Embryonic stem (ES) cells can be maintained in an

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by elevation of plasma levels of total IgE, infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 T cells. However, the role of Th2 cells in the pathogenesis of AD is not fully understood. In this study we examined the NC/Nga (NC) mouse model of AD and established STAT6-deficient (SATA6−/−) NC mice to investigate the relevance of IL-4-mediated immune responses. Surprisingly, these mice elicited AD-like skin lesions at equivalent frequency and time of onset compared with normal NC littermates. Histological features of the lesion in STAT6−/− NC mice fulfilled the criteria for the pathogenesis of AD, although these mice fail to produce IgE and Th2 cytokines. The lymph nodes proximal to the regions of skin that developed lesions exhibited massive enlargement elicited by the accumulation of activated IFN-γ-secreting T cells. Moreover, caspase I, IL-18, IL-12, and IFN-γ are found to be highly expressed at the skin lesion, occurring simultaneously with elevation of eotaxin 2 and CCR3 expression. Therefore, the Th2-mediated immune response is not necessary for the development of AD-like skin disease in NC mice. The skin microenvironment that favored IFN-γ production tightly correlates with the skin disease in NC mice through the infiltration of eosinophils.

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The ST2 gene, which is specifically induced by growth stimulation, encodes interleukin-1 receptor-related proteins. Using the RT-PCR method, we found that the ST2 gene was broadly expressed in hematopoietic cell lines. It was also expressed specifically in helper T cell lines among lymphocytic cell lines. We analyzed the expression of ST2 in mouse helper T cell subsets with Northern blotting analysis. Mouse Th1 cell lines so far studied did not express ST2 mRNAs. On the other hand, one of the Th2 cell lines, D10, expressed ST2L (transmembrane form) without stimulation, while co-stimulation by PMA and A23187 induced ST2 (soluble form) mRNA. These results suggest that the ST2 gene is involved in the regulation of the immune system. IL-1 alpha, IL-1 beta, and receptor antagonist did not bind to ST2L protein, which prompted us to search for the specific ligand of ST2. The recombinant human ST2 protein was purified and labeled with FITC. The labeled human ST2 protein bound with myeloma-derived RPMI8226 cells among the various B-cell lines, indicating possible involvement of ST2 in T-cell/B-cell interaction.