Research projects

動物の骨格の左右対称性は、歩行や走行、遊泳、飛行の基盤となる。「骨格パターン形成の段階」では、骨芽細胞の細胞キラリティ（互いに鏡像の左手型や右手型細胞のこと）と左右対称性形態形成の関係を解明する。次に「骨の変容成長の段階」では、皮質骨が周囲の臓器の変化に応じて吸収され、対面の皮質骨が形成される「内形成性トランスペアリング」が起きるメカニズムを解明し、鏡像的な骨周囲環境で骨格が左右対称になることを示す。

我々が行った予備実験の結果、脊索腫細胞そのものが、骨浸潤部位において破骨細胞様のふるまいをし、酸性環境を作り出すことで骨破壊を引き起こしている可能性を見出した。本研究では、骨代謝学において蓄積のある破骨細胞研究の知見を元に、「脊索腫細胞自身が直接骨破壊を引き起こす」という仮説を検証することで、脊索腫における骨破壊の機序を解明し、それを標的とした新たな治療戦略の提言を目指す。

Based on the histological analysis of endochondral ossification, we found that calcified cartilage function as a superior scaffold for bone formation by osteoblasts. So, we performed screening of molecules enhance bone formation from components of calcified cartilage and applied to coating. We identified several candidate molecules and these coating on culture dish significantly enhanced in vitro bone formation by osteoblasts. In addition, we designed a surface micropattern inspired by characteristic structures of calcified cartilage. We also developed assay methods to evaluate the surface properties by using kinetics of osteoblast migration. The micropattern changed in vitro bone formation rate and cell migration speed of osteoblasts. From these examinations, we suggest that the combination of novel coating and surface micropatterns provide effective surface properties for implants to achieve high biological compatibility and secure engraftment.

IRBIT was in vitro phosphorylated by PKA on Ser62, Ser64, and Ser66, and the phosphorylations might be involved in the high affinity of high-phosphorylated IRBIT for IP3R. Indeed, IRBIT phosphorylated on Ser68, Ser71, Ser74 and Ser77 as well as on Ser62, Ser64,and Ser66 showed high-phosphorylated pattern revealed on Phos-Tag SDS-PAGE analysis. However, this densely phosphorylated IRBIT did not show high binding affinity to IP3R suggesting that additional phosphorylations on other sites or unidentified modifications are necessary for getting high affinity to IP3R. In SLC4A4 deficient mice, an expression level of IRBIT protein was drastically reduced, and IRBIT in a soluble fraction was increased. Moreover, IRBIT was massively phosphorylated showing high affinity for IP3R. These results suggested that IRBIT was indeed phosphorylated in an NBCe1 activity dependent manner and could couple the intracellular pH homeostasis and Ca2+ dynamics.

An IP_3R binding protein termed IRBIT(IP_3R binding protein released with inositol 1, 4, 5-trisphosphate) that interacts with the IP_3 binding core domain of IP_3R and regulate the IP_3 sensitivity of IP_3R. Recently, we identified calcium/calmodulin-dependent kinase II alpha(CaMKIIα) as an IRBIT binding protein. In this study, we investigated the effect of IRBIT on the CaMKIIαactivity and analyzed IRBIT KO mice to explain the function of IRBIT in the central nervous system.

We identified anion exchanger (AE) and calcium dependent chloride channel (CaCC) as novel IRBIT family interacting molecule and found that IRBIT family regulates the activity of AE and CaCC. IRBIT, Long-IRBIT, AE, and CaCC were expressed in hippocampal neurons. We also found that the intracellular pH and chloride changes were impaired in hippocampal neurons, which were derived from IRBIT or Long-IRBIT knockout mouse. In addition, we found that IRBIT and Long-IRBIT splicing variants formed heteromultimers and N-terminal variation of Long-IRBIT by splicing affected protein stability and target selectivity. Thus, N-terminal variation of IRBIT family members mediates the regulation of multiple signaling pathways.

An IP3R binding protein termed IRBIT (IP3R binding protein released with inositol 1,4,5-trisphosphate) that interacts with the IP3 binding core domain of IP3R and regulate the IP3 sensitivity of IP3R. Recently, we identified calcium/calmodulin-dependent kinase II alpha (CaMKIIalpha) as an IRBIT binding protein. In this study, we investigated the effect of IRBIT deletion on the monoamine (dopamine and norepinephrine) synthesis and intracellular pH regulation to explain the function of IRBIT in the central nervous system. We found that IRBIT regulated the phosphoryaltion state of TH by CaMKIIalpha. In addition, IRBIT contributed the regulation of intracellular pH by NBC1 in the neuron and astrocyte.

We found that a catalytic subunit of calcineurin binds to the C terminus of NBCe1-C in the mouse cerebellum. Heterologous-coexpression experiments revealed that calcineurin binds to NBCe1-C via a “PQIRIE” motif at its C terminus. The interaction enhanced cell surface expression of NBCe1-C, resulting in an increase of its transporter activity, for which the phosphatase activity of calcineurin was essential. When NBCe1-C was stably expressed in HeLa cells, its cell surface expression was enhanced by an intracellular Ca2+ concentration increase and was suppressed by FK506, a specific inhibitor of calcineurin. These mechanisms of surface expression and transport activity of NBCe1-C regulated by the Ca2+-CaM-calcineurin axis indicate specialized functions of NBCe1-C in the brain.