Masataka Haneda

The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage‐ and tissue‐specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34−/− mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 α (eIF2α) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34−/− MEF, although following treatment with tunicamycin, the eIF2α phosphorylation level did not change in either GADD34+/+ or GADD34−/− cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild‐type mice. These expressions were strongly reduced in GADD34−/− MEF, which suggests that GADD34 up‐regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Induction of matrix metalloproteinases (MMP3, MMP12 and MMP13) expression in the microglia by amyloid-β stimulation via the PI3k/Akt pathway

Alzheimers disease is characterized by the presence of senile plaques in the brain composed primarily of amyloid-beta peptide. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. We herein analyzed the process of expression of Matrix metalloproteinases induced by Abeta stimulation. We found that Abeta1-42 induces a high level of MMP3, MMP12 and MMP13 in the microglia. The signal transduction pathway for the expression of these MMPs mRNA induced by Abeta1-42 depends on PI3K/Akt.

Potential value of human thrombomodulin and DAF expression for coagulation control in pig‐to‐human xenotransplantation

Miwa Y, Yamamoto K, Onishi A, Iwamoto M, Yazaki S, Haneda M, Iwasaki K, Liu D, Ogawa H, Nagasaka T, Uchida K, Nakao A, Kadomatsu K, Kobayashi T. Potential value of human thrombomodulin and DAF expression for coagulation control in pig‐to‐human xenotransplantation. Xenotransplantation 2010; 17: 26–37.

GADD34 induces p53 phosphorylation and p21/WAF1 transcription

Recently, others and we have shown that one of the functions of GADD34 is a recovery from a shutoff of protein synthesis induced by endoplasmic reticulum stress. GADD34 has been shown to induce growth arrest and apoptosis. Main protein of apoptosis is p53, especially phosphorylation of p53. And one of the main proteins of growth arrest is p21/WAF1. Here we analyzed the effects of GADD34 on p53 phosphorylation and p21/WAF1 transcription. Transfected Myc‐tagged p53 was dose‐dependently phosphorylated at Ser15 by increasing the amount of GADD34. Transfection of GADD34 also induced the endogenous phosphorylation of p53 and enhanced p21 protein expression. Transfection of GADD34 induced p21/WAF1 promoter activity. This activity was dependent on p53, because GADD34 transfection to p53‐deficient cells produced only a slight increase of p21/WAF1 promoter activity. The p21/WAF1 promoter activity was greatly enhanced by the transfection of p53. Both GADD34 and p53 transfection induced much higher p21/WAF1 promoter activity. The promoter activity of p21/WAF1 was very low in GADD34 deficient MEF. The transfection of GADD34 increased the p21/WAF1 promoter activity in GADD34 deficient MEF.

Clinical significance of regulatory T-cell-related gene expression in peripheral blood after renal transplantation.

Background. Regulatory T cells (Tregs) have been suggested to be deeply associated with immune tolerance and long-term graft survival in transplantation. Some recipients with stable graft function (ST) could possibly minimize immunosuppression during the maintenance period. However, effective assays for assessing the suitability of patients have yet to be established. The purpose of this study was to elucidate the clinical relevance of Treg-related gene expression such as forkhead box P3 (Foxp3) in peripheral blood after renal transplantation. Methods. Several key molecules related to the function of immune cells such as Treg, including Foxp3, transforming growth factor-&bgr;, cytotoxic T-lymphocyte antigen-4, chemokine receptor 7, toll-like receptor 4, granzyme B, T-bet, GATA3, RORC, &agr;1,2-mannosidase, and proteasome subunit &bgr; 10 were examined in the peripheral blood of 272 renal transplant recipients by quantitative real-time reverse-transcriptase polymerase chain reaction. The expression levels were compared between recipients with chronic rejection and ST. Results. Foxp3 messenger RNA (mRNA) levels were reduced immediately after transplantation and gradually recovered. Pretransplantation levels were closely correlated with 1 year posttransplantation levels. Recipients with chronic rejection had significantly lower levels of Foxp3, chemokine receptor 7, and granzyme B mRNA, and higher levels of toll-like receptor 4 and proteasome subunit &bgr; 10 mRNA compared with those with ST, although Foxp3 was the most relevant marker. Conclusion. There is a possibility that monitoring mRNA expression levels of Treg-related molecules in peripheral blood might offer useful information on patient selection and early detection of rejection when immunosuppression minimization strategy is implemented in renal transplantation.

Protein phosphatase 1, but not protein phosphatase 2A, dephosphorylates DNA-damaging stress-induced phospho-serine 15 of p53

Okadaic acid (OA) is a protein phosphatase (PP) inhibitor and induces hyperphosphorylation of p53. We investigated whether the inhibition of PP1 by OA promotes the phosphorylation of the serine 15 of p53. In vitro dephosphorylation assay showed that PP1 dephosphorylated ultraviolet C (UVC)‐induced phospho‐ser15 of p53, and that OA treatment inhibited it. One of the PP1 regulators, growth arrest and DNA damage 34 (GADD34), disturbed PP1 binding with p53, interfered with the dephosphorylation of p53 and increased the amount of phospho‐p53 after UVC‐treatment. This report provides the first evidence that PP1, but not PP2A, dephosphorylates phospho‐serine 15 of p53.

Comparative study on signal transduction in endothelial cells after anti-a/b and human leukocyte antigen antibody reaction: implication of accommodation.

Background. Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. Methods. Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. Results. Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. Conclusion. Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

Suppression of viral replication by stress-inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR pathway.

ABSTRACT GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2α nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.

Amyloid-β peptides induce cell proliferation and macrophage colony-stimulating factor expression via the PI3-kinase/Akt pathway in cultured Ra2 microglial cells

Alzheimers disease is characterized by numerous amyloid‐β peptide (Aβ) plaques surrounded by microglia. Here we report that Aβ induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony‐stimulating factor (M‐CSF). We examined signal cascades for Aβ‐induced M‐CSF mRNA expression. The induction of M‐CSF was blocked by a phosphatidylinositol 3 kinase (PI3‐kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Aβ enhanced NF‐κB binding activity to the NF‐κB site of the mouse M‐CSF promoter, which was blocked by LY294002. These results indicate that Aβ induces M‐CSF mRNA expression via the PI3‐kinase/Akt/NF‐κB pathway.

Comparative analysis of drug action on B-cell proliferation and differentiation for mycophenolic acid, everolimus, and prednisolone.

Background Although more attention has been paid recently to B-cell immunity, assay for B-cell analysis has yet to be clinically applicable because, unlike T cell, a B-cell culture system has not been well established. Methods We attempted to develop an in vitro culture system for the proliferation and differentiation of peripheral B cells into plasma cells, and to analyze the action of everolimus (EVR), mycophenolic acid (MPA), and prednisolone (PRD). Results Using a three-step culture system, peripheral CD19+ B cells could differentiate into plasma cells and produce IgG antibody. Activated B cells (CD19(hi)CD38(lo)IgD(−)), plasmablasts (CD19(hi)CD38(hi)IgD(−)), and plasma cells (CD19(lo/−)CD38(hi)IgD(−)) were observed as a main cell subset in step 1 (day 0–4), 2 (day 4–7), and 3 (day 7–10), respectively. IgG production on day 10 was significantly suppressed by EVR, MPA, and PRD, but not cyclosporine. Although both EVR and MPA inhibited B-cell proliferation and differentiation in step 1, EVR suppressed B-cell differentiation in step 2. Only a high concentration of PRD significantly inhibited B-cell proliferation, differentiation, and IgG production in step 3. Conclusions Although both MPA and EVR efficiently suppressed cell proliferation during the early phase of B-cell immune reaction, EVR could act in a later phase than MPA. PRD at a high concentration worked even in the last phase. An in vitro B-cell culture system would clarify the mode of drug action during B-cell differentiation and provide useful information on the effective selection or combination of immunosuppressive agents.