Doxorubicin enhances TRAIL-induced cell death via ceramide-enriched membrane platforms

Previous studies indicated that signalling via CD95 and DR5 is greatly enhanced by the formation of ceramide-enriched membrane platforms. Here, we employed this concept to convert doses of subtherapeutic TRAIL that were unable to release ceramide and kill leukemic B-cells or ex vivo T lymphocytes, into a very effective apoptotic stimulus. Ceramide production was induced by application of sub-toxic doses of doxorubicin that resulted in an activation of the acid sphingomyelinase (ASM), release of ceramide and formation of ceramide-enriched membrane platforms. The latter served DR5 to cluster after application of very low doses of TRAIL in combination with doxorubicin. Genetic deficiency of the ASM abrogated doxorubicin-induced ceramide release, as well as clustering of DR5 and apoptosis induced by the combined treatment of doxorubicin and TRAIL. These data show that local release of ceramide potentiates very low, otherwise inactive doses of TRAIL that may represent a novel therapeutic concept to treat tumors.     

Biological aspects of ceramide-enriched membrane domains

Ceramide has been shown to be critically involved in many aspects of cellular responses to receptor-dependent and -independent stimuli. For instance, ceramide was demonstrated to be a central component of the signaling cascades mediating apoptosis after death receptor stimulation, treatment with chemotherapy or exposure to gamma-irradiation or UV-A light. Further studies indicated the importance of ceramide for the infection of mammalian cells with bacterial, viral and parasitic pathogens. Ceramide is released by the activity of acid, neutral or alkaline sphingomyelinases or de novo synthesized. A concept unifying the diverse biological functions of ceramide indicates that ceramide forms distinct membrane domains, named ceramide-enriched membrane domains or platforms. These domains serve the clustering of receptor molecules, the re-organization of signaling proteins, the exclusion of inhibitory signals and, thus, initiate and greatly amplify a primary signal. In addition, ceramide directly interacts with and stimulates intracellular enzymes that may act together with signals initiated in ceramide-enriched membrane domains to transmit signals into a cell.     

Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes

CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca(2+) signaling by the production of cyclic ADP-ribose (cADPR), but the mechanisms by which the agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid-raft (LR) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to muscarinic type 1 (M(1)) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M(1) receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM(1), acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 microM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-beta-cyclodextrin (MCD), or filipin. With the use of a fluorescence resonance energy transfer (FRET) technique, 15.5+/-1.9% energy transfer rate (vs. 5.3+/-0.9% of control) between CD38 and LR component, ganglioside M(1) was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by Oxo. Moreover, MCD or filipin attenuated Oxo-induced production of cADPR via CD38. Functionally, Oxo-induced intracellular Ca(2+) release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for Oxo-induced activation of CD38 to produce cADPR in CAMs, and these lipid macrodomains mediate transmembrane signaling of M(1) receptor activation to produce second messenger cADPR.     

Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells

Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.     

AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells

αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl β-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.     

Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes

Background

Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis.

Methodology and Principal Findings

Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells.

Conclusions and Significance

Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.     

Differential ability of human immunodeficiency virus isolates to productively infect human cells

Isolates of HIV showed distinct differences in the ability to replicate in continuous human hematopoietic cell lines. Moreover, although all PMC cultures obtained from healthy individuals could be infected with HIV, considerable variation in the amount of virus released from different PMC cultures was observed. These biological properties of HIV could not be correlated with clinical state, binding properties of the virus isolates to target cells, or differences in target cell CD4 antigen expression. Some isolates of HIV that could not directly infect the HUT-78 cell line showed productive infection when PMC infected with these viruses were added to this human T cell line. These observations emphasize the importance of cell to cell contact in the spread of virus. The results demonstrate for the first time the differences in the host range specificity of HIV isolates in several individual PMC cultures, and indicate that the optimal isolation of HIV is achieved with normal human PMC rather than established human cell lines.     

Lateral membrane biogenesis in human bronchial epithelial cells requires 190-kDa ankyrin-G

Ankyrin-G polypeptides are required for restriction of voltage-gated sodium channels, L1 cell adhesion molecules, and beta IV spectrin to axon initial segments and are believed to couple the Na/K-ATPase to the spectrin-actin network at the lateral membrane in epithelial cells. We report here that depletion of 190-kDa ankyrin-G in human bronchial epithelial cells by small interfering RNA results in nearly complete loss of lateral plasma membrane in interphase cells, and also blocks de novo lateral membrane biogenesis following mitosis. Loss of the lateral membrane domain is accompanied by an expansion of apical and basal plasma membranes and preservation of apical-basal polarity. Expression of rat 190-kDa ankyrin-G, which is resistant to human small interfering RNA, prevents loss of the lateral membrane following depletion of human 190-kDa ankyrin-G. Human 220-kDa ankyrin-B, a closely related ankyrin isoform, is incapable of preserving the lateral membrane following 190-kDa ankyrin-G depletion. Moreover, analysis of rat 190-kDa ankyrin G/ankyrin B chimeras shows that all three domains of 190-kDa ankyrin-G are required for preservation of the lateral membrane. These results demonstrate that 190-kDa ankyrin-G plays a pleiotropic role in assembly of lateral membranes of bronchial epithelial cells.     

Protein 4.1-mediated membrane targeting of human discs large in epithelial cells

Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R.     

Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells

The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2‐C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole‐cell patch‐clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non‐malignant human tissues.     

Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na(+)/H(+) exchangers (NHE) and Cl(-)/OH(-) exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 muM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca(2+) and PKCalpha inhibition (Go6976). In vitro assays revealed that PKCalpha interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca(2+) and PKCalpha-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.     

Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells

A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [35S]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.     

Glutamine inhibits cytokine-induced apoptosis in human colonic epithelial cells via the pyrimidine pathway

Glutamine (Gln) prevents apoptosis in intestinal epithelial cells, but the mechanism(s) remain unknown. Gln-derived metabolites include ammonia, glutamate (Glu), glutathione (GSH), and nucleotides. We previously showed that Gln potently inhibited apoptosis in cytokine-treated human colonic HT-29 cells; this effect was specific to Gln, unaffected by Glu, and unrelated to intracellular GSH. The current research examines mechanism(s) for Gln-induced antiapoptotic effects in HT-29 cells treated with TNF-alpha-related apoptosis-inducing ligand (TRAIL). Proliferating cells were treated with Gln or selected Gln metabolites for 24 h. Cells were then treated with TRAIL and Gln or its downstream metabolites, and apoptosis was assessed at 8 h after treatment. The purine and pyrimidine precursors inosine and orotate inhibited TRAIL-induced apoptosis. However, inhibition of purine synthesis with azaserine did not alter the potent antiapoptotic effect of Gln. In contrast, the pyrimidine synthesis inhibitor, acivicin, completely prevented this response. Supplementation with the pyrimidine uracil or the pyrimidine precursor orotate rescued the acivicin-induced blockade of Gln antiapoptotic action. Removal of bicarbonate, a substrate for pyrimidine synthesis, also inhibited the antiapoptotic effects of Gln. Uracil and thymine alone also significantly decreased TRAIL-induced apoptosis. The antiapoptotic effects of Gln were independent of DNA/RNA synthesis as measured by flow cytometry and bromodeoxyuridine incorporation. In conclusion, Gln prevents TRAIL-induced apoptosis in HT-29 cells through a mechanism involving the pyrimidine pathway. Our data also demonstrate the novel antiapoptotic effects of pyrimidine bases and their precursor orotate in these human intestinal cells.     

The binding of Pseudomonas aeruginosa outer membrane ghosts to human buccal epithelial cells

The binding of outer membrane (OM) ghosts derived from Pseudomonas aeruginosa strain 492c to human buccal epithelial cells (BECs) was examined. Electron microscopic examination of the binding of OM ghosts to BECs revealed direct OM ghost-BEC interaction. Equilibrium analysis of the binding of OM ghosts to trypsinized BECs employing the Langmuir adsorption isotherm indicated the number of binding sites (N) to be 1.3 X 10(-4) micrograms protein per BEC with an apparent association constant (Ka) of 3.4 X 10(-2) mL/microgram protein. The Langmuir analysis of binding of OM ghosts to untrypsinized BECs was complex, suggesting two possible classes of receptors, a high affinity-low copy number class (Ka, 7.8 X 10(-2)mL/microgram protein; N, 8.6 X 10(-5) microgram protein per BEC) and a low affinity-high copy number class (Ka, 3.7 X 10(-3)mL/microgram protein; N, 9.2 X 10(-4)microgram protein per BEC). Sugar inhibition studies incorporating D-galactose enhanced binding to each BEC type. N-Acetylneuraminic acid and N-acetylglucosamine both enhanced binding of OM ghosts to untrypsinized BECs, while inhibiting binding to trypsinized BECs. D-Arabinose inhibited binding to both BEC types. Binding of OM ghosts to both BEC types was greatly inhibited by D-fucose, while L-fucose only greatly inhibited binding to untrypsinized BECs. These sugar inhibition data demonstrated a difference in the binding of OM ghosts to trypsinized and untrypsinized BECs and possibly reveal the nature of the receptor(s), free of possible bacterial metabolic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium selectivity of Reissner's membrane epithelial cells

Background  

Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K⁺ absorption via amiloride- and benzamil-sensitive electrogenic pathways.     

Basement membrane and epithelial features of fetal-type Leydig cells in rat and human testis

The basement membranes of developing Leydig cells in fetal and newborn testis of rat were studied by ultrastructural and immunocytochemical methods. Fetal-type Leydig cells in prenatal rats were organized in irregularly outlined groups in the interstitium and were extensively surrounded by ultrastructurally identifiable basement membranes and immunocytochemically localized laminin and collagen type IV. Prenatal Leydig cell precursors had small patches of laminin and collagen type IV on their surfaces, which indicated that changes in extracellular matrix took place during their differentiation to mature fetal-type Leydig cells. Additionally, ultrastructural evidence was obtained for a basement membrane surrounding the fetal human Leydig cells similar to that in fetal rats. Soon after birth the rat fetal-type cells gathered into distinct clusters surrounded by delicate envelope cells and a discontinuous basement membrane. Basement-membrane structures, laminin, and collagen type IV were observed between the clustered cells as well. The basement membranes covering large cell surface areas of the fetal-type Leydig cells in fetal and newborn rats differed from those of the adult-type cells, which, according to our earlier study, are covered only by small patches of basement membrane. The difference between the basement membranes of the fetal- and adult-type rat Leydig cells further supports the concept of two different Leydig cell populations. The earlier findings of the epithelial nature of the Leydig cells agree with the observation of basement membranes in the Leydig cells.     

Determination of electrical parameters of the membrane of human amniotic epithelial cells in vitro

Electrical parameters determination of epithelial amniotic cells membrane shows that the temperature increase alters membrane potential and input resistance, that the transcellular pathway of monovalent cations is less important than the paracellular pathway and that there is an intercellular electrical coupling.     

Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect epithelial cells

Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.