Methamphetamine enhances caveolar transport of therapeutic agents across the rodent blood-brain barrier.

The blood-brain barrier (BBB) restricts clinically relevant accumulation of many therapeutics in the CNS. Low-dose methamphetamine (METH) induces fluid-phase transcytosis across BBB endothelial cells in vitro and could be used to enhance CNS drug delivery. Here, we show that low-dose METH induces significant BBB leakage in rodents ex vivo and in vivo. Notably, METH leaves tight junctions intact and induces transient leakage via caveolar transport, which is suppressed at 4°C and in caveolin-1 (CAV1) knockout mice. METH enhances brain penetration of both small therapeutic molecules, such as doxorubicin (DOX), and large proteins. Lastly, METH improves the therapeutic efficacy of DOX in a mouse model of glioblastoma, as measured by a 25% increase in median survival time and a significant reduction in satellite lesions. Collectively, our data indicate that caveolar transport at the adult BBB is agonist inducible and that METH can enhance drug delivery to the CNS.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Adhesion-dependent Caveolin-1 Tyrosine-14 phosphorylation is regulated by FAK in response to changing matrix stiffness.

Integrin-mediated adhesion regulates cellular responses to changes in the mechanical and biochemical properties of the extracellular matrix. Cell-matrix adhesion regulates caveolar endocytosis, dependent on caveolin 1 (Cav1) Tyr14 phosphorylation (pY14Cav1), to control anchorage-dependent signaling. We find that cell-matrix adhesion regulates pY14Cav1 levels in mouse fibroblasts. Biochemical fractionation reveals endogenous pY14Cav1 to be present in caveolae and focal adhesions (FA). Adhesion does not affect caveolar pY14Cav1, supporting its regulation at FA, in which PF-228-mediated inhibition of focal adhesion kinase (FAK) disrupts. Cell adhesion on 2D polyacrylamide matrices of increasing stiffness stimulates Cav1 phosphorylation, which is comparable to the phosphorylation of FAK. Inhibition of FAK across varying stiffnesses shows it regulates pY14Cav1 more prominently at higher stiffness. Taken together, these studies reveal the presence of FAK-pY14Cav1 crosstalk at FA, which is regulated by cell-matrix adhesion.

Lentinan-functionalized Selenium Nanoparticles target Tumor Cell Mitochondria via TLR4/TRAF3/MFN1 pathway.

Rationale: Malignant ascites caused by cancer cells results in poor prognosis and short average survival time. No effective treatment is currently available for malignant ascites. In this study, the effects of lentinan (LNT)-functionalized selenium nanoparticles (Selene) on malignant ascites were evaluated. Furthermore, the mechanism of Selene targeting mitochondria of tumor cells were also investigated. Methods: Selene were synthesized and characterized by TEM, AFM and particle size analysis. The OVCAR-3 and EAC cells induced ascites models were used to evaluate the effects of Selene on malignant ascites. Proteomic analysis, immunofluorescence, TEM and ICP-MS were used to determine the location of Selene in tumor cells. Mitochondrial membrane potential, ROS, ATP content, and caspase-1/3 activity were detected to evaluate the effect of Selene on mitochondrial function and cell apoptosis. Immunofluorescence, Co-IP, pull-down, duolink, Western blot, and FPLC were used to investigate the pathway of Selene targeting mitochondria. Results: Selene could effectively inhibit ascites induced by OVCAR-3 and EAC cells. Selene was mainly located in the mitochondria of tumor cells and induced apoptosis of tumor cells. The LNT in Selene was involved in caveolae-mediated endocytosis through the interaction between toll-like receptor-4 (TLR4) and caveolin 1 (CAV1). Furthermore, the Selene in the endocytic vesicles could enter the mitochondria via the mitochondrial membrane fusion pathway, which was mediated by TLR4/TNF receptor associated factor 3 (TRAF3)/mitofusin-1 (MFN1) protein complex. Conclusion: Selene is a candidate anticancer drug for the treatment of malignant ascites. And TLR4/TRAF3/MFN1 may be a specific nano-drug delivery pathway that could target the mitochondria.

Understanding SARS-CoV-2 endocytosis for COVID-19 drug repurposing.

The quest for the effective treatment against coronavirus disease 2019 pneumonia caused by the severe acute respiratory syndrome (SARS)-coronavirus 2(CoV-2) coronavirus is hampered by the lack of knowledge concerning the basic cell biology of the infection. Given that most viruses use endocytosis to enter the host cell, mechanistic investigation of SARS-CoV-2 infection needs to consider the diversity of endocytic pathways available for SARS-CoV-2 entry in the human lung epithelium. Taking advantage of the well-established methodology of membrane trafficking studies, this research direction allows for the rapid characterisation of the key cell biological mechanism(s) responsible for SARS-CoV-2 infection. Furthermore, 11 clinically approved generic drugs are identified as potential candidates for repurposing as blockers of several potential routes for SARS-CoV-2 endocytosis. More broadly, the paradigm of targeting a fundamental aspect of human cell biology to protect against infection may be advantageous in the context of future pandemic outbreaks.

Activation of ß1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival.

The tissue factor/coagulation factor VIIa (TF/FVIIa) complex induces transactivation of the IGF-1 receptor (IGF-1R) in a number of different cell types. The mechanism is largely unknown. The transactivation leads to protection from apoptosis and nuclear translocation of the IGF-1R. The aim of this study was to clarify the signaling pathway between TF and IGF-1R after FVIIa treatment with PC3 and DU145 prostate or MDA-MB-231 breast cancer cells as model systems. Protein interactions, levels, and phosphorylations were assessed by proximity ligation assay or flow cytometry in intact cells and by western blot on cell lysates. The transactivation of the IGF-1R was found dependent on TF/FVIIa-induced activation of ß1-integrins. A series of experiments led to the conclusion that the caveolae protein caveolin-1 prevented IGF-1R activation in resting cells via its scaffolding domain. TF/FVIIa/ß1-integrins terminated this inhibition by activation of Src family kinases and subsequent phosphorylation of caveolin-1 on tyrosine 14. This phosphorylation was not seen after treatment with PAR1 or PAR2 agonists. Consequently, the protective effect of FVIIa against apoptosis induced by the death receptor agonist TRAIL and the de novo synthesis of cyclin D1 induced by nuclear IGF-1R accumulation were both significantly reduced by down-regulation of ß1-integrins or overexpression of the caveolin-1 scaffolding domain. In conclusion, we present a plausible mechanism for the interplay between TF and IGF-1R involving FVIIa, ß1-integrins, Src family proteins, and caveolin-1. Our results increase the knowledge of diseases associated with TF and IGF-1R overexpression in general but specifically of TF-mediated signaling with focus on cell survival.

Salvianolic acid A increases the accumulation of doxorubicin in brain tumors through Caveolae endocytosis.

Brain glioma is one of the most common brain tumors in the central nervous system (CNS). The blood-brain tumor barrier (BTB) restricts the delivery of anti-tumor drugs into tumor tissue in the brain. Therefore, improving the transportation of antineoplastic drugs across the BTB is essential to ameliorate treatment of brain tumors. The present study was performed to explore the effect and mechanism of salvianolic acid A (Sal A) on transportation of doxorubicin (Dox) across the BTB in vivo and in vitro. By creating a brain C6 glioma model in rats, we demonstrated that Sal A significantly increased the level of Dox in brain tumor tissue as shown by liquid chromatograph mass spectrometry. Interestingly, we found that Sal A increased transendothelial electrical resistance (TEER) values of the BTB and decreased the permeability of FITC-Dextran (4kD) across the BTB in vitro. Furthermore, the expression of tight junction proteins (TJs) in glioma endothelial cells (GECs) and brain tumor microvessels were also increased, suggesting that Sal A enhanced delivery of Dox across the BTB independent of the paracellular pathway. Next, we detected that Sal A had an effect on transcellular transport of compounds across the BTB. The accumulation of FITC-labeled bovine serum albumin (FITC-BSA) was significantly increased in GECs after treatment with Sal A (10 µM) for 6h, which was inhibited after pre-treatment with methyl-ß-cyclodextrin (MßCD) for 30 min. The increased delivery of Dox across the BTB was also reduced after treatment with MßCD. In addition, phosphorylation levels of protein kinase B (PKB) and tyrosine protein kinase-Src family (Src) were increased in the Sal A treatment group. Sal A up-regulated the expression level of the phosphorylation of Caveolin-1 (pCaveolin-1), and this effect was reversed by a PKB or Src inhibitor. Taken together, our study showed for the first time that Sal A facilitated the delivery of antitumor drugs into brain tumor tissues by targeting the PKB/Src/Caveolin-1 signaling pathway.

Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide and Its Immunological Effect on Macrophages.

BACKGROUND: Rehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages. METHODS: pRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry. RESULTS: The lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG2000 of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells. CONCLUSION: Our study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.

Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL.

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma.

Lifelong n-3 Polyunsaturated Fatty Acid Exposure Modulates Size of Mammary Epithelial Cell Populations and Expression of Caveolae Resident Proteins in Fat-1 Mice.

Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been associated with reduced breast cancer risk; however, the exact mechanism remains elusive. Female wildtype (WT) and fat-1 mice were fed a 10% safflower diet until 6 weeks of age. Mammary gland epithelial cells (EC) were isolated and EC populations were determined by CD24 surface expression. Fat-1 mice expressed 65%, 20%, and 15% while WT mice expressed 65%, 26% and 9% for non-, myo- and luminal ECs, respectively. The luminal EC population was significantly greater in fat-1 mice (p ≤ 0.05), while the total number of mammary ECs were similar between groups (p = 0.79). Caveolae was isolated from ECs and Her-2/neu, ER-α and cav-1 protein expression was determined by Western blotting. Fat-1 mice had a two-fold greater ER-α (p ≤ 0.05) and a 1.5-fold greater cav-1 (p ≤ 0.05) expression than WT with a similar amount of Her-2/neu protein (p = 0.990) between groups. Overall, this study provides novel mechanistic evidence by which n-3 PUFA modifies early mammary gland development that may potentially reduce breast cancer risk later in life.

Sertraline Reduces Albuminuria by Interfering with Caveolae-Mediated Endocytosis through Glomerular Endothelial and Epithelial Cells.

INTRODUCTION: Previously, we reported the caveolae-mediated intracellular trafficking pathway of albumin through glomerular endothelial cells (GEnCs) as a new etiological hypothesis of urinary albumin excretion. The selective serotonin reuptake inhibitor, sertraline (Ser), inhibits dynamin, which plays a pivotal role in the fission of caveolae from the cell membrane during caveolae endocytosis. OBJECTIVE: In this study, we evaluated whether Ser reduces albuminuria levels by interfering with albumin endocytosis through caveolae into GEnCs and podocytes as a novel treatment for glomerulonephritis. METHODS: After treating the cells with Ser, albumin and caveolin-1 (Cav-1) expression levels were evaluated by immunofluorescence (IF) and western blot (WB) analyses. The albuminuria level was determined by histology in a puromycin aminonucleoside (PAN)-induced nephrotic syndrome mouse model (PAN mice) treated with or without Ser. RESULTS: IF and WB analyses showed that the albumin expression level was significantly decreased by Ser treatment; however, Cav-1 expression was not decreased in GEnCs or podocytes based on the IF results. In PAN mice treated with or without Ser, Cav-1 expression increased, and the foot process effacement of podocytes and swelling of GEnCs were observed. However, proteinuria levels were not increased in PAN mice treated with Ser relative to that in normal control mice (p = 0.17), and a significant increase was observed in PAN mice without Ser treatment (p = 0.0027). CONCLUSIONS: Ser interfered with albumin internalization through the caveolae into GEnCs and podocytes and reduced albuminuria. Dynamin inhibitors may serve as a novel therapeutic option for reducing albuminuria in glomerulonephritis.

Endocytosis of GM-CSF receptor ß is essential for signal transduction regulating mesothelial-macrophage transition.

During Freund's adjuvant induced inflammation rat mesenteric mesothelial cells transdifferentiate into mesenchymal cell. They express macrophage markers, inflammatory cytokines (TGF-ß, TNFα, IL-6), and specific receptors. When primary mesenteric cultures were treated with GM-CSF and/or TGF-ß (in vitro), similar phenotypic and biological changes were induced. It seemed likely that GM-CSF receptor-ligand complex should be internalized to initiate mesothelial-macrophage transition. To follow the intracellular route of GM-CSF receptor ß, we co-localized this receptor with various endocytic markers (Cav-1, EEA1, Rab7, and Rab11a), and carried out detailed immunocytochemical, statistical and biochemical analyses. Since STAT5 is one of the downstream element of GM-CSF signaling, we followed the expression and phosphorylation level of this transcription factor. Our results showed that in mesenteric mesothelial cells GM-CSF receptor ß is internalized by caveolae, delivered into early endosomes where the signaling events occur, STAT5A is phosphorylated by JAK2, and then translocated into the nucleus. When dynamin-dependent endocytosis of GM-CSFR ß is inhibited by dynasore, phosphorylation of STAT5A is not occurred, confirming, that the internalization of receptor ß is indispensable for signal transduction. At the early time of inflammation a significant receptor recycling can be found to the plasma membrane. Later (day 8) the receptor is delivered into late endosomes, indicating that its degradation has already started, and the regeneration of mesothelial cells can start. All of these data strongly support that the internalization of GM-CSF receptor ß is required and essential for signal transduction.

Helium-Induced Changes in Circulating Caveolin in Mice Suggest a Novel Mechanism of Cardiac Protection.

The noble gas helium (He) induces cardioprotection in vivo through unknown molecular mechanisms. He can interact with and modify cellular membranes. Caveolae are cholesterol and sphingolipid-enriched invaginations of the plasma-membrane-containing caveolin (Cav) proteins that are critical in protection of the heart. Mice (C57BL/6J) inhaled either He gas or adjusted room air. Functional measurements were performed in the isolated Langendorff perfused heart at 24 h post He inhalation. Electron paramagnetic resonance spectrometry (EPR) of samples was carried out at 24 h post He inhalation. Immunoblotting was used to detect Cav-1/3 expression in whole-heart tissue, exosomes isolated from platelet free plasma (PFP) and membrane fractions. Additionally, transmission electron microscopy analysis of cardiac tissue and serum function and metabolomic analysis were performed. In contrast to cardioprotection observed in in vivo models, the isolated Langendorff perfused heart revealed no protection after He inhalation. However, levels of Cav-1/3 were reduced 24 h after He inhalation in whole-heart tissue, and Cav-3 was increased in exosomes from PFP. Addition of serum to muscle cells in culture or naïve ventricular tissue increased mitochondrial metabolism without increasing reactive oxygen species generation. Primary and lipid metabolites determined potential changes in ceramide by He exposure. In addition to direct effects on myocardium, He likely induces the release of secreted membrane factors enriched in caveolae. Our results suggest a critical role for such circulating factors in He-induced organ protection.

Ganglioside GM1 promotes contact inhibition of growth by regulating the localization of epidermal growth factor receptor from glycosphingolipid-enriched microdomain to caveolae.

OBJECTIVES: Accumulating data show that gangliosides are involved in regulation of cell proliferation. Specific changes in gangliosides expression associated with growth density of cells have been documented in several cell lines. However, the function and the potential mechanism of ganglioside GM1 in contact inhibition of growth are not clear. MATERIALS AND METHODS: EdU incorporation assay and western blot were applied to detect the contact inhibition of growth in human mammary epithelial cells. GM1 manipulation of cell proliferation and epidermal growth factor receptor (EGFR) activation was investigated by immunoprecipitation, OptiPrep density gradient centrifugation and immunofluorescence. The function of GM1 on contact inhibition of growth was further studied by using GM1 stably knockdown and overexpression cells. RESULTS: MCF-10A, MCF-7 and MDA-MB-231 cells showed contact inhibition of growth in high-density condition. Exogenous addition of GM1 to high-density cells clearly inhibited cell growth and deactivated EGFR signalling. Compared to normal-density cells, distribution of EGFR in high-density cells was decreased in glycosphingolipid-enriched microdomain (GEM), but more concentrated in caveolae, and incubation with GM1 obviously promoted this translocation. Furthermore, the cell growth and EGFR activation were increased in GM1 stably knockdown cells and decreased in GM1 stably overexpression cells when cultured in high density. CONCLUSIONS: Our results demonstrated that GM1 suppressed EGFR signalling and promoted contact inhibition of growth by changing the localization of EGFR from GEM to caveolae.

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands.

Enhancement of Vascular Smooth Muscle Contractility by Alterations of Membranous Architecture.

Plasma membrane (PM) of smooth muscle cells hosts channel molecules regulating the flow of various ions. An intact architecture of PM is essential to orchestrate proper channel functions in order to complete agonist-mediated contraction, which includes Ca2+ release from the sarcoplasmic reticulum (SR) to initiate contraction, and subsequent Ca2+ refilling into SR through PM to sustain muscle contraction. The Junctional Complex (JC), comprising of junctional SR, and its apposing PM and neighboring caveolae, provides a quasi-enclosed microdomain housing receptors as well as ion channels and also restricting ion diffusions into the cytosol so the cell achieves optimal performance. The spatial arrangement of the JC is believed to ensure an uninterrupted Ca2+ cycling route. Full understanding of the functional role of the JC is the key to elucidating the contractile mechanisms of vascular smooth muscle and the physiological function of vessel contraction. The JC can be further divided into two sub-divisions, namely the PM-SR and caveolar regions. Previously, we demonstrated the role of the PM-SR region in the initiation of muscle contraction using pharmacological tools on the inferior vena cava (IVC) of rabbit. In the current study, we further dissected the caveolar region using a cholesterol-disrupting agent to investigate the role of the caveolar region. We conclude that disruption of the caveolar region in rabbit IVC smooth muscle results in augmented muscle contraction in response to adrenergic stimulation and the altered Ca2+ signaling may underlie the augmented contractility. Anat Rec, 302:186-192, 2019. © 2018 Wiley Periodicals, Inc.

Urea-modulated UT-B urea transporter internalization is clathrin- and caveolae-dependent in infantile hemangioma-derived vascular endothelial cells.

The aim of this study was to investigate the manner of urea-modulated UT-B urea transporter (UT) internalization in infantile hemangioma-derived vascular endothelial cells (HemECs). The immunohistochemistry assay was performed to identify infancy hemangioma-derived endothelial cell line (XPTS-1) cells. Cell toxicity was detected with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Quantitative real-time polymerase chain reaction and Western blot analysis were measured to analyze the expression of UT-B. UT-B internalization was observed by confocal microscopy. The clathrin inhibitor chlorpromazine (CPZ) and caveolin endocytic disrupter methyl-ß-cyclodextrin (MßCD) were used in XPTS-1 cells transfected with UT-B-GFP to repress endocytosis. Urea-promoted UT-B expression in a concentration-dependent manner in an infantile XPTS-1 cell line. CPZ and MßCD significantly inhibited UT-B protein internalization. The pretreatment of UT-B-GFP cells with adaptor protein2 (AP2)-µ2-siRNA and caveolin-siRNA significantly inhibited UT-B protein internalization. Our findings suggested that urea-mediated UT-B UT internalization is clathrin and caveolae dependent in infantile HemECs.

Signaling of Macrophage Inflammatory Protein (MIP)-3ß Facilitates Dengue Virus-Induced Microglial Cell Migration.

The infection by dengue virus (DENV) of microglia causes cell activation and migration via a mechanism involving viral entry, RNA release, and Toll-like receptor 3 signaling. In this study, we demonstrated that secreted chemotactic factors present in microglial conditioned medium (MCM) facilitated cell motility in the murine BV2 microglial cells. The pharmacological disruption of lipid rafts/caveolae reduced DENV- and ultraviolet (UV)-inactivated MCM-induced microglial cell migration. An antibody-based cytokine/chemokine array showed an increase in macrophage inflammatory protein (MIP)-3ß in MCM produced using DENV-infected cells. The pharmacological inhibition of c-Jun N-terminal kinase (JNK) retarded UV-MCM-induced microglial cell migration. These results demonstrate that secreted MIP-3ß and its effect on the JNK signaling pathways mediates DENV-induced BV2 microglial cell migration.

Chemotherapy induced PRL3 expression promotes cancer growth via plasma membrane remodeling and specific alterations of caveolae-associated signaling.

BACKGROUND: The outcome of cancer therapy is greatly defined by the ability of a tumor cell to evade treatment and re-establish its bulk mass after medical interventions. Consequently, there is an urgent need for the characterization of molecules affecting tumor reoccurrence. The phosphatase of regenerating liver 3 (PRL3) protein was recently emerged among the targets that could affect such a phenomenon. METHODS: The expression induction of PRL3 in melanoma cells treated with chemotherapeutic agents was assessed by western blotting. The effect of PRL3 expression on cancer growth was investigated both in vitro and in vivo. The association of PRL3 with the caveolae structures of the plasma membrane was analyzed by detergent free raft purification. The effect of PRL3 expression on the membrane organization was assayed by electron microscopy and by membrane biophysical measurements. Purification of the plasma membrane fraction and co-immunoprecipitation were used to evaluate the altered protein composition of the plasma membrane upon PRL3 expression. RESULTS: Here, we identified PRL3 as a genotoxic stress-induced oncogene whose expression is significantly increased by the presence of classical antitumor therapeutics. Furthermore, we successfully connected the presence of this oncogene with increased tumor growth, which implies that tumor cells can utilize PRL3 effects as a survival strategy. We further demonstrated the molecular mechanism that is connected with the pro-growth action of PRL3, which is closely associated with its localization to the caveolae-type lipid raft compartment of the plasma membrane. In our study, PRL3 was associated with distinct changes in the plasma membrane structure and in the caveolar proteome, such as the dephosphorylation of integrin ß1 at Thr788/Thr789 and the increased partitioning of Rac1 to the plasma membrane. These alterations at the plasma membrane were further associated with the elevation of cyclin D1 in the nucleus. CONCLUSIONS: This study identifies PRL3 as an oncogene upregulated in cancer cells upon exposure to anticancer therapeutics. Furthermore, this work contributes to the existing knowledge on PRL3 function by characterizing its association with the caveolae-like domains of the plasma membrane and their resident proteins.

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-ß-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.