"LIVING IN SPACE" - Integral Understanding of life-regulation mechanism from "SPACE"

金沢大学医薬保健研究域薬学系生体間葉系幹細胞は、骨組織において骨芽細胞や軟骨細胞の供給源であり、さらに、ニッチ細胞として血液細胞の機能調節にも関与することが報告されている。宇宙飛行士や長期臥床者には骨量の急激な減少とともに、免疫機能の低下も観察される。近年、間葉系幹細胞の幹細胞特性と重力環境変動の関連性が報告されているが、微小重力環境下で発症する機能異常における間葉系幹細胞の役割は未解明である。私たちはこれまでに、MAPKの一つであるExtracellular signal-regulated kinase 5（Erk5）が、間葉系幹細胞の幹細胞性維持機構に必須であることを見出した。本研究では間葉系幹細胞に発現するErk5の骨環境維持機構における役割を探索し、以下の点を見出した。①：模擬微小重力モデルである尾部懸垂を負荷したマウスの骨組織において、間葉系幹細胞のErk5の活性化（リン酸化）低下が認められた。②：間葉系幹細胞細胞特異的Erk5欠損マウスでは、骨環境変化（骨髄内の骨量の著明な上昇とB細胞の上昇）が認められた。③：転写制御因子Sox9欠損により、間葉系幹細胞特異的Erk5不活性化マウスで観察された骨環境変化が有意に抑制された。④：E3ユビキチンリガーゼSmurf2活性化により、間葉系幹細胞特異的Erk5不活性化マウスで観察された骨環境変化が有意に抑制された。以上の4点から、生体骨髄内の間葉系幹細胞のErk5は、微小重力に応答し、Smurf2とSox9を介して骨環境の恒常性維持に重要な役割を果たしている可能性が示唆された。研究課題/領域番号:18H04971, 研究期間(年度):2018-04-01 – 2020-03-31出典：研究課題「メカノセンサーとしての間葉系幹細胞 」課題番号18H04971（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PUBLICLY-18H04971/）を加工して作

細胞性粘菌をモデル生物とするグルタメートレセプターの比較ゲノム研究

金沢大学医薬保健研究域薬学系細胞性粘菌(Dictyostelium discoideum以下粘菌)は、飢餓に伴い単細胞生物から多細胞生物へと変換されるとともに、柄と胞子の二種類の細胞集団に分化して子実体を形成するので、細胞分化機構解析のための生物モデルとして非常に有効な生物である。一方、グルタミン酸(Glu)は、高等生物の中枢神経系において重要な興奮性の神経伝達物質であり、このシグナル伝達の一部は代謝型Glu受容体(mGluR)を介して行われる。mGluRは、抑制性神経伝達物質Y-アミノ酪酸(GABA)の代謝型受容体であるGABA_B受容体と相同性が高いため、mGluRはGABA_B受容体と同じグループに分類されることが多い。本研究では、粘菌mGluRの同定と、mGlURによる細胞分化制御機構の解析を試みた。Glu含有寒天培地にて細胞性粘菌Ax2株の分化を誘導し、4時間ごとに分化形態を観察した。また、細胞性粘菌を栄養不含寒天培地にて分化を誘導し、4時間ごとに全RNAを抽出した。ゲノムプロジェクトデータベースからGluR様遺伝子の塩基配列を検索し、それを基に設計したプライマーを用いて半定量RT-PCRを行った。細胞性粘菌の分化形態を観察した結果、1mMのGlu存在下で分化速度の遅延が認められたが、同濃度のGABA存在下では分化速度に著変は見られなかった。RT-PCRにより、分化に伴い転写量が変動するmGluR様遺伝子の存在が明らかとなった。同遺伝子の推定塩基配列からhydropathyを求めた結果、哺乳動物のGluRと同様に7つの疎水性領域をもち、これらは膜貫通領域であると推定された。細胞性粘菌にはGluシグナルによる分化制御機構が存在し、その形態分化に伴って特定のGluRが発現する可能性が示唆される。研究課題/領域番号:15013219, 研究期間(年度):2003出典：「細胞性粘菌をモデル生物とするグルタメートレセプターの比較ゲノム研究」研究成果報告書　課題番号15013219（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-15013219/)を加工して作

Daily intake of β-cryptoxanthin prevents bone loss by preferential disturbance of osteoclastic activation in ovariectomized mice

AbstractAlthough β-cryptoxanthin, a xanthophyll carotenoid, has been shown to exert an anabolic effect on bone calcification, little attention has been paid thus far to the precise mechanism of bone remodeling. Daily oral administration of β-cryptoxanthin significantly inhibited osteoclastic activation as well as reduction of bone volume in ovariectomized mice. In vitro studies revealed that β-cryptoxanthin inhibited differentiation and maturation of osteoclasts by repression of the nuclear factor-κB-dependent transcriptional pathway. Our results suggest that supplementation with β-cryptoxanthin would be beneficial for prophylaxis and for therapy of metabolic bone diseases associated with abnormal osteoclast activation

Possible expression of functional glutamate transporters in the rat testis

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [3H]Glu was accumulated in testicular crude mitochondrial fractions in a temperature-and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed. © 2004 Society for Endocrinology

Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro

AbstractWe have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2’-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells

The sympathetic tone mediates leptin's inhibition of insulin secretion by modulating osteocalcin bioactivity

The osteoblast-secreted molecule osteocalcin favors insulin secretion, but how this function is regulated in vivo by extracellular signals is for now unknown. In this study, we show that leptin, which instead inhibits insulin secretion, partly uses the sympathetic nervous system to fulfill this function. Remarkably, for our purpose, an osteoblast-specific ablation of sympathetic signaling results in a leptin-dependent hyperinsulinemia. In osteoblasts, sympathetic tone stimulates expression of Esp, a gene inhibiting the activity of osteocalcin, which is an insulin secretagogue. Accordingly, Esp inactivation doubles hyperinsulinemia and delays glucose intolerance in ob/ob mice, whereas Osteocalcin inactivation halves their hyperinsulinemia. By showing that leptin inhibits insulin secretion by decreasing osteocalcin bioactivity, this study illustrates the importance of the relationship existing between fat and skeleton for the regulation of glucose homeostasis

An Osteoblast-dependent Mechanism Contributes to the Leptin Regulation of Insulin Secretion

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72661/1/j.1749-6632.2009.05061.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/72661/2/NYAS_5061_sm_SuppMat.pd

Positive Regulation by GABABR1 Subunit of Leptin Expression through Gene Transactivation in Adipocytes

Background: The view that c-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes. Methodology/Principal Findings: GABAB receptor 1 (GABABR1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA BR ligands. However, no prominent expression was seen with mRNA for GABA BR2 subunit required for heteromeric orchestration of the functional GABABR by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA BR1-null mice than in wild-type mice. Knockdown by siRNA of GABA BR1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells. Conclusions/Significance: Our results indicate that GABABR1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner o

Bioinformatic analysis reveals potential relationship between chondrocyte senescence and protein glycosylation in osteoarthritis pathogenesis

Osteoarthritis (OA) is the most common degenerative and progressive joint disease. Cellular senescence is an irreversible cell cycle arrest progressive with age, while protein glycosylation is the most abundant post-translational modification, regulating various cellular and biological pathways. The implication of either chondrocyte senescence or protein glycosylation in the OA pathogenesis has been extensively and individually studied. In this study, we aimed to investigate the possible relationship between chondrocyte senescence and protein glycosylation on the pathogenesis of OA using single-cell RNA sequencing datasets of clinical OA specimens deposited in the Gene Expression Omnibus database with a different cohort. We demonstrated that both cellular senescence signal and protein glycosylation pathways in chondrocytes are validly associated with OA pathogenesis. In addition, the cellular senescence signal is well-connected to the O-linked glycosylation pathway in OA chondrocyte and vice-versa. The expression levels of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family, which is essential for the biosynthesis of O-Glycans at the early stage, are highly upregulated in OA chondrocytes. Moreover, the expression levels of the GALNT family are prominently associated with chondrocyte senescence as well as pathological features of OA. Collectively, these findings uncover a crucial relationship between chondrocyte senescence and O-linked glycosylation on the OA pathophysiology, thereby revealing a potential target for OA