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Gut bacteria-associated sepsis is a serious concern in patients with gastrointestinal acute radiation syndrome (GIARS). In our previous studies, gut bacteria-associated sepsis caused high mortality rates in mice exposed to 6-9 Gy of γ-rays. IL-12+CD38+ iNOS+ Mϕ (M1Mϕ) located in the bacterial translocation site (mesenteric lymph nodes [MLNs]) of unirradiated mice were characterized as host defense antibacterial effector cells. However, cells isolated from the MLNs of GIARS mice were mostly CCL1+IL-10+LIGHT+miR-27a+ Mϕ (M2bMϕ, inhibitor cells for the M1Mf polarization). Reduced long noncoding RNA Gas5 and increased miR-222 expression in MLN-Mϕ influenced by the irradiation were shown to be associated with M2bMf polarization. In this study, the mortality of mice exposed to 7 Gy of gγ-rays (7 Gy GIARS mice) was completely controlled after the administration of glycyrrhizin (GL), a major active ingredient in licorice root (Glycyrrhiza glabra). Bacterial translocation and subsequent sepsis were minimal in 7 Gy GIARS mice treated with GL. Increased Gas5 RNA level and decreased miR-222 expression were shown in MLN-Mϕ isolated from 7 Gy GIARS mice treated with GL, and these macrophages did not display any properties of M2bMϕ. These results indicate that gut bacteria-associated sepsis in 7 Gy GIARS mice was controlled by the GL through the inhibition of M2bMϕ polarization at the bacteria translocation site. Expression of Ccl1, a gene required for M2bMϕ survival, is silenced in the MLNs of 7 Gy GIARS mice because of Gas5 RNA, which is increased in these cells after the suppression of miR-222 (a Gas5 RNA expression inhibitor) by the GL. [ABSTRACT FROM AUTHOR]

Macrophages (Mϕ) are highly plastic and change their functional phenotypes depending on microenvironmental signals. Recent studies have shown that microRNAs are involved in the polarization of Mϕ. In this study, we demonstrated that the phenotype of M2bMϕ [CCL1(+) IL-10(+) LIGHT(+)] switches to other phenotypes with interchangeability attained through the increased expression of growth arrest-specific 5 RNA (GAS5 RNA), a long noncoding RNA. GAS5 RNA has been described as a silencer of the CCL1 gene. Various phenotypes of Mϕ were prepared from bone marrow-derived Mϕ (BMDMϕ) after stimulation with IFNγ [M(IFNγ)/M1Mϕ], IL-4 [M(IL-4)/M2aMϕ], LPS and immobilized IgG [M(LPS + IC)/M2bMϕ], and IL-10 [M(IL-10)/M2cMϕ]. BMDMϕ cultured with medium [M(no)/quiescent Mϕ] were used as a control. As compared to Μ(no), M(IFNγ), M(IL-4) and M(IL-10), the reduced level of GAS5 RNA was shown in M(LPS + IC). CCL1 and LIGHT mRNAs (typical biomarkers of M2bMϕ) were not expressed by M(LPS + IC) transduced with a GAS5 gene using lentiviral vector. The reduction of GAS5 RNA in M(LPS + IC) was mediated by the activation of nonsense-mediated RNA decay (NMD) pathway. BMDMϕ overexpressed with GAS5 RNA after GAS5 gene transduction did not polarize to M2bMϕ even though they were stimulated with LPS and IC in combination. These results indicate that the reduction of GAS5 RNA influenced by the NMD pathway activation leads to the Mϕ polarization stimulated with LPS and IC in combination. [ABSTRACT FROM AUTHOR]

Mortality associated with Staphylococcus aureus infection remains high during the sub‐acute phase of burn injury. In this study, we aimed to improve antibacterial resistance of sub‐acutely burned mice through macrophage polarization. Sepsis did not develop in mice at the sub‐acute phase of 5% total body surface area (TBSA) burn after being infected with methicillin‐resistant S. aureus (MRSA), and M1 macrophages (interleukin (IL)‐10−IL‐12+ inducible nitric oxide synthase+ Mφ) were isolated from these mice. In contrast, predominantly M2b macrophages (C‐C motif chemokine ligand 1 (CCL1)+IL‐10+IL‐12− Mφ) were isolated from mice with >15% TBSA burn, and all of these mice died after the same MRSA infection. Comparing NOD scid gamma mice inoculated with Mφ with 25% TBSA burns, all mice treated with CCL1 antisense oligodeoxynucleotide (ODN) survived after MRSA infection, whereas all untreated mice given the same infection died within 4 days. CCL1 antisense ODN has been characterized as a specific polarizer of M2bMφ. M1Mφ were isolated from MRSA‐infected mice with 25% TBSA burn after treatment with CCL1 antisense ODN, and these mice were shown to be resistant against a lethal dose of MRSA infection. M1Mφ were also isolated from 25% TBSA‐burned mice infected with MRSA when the ODN was administered therapeutically, and subsequent sepsis was effectively controlled in these mice. These results indicate that the M2bMφ polarizer is beneficial for controlling MRSA infection in mice at the sub‐acute phase of severe burn injury. [ABSTRACT FROM AUTHOR]

Orosomucoid (ORM, composed of two isoforms, ORM1 and ORM2) has been described as an inducer of M2 macrophages, which are cells that decrease host antibacterial innate immunities. However, it is unknown which phenotypes of M2 macrophages are induced by ORM. In this study, healthy donor monocytes stimulated with ORM (ORM-monocytes) were characterized phenotypically and biologically. CCL1 (a biomarker of M2b macrophages) and IL-10 were detected in monocyte cultures supplemented with ORM1; however, CCL17 (a biomarker of M2a macrophages) and CXCL13 (a biomarker of M2c macrophages) were not produced in these cultures. All of these soluble factors were not detected in the culture fluids of monocytes stimulated with ORM2. Monocytes stimulated with ORM1 were characterized as CD64 − CD209 − CD163 + CCL1 + cells. MRSA and Enterococcus faecalis infections were accelerated in chimeras (NOD/scid IL-2Rγ null mice reconstituted with white blood cells) after inoculation with monocytes stimulated with ORM1 or treatment with ORM1; however, the infections were greatly mitigated in both chimeras inoculated with ORM1-stimulated monocytes and treated with ORM1, after an additional treatment with an inhibitor of M2b macrophages (CCL1 antisense ODN). These results indicate that ORM1 stimulates quiescent monocytes to polarize to M2b monocytes. The regulation of M2b macrophages may be beneficial in controlling opportunistic infections in patients with a large amount of plasma ORM1. [ABSTRACT FROM AUTHOR]

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-β-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-β-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β-SMAD signal transduction. Since TGF-β-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia. [ABSTRACT FROM AUTHOR]

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D3-bound [1,25(OH)2D3-bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH)2D3 treatment prevented renal fibrosis through the suppression of TGF-β-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH)2D3-dependent suppression of TGF-β-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β-SMAD signal transduction. Since TGF-β-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia. [ABSTRACT FROM AUTHOR]

Abstract: Non-secosteroidal ligands for vitamin D receptor (VDR) have been developed for the agonist with non-calcemic profiles. Here, we provide the structural mechanism of VDR agonism by novel non-secosteroidal ligands. All ligands had the similar efficacy, while two had the higher potency. Crystallographic analyses revealed that all ligands interacted with helix H10 and the loop between helices H6 and H7 in a similar manner, but also that the two ligands with higher potency had different interaction modes. This study suggests that distinct ligand potency depend upon differences in the formation and rearrangement of hydrogen-bond networks induced by each ligand. [Copyright &y& Elsevier]

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERα and ERβ, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-β acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERa and TGF-β/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERα inhibits TGF-β signaling by decreasing Smad protein levels. ERα-mediated reductions in Smad levels did not require the DNA binding ability of ERα, implying that ERα opposes the effects of TGF-β via a novel non-genomic mechanism. Our analysis revealed that ERα formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERα, [ABSTRACT FROM AUTHOR]

Abstract: PPARγ is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARγ ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARγ ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein. [Copyright &y& Elsevier]

High mobility group box 1 (HMGB1) protein plays multiple roles in transcription, replication, and cellular differentiation. HMGB1 is also secreted by activated monocytes and macrophages and passively released by necrotic or damaged cells, stimulating inflammation. HMGB1 is a novel antigen of anti-neutrophil cytoplasmic antibodies (ANCA) observed in the sera of patients with ulcerative colitis and autoimmune hepatitis, suggesting that HMGB1 is secreted from neutrophils to the extracellular milieu. However, the actual distribution of HMGB1 in the cytoplasm of neutrophils and the mechanisms responsible for it are obscure. Here we show that HMGB1 in neutrophils is post-translationally mono-methylated at Lys42. The methylation alters the conformation of HMGB1 and weakens its DNA binding activity, causing it to become largely distributed in the cytoplasm by passive diffusion out of the nucleus. Thus, post-translational methylation of HMGB1 causes its cytoplasmic localization in neutrophils. This novel pathway explains the distribution of nuclear HMGB1 to the cytoplasm and is important for understanding how neutrophils release HMGB1 to the extracellular milieu. [ABSTRACT FROM AUTHOR]

HMGB1 and HMGB2 are abundant nonhistone chromosomal proteins in eukaryotic organisms. Their respective primary sequences are highly conserved. Our previous studies showed that these proteins are novel autoantigens of anti-neutrophil cytoplasmic antibodies in sera from patients with ulcerative colitis (UC), rheumatic disease and autoimmune hepatitis (AIH). In the present paper, we showed that anti-HMGB1 and HMGB2 antibodies in sera of patients with UC do not recognize HMGB1 in neutrophils while they recognize the protein in lymphocytes. Anti-HMGB2 monoclonal antibody FBH7, recognizing HMGB1 in lymphocytes, showed a similar profile to the antibodies in the patients’ sera. In order to elucidate the difference in immunoreactivity to HMGB1 between neutrophils and lymphocytes, we mapped the epitope for FBH7 by means of several methods. The results showed that FBH7 recognizes the intact conformation composed of 52–56 residues of HMGB1 in lymphocytes. This suggested that HMGB1 in neutrophils is conformationally changed in the epitope or the peripheral structure of the epitope from the protein in lymphocytes. The apparent conformational change of HMGB1 between neutrophils and lymphocytes will be important for understanding the functional difference of HMGB1 in these cells. [ABSTRACT FROM PUBLISHER]

The high mobility group box 1 (HMGB1) protein is a non-histone chromosomal protein that acts as a potent proinflammatory cytokine when actively secreted from LPS- or TNF-activated macrophages, monocytes, and other cells. Anti-HMGB1/2 antibodies have been previously identified in sera from a high proportion of patients with autoimmune diseases. In this study, we examined anti-HMGB1 antibody titers in sera of patients with systemic rheumatic diseases and the correlations between the presence of anti-HMGB1 antibodies and disease activity in systemic lupus erythematosus (SLE) patients by enzyme-linked immunosorbent assay and western blotting. We detected increases in both the levels and the frequency of anti-HMGB1 antibodies in sera from SLE and polymyositis/dermatomyositis (PM/DM) patients, and observed that the presence of anti-HMGB1 antibodies positively correlates with SLE disease activity index. Through epitope mapping, we found that multiple HMGB1 epitopes were recognised in SLE sera, with the major epitope mapping to box A. Another epitope, the joiner region of HMGB1, was preferentially recognized by SLE sera, but not by PM/DM sera. Collectively, these observations suggest that the presence of anti-HMGB1 antibodies correlates with disease activity in SLE patients. [ABSTRACT FROM AUTHOR]

The active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3, acts as a ligand for the vitamin D receptor ( VDR) and activates VDR-mediated gene expression. Recently, we characterized 1α,25-dihydroxyvitamin D3-26,23-lactams ( DLAMs), which mimic vitamin D3 metabolites, as noncalcemic VDR ligands that barely activate the receptor. In this study, we present structural insights onto the regulation of VDR function by DLAMs. X-ray crystallographic analysis revealed that DLAMs induced a large conformational change in the loop region between helices H6 and H7 in the VDR ligand-binding domain. Our structural analysis suggests that targeting of the loop region may be a new mode of VDR regulation. [ABSTRACT FROM AUTHOR]

Background: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. Methodology/Result: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24±0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX. Conclusions/Significance: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX. [ABSTRACT FROM AUTHOR]

CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins. The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs 13, 14, 15, 16, 17, 18, 19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3. [ABSTRACT FROM AUTHOR]