The EWSR1 (EWS RNA binding protein 1) was originally discovered in Ewing sarcoma, a pediatric bone cancer. In a previous study, we identified zebrafish ewsr1a, the homologue of human EWSR1. Using a zebrafish mutant for ewsr1a, we demonstrated that Ewsr1a promotes differentiation of chondrogenesis in Meckel's cartilage, a component of the facial skeletal elements. Furthermore, we demonstrated that Ewsr1a interacts with the transcription factor SRY-BOX transcription factor 9 (SOX9) and modulates the transcription of its target genes. In this study, the role of Ewsr1a was further investigated in axial skeletogenesis. The maternal zygotic (MZ) ewsr1a/ewsr1a zebrafish display curved spines derived from irregularly spaced intervertebral discs (IVDs) and aberrant differentiation of nucleus pulposus cells. Using an in vitro cell culture system, we further show that the N-terminus of Ewsr1a is required for the interaction with Sox9. In zebrafish, the Ewsr1a also interacts with a Sox9 target gene, specifically the first intron of col2a1a. Zebrafish with the MZ ewsr1a/ewsr1a genotype display an increased level of collagen type II protein in the notochord starting at 36 h post fertilization. We propose that Ewsr1a contributes to IVD formation by regulating the expression of col2a1a.

Type 1 diabetes (T1D) is frequently associated with other autoimmune diseases. Most T1D patients' sera contain two distinct glutamic acid decarboxylase (GAD) antibody specificities, of which one targets an epitope region in the middle-third of GAD65 (amino acids 221-359) and the other targets the carboxy-third of GAD65 (amino acids 453-569). Using five chimeric GAD65/GAD67 proteins to maintain conformation-dependent epitopes of GAD65, we compared the humoral repertoire of antibodies from 127 T1D patients with and without autoimmune thyroid diseases (ATD). Thirty-one patients with T1D (24%) expressed antithyroid autoantibodies ATA and 22 patients (17%) had ATD in comparison to 6% of age-matched controls having ATA. GAD65-antibody-positive patients much more often (28% versus 5%, P < 0.0004) had ATD. Of 66 GAD65-autoantibody-positive T1D patients, 34 had autoantibodies reacting with both middle and carboxy epitopes. Autoantibodies of the other 32 reacted with middle, carboxy, or other epitopes but not with both middle- and carboxy-third. Those with GAD65 autoantibodies reacting with both middle- and carboxy-third had less ATD. Of 22 (23%) patients with ATD, 5 compared to 29 of 47 (62%) T1D patients without ATD had GAD65 autoantibodies reacting with both middle- and carboxy-third (relative risk = 0.2, P < 0.01). These results indicate that there are both similarities and differences in the humoral response to GAD65 in ATD and T1D, and expression of antibodies to middle- and carboxy-third at the same time is a feature specific to T1D.

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1β and TNF-α, and the activation of NF-κB. Functional blocking of IL-1β and TNF-α using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.

Receptor activator of NFκB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFα, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-γ, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.

Ceramide generated from sphingomyelin in response to ionizing radiation has been implicated as a second messenger to induce cellular proapoptotic signals. Both ceramide and its metabolic inhibitor, N, N-dimethyl-D-erythro-sphingosine (DMS), might lead to sustained ceramide accumulation in cells more efficiently, thereby sensitizing them to γ-radiation-induced cell death. To delineate this problem, the clonogenic survival of Lewis lung carcinoma (LLC) cells was evaluated following exposure to radiation together with or without C2-ceramide, DMS, or both. The treatment of ceramide/DMS synergistically decreased the survival of the irradiated cells compared with treatment with ceramide or DMS alone. Ceramide/DMS-treated cells displayed several apoptotic features after γ-irradiation, including increased sub G1 population, TUNEL-positive fraction, and poly-(ADP-ribose) polymerase (PARP) cleavage. We also observed ceramide/ DMS induced disruption of mitochondrial membrane potential (MMP) and activation of caspase- 9 and -3 in a radiation-dose-dependent manner. Furthermore, pretreatment of LLC cells with ceramide/DMS not only increased the protein expression level of Bax, but also decreased Bcl-2 after γ-irradiation. Taken together, the present study indicates that the radiosensitizing activity of ceramide/DMS on LLC cells most likely reflects the dominance of pro-apoptotic signals related to the mitochondria-dependent pathway.

Abstract: The SOX13, one of the family of transcription factors that play key roles in organ development, is reported to be a diabetes autoantigen, islet cell antigen 12 (ICA12). Recently, a study of antibodies to SOX13 was conducted in patients with type 1 diabetes mellitus (T1DM) indicating that these antibodies potentially identified patients without antibodies to the major T1DM‐associated autoantigens, insulin, GAD, or IA‐2. We know that the prevalence of islet‐specific autoantibodies (GAD, IA‐2) in Korean patients is much lower than that in white patients. It may be possible that other autoantibodies that could be directed to as yet unknown antigen may play a role in Korean T1DM patients. To investigate this, we measured SOX13 autoantibodies applying a radioligand binding assay using in vitro transcribed and translated antigen in 188 T1DM patients (mean duration, 4.2 years) and 64 T2DM patients and compared the results with those of 101 healthy control subjects. SOX13 autoantibodies occurred at a significantly higher frequency among T1DM patients (55/188, 29.3%) than among T2DM patients (4/64, 6.2%) or healthy adult controls (1/101, 1%). The 55 patients with positive SOX13 antibodies had significantly shorter duration of diabetes than SOX13 antibody‐negative patients (3.6 ± 2.8 vs. 4.5 ± 3.9 years; p < 0.05). We could detect a prevalence similar to control in patients with Hashimoto's thyroiditis (4.9%, n= 101) and rheumatoid arthritis (6.7%, n= 89). As a whole, 44 of the 55 patients with SOX13 antibodies had at least one or more other autoantibodies to the major T1DM‐associated autoantigens. However, SOX13 antibodies were the only antibodies detected as positive in 1 of the 11 new‐onset patients. We conclude, therefore, that these antibodies are likely to be one of several epitope‐spreading responses to islet‐ or nonislet‐specific autoantigens seen in the development of T1DM, and they may be used as a supplementary marker for investigating T1DM in Korea.

Abstract: Host nonspecific β cell injury by cytokines has been implicated in the process of early islet graft dysfunction. Islet transplant destruction by cytokines released from inflammatory cells that infiltrate the graft is thought to be mainly mediated by NF‐κB‐dependent nitric oxide (NO) production by the islet. In this study, we aimed to evaluate the role of IL‐6 in making a β cell resistant to cytokine‐induced apoptosis by decreasing the NF‐κB‐dependent NO production and compared the result with that of the expression of a dominant negative inhibitor of NF‐κB (DN NF‐κB). Incubation of MIN6 cells with IL‐1β, IFN‐γ, and TNF‐α (cytomix) increased production of nitrites, with increased expression of iNOS mRNA. When treated with cytomix, the DN NF‐κB‐transfected mutant demonstrated significantly less nitrite production and apoptosis than parent MIN6. NO production was effectively blocked by IL‐6 as well as by N‐monomethyl‐l‐arginine (l‐NMMA). Inhibition of the NO production led to decreased rate of apoptosis accompanied by downregulation of the proapoptotic molecule Bax and increased expression of the antiapoptotic molecule Bcl‐2 and Bcl‐xL. These data indicate that cytokine‐induced cell death in the MIN6 β cell line involves mechanisms that are, in part, NF‐κB‐ and NO‐dependent. Inhibition of the NO production by the incubation of the MIN6 cells by the pretreatment of IL‐6 or l‐NMMA is cytoprotective and can be used as a substitute for the expression of DN NF‐κB.