Atsuhisa Ueda

Transcriptional Regulation of The Human Monocyte Chemoattractant Protein-1 Gene COOPERATION OF TWO NF-κB SITES AND NF-κB/Rel SUBUNIT SPECIFICITY

Human monocyte chemoattractant protein-1 (human MCP-1) mRNA accumulated in THP-1 cells 2 h after lipopolysaccharide (LPS) stimulation. DNase I footprinting revealed that LPS stimulation induced protein binding to the two closely located NF-κB sites, A1 and A2. By electrophoretic gel mobility shift assay and supershift assay, the binding of (p65)2, c-Rel/p65, p50/p65, and p50/c-Rel to the A2 oligonucleotide probe was detected after LPS stimulation. In contrast, 12-o-tetradecanoylphorbol 13-acetate did not induce a significant amount of MCP-1 mRNA in THP-1 cells 2 h after stimulation, and only p50/p65 bound to the A2 probe.trans-Activity of each NF-κB/Rel dimer was investigated by transfecting P19 cells with p65, p50, and/or c-Rel expression vectors, and a luciferase construct containing the enhancer region of the human MCP-1 gene. Expression of recombinant p65 or p65 and c-Rel resulted in elevated luciferase activities, indicating that (p65)2 and c-Rel/p65 had trans-activity. The binding of (p65)2 and/or c-Rel/p65 to the A2 probe was also detected from 12-o-tetradecanoylphorbol 13-acetate-stimulated HeLa, HOS, and A172 cells in which expression of MCP-1 mRNA was elevated. Finally, the role of the A1 site was investigated. Both (p65)2 and c-Rel/p65 bound to the A1 probe by electrophoretic mobility shift assay and a mutation in the A1 or A2 site resulted in a loss of the enhancer activity. These results suggest that the binding of (p65)2 and c-Rel/p65 to the A1 and A2 sites of this gene is important for the tissue- and stimulus-specific transcription of the human MCP-1gene.

Genome-wide association analysis identifies new susceptibility loci for Behcet's disease and epistasis between HLA-B*51 and ERAP1.

Individuals with Behçets disease suffer from episodic inflammation often affecting the orogenital mucosa, skin and eyes. To discover new susceptibility loci for Behçets disease, we performed a genome-wide association study (GWAS) of 779,465 SNPs with imputed genotypes in 1,209 Turkish individuals with Behçets disease and 1,278 controls. We identified new associations at CCR1, STAT4 and KLRC4. Additionally, two SNPs in ERAP1, encoding ERAP1 p.Asp575Asn and p.Arg725Gln alterations, recessively conferred disease risk. These findings were replicated in 1,468 independent Turkish and/or 1,352 Japanese samples (combined meta-analysis P < 2 × 10−9). We also found evidence for interaction between HLA-B*51 and ERAP1 (P = 9 × 10−4). The CCR1 and STAT4 variants were associated with gene expression differences. Three risk loci shared with ankylosing spondylitis and psoriasis (the MHC class I region, ERAP1 and IL23R and the MHC class I–ERAP1 interaction), as well as two loci shared with inflammatory bowel disease (IL23R and IL10) implicate shared pathogenic pathways in the spondyloarthritides and Behçets disease.

Adenovirus-Mediated Transfer and Overexpression of Heme Oxygenase 1 cDNA in Lung Prevents Bleomycin-Induced Pulmonary Fibrosis via a Fas–Fas Ligand-Independent Pathway

Heme oxygenase 1 (HO-1) is an inducible enzyme that catalyzes heme to generate bilirubin, ferritin, and carbon monoxide. Because enhanced expression of HO-1 confers protection against many types of cell and tissue damage by modulating apoptotic cell death or cytokine expression profiles, we hypothesized that adenovirus-mediated transfer of HO-1 cDNA and subsequent overexpression of the protein in lung would provide therapeutic benefit in a murine model of bleomycin-induced pulmonary fibrosis. In C57BL/6 mice, HO-1 overexpression clearly suppressed the development of fibrotic changes and was associated with enhanced interferon gamma production in lung and reduced numbers of respiratory epithelial cells with damaged DNA. However, HO-1 overexpression did not prevent pulmonary fibrosis induced by agonistic anti-Fas antibody inhalation in C57BL/6 or ICR mice, a strain known to develop pulmonary fibrosis via the Fas-Fas ligand (FasL) pathway. Consistent with the concept that HO-1 overexpression prevents fibrosis via a pathway independent of Fas-FasL interaction, Ad.HO-1 administration prevented bleomycin-induced pulmonary fibrosis in gld/gld mice, which express nonfunctional FasL. These observations suggest that using HO-1 overexpression strategies to treat idiopathic pulmonary fibrosis, or fibrotic disorders of other target organs, by attenuating apoptotic cell death likely would be effective in clinical situations.

Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behçet disease

Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behçet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were evaluated for BD association. A differential distribution of the rare and low-frequency NSVs of a gene in 2,461 BD cases compared with 2,458 controls indicated their collective association with disease. By stringent criteria requiring at least a single burden test with study-wide significance and a corroborating test with at least nominal significance, rare and low-frequency NSVs in one GWAS-identified gene, IL23R (P = 6.9 × 10−5), and one gene involved in innate immunity, TLR4 (P = 8.0 × 10−4), were associated with BD. In addition, damaging or rare damaging NOD2 variants were nominally significant across all three burden tests applied (P = 0.0063–0.045). Furthermore, carriage of the familial Mediterranean fever gene (MEFV) mutation Met694Val, which is known to cause recessively inherited familial Mediterranean fever, conferred BD risk in the Turkish population (OR, 2.65; P = 1.8 × 10−12). The disease-associated NSVs in MEFV and TLR4 implicate innate immune and bacterial sensing mechanisms in BD pathogenesis.

Characterization of cis-Acting Elements of the Gene for Macrophage-stimulating Protein from the Human THE INVOLVEMENT OF POSITIVE AND NEGATIVE REGULATORY ELEMENTS

To analyze the promotor region of the human macrophage-stimulating protein (MSP) gene, the 5′-flanking region of this gene was cloned. The major initiation site was determined at T located 49 base pairs upstream of the translation initiation site by primer extention with mRNA from HepG2 and Hep3B cells. There was no TATA sequence in this region. Transient transfection assay with 5′-deletion constructs showed that the transcription of this gene was regulated by positive and negative regulatory elements (PRE and NRE). The PRE (−34 to +2) was essential for the maximal transcription of this gene, and the NRE (−141 to −34) appeared to be responsible for the tissue-specific expression of the gene. The PRE contained the CCAAT sequence and a mutation from CCAAT to CTGAT resulted in a significant loss of the transcriptional activity. Electrophoretic mobility shift assay suggested that two different proteins bound to the PRE (MSP-PRE-binding protein-1 (MSP-PREB1) and 2). MSP-PREB1 and 2 were detected in various cell types, and the CCAAT sequence was involved in these bindings. These findings indicate that MSP-PREB1 and 2 are positive regulators. Further characterization also revealed that MSP-PREB2 was identical to CCAAT binding transcription factor, also known as NF-Y.

Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM.

RanBPM is a RanGTP-binding protein required for correct nucleation of microtubules. To characterize the mechanism, we searched for RanBPM-binding proteins by using a yeast two-hybrid method and isolated a cDNA encoding the ubiquitin-specific protease USP11. The full-length cDNA of USP11 was cloned from a Jurkat cell library. Sequencing revealed that USP11 possesses Cys box, His box, Asp and KRF domains, which are highly conserved in many ubiquitin-specific proteases. By immunoblotting using HeLa cells, we concluded that 921-residue version of USP11 was the predominant form, and USP11 may be a ubiquitous protein in various human tissues. By immunofluorescence assay, USP11 primarily was localized in the nucleus of non-dividing cells, suggesting an association between USP11 and RanBPM in the nucleus. Furthermore, the association between USP11 and RanBPM in vivo was confirmed not only by yeast two-hybrid assay but also by co-immunoprecipitation assays using exogenously expressed USP11 and RanBPM. We next revealed proteasome-dependent degradation of RanBPM by pulse-chase analysis using proteasome inhibitors. In fact, ubiquitinated RanBPM was detected by both in vivo and in vitro ubiquitination assays. Finally, ubiquitin conjugation to RanBPM was inhibited in a dose-dependent manner by the addition of recombinant USP11. We conclude that RanBPM was the enzymic substrate for USP11 and was deubiquitinated specifically.

Transactivation of MCP-1/CCL2 by beta-catenin/TCF-4 in human breast cancer cells.

The loss of E‐cadherin expression and the translocation of β‐catenin to the nucleus are frequently associated with the metastatic conversion of epithelial cells. In the nucleus, β‐catenin binds to the TCF/LEF‐1 (T‐cell factor/ lymphoid enhancer factor) transcription factor family resulting in the activation of several genes, some of them having important implications in tumour progression. In our study, we investigated the potential regulation of monocyte chemotactic protein‐1 (MCP‐1/CCL2) expression by the β‐catenin/TCF pathway. This CC‐chemokine has been implicated in tumour progression events such as angiogenesis or tumour associated macrophage (TAM) infiltration. We thus demonstrated that MCP‐1 expression correlates with the reorganization of the E‐cadherin/β‐catenin complexes. Indeed, MCP‐1 was expressed by invasive breast cancer cells (MDA‐MB‐231, BT549 and Hs578T), which do not express E‐cadherin but was not produced by noninvasive breast cancer cell lines (MCF7 and T47D) expressing high level of E‐cadherin. In addition, the MCP‐1 promoter was activated in BT549 breast cancer cells transfected with β‐catenin and TCF‐4 cDNAs. The MCP‐1 mRNA level was similarly upregulated. Moreover, we showed that MCP‐1 mRNA was downregulated after transfection with a siRNA against β‐catenin in both BT549 and Hs578T cells. Our results therefore identify MCP‐1 as a target of the β‐catenin/TCF/LEF pathway in breast tumour cells, a regulation which could play a key role in breast tumour progression.

Chemical induction of HO-1 suppresses lupus nephritis by reducing local iNOS expression and synthesis of anti-dsDNA antibody.

There is accumulating evidence that haem oxygenase (HO)‐1 plays a protective role in various disorders. The beneficial efficacy of HO‐1 induction therapy has been shown in renal diseases such as glomerulonephritis, interstitial nephritis and drug induced nephrotoxicity. However, involvement of HO‐1 in the development of autoimmune renal diseases remains uncertain. To assess the clinical efficacy of HO‐1 induction therapy for lupus glomerulonephritis, MRL/lpr mice were intraperitoneally injected with 100 µmol/kg hemin, a potent HO‐1 inducer, or PBS as controls, once a week from 6 weeks of age to 21–24 weeks‐old. We found that treatment with hemin led to a significant reduction of proteinuria and remarkable amelioration of glomerular lesions accompanied by decreased immune depositions. In addition, the circulating IgG anti‐double‐stranded DNA antibody level was significantly decreased in hemin treated mice when compared with controls. A single intraperitoneal injection with hemin resulted in reduction of inducible nitric oxide synthase expression in the kidney and spleen, and serum interferon‐γ level. Our results suggest that HO‐1 induction therapy ameliorates lupus nephritis by suppressing nitric oxide (NO) dependent inflammatory responses and attenuating production of pathogenic autoantibodies.

Ultrasonography is a potent tool for the prediction of progressive joint destruction during clinical remission of rheumatoid arthritis

ObjectivesAlthough “clinical remission” has been a realistic goal of treatment in rheumatoid arthritis (RA), there is evidence that subclinical synovitis is associated with ongoing structural damage even after clinical remission is achieved. In the study reported here, we assessed whether ultrasonography (US) can predict progressive joint destruction during clinical remission of RA.MethodsThirty-one patients with RA in clinical remission based on the disease activity score in 28 joints were recruited for this study. Bilateral wrists and all of the metacarpophalangeal and proximal interphalangeal (PIP) joints were examined by power Doppler (PD) ultrasonography (US), and the PD signals were scored semiquantitatively in each joint. The total PD score was calculated as the sum of individual scores for each joint.ResultsAmong 22 RA patients who maintained clinical remission during the 2-year follow-up period, seven showed radiographic progression. Radiographic progression was strongly associated with total PD score at entry, with all patients showing radiographic progression having a total PD score of ≥2 at entry and none of the patients with a total PD score of ≤1 showing any radiographic progression. There was no significant association of therapeutic agents with progressing or non-progressing cases.ConclusionsPD-US detects synovitis causing joint destruction even when the patient is in clinical remission. Thus, remission visible on US is essential to reach “true remission” of RA.

Increased serum HO-1 in hemophagocytic syndrome and adult-onset Still's disease: use in the differential diagnosis of hyperferritinemia

Heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, is expressed by macrophages and endothelial cells in response to various stresses. Because ferritin synthesis is stimulated by Fe2+, which is a product of heme degradation, we examined the relation between HO-1 and ferritin levels in the serum of patients with hemophagocytic syndrome (HPS), adult-onset Stills disease (ASD), and other diseases that may cause hyperferritinemia. Seven patients with HPS, 10 with ASD, 73 with other rheumatic diseases, 20 with liver diseases, 10 recipients of repeated blood transfusion because of hematological disorders, and 22 healthy volunteers were enrolled. Serum HO-1 and ferritin levels were determined by ELISA. Expression of HO-1 mRNA and protein by peripheral blood mononuclear cells (PBMCs) was determined by real-time PCR and immunocytochemical techniques, respectively. Serum levels of HO-1 were significantly higher in patients with active HPS and ASD than in the other groups (P < 0.01). HO-1 levels were not elevated in patients with other causes of hyperferritinemia but were moderately elevated in patients with dermatomyositis/polymyositis. Among patients with HPS and ASD, serum HO-1 levels correlated closely with serum ferritin levels, and the levels of both returned to normal after therapy had induced remission. Increased expression of HO-1 mRNA was confirmed in PBMCs from some patients with HPS and ASD. Hyperferritinemia correlated closely with increased serum HO-1 in patients with HPS and ASD but not other conditions, indicating that measurement of serum HO-1 and ferritin levels would be useful in the differential diagnosis of hyperferritinemia and perhaps also in monitoring disease activity in HPS and ASD.