A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4.

Cancer cells secrete copious amounts of exosomes, and elevated intracellular Ca2+ is critical for tumor progression and metastasis, but the underlying cellular mechanisms are unknown. Munc13-4 is a Ca2+-dependent SNAP receptor- and Rab-binding protein required for Ca2+-dependent membrane fusion. Here we show that acute elevation of Ca2+ in cancer cells stimulated a fivefold increase in CD63+, CD9+, and ALIX+ exosome release that was eliminated by Munc13-4 knockdown and not restored by Ca2+ binding-deficient Munc13-4 mutants. Direct imaging of CD63-pHluorin exosome release confirmed its Munc13-4 dependence. Depletion of Munc13-4 in highly aggressive breast carcinoma MDA-MB-231 cells reduced the size of CD63+ multivesicular bodies (MVBs), indicating a role for Munc13-4 in MVB maturation. Munc13-4 used a Rab11-dependent trafficking pathway to generate MVBs competent for exosome release. Membrane type 1 matrix metalloproteinase trafficking to MVBs by a Rab11-dependent pathway was also Munc13-4 dependent, and Munc13-4 depletion reduced extracellular matrix degradation. These studies identify a novel Ca2+- and Munc13-4-dependent pathway that underlies increased exosome release by cancer cells.

Small molecules that inhibit the late stage of Munc13-4-dependent secretory granule exocytosis in mast cells.

Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain-containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4-membrane interactions and thereby inhibit Munc13-4-dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 µm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of ß-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells.

Induction of IL-8 expression by bacterial flagellin is mediated through lipid raft formation and intracellular TLR5 activation in A549 cells.

We investigated the mechanism for the induction of a chemokine, IL-8, by bacterial flagellins in the human alveolar type II epithelial cell line, A549. Bacterial flagellin induced expression of IL-8 mRNA and protein in dose- and time-dependent manners. IL-8 expression was inhibited by nystatin (a lipid rafts inhibitor) but not by chlorpromazine (a clathrin-coated pits inhibitor). Interestingly, Toll-like receptor 5 (TLR5) recognizing flagellins was found in the intracellular compartment of A549 but rarely on the cell surface. Flagellin-induced IL-8 expression appears to be mediated through TLR5 as determined by in vitro transient transfection experiment in HEK-293 cells expressing TLR5 using a reporter gene construct containing IL-8 promoter. IL-8 expression was attenuated by inhibitors for protein kinase C (PKC) and mitogen-activated protein (MAP) kinases. Furthermore, NF-kappaB and NF-IL6 transcription factors played an important role in the flagellin-induced IL-8 gene expression in A549 cells. Collectively, these results suggest that flagellin-induced IL-8 expression requires formation of lipid rafts, intracellular TLR activation, and subsequent activation of PKC and MAP kinases leading to the activation of the transcription factors NF-kappaB and NF-IL6 in human alveolar type II epithelial cells.

Bacillus anthracis lethal toxin attenuates lipoteichoic acid-induced maturation and activation of dendritic cells through a unique mechanism.

Lethal toxin (LT), produced by the gram-positive bacterium Bacillus anthracis, was identified as a major etiologic agent causing anthrax due to its strong immunotoxicity. Gram-positive bacteria express lipoteichoic acid (LTA), which is considered as a counterpart to lipopolysaccharide (LPS) of gram-negative bacteria, but differs from LPS in the structure and function. Since dendritic cells (DCs) are essential for the appropriate initiation of immune response, we investigated the effect of LT on LTA-induced DC maturation using immature DCs prepared by differentiation of C57BL/6 mouse bone marrow cells. When immature DCs were matured with LTA in the presence of LT, the expression of representative markers for DC maturation such as CD80, CD83, and CD86 together with MHC class I and II molecules was inhibited. LT ameliorated the attenuation of endocytic capacity during DC maturation by LTA while such effect was not observed in LPS-matured DCs. Furthermore, exposure to LT resulted in a decrease in the expression of pro-inflammatory cytokines including IL-6, TNF-alpha, and IL-12p40 in LTA-stimulated DCs as in LPS-stimulated DCs. Interestingly, LT showed a minimal change in LTA-induced IL-1beta expression while LT highly enhanced the LPS-induced IL-1beta expression. Those inhibitory effects might be associated with LT interference of LTA-signaling pathways mediated through mitogen-activated protein kinases (MAPKs) since LT suppressed phosphorylation of MAPK, which was induced by LTA. Meanwhile, no change was observed in the expression of putative anthrax toxin receptors, TEM8 and CMG2, or Toll-like receptor 2. These results suggest that LT suppresses the maturation and activation of DCs stimulated with LTA, similar to the suppression in the LPS-stimulated DCs, but via a distinct mechanism.

A food-born heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), suppresses tumor necrosis factor-alpha expression in lipoteichoic acid-stimulated RAW 264.7 cells.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine with strong carcinogenic and mutagenic potential, is created abundantly in the overcooking of meat and fish. Carcinogenic toxicants are often implicated in immunosuppression, where cancer cells are not easily eliminated by the host immune system. Here, we investigated the effect of PhIP on tumor necrosis factor-alpha (TNF-alpha) expression by murine macrophage-like cells (RAW 264.7) stimulated with lipoteichoic acid (LTA), a major virulence factor of Gram-positive bacteria. Upon exposure to LTA purified from Staphylococcus aureus, TNF-alpha expression was substantially induced, whereas pretreatment with PhIP significantly inhibited LTA-induced TNF-alpha expression. LTA is known to activate Toll-like receptor 2 (TLR2) and NF-kappaB, resulting in TNF-alpha expression. Interestingly, PhIP did not interfere with LTA-binding to TLR2, its stimulation of TLR2, or the DNA-binding activity of NF-kappaB. However, treatment with actinomycin D facilitated the PhIP-induced attenuation of TNF-alpha mRNA expression, implying that PhIP might decrease TNF-alpha mRNA stability rather than its biosynthesis. Furthermore, Western blot analysis demonstrated that PhIP reduced the phosphorylation of ERK1/2 and JNK but not p38 kinase in LTA-stimulated cells. The addition of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, rescued PhIP-inhibited TNF-alpha expression in LTA-stimulated cells. These results suggest that PhIP downregulates TNF-alpha expression in LTA-stimulated macrophages by decreasing TNF-alpha mRNA stability and signaling pathways related to PKC, ERK1/2, and JNK activation.

Peptidoglycan-mediated IL-8 expression in human alveolar type II epithelial cells requires lipid raft formation and MAPK activation.

Staphylococcus aureus, a major sepsis-causing Gram-positive bacterium, invades pulmonary epithelial cells and causes lung diseases. In the lung, alveolar type II epithelial cells play an important role in innate immunity by secreting chemokines and antimicrobial peptides upon bacterial infection whereas type I cells mainly function in gas-exchange. In this study, we investigated the ability of S. aureus peptidoglycan (PGN) to induce expression of a chemokine, IL-8, in a human alveolar type II epithelial cell line, A549. PGN induces IL-8 mRNA and protein expression in a dose- and time-dependent manner. Supplementation of soluble CD14 further enhanced the PGN-induced IL-8 expression. Interestingly, PGN-induced IL-8 expression was inhibited by nystatin, a specific inhibitor for lipid rafts, but not by chlorpromazine, a specific inhibitor for clathrin-coated pits. Furthermore, PGN-induced IL-8 expression was attenuated by inhibitors for MAP kinases such as ERK, p38 kinase, and JNK/SAPK, whereas no inhibitory effect was observed by inhibitors for reactive oxygen species or protein kinase C. Electrophoretic mobility shift assay demonstrates that PGN increased the DNA binding of the transcription factors, AP-1 and NF-kappaB while minimally, NF-IL6, all of which are involved in the transcription of IL-8. Taken together, these results suggest that PGN induces IL-8 expression in a CD14-enhanced manner in human alveolar type II epithelial cells, through the formation of lipid rafts and the activation of MAP kinases, which ultimately leads to activation of AP-1, NF-kappaB, and NF-IL6.

Human placenta promotes IL-8 expression through activation of JNK/SAPK and transcription factors NF-kappaB and AP-1 in PMA-differentiated THP-1 cells.

Human placenta is a rich reservoir of diverse bioactive molecules with therapeutic potential against certain diseases such as immune disorders. In the present study, we investigated the ability of human placenta extract (HPE) to induce expression of a CXC chemokine, interleukin-8 (IL-8), in a human monocytic cell line, THP-1, differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA). HPE significantly induced IL-8 mRNA and protein expressions in a dose-dependent manner. HPE-induced IL-8 expression was inhibited by a selective inhibitor of JNK/SAPK, but not by inhibitors of p38 kinase or ERK. Since IL-8 transcription is known to be regulated by nuclear factor (NF)-kappaB, activating protein (AP)-1 and NF for IL-6 (NF-IL6), an electrophoretic mobility shift assay was performed to examine the DNA-binding activities of these transcription factors. The DNA-binding activities of NF-kappaB and AP-1 increased in cells treated with HPE in a dose-dependent manner, while no change was observed in NF-IL6 binding activity under the same conditions. Taken together, these results suggest that HPE-induced IL-8 secretion occurs via activation of JNK/SAPK and transcription factors NF-kappaB and AP-1 in PMA-differentiated THP-1 cells.

Lipoteichoic acid and muramyl dipeptide synergistically induce maturation of human dendritic cells and concurrent expression of proinflammatory cytokines.

Maturation is an important process by which dendritic cells (DC) develop the potent antigen-presentation capacity necessary for efficient activation of adaptive immunity. Here, we have investigated the ability of lipoteichoic acid (LTA) and muramyl dipeptide (MDP; the minimal structural unit of peptidoglycan with immunostimulating activity) to induce maturation of human immature DC (iDC), derived from peripheral blood CD14-positive cells, and the production of proinflammatory cytokines. Exposure of iDC to staphylococcal LTA (StLTA) at 1 or 10 microg/ml or MDP at 0.1 or 1 microg/ml alone had little effect on the expression of CD80 and CD83, with a minor increase in expression of CD86, all of which are indicative of cell surface markers for maturation. However, there was a synergistic expression of these molecules when iDC were stimulated with StLTA and MDP together. It is interesting that selective induction of MHC Class II expression was observed during the DC maturation, only when costimulated with LTA plus MDP, and Escherichia coli LPS induced dramatic expression of MHC Classes I and II. Endocytosis assay using Dextran-FITC showed that costimulation with StLTA and MDP attenuated the endocytic capacity of the DC, which is a typical phenomenon of DC maturation. Concomitantly, increased expression of DEC-205, but decreased expression of CD206, was observed under the same costimulating condition. Furthermore, ELISA showed that secretions of TNF-alpha and IL-12 p40, but not IL-10, were induced in iDC by the costimulation. These results suggest that StLTA and MDP synergistically induce maturation and activation of human DC.