SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.     

Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin alphavbeta6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37 degrees C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin alphavbeta6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.     

Modulation of chicken macrophage effector function by T(H)1/T(H)2 cytokines

Regulation of macrophage activity by T(H)1/2 cytokines is important to maintain the balance of immunity to provide adequate protective immunity while avoiding excessive inflammation. IFN-γ and IL-4 are the hallmark T(H)1 and T(H)2 cytokines, respectively. In avian species, information concerning regulation of macrophage activity by T(H)1/2 cytokines is limited. Here, we investigated the regulatory function of chicken T(H)1 cytokines IFN-γ, IL-18 and T(H)2 cytokines IL-4, IL-10 on the HD11 macrophage cell line. Chicken IFN-γ stimulated nitric oxide (NO) synthesis in HD11 cells and primed the cells to produce significantly greater amounts of NO when exposed to microbial agonists, lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-ODN, and poly I:C. In contrast, chicken IL-4 exhibited bi-directional immune regulatory activity: it activated macrophage NO synthesis in the absence of inflammatory agonists, but inhibited NO production by macrophages in response to microbial agonists. Both IFN-γ and IL-4, however, enhanced oxidative burst activity of the HD11 cells when exposed to Salmonella enteritidis. IL-18 and IL-10 did not affect NO production nor oxidative burst in HD11 cells. Phagocytosis and bacterial killing by the HD11 cells were not affected by the treatments of these cytokines. Infection of HD11 cells with S.enteritidis was shown to completely abolish NO production regardless of IFN-γ treatment. This study has demonstrated that IFN-γ and IL-4 are important T(H)1 and T(H)2 cytokines that regulate macrophage function in chickens.     

RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent endocytosis pathway

Regulated endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, the specific combination of clathrin-dependent and -independent mechanisms that mediate AMPAR trafficking in vivo have not been fully characterized. Here, we examine the trafficking of the AMPAR subunit GLR-1 in Caenorhabditis elegans. GLR-1 is localized on synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit. Animals lacking RAB-10, a small GTPase required for endocytic recycling of intestinal cargo, are similar in phenotype to animals lacking LIN-10, a postsynaptic density 95/disc-large/zona occludens-domain containing protein: GLR-1 accumulates in large accretions and animals display a decreased frequency of reversals. Mutations in unc-11 (AP180) or itsn-1 (Intersectin 1), which reduce clathrin-dependent endocytosis, suppress the lin-10 but not rab-10 mutant phenotype, suggesting that LIN-10 functions after clathrin-mediated endocytosis. By contrast, cholesterol depletion, which impairs lipid raft formation and clathrin-independent endocytosis, suppresses the rab-10 but not the lin-10 phenotype, suggesting that RAB-10 functions after clathrin-independent endocytosis. Animals lacking both genes display additive GLR-1 trafficking defects. We propose that RAB-10 and LIN-10 recycle AMPARs from intracellular endosomal compartments to synapses along distinct pathways, each with distinct sensitivities to cholesterol and the clathrin-mediated endocytosis machinery.     

Dual role of inflammatory stimuli in activation-induced cell death of mouse microglial cells. Initiation of two separate apoptotic pathways via induction of interferon regulatory factor-1 and caspase-11

We have previously shown that mouse microglial cells undergo apoptosis upon inflammatory activation and that nitric oxide (NO) is the major autocrine mediator in this process (Lee, P., Lee, J., Kim, S., Yagita, H., Lee, M. S., Kim, S. Y., Kim, H., and Suk, K. (2001) Brain Res. 892, 380-385). Here, we present evidence that interferon regulatory factor-1 (IRF-1) and caspase-11 are the essential molecules in activation-induced cell death of microglial cells. The apoptogenic action of inflammatory stimuli such as lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) was mediated through the induction of IRF-1 and caspase-11 expression in two separate events. Although IRF-1 was required for NO synthesis, caspase-11 induction was necessary for NO-independent apoptotic pathway. Microglial cells from IRF-1-deficient mice showed markedly decreased NO production, and they were partially resistant to apoptosis in response to LPS/IFNgamma but were sensitive to NO donor exposure. LPS/IFNgamma treatment resulted in the induction of caspase-11 followed by activation of caspase-11, -1, and -3. Inactivation of caspase-11 by the transfection of dominant-negative mutant or treatment with the caspase inhibitors rendered microglial cells partially resistant to LPS/IFNgamma-induced apoptosis. Inhibition of both NO synthesis and caspase-11 completely blocked LPS/IFNgamma-induced cytotoxicity. These results indicated that LPS/IFNgamma not only induced the production of cytotoxic NO through IRF-1 but also initiated the NO-independent apoptotic pathway through the induction of caspase-11 expression.     

Chlamydia trachomatis induces expression of IFN-gamma-inducible protein 10 and IFN-beta independent of TLR2 and TLR4, but largely dependent on MyD88

IFN-gamma-inducible protein 10 (IP-10) is a chemokine important in the attraction of T cells, which are essential for resolution of chlamydial genital tract infection. During infections with Gram-negative bacteria, the IP-10 response mediated through type I IFNs usually occurs as a result of TLR4 stimulation by bacterial LPS. However, we found that levels of IP-10 in genital tract secretions of Chlamydia trachomatis-infected female wild-type mice were similar to those of infected TLR2- and TLR4-deficient mice but significantly greater than those of infected MyD88-deficient mice. We investigated the mechanism of IP-10 and IFN-beta induction during chlamydial infection using mouse macrophages and fibroblasts infected ex vivo. The induction of IP-10 and IFN-beta was unchanged in Chlamydia-infected TLR2- and TLR4-deficient cells compared with wild-type cells. However, infection of MyD88-deficient cells resulted in significantly decreased responses. These results suggest a role for MyD88-dependent pathways in induction of IP-10 and IFN-beta during chlamydial infection. Furthermore, treatment of infected macrophages with an endosomal maturation inhibitor significantly reduced chlamydial-induced IFN-beta. Because endosomal maturation is required for MyD88-dependent intracellular pathogen recognition receptors to function, our data suggest a role for the intracellular pathogen recognition receptor(s) in induction of IFN-beta and IP-10 during chlamydial infection. Furthermore, the intracellular pathways that lead to chlamydial-induced IFN-beta function through TANK-binding kinase mediated phosphorylation and nuclear translocation of IFN regulatory factor-3.     

A comparative analysis of cytokine responses, cell surface marker expression and MAPKs in DCs matured with LPS compared with a panel of TLR ligands

Dendritic cells (DCs) are professional antigen-presenting cells that play a vital role in shaping adaptive immunity. DC maturation begins when exogenous danger signals bind to the appropriate toll-like receptor (TLR) and initiate expression of cell surface markers and the secretion of cytokines. This process occurs through defined mitogen-activated protein kinase (MAPK) signalling pathways. Of the 13 known mammalian TLRs, lipopolysaccharide (LPS), which activates TLR4, is the most commonly used ligand for the maturation of DCs in vitro. This comprehensive study measures cytokine secretion and cell surface marker expression in murine bone-marrow-derived DCs following maturation with LPS compared to DCs matured with a panel of other TLR-ligands (zymosan A (TLR2/6), PGN (TLR2), poly(I:C) (TLR3), flagellin (TLR5) and CpG-ODN1826 (TLR9)). The role of MAPK signalling pathways in the maturation process was also examined. Results demonstrate that zymosan A and CpG induce comparable cytokine and cell surface marker profiles to LPS. The remaining ligands differed significantly for cytokine and CD40 expression, but not for CD80 and CD86 expression. While there were differences for MAPK signalling pathways for all ligands, the effect of the inhibitors were broadly similar. These findings broaden our knowledge of TLR ligand-matured DCs.     

Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity

Recognition of pathogen-associated molecular patterns by innate immune receptors is essential for host defense responses. Although extracellular stress proteins are considered as indicators of the stressful conditions (e.g., infection or cell injury), the exact roles of these molecules in the extracellular milieu remain less defined. We found that glucose-regulated protein 170 (Grp170), the largest stress protein and molecular chaperone, is highly efficient in binding CpG oligodeoxynucleotides (CpG-ODN), the microbial DNA mimetic sensed by toll-like receptor 9 (TLR9). Extracellular Grp170 markedly potentiates the endocytosis and internalization of CpG-ODN by mouse bone marrow-derived macrophages and directly interacts with endosomal TLR9 on cell entry. These molecular collaborations result in the synergistic activation of the MyD88-dependent signaling and enhanced production of proinflammatory cytokines and nitric oxide in mouse primary macrophages as well as human THP-1 monocyte-derived macrophages, suggesting that Grp170 released from injured cells facilitates the sensing of pathogen-associated "danger" signals by intracellular receptors. This CpG-ODN chaperone complex-promoted innate immunity confers increased resistance in mice to infection of Listeria monocytogenes compared with CpG-ODN treatment alone. Our studies reveal a previously unrecognized attribute of Grp170 as a superior DNA-binding chaperone capable of amplifying TLR9 activation on pathogen recognition, which provides a conceptual advance in understanding the dynamics of ancient chaperoning functions inside and outside the cell.     

Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.     

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.     

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.     

Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.     

Contrasting roles of endosomal pH and the cytoskeleton in infection of human glial cells by JC virus and simian virus 40

Infection of eukaryotic cells by pathogens requires the efficient use of host cell endocytic and cytoplasmic transport mechanisms. Understanding how these cellular functions are exploited by microorganisms allows us to better define the basic biology of pathogenesis while providing better insight into normal cellular functions. In this report we compare and contrast intracellular transport and trafficking of the human polyomavirus JC virus (JCV) with that of simian virus 40 (SV40). We have previously shown that infection of human glial cells by JCV requires clathrin-dependent endocytosis. In contrast, infection of cells by SV40 proceeds by caveola-dependent endocytosis. We now examine the roles of endosomal pH and the cellular cytoskeleton during infection of glial cells by both viruses. Our results demonstrate that JCV infection is sensitive to disruption of endosomal pH, whereas SV40 infection is pH independent. Infection by JCV is inhibited by treatment of glial cells with cytochalasin D, nocodazole, and acrylamide, whereas SV40 infection is affected only by nocodazole. These data point to critical differences between JCV and SV40 in terms of endocytosis and intracellular trafficking of their DNA genomes to the nucleus. These data also suggest a unique sequential involvement of cytoskeletal elements during infection of glial cells by JCV.     

Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.     

Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.

The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.     

Anti-inflammatory effect of heme oxygenase 1: glycosylation and nitric oxide inhibition in macrophages

Flavonoids including the aglycones, hesperetin (HT; 5,7,3'-trihydroxy-4'-methoxy-flavanone), and naringenin (NE; 5,7,4'-trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3'-trihydroxy-4'-methoxy-flavanone 7-rhamnoglucoside) and naringin (NI; 5,7,4'-trihydroxy flavanone 7-rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO-1) protein expression in the presence or absence of LPS and showed time and dose-dependent inhibition of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate-elicited peritoneal macrophages. Additive inhibitory effect of an HO-1 inducer hemin and NE or NI on LPS-induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS-induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS-induced NO production, according to the production of the aglycones, HT and NE, by high-performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO-1-overexpressing cells (HO-1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS-induced NO production, and that HO-1 is involved in the inhibitory mechanism of flavonoids on LPS-induced iNOS and NO production.     

Transfer of M2 muscarinic acetylcholine receptors to clathrin-derived early endosomes following clathrin-independent endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as beta(2)-adrenergic receptors are internalized via beta-arrestin- and clathrin-dependent mechanisms, whereas others, like M(2) muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M(2) mAChR endocytosis, we investigated the post-endocytic trafficking of M(2) mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M(2) mAChR internalization. Here, we report that M(2) mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Galpha(q) Q209L. In contrast, M(2) mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M(2) mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M(2) mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.