Tadashi Nishiya

TLR3 and TLR7 Are Targeted to the Same Intracellular Compartments by Distinct Regulatory Elements

Toll-like receptor (TLR) 3 and TLR7 are indispensable for host defense against viral infection by recognizing virus-derived RNAs and are localized to intracellular membranes via an unknown mechanism. We recently reported experiments with chimeric Toll-like receptors that suggested that the subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains. Here we demonstrate that the intracellular localization of TLR3 is achieved by a 23-amino acid sequence (Glu727 to Asp749) present in the linker region between the transmembrane domain and Toll-interleukin 1 receptor resistance (TIR) domain. In contrast, the intracellular localization of TLR7 is achieved by its transmembrane domain. These elements also targeted a heterologous type I transmembrane protein CD25 to the intracellular compartment that contained TLR3 and TLR7. Despite their using distinct regulatory elements for intracellular localization, TLR3 was found to co-localize with TLR7. In addition, TLR3 and TLR7 were preferentially localized near phagosomes containing apoptotic cell particles. These findings reveal that TLR3 and TLR7 contain unique targeting sequences, which differentially lead them to the same intracellular compartments and adjacent to phagosomes containing apoptotic cell particles, where these receptors may access their ligands for the induction of immune responses against viral infection.

On–off system for PI3-kinase–Akt signaling through S-nitrosylation of phosphatase with sequence homology to tensin (PTEN)

Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an “on–off” pattern of control of Akt signaling.

Herbimycin A suppresses NF-κB activation and tyrosine phosphorylation of JAK2 and the subsequent induction of nitric oxide synthase in C6 glioma cells

Herbimycin A, a potent tyrosine kinase inhibitor, suppressed nitric oxide synthase (NOS) induced by lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) in C6 glial cells. LPS activated NF‐κB, and this effect was inhibited by pretreatment with herbimycin A. In addition, IFN‐γ activated the tyrosine protein kinase, JAK2, and tyrosine‐phosphorylation by itself was also inhibited by herbimycin A. These results suggest that herbimycin A suppresses iNOS induction by inhibition of both NF‐κB activation caused by LPS, and tyrosine‐phosphorylation of JAK2 caused by IFN‐γ in C6 glioma cells.

Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-γ-induced MAPK activation that is mediated by p21ras

Rat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon‐γ (IFN‐γ) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant‐negative form of c‐Ha‐Ras (Asn‐17) abrogated IFN‐γ‐induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21 ras and MEK1 are required for IFN‐γ‐induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1s DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras‐MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn‐17 Ras expression nor concentrations of PD98059, which completely abrogated IFN‐γ‐induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras‐MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells.

Transient Nuclear Factor κB (NF-κB) Activation Stimulated by Interleukin-1β May Be Partly Dependent on Proteasome Activity, but Not Phosphorylation and Ubiquitination of the IκBα Molecule, in C6 Glioma Cells REGULATION OF NF-κB LINKED TO CHEMOKINE PRODUCTION

We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor κB (NF-κB)-dependent manner. In this study, we focused further on the regulation of NF-κB. The activation of NF-κB and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1β (IL-1β) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-κB into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IκBα, a regulatory protein of NF-κB. Nascent IκBα began to degrade 5 min after treatment with IL-1β and disappeared completely after 15 min. However, IκBα returned to basal levels after 45–60 min. Interestingly, resynthesized IκBα was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-κB peaks at 15 min; and 2) the regulated protein(s) acts downstream of IκBα phosphorylation. Western blotting showed that the resynthesized and phosphorylated IκB molecules were also upward-shifted by multi-ubiquitination in response to IL-1β treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1β for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IκBα. These results indicate that the transient translocation of NF-κB in response to IL-1β may be partly dependent on transient proteasome activation.

Regulation of inducible nitric oxide synthase by the SPRY domain- and SOCS box-containing proteins

Inducible nitric-oxide synthase (iNOS, NOS2) plays a prominent role in macrophage bactericidal and tumoricidal activities. A relatively large amount of NO produced via iNOS, however, also targets the macrophage itself for apoptotic cell death. To uncover the intrinsic mechanisms of iNOS regulation, we have characterized the SPRY domain- and SOCS box-containing protein 1 (SPSB1), SPSB2, and SPSB4 that interact with the N-terminal region of iNOS in a D-I-N-N-N sequence-dependent manner. Fluorescence microscopy revealed that these SPSB proteins can induce the subcellular redistribution of iNOS from dense regions to diffused expression in a SOCS box-dependent manner. In immunoprecipitation studies, both Elongin C and Cullin-5, components of the multi-subunit E3 ubiquitin ligase, were found to bind to iNOS via SPSB1, SPSB2, or SPSB4. Consistently, iNOS was polyubiquitinated and degraded in a proteasome-dependent manner when SPSB1, SPSB2, or SPSB4 was expressed. SPSB1 and SPSB4 had a greater effect on iNOS regulation than SPSB2. The iNOS N-terminal fragment (residues 1–124 of human iNOS) could disrupt iNOS-SPSB interactions and inhibit iNOS degradation. In lipopolysaccharide-treated macrophages, this fragment attenuated iNOS ubiquitination and substantially prolonged iNOS lifetime, resulting in a corresponding increase in NO production and enhanced NO-dependent cell death. These results not only demonstrate the mechanism of SPSB-mediated iNOS degradation and the relative contributions of different SPSB proteins to iNOS regulation, but also show that iNOS levels are sophisticatedly regulated by SPSB proteins in activated macrophages to prevent overproduction of NO that could trigger detrimental effects, such as cytotoxicity.

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.

MyD88 is a cytoplasmic adaptor protein that is critical for Toll‐like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non‐TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll‐interleukin I receptor‐resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non‐TIR regions in the subcellular localization and signaling properties of MyD88.

Sequential modifications in class II transactivator isoform 1 induced by lipopolysaccharide stimulate major histocompatibility complex class II transcription in macrophages.

By presenting antigenic peptides on major histocompatibility complex class (MHC) II determinants to CD4+ T cells, macrophages help to direct the establishment of adaptive immunity. We found that in these cells, lipopolysaccharide stimulates the expression of MHC II genes via the activation of Erk1/2, which is mediated by Toll-like receptor 4. Erk1/2 then phosphorylates the serine at position 357, which is located in a degron of CIITA isoform 1 that leads to its monoubiquitylation. Thus modified, CIITA isoform 1 binds P-TEFb, which mediates the elongation of RNA polymerase II and co-transcriptional processing of nascent transcripts. This induction leads to the expression of MHC II genes. Subsequent polyubiquitylation results in the degradation of CIITA isoform 1. Thus, the signaling cascade from Toll-like receptor 4 to CIITA isoform 1 represents one connection between innate and adaptive immunity in macrophages.

Kyotorphin (l-tyrosyl-l-arginine) as a possible substrate for inducible nitric oxide synthase in rat glial cells

L-Arginine (L-Arg) is an endogenous substrate for nitric oxide synthase (NOS). In the present study, we examined whether L-tyrosyl-L-Arg (kyotorphin), an endogenous analgesic neuropeptide, might be a substrate for inducible NOS (iNOS) in the brain. Both kyotorphin and L-Arg caused an accumulation of nitrites in lipopolysaccharide (LPS)-treated glial cells cultured from infant rat brains. However, such accumulation of nitrites was not induced by NG-nitro-L-Arg (a NOS inhibitor), L-tyrosyl-D-Arg (D-kyotorphin) or D-Arg. L-Leucyl-L-Arg (an antagonist for kyotorphin receptors) or bestatin (an inhibitor for kyotorphin-hydrolyzing peptidase) did not inhibit the kyotorphin-induced accumulation of nitrites in LPS-treated cells. On the contrary, L-Leucyl-L-Arg caused an accumulation of nitrites in a concentration-dependent manner. The results indicate that nitric oxide (NO) is produced in LPS-treated glial cells directly from kyotorphin through the catalytic action of iNOS.

Adenylate cyclase/cAMP/protein kinase A signaling pathway inhibits endothelin type A receptor-operated Ca²⁺ entry mediated via transient receptor potential canonical 6 channels.

Receptor-operated Ca2+ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca2+ concentration triggered by the activation of Gq protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of Gq protein-coupled endothelin type A receptor (ETAR)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ETAR and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca2+ measurements with thapsigargin-induced Ca2+-depletion/Ca2+-restoration protocol to estimate ROCE showed that the stimulation of ETAR induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6S28A) but not Thr69 (TRPC6T69A) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6S28A and TRPC6T69A) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.