Norimitsu Saitoh

Identification of functional domains and novel binding partners of STIM proteins

With a signal trap method, we previously identified stromal interaction molecule (STIM: originally named as SIM) as a protein, which has a signal peptide in 1996. However, recent works have accumulated evidences that STIM1 and STIM2 reside in endoplasmic reticulum (ER) and that both mainly sense ER Ca2+ depletion, which plays an essential role in store operated calcium entry. In the present study, we extensively analyzed the domain functions and associated molecules of STIMs. A STIM1 mutant lacking the coiled‐coil domains was massively expressed on the cell surface while mutants with the coiled‐coil domains localized in ER. In addition, STIM1 mutants with the coiled‐coil domains showed a longer half‐life of proteins than those without them. These results are likely to indicate that the coiled‐coil domains of STIM1 are essential for its ER‐retention and its stability. Furthermore, we tried to comprehensively identify STIM1‐associated molecules with mass spectrometry analysis of co‐immunoprecipitated proteins for STIM1. This screening clarified that both STIM1 and STIM2 have a capacity to bind to a chaperone, calnexin as well as two protein‐transporters, exportin1 and transportin1. Of importance, our result that glycosylation on STIM1 was not required for the association between STIM1 and calnexin seems to indicate that calnexin might function on STIM1 beyond a chaperone protein. Further information concerning regulatory mechanisms for STIM proteins including the data shown here will provide a model of Ca2+ control as well as a useful strategy to develop therapeutic drugs for intracellular Ca2+‐related diseases including inflammation and allergy. J. Cell. Biochem. 112: 147–156, 2011.

Regulation of human B lymphopoiesis by the transforming growth factor-β superfamily in a newly established coculture system using human mesenchymal stem cells as a supportive microenvironment

OBJECTIVES To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology. MATERIALS AND METHODS We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development. RESULTS Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A > TGF-beta1 > BMP-4. None of these three factors influenced the emergence of IgM(+) cells. CONCLUSIONS hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.

Signal-transducing adaptor protein-2 regulates macrophage migration into inflammatory sites during dextran sodium sulfate induced colitis.

Signal‐transducing adaptor protein‐2 (STAP‐2) was cloned as a c‐fms/M‐CSF receptor interacting protein. STAP‐2 is an adaptor protein carrying pleckstrin homology and Src homology 2 like domains, as well as a YXXQ motif. STAP‐2 has been indicated to have an ability to bind and modulate a variety of signaling and transcriptional molecules. Especially, our previous in vitro studies showed that STAP‐2 is crucial for immune and/or inflammatory responses. Here, we have investigated the role of STAP‐2 in intestinal inflammation in vivo. The disruption of STAP‐2 attenuates dextran sodium sulfate induced colitis via inhibition of macrophage recruitment. To study whether hematopoietic or epithelial cell derived STAP‐2 is required for this phenomenon, we generated BM chimeric mice. STAP‐2‐deficient macrophages impair the ability of CXCL12‐induced migration. Intriguingly, STAP‐2 also regulates production of proinflammatory chemokines and cytokines such as CXCL1 and TNF‐α from intestinal epithelial cells. Therefore, STAP‐2 has a potential to regulate plural molecular events during pathological inflammatory responses. Furthermore, our findings not only indicate that STAP‐2 is important in regulating intestinal inflammation, but also provide new insights toward the development of novel therapeutic approaches.