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 antineutrophil 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 Lys 42 . 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. High mobility group box 1 (HMGB1)2 protein is one of the most abundant nonhistone chromosomal proteins in eukaryotic organisms. The primary sequences of HMGB1 in various higher organisms, from birds to mammals, show more than 90% homology with each other (1). The protein has multiple roles in transcription, replication, and cellular differentiation (2, 3). HMGB1 interacts with several transcription factors, thereby allowing them to perform their cellular roles. The phenotype of Hmgb1 knock-out mice confirmed the functional importance of HMGB1 as a regulator of transcription: they die shortly after birth and show a defect in the transcriptional control exerted by the glucocorticoid receptor (4). The subcellular distribution of the protein is tissue-specific: HMGB1 is located in both the nuclei and the cytoplasm of different tissues, such as lymphoid tissue, testis, neurons, and hepatocytes (5). Wang et al. (6) identified HMGB1 as a late mediator of endotoxin lethality in mice and showed that monocytes and macrophages stimulated by lipopolysaccharide (LPS), tumor necrosis factor (TNF) or interleukin-1 (IL-1) secrete HMGB1 in a delayed response. Patients with sepsis show an increased serum level of HMGB1, which is correlated with the severity of infection (7). Moreover, HMGB1 in monocytes and macrophages is extensively acetylated upon activation by LPS, causing localization of the protein to the cytosol (8). Cytosolic HMGB1 is then concentrated into secretory lysosomes and secreted when the cells receive an appropriate second signal (9). The recent discovery of extracellular HMGB1 as a proinflammatory mediator has been supported by a number of studies. In addition, HMGB1 is passively released from the nucleus to the extracellular milieu by cells that die as a result of necrosis or damage (10).Our previous studies showed that HMGB1 and HMGB2 are novel antigens of a...

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.

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ER␣ and ER␤, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-␤ 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/ER␣ and TGF-␤/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ER␣ inhibits TGF-␤ signaling by decreasing Smad protein levels. ER␣-mediated reductions in Smad levels did not require the DNA binding ability of ER␣, implying that ER␣ opposes the effects of TGF-␤ via a novel non-genomic mechanism. Our analysis revealed that ER␣ 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 ER␣.

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 D 3 -bound [1,25(OH) 2 D 3 -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) 2 D 3 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) 2 D 3 -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.

Alcohol abuse was found to predispose persons to opportunistic infections. In this study, we tried to improve the host antibacterial resistance of chronic alcohol-consuming (CAC) mice to opportunistic infections. Bactericidal macrophages with functions to produce IL-12 and to express mRNAs for CXCL9 and inducible nitric oxide synthase (M1 macrophages) were characterized as the main effector cells in host antibacterial innate immunities against infections with opportunistic pathogens. However, CAC mice were found to be carriers of M2b macrophages [macrophages with functions to produce IL-10 and to express mRNAs for CD163, chemokine ligand (CCL)1, and LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for high-voltage electron microscopy on T cells)], which were inhibitory on macrophage conversion from resident macrophages to M1 macrophages. Under treatment with CCL1 antisense oligodeoxynucleotides, a specific inhibitor of M2b macrophages, CAC mouse macrophages reverted to resident macrophages, and M1 macrophages were induced by a bacterial antigen from macrophages of CAC mice that were previously treated with the oligodeoxynucleotides. Opportunistic infections (enterococcal translocation and Klebsiella pneumonia) in CAC mice were completely controlled by CCL1 antisense oligodeoxynucleotides. These results indicate that certain opportunistic infections in alcoholics are controllable through the modulation of M2b macrophages.

Introduction Recent studies identified STAT4 (signal transducers and activators of transcription-4) as a susceptibility gene for systemic lupus erythematosus (SLE). STAT1 is encoded adjacently to STAT4 on 2q32.2-q32.3, upregulated in peripheral blood mononuclear cells from SLE patients, and functionally relevant to SLE. This study was conducted to test whether STAT4 is associated with SLE in a Japanese population also, to identify the risk haplotype, and to examine the potential genetic contribution of STAT1. To accomplish these aims, we carried out a comprehensive association analysis of 52 tag single nucleotide polymorphisms (SNPs) encompassing the STAT1-STAT4 region.

Kruppel-like factor 4 (KLF4) is a transcription factor that participates in both tumor suppression and oncogenesis. To determine the association of KLF4 with tumorigenesis, we integrated data assembled in the Oncomine database and discovered a decrease in KLF4 gene transcripts in breast cancers. Further analysis of the database also showed a correlation between KLF4 expression and estrogen receptor-a (ERa) positivity. Knockdown of KLF4 in MCF-7 cells elevated the growth rate of these cells in the presence of estrogen. Therefore, we examined the interaction between KLF4 and ERa, and found that KLF4 bound to the DNA-binding region of ERa. KLF4 thus inhibits the binding of ERa to estrogen response elements in promoter regions, resulting in a reduction in ERa target gene transcription. Earlier studies have reported that KLF4 is transcriptionally activated by p53 following DNA damage. We also showed that activation of p53 decreased the transcriptional activity of ERa by elevating KLF4 expression. Our studies discovered a novel molecular network between p53, KLF4 and ERa. As both p53 and ERa are involved in cell growth and apoptosis, these results may explain why KLF4 possesses both tumor suppressive and oncogenic functions in breast cancers.

Objective. Interferon regulatory factor 5, an established susceptibility factor for systemic lupus erythematosus (SLE), plays a role in type I interferon and proinflammatory cytokine induction. A recent study showed association of a functional single-nucleotide polymorphism (SNP) in intron 1 of IRF5, rs2004640, with systemic sclerosis (SSc) in a European French population. We undertook the present study to determine whether IRF5 polymorphisms are also associated with a predisposition to SSc in Japanese.Methods. A case-control association study was performed for rs2004640 as well as for rs10954213 and rs2280714, all of which were previously reported to be associated with SLE, in 281 SSc patients and 477 healthy controls. Patients with SSc complicated by SLE or Sjögren's syndrome were excluded. Association of the rs2280714 genotype with messenger RNA (mRNA) levels of IRF5 and adjacently located transportin 3 (TNPO3) was examined using the GENEVAR database.Results. All 3 SNPs were significantly associated with SSc, with the rs2280714 A allele having the strongest association (allele frequency P ‫؍‬ 0.0012, odds ratio 1.42 [95% confidence interval 1.15-1.75]). Association was preferentially observed in subsets of patients with diffuse cutaneous SSc (dcSSc) and anti-topoisomerase I antibody positivity. Conditional analysis revealed that rs2280714 could account for most of the association of these SNPs, while an additional contribution of rs2004640 was also suggested for dcSSc. The genotype of rs2280714 was strongly associated with IRF5 mRNA expression, while only marginal association was detected with TNPO3 mRNA expression.Conclusion. Association of IRF5 with SSc was replicated in a Japanese population. Whether the causal SNP is different among populations requires further investigation.