Takamitsu Mizobe

Intracellular galectin‐9 activates inflammatory cytokines in monocytes

Whether galectin‐9 plays a role in inflammatory responses remains elusive. The present study was designed to determine the role of intracellular galectin‐9 in activation of inflammatory cytokine genes in human monocytes. Galectin‐9 expression vector pBKCMV3‐G9 was transiently co‐transfected into THP‐1 monocytic cells along with luciferase reporters carrying gene promoters of IL‐1α (IL1A), IL‐1β (IL1B) and IFNγ. Transient transfection studies showed that galectin‐9 over‐expression activated all three gene promoters, suggesting that intracellular galectin‐9 induces inflammatory cytokine genes in monocytes. Galectin‐9 over‐expression also activated NF‐IL6 (C/EBP β) and AP‐1, but not NF‐κB. In contrast, extracellular galectin‐9 is not involved in regulation of inflammatory cytokines. Immunoprecipitation/Western blotting, using anti‐galectin‐9 Ab and anti‐NF‐IL6 Ab, showed physical association of intracellular galectin‐9 with NF‐IL6. RT‐PCR confirmed that galectin‐9 over‐expression increased IL‐1α and IL‐1β mRNA levels in THP‐1 cells. The interaction of galectin‐9 with NF‐IL6 was enhanced following LPS treatment in THP‐1 cells. Intracellular galectin‐9 synergized with LPS to activate NF‐IL6. Nuclear translocation of galectin‐9 was also observed in THP‐1 cells treated with LPS. Our results indicate that galectin‐9 is a LPS‐responsive factor, and further demonstrate that intracellular galectin‐9 transactivates inflammatory cytokine genes in monocytes through direct physical interaction with NF‐IL6.

Intracellular HMGB1 transactivates the human IL1B gene promoter through association with an Ets transcription factor PU.1

High mobility group box 1 protein (HMGB1), originally described as a non‐histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte‐specific E26 transformation‐specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31‐kDa proIL‐1β protein. The −131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S‐transferase‐pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix‐turn‐helix DNA‐binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1‐binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1‐deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.

Growth suppression of human mast cells expressing constitutively active c-kit receptors by JNK inhibitor SP600125

Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 showed constitutive activation of JNK/c-Jun, and the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G1 phase and apoptosis accompanied by the cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis. Following Sp600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Thus, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with mutations in the catalytic domain of c-kit.

Constitutive tyrosine and serine phosphorylation of STAT4 in T‐cells transformed with HTLV‐I

STAT4 is a critical mediator of IL‐12‐stimulated gene regulation in T‐helper type 1 (Th1) cell. IL‐12 activates the Janus family tyrosine kinases JAK2 and Tyk2, which in turn phosphorylate STAT4 on tyrosine 693. The p38 mitogen‐activated protein kinase (MAPK) signaling pathway is also activated in response to IL‐12, followed by phosphorylation of STAT4 on serine 721, which is required for STAT4 full transcriptional activity. In the present study, we demonstrated constitutive activation of STAT4 in HTLV‐I‐transformed T‐cell lines MT‐2, MT‐4 and HUT102 by immunoprecipitation, Western blotting and electrophoretic mobility shift assay (EMSA). In HTLV‐I‐transformed T‐cell lines, STAT4 was constitutively phosphorylated not only on tyrosine 693 but also on serine 721, and formed a heterodimer with STAT3. Constitutive phosphorylation of its upstream activators, JAK2, Tyk2 and p38 MAPK was also observed in the cells. EMSA and transient transfection studies further showed that the high‐affinity sis‐inducible element (hSIE) preferentially binds the STAT3/STAT4 heterodimer and is constitutively transactivated in MT‐2 cells in the absence of exogenous cytokine stimulation. When STAT4 expression vector was cotransfected along with STAT3 expression vector into MT‐2 cells, STAT4 significantly synergized with STAT3 to transactivate hSIE, showing the functional importance of heterodimer formation between STAT4 and STAT3.

Clinical Profiles and Outcomes of End-Stage Renal Failure Patients with Late Initiation of Renal Replacement Therapy Based on Uremic Symptoms under Intensive Renoprotective Therapies

Aim: This study describes the clinical profiles and outcomes of renal failure patients with late initiation of renal replacement therapies (RRT) based on uremic symptoms under intensive treatment prior to the start of RRT (IT). Methods: Thirteen patients (male 10, female 3) with end-stage renal disease who preferred to wait for the initiation of RRT until uremic symptoms appeared regardless of serum creatinine (s-Cr) and 24-hour creatinine clearance (24-hour Ccr) were chosen. All patients received IT including a low-protein diet, antihypertensive drugs including enalapril, erythropoietin and others to prevent and manage uremic states until the initiation of RRT. Clinical findings at the initiation of RRT and the outcomes after the start of RRT were examined. Results: RRT was initiated 23.6 ± 16.9 months after IT without any complication in all patients when mild uremic symptoms appeared. Uremic symptoms, blood pressure, serum albumin, potassium, calcium and urinary Cr excretion were well controlled except inorganic phosphate, hemoglobin and cardiac size. 24-hour Ccr and s-Cr were 3.4 ± 0.7 ml/min and 17.4 ± 3.8 mg/dl at initiation of RRT. The outcomes of all the patients were all well during chronic RRT. Conclusion: Intensive treatment prior to the start of RRT can diminish uremic symptoms and complications so that RRT might be initiated safely and with fewer problems, even in the face of lower 24-hour Ccr and markedly higher s-Cr.