Yoshinao Muro

A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

Anti-MDA5 and anti-TIF1-γ antibodies have clinical significance for patients with dermatomyositis

OBJECTIVES Myositis-specific autoantibodies are useful for diagnosing PM/DM. Recently, two new myositis-specific autoantibodies against melanoma differentiation-associated gene 5 (MDA5) and transcriptional intermediary factor 1-gamma (TIF1-gamma) were identified in DM. Here, we detected these autoantibodies in patient sera using new assays with recombinant MDA5 and TIF1-gamma, and associated clinical features with the presence of anti-MDA5 or anti-TIF1-gamma antibodies. METHODS We screened 135 Japanese patients with various CTDs, including 82 with DM. DM patients were classified as clinically amyopathic DM (CADM), cancer-associated DM or classical DM without cancer. Anti-MDA5 and anti-TIF1-gamma antibodies were detected by their ability to immunoprecipitate biotinylated recombinant proteins. RESULTS Sera from 21 (26%) of 82 DM patients immunoprecipitated MDA5, and every anti-MDA5-positive patient had DM (except one patient with SSc). Sera from 20 (65%) of 31 CADM patients reacted with MDA5. Notably, anti-MDA5-positive DM patients had significantly more interstitial lung disease than anti-MDA5-negative DM patients (95 vs 32%, P < 0.001). Sera from 12 (15%) of 82 DM patients immunoprecipitated TIF1-gamma, and anti-TIF1-gamma antibodies were only detected in DM patients. Strikingly, 7 (58%) of 12 patients with cancer-associated DM had sera that reacted with TIF1-gamma. Anti-TIF1-gamma-positive DM patients had significantly more internal malignancies than anti-TIF1-gamma-negative DM patients (58 vs 9%, P < 0.001). CONCLUSIONS Anti-MDA5 and anti-TIF1-gamma antibodies were confirmed to be serological DM subset markers. Anti-MDA5 and anti-TIF1-gamma antibodies were detected based on their ability to immunoprecipitate biotinylated recombinant MDA5 and TIF1-gamma, and were closely associated with life-threatening complications in DM.

Clinical Correlations With Dermatomyositis-Specific Autoantibodies in Adult Japanese Patients With Dermatomyositis: A Multicenter Cross-sectional Study

OBJECTIVE To clarify the association of clinical and prognostic features with dermatomyositis (DM)-specific autoantibodies (Abs) in adult Japanese patients with DM. DESIGN Retrospective study. SETTING Kanazawa University Graduate School of Medical Science Department of Dermatology and collaborating medical centers. Patients A total of 376 consecutive adult Japanese patients with DM who visited our hospital or collaborating medical centers between 2003 and 2008. MAIN OUTCOME MEASURES Clinical and laboratory characteristics of adult Japanese patients with DM and DM-specific Abs that include Abs against Mi-2, 155/140, and CADM-140. RESULTS In patients with DM, anti-Mi-2, anti-155/140, and anti-CADM-140 were detected in 9 (2%), 25 (7%), and 43 (11%), respectively. These DM-specific Abs were mutually exclusive and were detected in none of 34 patients with polymyositis, 326 with systemic sclerosis, and 97 with systemic lupus erythematosus. Anti-Mi-2 was associated with classical DM without interstitial lung disease or malignancy, whereas anti-155/140 was associated with malignancy. Patients with anti-CADM-140 frequently had clinically amyopathic DM and rapidly progressive interstitial lung disease. Cumulative survival rates were more favorable in patients with anti-Mi-2 compared with those with anti-155/140 or anti-CADM-140 (P < .01 for both comparisons). Nearly all deaths occurred within 1 year after diagnosis in patients with anti-CADM-140. Conclusion Dermatomyositis-specific Abs define clinically distinct subsets and are useful for predicting clinical outcomes in patients with DM.

The Majority of Generalized Pustular Psoriasis without Psoriasis Vulgaris Is Caused by Deficiency of Interleukin-36 Receptor Antagonist

Generalized pustular psoriasis (GPP) is a rare inflammatory skin disease that can be life-threatening. Recently, it has been reported that familial GPP is caused by homozygous or compound heterozygous mutations of IL36RN. However, the majority of GPP cases are sporadic and it is controversial whether IL36RN mutations are a causative/predisposing factor for sporadic GPP. We searched for IL36RN mutations in two groups of GPP patients in the Japanese population in this study: GPP without psoriasis vulgaris (PV), and GPP with PV. Eleven cases of GPP without PV (GPP alone) and 20 cases of GPP accompanied by PV (GPP with PV) were analyzed. Surprisingly, 9 out of 11 cases of GPP alone had homozygous or compound heterozygous mutations in IL36RN. In contrast, only 2 of 20 cases of GPP with PV had compound heterozygous mutations in IL36RN. The two cases of GPP with PV who had compound heterozygous mutations in IL36RN are siblings, and both cases had PV-susceptible HLA-A*0206. We determined that GPP alone is a distinct subtype of GPP and is etiologically distinguished from GPP with PV, and that the majority of GPP alone is caused by deficiency of the interleukin-36 receptor antagonist due to IL36RN mutations.

Identification of a novel nuclear speckle-type protein, SPOP

A novel antigen recognized by serum from a scleroderma patient was identified by expression cloning from the HeLa cell cDNA library. The cloned cDNA encoded a 374‐amino acid protein with a relative molecular mass of 47 000 and a predicted amino acid sequence 62.7% identical to the hypothetical protein of Caenorhabditis elegans, T16H12.5. The deduced amino acid sequence had a typical POZ domain and an unidentified region conserved during evolution. No zinc finger or RNA recognition motifs were found in this clone. The 2 kbp mRNA encoding the novel clone SPOP (speckle‐type POZ protein) was found to be expressed in all human tissues examined. HA‐tagged SPOP, transfected and overexpressed in COS7 cells, exhibited a discrete speckled pattern in the nuclei and was co‐localized with the splicing factor, snRNP B′/B. Deletion analysis revealed that both the POZ domain and the evolutionarily conserved region at the amino‐terminus are required for the nuclear speckled accumulation of SPOP.

Disappearance of anti-MDA-5 autoantibodies in clinically amyopathic DM/interstitial lung disease during disease remission

OBJECTIVE Autoantibodies against melanoma differentiation-associated gene 5 (MDA-5) are one of the serological markers for DM. Anti-MDA-5 antibodies are especially associated with rapidly progressive interstitial lung disease (ILD) in amyopathic DM (ADM). It is known that the antibody status of anti-ENAs does not generally change significantly with disease course. For anti-MDA-5 antibodies, however, few longitudinal studies have investigated such changes. This study aimed to establish a quantitative assay for anti-MDA-5 antibodies towards assessing the long-term outcome of ADM patients who had anti-MDA-5 antibodies. METHODS We established ELISA for measuring anti-MDA-5 antibody levels using in vitro transcription and translation recombinant protein. The antibody levels were measured at different time points in 11 clinically ADM patients who tested positive for the anti-MDA-5 antibody on their first visit (range of follow-up 3 months to 16 years). RESULTS At the stage of clinical remission, six patients received no medication and the four others received low-dose CS. ELISA showed that anti-MDA-5 antibodies disappeared in nine of the patients and fell to just above the cut-off in one patient; in the patient who died, the antibodies remained. CONCLUSION Our results suggest that anti-MDA-5 antibodies may be useful as a marker for monitoring disease activity in ILD complicated with ADM. Serial monitoring at short intervals is required to evaluate whether anti-MDA-5 antibody levels correlate with ADM disease activity.

A novel IL36RN/IL1F5 homozygous nonsense mutation, p.Arg10X, in a Japanese patient with adult‐onset generalized pustular psoriasis

homozygous, asymptomatic individual with porphyrin levels 10 times the upper limit of normal in the report by Ged et al. Simultaneous occurrence of thalassaemia and CEP due to a single trans-acting mutation has been previously reported. We herein report the occurrence of thalassaemia trait and CEP due to two independent mutations. Thalassaemia is endemic to our region and the co-occurrence is probably a coincidence; however, it allows us to examine the existence of any interaction. Despite reported exceptions, the S47P generally displays a severe phenotype, as is the case in our patient. Our patient is also heterozygous for a severe b-chain mutation and an interaction cannot be ruled out. Thalassaemia might increase the severity of the phenotype by increasing the demand for haem products through ineffective erythropoiesis. This remains unknown, but it is a theory that merits consideration.

The Unfolded Protein Response Is Activated in Differentiating Epidermal Keratinocytes

The unfolded protein response (UPR), which is induced by stress to the endoplasmic reticulum (ER), is involved in the functional alteration of certain cells, such as the differentiation of B cells to plasma cells. The aim of this study is to determine whether the UPR is activated during epidermal keratinocyte (KC) differentiation. Here, we show that the expression of the UPR-induced proteins Bip/GRP78 and HRD1 was increased in cells in the supra-basal layers of normal human epidermis that contain KCs undergoing differentiation as well as in skin-equivalent cultured KCs. However, Bip/GRP78 and HRD1 were poorly expressed in proliferating KCs in squamous cell carcinoma and psoriasis vulgaris tissues. The epidermal growth factor receptor tyrosine kinase inhibitor, PD153035, which induces KC differentiation, upregulated UPR-induced marker mRNAs and proteins. Furthermore, microarray analyses and quantitative PCR revealed that ER stress-inducing reagents, tunicamycin (TU), thapsigargin, and brefeldin A, altered the expression of genes essential for human epidermal KC differentiation, including C/EBPbeta, KLF4, and ABCA12 in vitro. However, ABCA12 and KLF4 mRNA did not increase with TU treatment after siRNA-mediated knockdown of XBP-1. Taken together, our findings strongly suggest that the UPR is activated during normal epidermal KC differentiation and induces C/EBPbeta, KLF4, and ABCA12 mRNAs.

Low prevalence of anti-small ubiquitin-like modifier activating enzyme antibodies in dermatomyositis patients

Background. The myositis-specific autoantibodies that characterize certain forms of inflammatory myopathy are useful in diagnosing dermatomyositis (DM) / polymyositis and predicting its prognosis. Autoantibodies to small ubiquitin-like modifier activating enzyme (SAE) have been identified as a DM-marker antibody in European Caucasians. Objective and methods. This study investigates the frequency and clinical characteristics of anti-SAE autoantibodies in Japanese patients with DM. Sera from 110 Japanese patients, including 13 with juvenile DM, were screened for anti-SAE antibodies by enzyme-linked immunosorbent assays. Positive sera were further examined by immunoblotting of the immunoprecipitates. Results. Only two patients (1.8%) were confirmed to have anti-SAE antibodies, and neither of these two patients had amyopathic or juvenile DM. One patient with anti-SAE had DM complicated with pulmonary arterial hypertension, and the other had cancer-associated DM. Both had hallmark cutaneous manifestations of DM. Conclusion. This is the first report of anti-SAE antibodies from an Asian single center cohort. Although Japanese patients with anti-SAE antibodies have a clinical phenotype similar to that of Caucasian patients, their frequency was lower in the Japanese patients than in the previously reported Caucasian patients.

Properties of CENP-B and Its Target Sequence in a Satellite DNA

The centromere plays an essential role in the proper segregation of eukaryotic chromosomes at mitosis and meiosis. The centromere is the multifunctional domain of chromosome responsible for sister chromatid association at the inner site and for microtubule attachment at the outer surface. It also acts as a mechanochemical motor for chromosome movement. These multiple centromere functions must, in some way, be directed by a cis-acting DNA sequence located in the centromere region. Indeed, specific centromere DNA sequences (CEN-DNA) were identified in two yeast species. In Saccharomyces cerevisiae, CEN-DNA consists of roughly 125 bp sequence composed of three conserved elements. In contrast, the centromere sequence of S. pombe is quite different from S. cerevisiae in length and sequence organization (Chikasige et al, 1989; Hahnenberger et al, 1989; Clarke, 1990). The molecular bases for understanding the structure and function of the centromere/ kinetochore domain have not been elucidated in higher eukaryotes. In mammalian cells, satellite DNAs are localized in the centromeric hetero-chromatin or heterochromatic arm. In all human chromosomes, the alpha satellite or alphoid DNA family, a highly repetitive DNA composed of about 170 bp fundamental monomer repeating units, is found at the primary constriction (Willard, 1990), Its function, however, has not been established.