI3K/PKB/Src/Cav-1, thereby revealing a novel drug target for the treatment of brain gliomas.-Gu, Y., Cai, R., Zhang, C., Xue, Y., Pan, Y., Wang, J., Zhang, Z. miR-132-3p boosts caveolae-mediated transcellular transport in glioma endothelial cells by targeting PTEN/PI3K/PKB/Src/Cav-1 signaling pathway.

Abstract 5522: Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity

Abstract The tumor suppressor gene DLC1 encodes a multi-domain protein, including a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, RhoB, and RhoC, and has been hypothesized to be the basis of its tumor suppressor functions. Other well recognized DLC1 domains are an N-terminal sterile alpha motif (SAM) domain, and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. DLC1 is down-regulated in several malignancies by gene deletion, epigenetic promoter methylation, or post-translational mechanisms. In addition, we have recently determined, by analyzing the TCGA database that point mutations in the DLC1 gene occur frequently, and have provided evidence that most of the mutant proteins display attenuated tumor suppressor function. Moreover, the mutations are distributed along the entire coding region, implying that regions of DLC1 in addition to its RhoGAP domain contribute to its full tumor suppressor activity. Several DLC1 binding partners, in addition to Rho-GTP, have been identified. They include Caveolin-1, the principal structural component and marker of caveolae, which interacts with the START domain of DLC1. The distribution of caveolae is closely related to intracellular events, such as MAPK activation or the control of intracellular Ca2+ levels. Phospholipase C delta 1 (PLCD1) transforms PtdIns(4,5)P2 (PIP2) into inositol triphosphate (IP3) and diacyglycerol (DAG), which participate in intracellular Ca2+ mobilization and protein kinase C activation, respectively. p122RhoGAP, the rat homolog of DLC1, was initially cloned as a novel PLCD1-interacting protein, but the interacting region of PLCD1 with p122RhoGAP was not identified. In addition, others have concluded the function of PLCD1 is not affected by human DLC1. In the current study, we have characterized a colon cancer-associated DLC1 mutant (R947C) whose mutation lies in the START domain. The mutant has an attenuated tumor suppressor phenotype, as determined by deficiency in reduction of growth in soft agar and cell migration. Compared with the wild type, the binding of the mutant to PLCD1 and to Caveolin-1 is reduced, but its RhoGAP activity is intact. In addition, phosphorylation of Caveolin-1 by Src at Tyr 14 reduces its interaction with wild type DLC1, and Caveolin-1 and PLCD1 interact in vivo more efficiently with each other in the absence of DLC1. Moreover, wild type DLC1, Caveolin-1 and PLCD1 possess cooperative tumor suppressor activities in cells expressing all three genes. Overall, this study provides new insight into the role of the START domain of DLC1, and reinforces the Rho-GAP-independent regulation of tumor growth by DLC1. Citation Format: Beatriz Sanchez-Solana, Xiaolan Qian, Dunrui Wang, Marian Durkin, Brajendra Tripathi, Alex Papageorge, Douglas R. Lowy. Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5522.

Endothelial barrier function is differentially regulated by CEACAM1-mediated signaling.

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is known to be crucial to vasculogenesis and angiogenesis. Recently, CEACAM1 deficiency was shown to result in the formation of aortic plaque-like lesions, indicating a role for CEACAM1 in adult vessels as well. The underlying mechanisms remained largely elusive. Therefore, we aimed to elucidate the role of CEACAM1 in endothelial homeostasis. Here, we show that CEACAM1 deficiency causes subcellular eNOS redistribution in endothelial cells ( i.e., by eNOS depalmitoylation) and alters endothelial glycocalyx that confers antiadhesive properties to the endothelium ( i.e., by repression of glycocalyx-degrading enzymes). Accordingly, our analysis revealed an increased leukocyte-endothelial interaction in CEACAM1-deficient endothelium. In addition, CEACAM1 age dependently modulated basal and TNF-α-mediated endothelial barrier (EB) leakiness. In younger mice, CEACAM1 was protective for EB, whereas in aged mice it promoted EB leakiness. EB function depends on interendothelial adherence junctions formed by β-catenin/vascular endothelial-cadherin complexes. We show here that CEACAM1 influenced basal and TNF-α-mediated phosphorylation of β-catenin and caveolin-1, which are essential players in EB modulation. Both increased adhesiveness to leukocytes and EB modulation due to CEACAM1 deficiency may facilitate inflammatory cell transmigration into the vascular wall and subsequent plaque formation. Collectively, these results identify a crucial role for CEACAM1 in endothelial homeostasis of adult blood vessels.-Ghavampour, S., Kleefeldt, F., Bömmel, H., Volland, J., Paus, A., Horst, A., Pfeiffer, V., Hübner, S., Wagner, N., Rueckschloss, U., Ergün, S. Endothelial barrier function is differentially regulated by CEACAM1-mediated signaling.

Ox-LDL induces endothelial cell apoptosis and macrophage migration by regulating caveolin-1 phosphorylation.

Oxidative low-density lipoprotein (ox-LDL) is a risk factor for atherosclerosis. Ox-LDL leads to endothelial injury in the initial stage of atherosclerosis. In this study, we investigated the role of ox-LDL in endothelial injury and macrophage recruitment. We demonstrated that ox-LDL promoted a dose-dependent phosphorylation of caveolin-1 in human umbilical vein endothelial cells. Phosphorylated caveolin-1 increased ox-LDL uptake. Intracellular accumulation of ox-LDL induced NF-κB p65 phosphorylation, promoted HMGB1 translocation from nucleus to cytoplasm and cytochrome C release from mitochondria to cytoplasm, and activated caspase 3, resulting in cell apoptosis. NF-κB activation also facilitated cavolin-1 phosphorylation and HMGB1 expression. In addition, caveolin-1 phosphorylation favored HMGB1 release and nuclear translocation of EGR1. Nuclear translocation of EGR1 contributed to cytoplasmic translocation of HMGB1. The extracellular HMGB1 induced the migration of PMBC-derived macrophages toward HUVECs in a TLR4-dependent manner. Our results suggested that ox-LDL promoted HUVECs apoptosis and macrophage recruitment by regulating caveolin-1 phosphorylation.

Magnesium Reduces Blood-Brain Barrier Permeability and Regulates Amyloid-β Transcytosis.

Poor Mg status is a risk factor for Alzheimer's disease (AD), and the underlying mechanisms remain elusive. Here, we provided the first evidence that elevated Mg levels significantly reduced the blood-brain barrier (BBB) permeability and regulated its function in vitro. Transient receptor potential melastatin 7 (TRPM7) and magnesium transporter subtype 1 (MagT1) were two major cellular receptors mediating entry of extracellular Mg2+ into the cells. Elevated Mg levels also induced an accelerated clearance of amyloid-β peptide (Aβ) from the brain to the blood side via BBB transcytosis through low-density lipoprotein receptor-related protein (LRP) and phosphatidylinositol binding clathrin assembly protein (PICALM), while reduced the influx of Aβ from the blood to the brain side involving receptor for advanced glycation end products (RAGE) and caveolae. Mg enhanced BBB barrier properties and overall expression of LRP1 and PICALM whereas reduced that of RAGE and caveolin-1. Apical-to-basolateral and vice versa steady-state Aβ flux achieved an equilibrium of 18 and 0.27fmol/min/cm2, respectively, about 30min after the initial addition of physiological levels of free Aβ. Knockdown of caveolin-1 or disruption of caveolae membrane microdomains reduced RAGE-mediated influx significantly, but not LRP1-mediated efflux of Aβ. Stimulating endothelial cells with vascular endothelial growth factor (VEGF) enhanced caveolin-1 phosphorylation and RAGE expression. Co-immunoprecipitation demonstrated that RAGE, but not LRP1, was physically associated with caveolin-1. Thus, Mg can reduce BBB permeability and promote BBB clearance of Aβ from the brain by increasing the expression of LRP1 and PICALM while reducing the level of RAGE and caveolin-1.

Inhibition of neddylation facilitates cell migration through enhanced phosphorylation of caveolin-1 in PC3 and U373MG cells

BackgroundProtein neddylation is a post-translational modification by a covalent conjugation with the neural precursor cell expressed, developmentally downregulated 8 (NEDD8). Although this process has been reported to participate in diverse cellular signaling, little is known about its role in cancer cell migration. Given a recent proteomics report showing that NEDD8 is downregulated in prostate cancer tissues versus normal prostate tissues, we tested the possibility that neddylation plays a role in cancer evolution, and then tried to identify target proteins of the neddylation.MethodsThe neddylation process was inhibited by transfecting cancer cells with NEDD8-targeting siRNAs or by treating the cells with a NAE1 inhibitor MLN4924. Cell migration was evaluated by an in vitro wound-healing assay and a Transwell migration assay. His/NEDD8-conjugated proteins were pulled down with nickel-affinity beads under a denaturing condition, and identified by Western blotting. All data were processed using the Microsoft Excel program and analyzed statistically by two-sided, unpaired Student’s t-test.ResultsCaveolin-1, which plays a critical role in cell migration, was identified to be conjugated with NEDD8. When the neddylation was inhibited, the phosphorylation of caveolin-1 at Tyr14 was augmented in PC3 and U373MG cells, thereby leading to increased cell migration. Such consequences by neddylation inhibition were abolished in the presence of a Src family kinase inhibitor PP2.ConclusionsNEDD8 seems to inhibit the Src-mediated phosphorylation of caveolin-1 by modifying the structure of caveolin-1 protein, which blocks the migration of cancer cells. Although the neddylation process is currently regarded as an emerging target for cancer therapy, our results suggest the possibility that the inhibition of neddylation could facilitate cancer invasion or metastasis at least in some types of cancers.

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation.

The reperfusion of ischemic brain tissue following a cerebral stroke causes oxidative stress, and leads to the generation of reactive oxygen species (ROS). Apart from inflicting oxidative damage, the latter may also trigger the upregulation of aquaporin 4 (AQP4), a water-permeable channel expressed by astroglial cells of the blood-brain barrier (BBB), and contribute to edema formation, the severity of which is known to be the primary determinant of mortality and morbidity. The mechanism through which this occurs remains unknown. In the present study, we have attempted to address this question using primary astrocyte cultures treated with hydrogen peroxide (H2O2) as a model system. First, we showed that H2O2 induces a significant increase in AQP4 protein levels and that this is inhibited by the antioxidant N-acetylcysteine (NAC). Second, we demonstrated using cell surface biotinylation that H2O2 increases AQP4 cell-surface expression independently of it’s increased synthesis. In parallel, we found that caveolin-1 (Cav1) is phosphorylated in response to H2O2 and that this is reversed by the Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). PP2 also abrogated the H2O2-induced increase in AQP4 surface levels, suggesting that the phosphorylation of tyrosine-14 of Cav1 regulates this process. We further showed that dominant-negative Y14F and phosphomimetic Y14D mutants caused a decrease and increase in AQP4 membrane expression respectively, and that the knockdown of Cav1 inhibits the increase in AQP4 cell-surface, expression following H2O2 treatment. Together, these findings suggest that oxidative stress-induced Cav1 phosphorylation modulates AQP4 subcellular distribution and therefore may indirectly regulate AQP4-mediated water transport.

Snail-Induced Epithelial-to-Mesenchymal Transition Enhances P-gp-Mediated Multidrug Resistance in HCC827 Cells

Overexpression and activation of P-glycoprotein (P-gp), which mediates efflux transport of various anticancer drugs in cancer cells, is associated with multidrug resistance (MDR). On the other hand, malignant cancer cells frequently undergo epithelial-to-mesenchymal transition (EMT), thereby acquiring high migratory mobility and invasive ability. Snail is a transcriptional factor that represses multiple other factors, and its overexpression is a trigger of EMT. Because both P-gp and Snail are involved in malignant evolution of cancer, in this work, we evaluated whether EMT induced by overexpression of Snail influences P-gp expression and activity. Snail-overexpressing cells showed downregulation of epithelial markers, E-cadherin, occludin, and claudin-1, and upregulation of mesenchymal markers, vimentin and ZEB1. Although Western blot analysis showed that P-gp expression levels were similar in Mock and Snail-overexpressing cells, the results of P-gp functional assays with P-gp substrates rhodamine123 and paclitaxel indicated that P-gp is activated in Snail-overexpressing cells. Indeed, Snail-overexpressing cells showed greater viability than Mock cells in the presence of paclitaxel. We observed caveolin-1 dephosphorylation and decreased growth factor receptor-bound protein 2 (GRB2) expression in Snail-overexpressing cells. These findings suggest a novel pathway leading to cancer MDR, in which Snail induces EMT concomitantly with a decrease in GRB2-mediated caveolin-1 phosphorylation, resulting in activation of P-gp.

Caveolin 1 and G-Protein–Coupled Receptor Kinase-2 Coregulate Endothelial Nitric Oxide Synthase Activity in Sinusoidal Endothelial Cells

Liver injury leads to a vasculopathy in which post-translational modifications of endothelial nitric oxide synthase (eNOS) lead to impaired nitric oxide synthesis. We hypothesized that caveolin 1 (CAV1), a well-known eNOS interactor, regulates eNOS activity in sinusoidal endothelial cells (SECs) via its interaction with G-protein-coupled receptor kinase-2 (GRK2) that also post-translationally modifies eNOS. Liver injury with portal hypertension was established using bile duct ligation in rats. CAV1 function was modified using a CAV1 scaffolding domain construct and cDNAs encoding wild-type CAV1, and CAV1 phosphorylation was increased in injured SECs, resulting in increased GRK2-CAV1 interaction and decreased eNOS activity. In injured SECs, endothelin-1 blocked CAV1 phosphorylation induced by CAV1 scaffolding domain, indicating that CAV1 interaction with GRK2 is inversely regulated by endothelin-1 and CAV1 scaffolding domain after liver injury. In addition, after transduction with DNA encoding wild-type CAV1 into SECs isolated from Cav1-deficient mice, GRK2 association with CAV1 was evident, whereas transduction with a dominant negative CAV1 mutated at tyrosine 14 reduced the interaction. Finally, isoproterenol-induced GRK2 phosphorylation enhanced CAV1-GRK2 interaction and reduced eNOS activity. Our data suggest a novel mechanism and model in which CAV1 phosphorylation facilitates CAV1 scaffolding and GRK2-CAV1 interaction, thus clustering eNOS within a complex that inhibits eNOS activity. This process takes place in injured, but not in normal, SECs.

Inhibition of c-Src protects paraquat induced microvascular endothelial injury by modulating caveolin-1 phosphorylation and caveolae mediated transcellular permeability.

The mechanisms underlying paraquat induced acute lung injury (ALI) is still not clear. C-Src plays an important role in the regulation of microvascular endothelial barrier function and the pathogenesis of ALI. In the present study, we found that paraquat induced cell toxicity and an increase of reactive oxygen species (ROS) in endothelium. Paraquat exposure also induced significant increase of caveolin-1 phosphorylation, caveolae trafficking and albumin permeability in endothelial monolayers. C-Src depletion by siRNA significantly attenuate paraquat induced cell toxicity, caveolin-1 phosphorylation, caveolae formation and endothelial hyperpermeability. N-acetylcysteine (NAC) failed to protect endothelial monolayers against paraquat induced toxicity. Thus, our findings suggest that paraquat exposure increases paracellular endothelial permeability by increasing caveolin-1 phosphorylation in a c-Src dependant manner. The depletion of c-Src might protect microvascular endothelial function by regulating caveolin-1 phosphorylation and caveolae trafficking during paraquat exposure, and might have potential therapeutic effects on paraquat induced ALI.

Α-synuclein induces microglial cell migration through stimulating HIF-1α accumulation.

Microglial cell migration and infiltration plays a critical role in spinal cord injury after thoracoabdominal aortic surgery. In our previous study, α-synuclein, a presynaptic protein was shown to be released from injured neurons and cause microglial cell activation. Here, we aimed to explore the effect of α-synuclein on microglial cell migration. Primary microglial cells were isolated from Sprague-Dawley rats and then exposed different doses (0.2, 0.4, and 0.6 μM) of α-synuclein oligomers. The mRNA and protein levels of HIF-1α were then analyzed by qRT-PCR and Western blot. Cell migration was examined by a 96-well Boyden chamber. Moreover, toll-like receptor (TLR) 2-expression as well as TLR7/8-expression was inhibited by specific siRNA transfection. HIF-1α was overexpressed by Ad-HIF-1α transfection. In the results, α-synuclein was found to stimulate HIF-1α accumulation in microglial cells in a dose-dependent manner. Silencing HIF-1α expression dampened α-synuclein induced microglial cell migration. Furthermore, blockade of TLR7/8 expression but not TLR2 expression reduced HIF-1α accumulation in microglial cells. In addition, overexpressed HIF-1α, along with Src, prompted caveolin-1 expression and phosphorylation, as well as migration in microglial cells. Α-synuclein acts via TLR7/8 and enhances HIF-1α expression, which might play a regulatory role in microglial cell migration. © 2017 Wiley Periodicals, Inc.

Endothelial microparticles are increased in congenital heart diseases and contribute to endothelial dysfunction

BackgroundWe previously demonstrated that endothelial microparticles (EMPs) are increased in mitral valve diseases and impair valvular endothelial cell function. Perioperative systemic inflammation is an important risk factor and complication of cardiac surgery. In this study, we investigate whether EMPs increase in congenital heart diseases to promote inflammation and endothelial dysfunction.MethodsThe level of plasma EMPs in 20 patients with atrial septal defect (ASD), 23 patients with ventricular septal defect (VSD), and 30 healthy subjects were analyzed by flow cytometry. EMPs generated from human umbilical vascular endothelial cells (HUVECs) were injected into C57BL6 mice, or cultured with HUVECs without or with siRNAs targeting P38 MAPK. The expression and/or phosphorylation of endothelial nitric oxide synthase (eNOS), P38 MAPK, and caveolin-1 in mouse heart and/or in cultured HUVECs were determined. We evaluated generation of nitric oxide (NO) in mouse hearts, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cultured HUVECs and in mice.ResultsEMPs were significantly elevated in patients with ASD and VSD, especially in those with pulmonary hypertension when compared with controls. EMPs increased caveolin-1 expression and P38 MAPK phosphorylation and decreased eNOS phosphorylation and NO production in mouse hearts. EMPs stimulated P38 MAPK expression, TNF-α and IL-6 production, which were all inhibited by siRNAs targeting P38 MAPK in cultured HUVECs.ConclusionsEMPs were increased in adult patients with congenital heart diseases and may contribute to increased inflammation leading to endothelial dysfunction via P38 MAPK-dependent pathways. This novel data provides a potential therapeutic target to address important complications of surgery of congenial heart disease.

Aliskiren attenuates the effects of interleukin-6 on endothelial nitric oxide synthase and caveolin-1 in human aortic endothelial cells

Renin inhibitors enhance endothelial nitric oxide synthase (eNOS) bioavailability and have protective effects on endothelial function and atherosclerotic changes. This study was designed to investigate whether aliskiren attenuates the effects of interleukin-6 (IL-6) on eNOS and the eNOS-caveolin-1 interaction in human aortic endothelial cells (HAECs). In this study, we examined the effects of pretreatment with aliskiren on the changes of IL-6-induced expression and activation of eNOS and caveolin-1 in cultured HAECs. IL-6 inhibited and aliskiren increased the phosphorylation of eNOS at Ser1177; however, eNOS protein and mRNA expression were not changed. Pretreatment with aliskiren attenuated the inhibitory effects of IL-6 on eNOS phosphorylation and nitric oxide production. IL-6 increased the phosphorylation of caveolin-1 at Tyr14 without affecting the caveolin-1 protein and mRNA expression. Pretreatment with aliskiren attenuated the effects of IL-6 on caveolin-1 phosphorylation. The binding of eNOS and caveolin-1, as determined by a co-immunoprecipitation assay, was increased by IL-6 treatment and decreased by aliskiren pretreatment. Furthermore, treatment with short interfering RNA of the extracellular signal-regulated kinase gene reversed the effects of IL-6 and aliskiren on eNOS and caveolin-1. In conclusion, aliskiren attenuates the inhibitory effects of IL-6 on eNOS phosphorylation and nitric oxide production and IL-6 induced caveolin-1 phosphorylation. In addition, aliskiren reverses the effects of IL-6 on the eNOS-caveolin-1 interaction.

Protein Kinase C Signaling in Adenoviral Infection.

Activation of protein kinase C (PKC), a serine/threonine protein kinase, ubiquitously influences cellular signal transduction and has been shown to play a role in viral entry. In this study, we explored a role for PKC in human adenovirus type 37 infection of primary human corneal fibroblasts, a major target cell for infection. We sought evidence for an interaction between PKC activation and two potential downstream targets: cSrc kinase, shown previously to play a critical role in adenovirus signaling in these cells, and caveolin-1, reported earlier to be important to entry of adenovirus type 37. Infection of fibroblasts increased PKCα phosphorylation and translocation of PKCα from the cytosol to caveolin-1 containing vesicles. Virus-induced phosphorylation of both cSrc and AKT was abolished in cell lysates pretreated with calphostin C, a chemical inhibitor of PKC. Inhibition of PKC also reduced virus associated phosphorylation of caveolin-1, while inhibition of cSrc by the chemical inhibitor PP2 reduced only caveolin-1 phosphorylation, but not PKCα phosphorylation, in lipid rafts. These results suggest a role for PKCα upstream to both cSrc and caveolin-1. Phosphorylated PKCα was found in the same endosomal fractions as phosphorylated cSrc, and PKCα was present to a greater degree in caveolin-1 pull downs from virus infected than mock infected cell lysates. Calphostin C also reduced early viral gene expression, indicating that PKCα activity may be required for viral entry. PKCα plays a central role in adenovirus infection of corneal fibroblasts and regulation of downstream molecules, including the important lipid raft component caveolin-1.

Nucleoside diphosphate kinase B regulates angiogenic responses in the endothelium via caveolae formation and c-Src-mediated caveolin-1 phosphorylation

Nucleoside diphosphate kinase B (NDPK-B) is an enzyme required for nucleoside triphosphate homeostasis, which has been shown to interact with caveolin-1 (Cav-1). In endothelial cells (ECs), NDPK-B contributes to the regulation of angiogenesis and adherens junction (AJ) integrity. We therefore investigated whether an interaction of NDPK-B with Cav-1 in ECs is required for this regulation and the involvement of VEGF signaling herein. We report that simultaneous depletion of NDPK-B/Cav-1 in HUVECs synergistically impaired sprouting angiogenesis. NDPK-B depletion alone impaired caveolae formation, VEGF-induced phosphorylation of c-Src/Cav-1 but not of ERK1/2/AKT/eNOS. In vivo, Cav-1−/− mice showed impaired retinal vascularization at postnatal-day five, whereas NDPK-B−/− mice did not. Primary mouse brain ECs (MBMECs) from NDPK-B−/− mice showed no change in caveolae content and transendothelial-electrical resistance upon VEGF stimulation. Interestingly, NDPK-B−/− MBMECs displayed an accumulation of intracellular vesicles and increased Cav-1 levels. Dextran tracer analysis showed increased vascular permeability in the brain of NDPK-B−/− mice compared to wild type. In conclusion, our data indicate that NDPK-B is required for the correct localization of Cav-1 at the plasma membrane and the formation of caveolae. The genetic ablation of NDPK-B could partially be compensated by an increased Cav-1 content, which restored caveolae formation and some endothelial functions.

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells

Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.

4-cholesten-3-one suppresses lung adenocarcinoma metastasis by regulating translocation of HMGB1, HIF1α and Caveolin-1.

Metastasis is a great challenge in lung adenocarcinoma (ADC) therapy. Cholesterol has been implicated in ADC metastasis. 4-cholesten-3-one, as cholesterol metabolite and analog, can substitute membrane cholesterol and increase membrane fluidity. In this study, we explored the possibility that 4-cholesten-3-one inhibited ADC metastasis. Low-dose 4-cholesten-3-one significantly restrained ADC cells migration and invasion with little effects on cells viabilities. Further investigation showed that 4-cholesten-3-one promoted ROS generation, which transiently activated AMPKα1, increased HIF1α expression, reduced Bcl-2 expression and caused autophagy. AMPKα1 knockdown partly suppressed 4-cholesten-3-one-induced autophagy but, neither prevented 4-cholesten-3-one-induced upregulation of HIF1α or downregulation of Bcl-2. 4-cholesten-3-one-induced autophagy facilitated the release of HMGB1 from nuclei to cytoplasm, blocking nuclear translocation of HIF1α and activation of MMP2 and MMP9. Also, 4-cholesten-3-one induced time-dependent phosphorylation of caveolin-1, Akt and NF-κB. With increasing treatment time, 4-cholesten-3-one accelerated caveolin-1 internalization, but reduced the phosphorylation of Akt and NF-κB, and inhibited the expression of snail and twist. These data suggested that 4-cholesten-3-one could be a potential candidate for anti-metastasis of lung adenocarcinoma.