Type 1 interferon suppresses expression and glucocorticoid induction of glucocorticoid-induced leucine zipper (GILZ).

SLE is a systemic multi-organ autoimmune condition associated with reduced life expectancy and quality of life. Glucocorticoids (GC) are heavily relied on for SLE treatment but are associated with detrimental metabolic effects. Type 1 interferons (IFN) are central to SLE pathogenesis and may confer GC insensitivity. Glucocorticoid-induced leucine zipper (GILZ) mediates many effects of GC relevant to SLE pathogenesis, but the effect of IFN on GC regulation of GILZ is unknown. We performed in vitro experiments using human PBMC to examine the effect of IFN on GILZ expression. JAK inhibitors tofacitinib and tosylate salt were used in vivo and in vitro respectively to investigate JAK-STAT pathway dependence of our observations. ChiP was performed to examine glucocorticoid receptor (GR) binding at the GILZ locus. Several public data sets were mined for correlating clinical data. High IFN was associated with suppressed GILZ and reduced GILZ relevant to GC exposure in a large SLE population. IFN directly reduced GILZ expression and suppressed the induction of GILZ by GC in vitro in human leukocytes. IFN actions on GILZ expression were dependent on the JAK1/Tyk2 pathway, as evidenced by loss of the inhibitory effect of IFN on GILZ in the presence of JAK inhibitors. Activation of this pathway led to reduced GR binding in key regulatory regions of the GILZ locus. IFN directly suppresses GILZ expression and GILZ upregulation by GC, indicating a potential mechanism for IFN-induced GC resistance. This work has important implications for the ongoing development of targeted GC-sparing therapeutics in SLE.

Exposure of female NZBWF1 mice to imiquimod-induced lupus nephritis at an early age via a unique mechanism that differed from spontaneous onset.

Systemic lupus erythematosus (SLE) is a chronic inflammatory and representative autoimmune disease. Extremely complicated and multifactorial interactions between various genetic factors and individual susceptibility to environmental factors are involved in the pathogenesis of SLE. Several studies have reported that mutation and activation of toll-like receptor (TLR) 7 are involved in the onset of autoimmunity, including SLE. Thus, we investigated the response of SLE-prone mice to continuous environmental factors, particularly TLR7 agonist exposure, and changes in their phenotypes. Female and male NZBWF1 (BWF1) mice were treated from 20 weeks of age with a TLR7 agonist, imiquimod (IMQ), 3 times weekly for up to 12 weeks. IMQ-exposed female BWF1 mice showed worsened lupus nephritis. However, autoantibody production was not enhanced in IMQ-exposed female BWF1 mice. The Th1 cytokine expression was upregulated in the kidney of IMQ-treated mice. In IMQ-exposed BWF1 mice, neutralization of IFN-γ suppressed early-phase lupus nephritis. Additionally, in male BWF1 mice IMQ exposure induced minor aggravation of lupus nephritis. These results suggest that the induction of aggravated lupus nephritis by TLR7 agonist exposure was related to the expression of IFN-γ via acute TLR7 signal-induced renal inflammation, and that the involvement of genetic factors associated with a predisposition to SLE is also essential. Thus, the activation of TLR7 signaling by exposure to environmental factors may upset the balance of factors that maintain SLE remission. We hypothesize that the inhibition of TLR7 signaling and IFN-γ signaling is effective for preventing the onset and flare and maintaining remission of lupus nephritis.

Metformin repositioning in rheumatoid arthritis.

OBJECTIVES: Metformin is a known therapeutic agent for diabetes. Recently, several reports suggested the possibility of improvement in autoimmune disease and malignancy conditions through the effect of metformin on the immune system. Although there have been reports on the therapeutic effects of metformin on mouse models of collagen-induced arthritis, simulating human rheumatoid arthritis (RA), the effect of metformin on human RA remains unknown. Therefore, we investigated the inhibitory effect of metformin on the pathogenesis of human RA in vitro. METHODS: Osteoclastogenesis was evaluated with or without metformin. through tartrate-resistant acid phosphatase staining, osteoclast-specific enzyme expression analysis, and a bone resorption assay. Human fibroblast-like synoviocyte MH7A cells were stimulated with TNF-α, and the expression of proinflammatory cytokines and protease and growth factor genes was evaluated with or without metformin. Metformin has been used to evaluate their potential modulatory effects on cells treated with TNF-α. Moreover, we examined angiogenesis by performing a tube formation assay using human umbilical vein endothelial cells (HUVECs) with or without metformin. RESULTS: Osteoclastogenesis was suppressed in the presence of metformin, and the expression of osteoclast-specific genes was reduced. The TNF-α-induced expression of inflammatory cytokines and protease and growth factor genes in MH7A cells was downregulated by metformin. Additionally, the induced formation of tubular networks in HUVECs was also disrupted following treatment with metformin. CONCLUSIONS: These results suggest that metformin might improve the pathogenesis of RA, including joint inflammation and destruction. Thus, metformin might be utilised as a potential therapeutic agent in the treatment of RA.

Social defeat stress exacerbates atopic dermatitis through downregulation of DNA methyltransferase 1 and upregulation of C-C motif chemokine receptor 7 in skin dendritic cells.

DNA methylation is an epigenetic modification that regulates gene transcription. DNA methyltransferase 1 (DNMT1) plays an important role in DNA methylation. However, the involvement of DNMT1 and DNA methylation in the pathogenesis of atopic dermatitis (AD) remains unclear. In this study, microarray analysis revealed that peripheral blood mononuclear cells of AD patients with low DNMT1 expression (DNMT1-low) highly expressed dendritic cell (DC) activation-related genes. Also, DNMT1-low AD patients exhibited a higher itch score compared to AD patients with high DNMT1 expression (DNMT1-high). By using an AD-like mouse model induced by the application of Dermatophagoides farinae body ointment, we found that Dnmt1 expression was decreased, while the expression of C-C chemokine receptor type 7 (Ccr7) was upregulated in mouse skin DCs. Furthermore, mice exposed to social defeat stress exhibited Dnmt1 downregulation and Ccr7 upregulation in skin DCs. Additionally, dermatitis and itch-related scratching behavior were exacerbated in AD mice exposed to stress. The relationship between low DNMT1 and itch induction was found in both human AD patients and AD mice. In mouse bone marrow-derived DCs, Ccr7 expression was inhibited by 5-aza-2-deoxycytidine, a methylation inhibitor. Furthermore, in mouse skin DCs, methylation of CpG sites in Ccr7 was modified by either AD induction or social defeat stress. Collectively, these findings suggest that social defeat stress exacerbates AD pathology through Dnmt1 downregulation and Ccr7 upregulation in mouse skin DCs. The data also suggest a role of DNMT1 downregulation in the exacerbation of AD pathology.

MicroRNA-766-3p Contributes to Anti-Inflammatory Responses through the Indirect Inhibition of NF-κB Signaling.

MicroRNA (miRNA) is small RNA of 20 to 22 nucleotides in length and is stably present in plasma. Regulating the expression of miRNA taken into cells has been suggested as a general therapeutic approach. We identified the novel anti-inflammatory miRNA hsa-miR-766-3p and investigated its biological function in human rheumatoid arthritis (RA) fibroblast-like synoviocyte MH7A cells. To verify the function of the miRNA present in the plasma of RA patients, we performed a comprehensive analysis of the miRNA expression during abatacept treatment and identified eight miRNAs with significantly altered expression levels. Among these eight miRNAs, miR-766-3p was found to have a clear function. The expression of inflammatory genes in response to inflammatory stimuli was suppressed in MH7A transduced with miR-766-3p. We showed that miR-766-3p indirectly reduced the activation of NF-κB and clarified that this mechanism was partially involved in the reduction of the mineralocorticoid receptor expression. In addition, the inflammatory responses were suppressed in other types of cells. These results indicate the novel function of miR-766-3p, findings that may aid in the development of therapies to suppress inflammation, not only in RA but also in other diseases.

Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization.

BACKGROUND: Ras homolog gene family H (RhoH) is a membrane-bound adaptor protein involved in proximal T-cell receptor signaling. Therefore RhoH plays critical roles in the differentiation of T cells; however, the function of RhoH in the effecter phase of the T-cell response has not been fully characterized. OBJECTIVE: We sought to explore the role of RhoH in inflammatory immune responses and investigated the involvement of RhoH in the pathogenesis of psoriasis. METHODS: We analyzed effector T-cell and systemic inflammation in wild-type and RhoH-null mice. RhoH expression in T cells in human PBMCs was quantified by using RT-PCR. RESULTS: RhoH deficiency in mice induced TH17 polarization during effector T-cell differentiation, thereby inducing psoriasis-like chronic dermatitis. Ubiquitin protein ligase E3 component N-recognin 5 (Ubr5) and nuclear receptor subfamily 2 group F member 6 (Nr2f6) expression levels decreased in RhoH-deficient T cells, resulting in increased protein levels and DNA binding activity of retinoic acid-related orphan receptor γt. The consequential increase in IL-17 and IL-22 production induced T cells to differentiate into TH17 cells. Furthermore, IL-22 binding protein/Fc chimeric protein reduced psoriatic inflammation in RhoH-deficient mice. Expression of RhoH in T cells was lower in patients with psoriasis with very severe symptoms. CONCLUSION: Our results indicate that RhoH inhibits TH17 differentiation and thereby plays a role in the pathogenesis of psoriasis. Additionally, IL-22 binding protein has therapeutic potential for the treatment of psoriasis.

Connective Tissue Growth Factor Neutralization Aggravates the Psoriasis Skin Lesion: The Analysis of Psoriasis Model Mice and Patients.

BACKGROUND: Connective tissue growth factor (CTGF) is a multifunctional cellular protein and playing a role as a central mediator in tissue remodeling and fibrosis. The physiological function of CTGF in psoriasis is unknown. OBJECTIVE: The purpose of this study was to investigate the function of CTGF in psoriasis using the established imiquimod (IMQ)-induced psoriasis murine model and psoriasis patients. METHODS: Anti-CTGF monoclonal antibody was applied to IMQ induced psoriasis mice and those skin were clinically, pathologically and immunologically analyzed. Additionally, CTGF expression was analyzes using skin samples and plasma from psoriasis patients. RESULTS: CTGF expression was observed in the dermis from both IMQ-induced psoriatic mice and psoriasis patients. CTGF inhibition using an anti-CTGF antibody slightly worsened IMQ-induced dermatitis. In addition, the increase of CTGF showed tendency to suppress the psoriatic dermatitis through inhibition of suprabasal cells proliferation and macrophage infiltration in the skin. CTGF was also detected significantly higher in plasma from psoriasis patients comparing with healthy control. CONCLUSION: Our findings suggest that CTGF could contribute to the healing rather than the worsening of psoriasis skin lesions.

Circulating plasma microRNA profiling in patients with polymyositis/dermatomyositis before and after treatment: miRNA may be associated with polymyositis/dermatomyositis.

BACKGROUND: MicroRNAs (miRNAs) are involved in the regulation of key biological processes and have been implicated in various diseases, including autoimmune disorders. The pathogenesis of polymyositis (PM) and dermatomyositis (DM) is considered to be mediated by autoimmune reactions. To determine miRNA role in the development and progression of PM and DM, we performed plasma miRNA profiling in PM/DM patients before and after treatment. METHODS: Total RNA was isolated from plasma of 10 patients before and after treatment with prednisolone, or, in case of prednisolone resistance or complications, with the combination of calcineurin inhibitors (cyclosporine or tacrolims) and/or pulse intravenous cyclophosphamide. The expression of miRNAs was determined using miRNA microarray and validated by qRT-PCR. RESULTS: More differentially expressed miRNAs were found in plasma of DM patients compared to PM patients before and after treatment, and their profiles were different. Among the differentially expressed plasma miRNA identified by microarray, the levels of hsa-miR-4442 were confirmed by qRT-PCR to be significantly decreased by treatment. In addition, plasma hsa-miR-4442 content in active PM/DM significantly exceeded that in other active autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, as well as in healthy individuals. The level of plasma hsa-miR-4442 was positively correlated with Skeletal Disease Activity in MITAX (Myositis Intention to Treat Activity Index). CONCLUSION: This is the first report describing plasma miRNA expression profiles in PM/DM patients. The present data suggest that plasma levels of miRNAs may be associated with polymyositis/dermatomyositis and hsa-miR-4442 could be used as a biomarker for PM/DM diagnosis and/or disease activity.

JAK inhibitor has the amelioration effect in lupus-prone mice: the involvement of IFN signature gene downregulation.

BACKGROUND: We previously reported that JAK-STAT-pathway mediated regulation of IFN-regulatory factor genes could play an important role in SLE pathogenesis. Here, we evaluated the efficacy of the JAK inhibitor tofacitinib (TOFA) for controlling IFN signalling via the JAK-STAT pathway and as a therapeutic for SLE. RESULTS: We treated NZB/NZW F1 mice with TOFA and assessed alterations in their disease, pathological, and immunological conditions. Gene-expression results obtained from CD4+ T cells (SLE mice) and CD3+ T cells (human SLE patients) were measured by DNA microarray and qRT-PCR. TOFA treatment resulted in reduced levels of anti-dsDNA antibodies, decreased proteinuria, and amelioration of nephritis as compared with those observed in control animals. Moreover, we observed the rebalance in the populations of naïve CD4+ T cells and effector/memory cells in TOFA-treated mice; however, treatment with a combination of TOFA and dexamethasone (DEXA) elicited a stronger inhibitory effect toward the effector/memory cells than did TOFA or DEXA monotherapy. We also detected decreased expression of several IFN-signature genes Ifit3 and Isg15 in CD4+ from SLE-prone mice following TOFA and DEXA treatment, and IFIT3 in CD3+ T cells from human patients following immunosuppressant therapy including steroid, respectively. CONCLUSION: Modulation of type I IFN signalling via JAK-STAT inhibition may exert a beneficial effect in SLE patients, and our results suggest that TOFA could be utilised for the development of new SLE-specific therapeutic strategies.

Kinase inhibitors of the IGF-1R as a potential therapeutic agent for rheumatoid arthritis.

We have previously shown that the inhibition of connective tissue growth factor (CTGF) is a potential therapeutic strategy against rheumatoid arthritis (RA). CTGF consists of four distinct modules, including the insulin-like growth factor binding protein (IGFBP). In serum, insulin-like growth factors (IGFs) bind IGFBPs, interact with the IGF-1 receptor (IGF-1 R), and regulate anabolic effects and bone metabolism. We investigated the correlation between IGF-1 and the pathogenesis of RA, and the inhibitory effect on osteoclastogenesis and angiogenesis of the small molecular weight kinase inhibitor of the IGF-1 R, NVP-AEW541, against pathogenesis of RA in vitro. Cell proliferation was evaluated by cell count and immunoblotting. The expression of IGF-1 and IGF-1 R was evaluated by RT-PCR. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay, and osteoclast-specific enzyme production. Angiogenesis was evaluated by a tube formation assay using human umbilical vein endothelial cells (HUVECs). The proliferation of MH7A cells was found to be inhibited in the presence of NVP-AEW541, and the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was downregulated in MH7A cells. IGF-1 and IGF-1 R mRNA expression levels were upregulated during formation of M-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL)-mediated osteoclast formation. Moreover, osteoclastogenesis was suppressed in the presence of NVP-AEW541. The formation of the tubular network was enhanced by IGF-1, and this effect was neutralized by NVP-ARE541. Our findings suggest that NVP-AEW541 may be utilized as a potential therapeutic agent in the treatment of RA.

The effectiveness of new triple combination therapy using synthetic disease-modifying anti-rheumatic drugs with different pharmacological function against rheumatoid arthritis: the verification by an in vitro and clinical study.

The study aims to confirm the feasibility of new oral triple combination therapy using methotrexate (MTX), mizoribine (MZR), and tacrolimus (TAC) in patients with rheumatoid arthritis (RA) by in vitro and clinical analyses. Triple therapy with a combination of MTX, MZR, and TAC was used for an in vitro study with osteoclasts and a prospective clinical study in order to show the efficacy of these agents against refractory RA. In particular, low-dose TAC or MZR was added to treat 14 patients with RA that was resistant to MTX + MZR or MTX + TAC dual therapy. The combination of three pharmacological agents showed statistically significant differences to reduce differentiation induction and activity of osteoclasts compared with single and double agents. In clinical use, triple therapy showed a statistically significant difference in the improvement of Disease Activity Score-28-erythrocyte sedimentation rate and the Simple Disease Activity Index score at around 8 months. Additionally, the serum matrix metalloproteinase-3 level significantly decreased. No patients dropped out because of adverse effects. Based on this in vitro and prospective clinical study, oral triple therapy might be effective against refractory RA. Furthermore, this therapy might be safe and economical for clinical practice.

Access-Related Infections Involving the Buttonhole Technique.

PURPOSE: In this study, we discuss a mechanism of development of access-related Staphylococcus aureus infections in patients on buttonhole (BH) method and logically construct a measure to prevent such infections on the basis of the mechanism. SUMMARY: S. aureus can colonize a BH track. Once S. aureus colonizes a BH track, access-related infections may develop when the equilibrium is upset between the factors of host resistance and a level of bacterial growth in a BH track. Thus, the logically constructed measure to prevent access-related infections are as follows: (1) decolonization of S. aureus from a BH track by applying mupirocin ointment to a BH entry site when a patient has been proven to be a carrier of S. aureus in the track, (2) prevention of bacterial invasion of the BH track by a new method to remove a scab completely, and (3) control of bacterial growth in the BH track by disinfecting the site with diluted povidone-iodine solution (0.1% povidone-iodine solution) before access vessel cannulation.

Inhibition of each module of connective tissue growth factor as a potential therapeutic target for rheumatoid arthritis.

We previously reported the importance of connective tissue growth factor (CTGF) in rheumatoid arthritis (RA). CTGF contains four distinct modules connected in tandem, namely insulin-like growth factor-binding protein (IGFBP)-like, von Willebrand factor (vWF) type C repeat, thrombospondin type 1 (TSP-1) repeat, and carboxyl-terminal (CT) modules. The relationships between each of these modules of CTGF and RA remain unknown. Here, we analyzed how inhibition of each CTGF module affects the pathophysiology of RA. We conducted stimulation and suppression experiments on synovial cells (MH7A) obtained from patients with RA. Moreover, we examined angiogenesis by means of a tube-formation assay performed using human umbilical vein endothelial cells (HUVECs), and we used tartrate-resistant acid phosphatase (TRAP) staining to analyze osteoclastogenesis. Our results showed that M-CSF/RANKL-mediated osteoclastogenesis was enhanced when CTGF was added, but the effect of CTGF was neutralized by mAbs against CTGF modules 1-4. Furthermore, CTGF treatment of HUVECs induced formation of tubular networks, which resulted in acceleration of the angiogenesis of RA synoviocytes, and quantification showed that this tubular-network formation was also disrupted by anti-CTGF module 1-4 mAbs. Lastly, TNF-α enhanced the expression of CTGF and matrix metalloproteinase-3 (MMP3) in MH7A cells, and this enhancement was potently neutralized by mAbs against CTGF modules 1, 3 and 4. Thus, our results indicate that not only a mAb against CTGF but also mAbs against each specific module of CTGF might serve as potential therapeutic agents in the treatment of RA.

Application of Buttonhole Cannulation Technique to Surgically Superficialized Arteries.

In Japan, use of a surgically superficialized brachial artery is recommended for vascular access in patients who are either unable to tolerate hemodialysis because of reduced cardiac function or who do not have vessels suitable for creation of an arteriovenous fistula. Superficializing a brachial artery involves relocating a portion of the artery into subcutaneous tissue and immobilizing the artery at that location. Superficialized artery access can result in certain serious complications, such as an aneurysm and/or stenosis. In order to avoid such complications, we attempted applying the buttonhole method to this vascular access. A buttonhole track was created slightly distal from the center of the superficialized portion of a brachial artery approximately 2 weeks after superficialization. When arteriosclerosis was evident in that location, we tried to find a less sclerotic portion, under ultrasonography guidance, for creation of the arterial-side buttonhole track. For returning extracorporeal circulated blood, a normal vein on the arm with a superficialized brachial artery was cannulated with a sharp needle. Recently, however, we attempted to create a buttonhole track also on a vein for venous-side buttonhole cannulation. The brachial artery was superficialized in 5 patients. In all patients, buttonhole cannulation was successfully performed with the artery access. Buttonhole cannulation had been performed on these patients for 8-54 months. No serious complications such as a pseudoaneurysm were found in these patients. Serious complications specific to the superficialized artery access may be prevented by application of the buttonhole method.

Inhibition of the insulin-like growth factor system is a potential therapy for rheumatoid arthritis.

OBJECTIVE: We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). Insulin-like growth factor binding proteins (IGFBPs) are modules of CTGF. IGFBPs bind IGF-I and IGF-II. IGF-I plays a role in the regulation of immunity, bone metabolism and inflammation. Therefore, we investigated how the IGF system is associated with RA disease progression. METHODS: Serum samples were collected from RA patients. IGF-I and IGFBP-3 production were evaluated by enzyme-linked immunosorbent assay, real-time RT-PCR and indirect immunofluorescence microscopy. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay and osteoclast-specific enzyme production. Angiogenesis was examined by a tube formation assay using human umbilical vein endothelial cells. RESULTS: The serum concentrations of IGFBP-3 in RA patients were greater than those in normal controls. IGF-I and IGFBP-3 were produced primarily by macrophages in the RA synovium. Furthermore, tumor necrosis factor-α could induce aberrant IGF-I and IGFBP-3 production in synovial fibroblasts. IGF-I and IGFBP-3 promoted the induction of osteoclast generation and morphological changes, in combination with M-colony stimulating factor and the receptor activator of NF-κB ligand. In addition, IGF-I and IGFBP-3 induced angiogenesis, as determined by the tube formation assay. These effects were neutralized by anti-IGF-IR monoclonal antibody (mAb). CONCLUSIONS: These results indicate that aberrant IGF-I and IGFBP-3 production plays a role in abnormal osteoclastic activation and angiogenesis in RA. This work supports future clinical exploration of anti-IGF-IR mAb in drug repositioning as a new treatment for RA.

Zfat-deficiency results in a loss of CD3ζ phosphorylation with dysregulation of ERK and Egr activities leading to impaired positive selection.

The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its physiological roles in T cell development remain poorly understood. Here, we generated Zfat (f/f)-LckCre mice and demonstrated that T cell-specific Zfat-deletion in Zfat (f/f)-LckCre mice resulted in a reduction in the number of CD4(+)CD8(+)double-positive (DP) cells, CD4(+)single positive cells and CD8(+)single positive cells. Indeed, in Zfat (f/f)-LckCre DP cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of CD3ζ phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection through ERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide deeper insight into T cell development and immune regulation.

Differential requirement for RhoH in development of TCRαß CD8αα IELs and other types of T cells.

RhoH is a new member of the atypical G proteins exclusively expressed in hematopoietic lineage cells. It has been shown to act as an adaptor for ZAP70, Syk, Lck and Csk kinases in signal transduction, and is required for positive selection of thymocytes as well as activation of peripheral T cells and mast cells. In the present study, we showed that RhoH is required not only for positive selection but also for negative selection of thymocytes. Regarding development of unconventional T cell subsets, development of NKT and regulatory T cells was also inhibited, whereas development of TCRαß CD8αα intestinal intraepithelial lymphocytes (IEL) was not affected by the absence of RhoH. TCR-dependent in vitro activation of TCRαß CD8αα IEL required RhoH, suggesting that overall development of IEL does not critically depend on TCR signaling but more on cytokine-dependent expansion and survival in the periphery. Our current results indicate differential requirements for RhoH in the development of TCRαß CD8αα IELs compared to other subsets of T cells including agonist selected T cells.

Induction of CCAAT/enhancer-binding protein-homologous protein by cigarette smoke through the superoxide anion-triggered PERK-eIF2α pathway.

Cigarette smoke triggers apoptosis through oxidative stress- and endoplasmic reticulum (ER) stress-dependent induction of CCAAT/enhancer-binding protein-homologous protein (CHOP) (Tagawa et al., 2008. Free Radic. Biol. Med. 45, 50-59). We investigated roles of individual reactive oxygen/nitrogen species in the transcriptional induction of CHOP by cigarette smoke. Exposure of bronchial epithelial cells to O(2)(-), ONOO(-) or H(2)O(2) induced expression of CHOP, whereas NO alone did not. Induction of CHOP mRNA by cigarette smoke extract (CSE) was attenuated by scavengers for O(2)(-), ONOO(-) or NO, whereas scavenging H(2)O(2) did not affect the induction of CHOP. Like CSE, O(2)(-) and ONOO(-) caused activation of the CHOP gene promoter. Scavengers for O(2)(-), ONOO(-) or NO attenuated CSE-triggered activation of the CHOP gene promoter. CSE, O(2)(-) and ONOO(-) induced phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) and caused induction of downstream activating transcription factor 4 (ATF4). Scavengers for O(2)(-), ONOO(-) or NO attenuated induction of ATF4 by CSE. Furthermore, dominant-negative inhibition of the PERK-eIF2α pathway exclusively suppressed CSE-triggered induction of CHOP and consequent apoptosis. These results suggest that O(2)(-) and ONOO(-) are selectively involved in CSE-triggered induction of CHOP and that the PERK-eIF2α pathway plays a crucial role in the induction of CHOP and apoptosis downstream of the particular reactive oxygen species.

Selective abrogation of BiP/GRP78 blunts activation of NF-κB through the ATF6 branch of the UPR: involvement of C/EBPß and mTOR-dependent dephosphorylation of Akt.

Subtilase cytotoxin (SubAB) that selectively cleaves BiP/GRP78 triggers the unfolded protein response (UPR) and protects mice from endotoxic lethality and collagen arthritis. We found that pretreatment of cells with SubAB suppressed tumor necrosis alpha (TNF-α)-induced activation of NF-κB and NF-κB-dependent chemokine expression. To elucidate underlying mechanisms, the involvement of C/EBP and Akt, putative regulators of NF-κB, was investigated. Among members of the C/EBP family, SubAB preferentially induced C/EBPß. Overexpression of C/EBPß suppressed TNF-α-induced NF-κB activation, and knockdown of C/EBPß attenuated the suppressive effect of SubAB on NF-κB. We identified that the ATF6 branch of the UPR plays a crucial role in the induction of C/EBPß. In addition to this effect, SubAB depressed basal and TNF-α-induced phosphorylation of Akt via the UPR. It was mediated by the induction of ATF6 and consequent activation of mTOR that dephosphorylated Akt. Inhibition of Akt attenuated activation of NF-κB by TNF-α, suggesting that the mTOR-Akt pathway is another target for SubAB-initiated, UPR-mediated NF-κB suppression. These results elucidated that SubAB blunts activation of NF-κB through ATF6-dependent mechanisms, i.e., preferential induction of C/EBPß and mTOR-dependent dephosphorylation of Akt.