The scaffold protein JSAP1 regulates proliferation and differentiation of cerebellar granule cell precursors by modulating JNK signaling

金沢大学がん研究所がん分子細胞制御Cerebellar granule cell precursors (GCPs) proliferate in the outer part of the external granular layer (EGL). They begin their differentiation by exiting the cell cycle and migrating into the inner part of the EGL. Here we report that JSAP1, a scaffold protein for JNK signaling pathways, is expressed predominantly in the post-mitotic GCPs of the inner EGL. JSAP1 knockdown or treatment with a JNK inhibitor enhances the proliferation of cultured GCPs, but the overexpression of wild-type JSAP1 leads to increased proportions of p27Kip1- and NeuN-positive cells, even with saturating concentrations of Sonic hedgehog (Shh), a potent GCP mitogen. However, these differentiation-promoting effects on GCPs are attenuated significantly in cells overexpressing a mutant JSAP1 that lacks the JNK-binding domain. Together, these data suggest that JSAP1 antagonizes the mitogenic effect of Shh on GCPs and promotes their exit from the cell cycle and differentiation, by modulating JNK activity. © 2008 Elsevier Inc. All rights reserved

Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice

BackgroundBeta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME.MethodsTo study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach.ResultsB4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL.ConclusionOur study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment

S1P2, the G protein-coupled receptor for sphingosine-1- phosphate, negatively regulates tumor angiogenesis and tumor growth in vivo in mice

金沢大学医薬保健研究域医学系Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the Gi-coupled chemotactic receptor S1P1. Here, we report that the distinct receptor S1P2 is responsible for mediating the G12/13/Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P2LacZ/+ mice, we found that S1P2 was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B16 melanoma cells implanted in S1P2-deficient (S1P2-/-) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S1P2-/- ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S1P2-/- ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S1P2-/- mice were also more efficient at recruiting CD11b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S1P2 acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S1P1, which stimulates tumor angiogenesis, S1P 2 on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment. ©2010 AACR

Ablation of the scaffold protein JLP causes reduced fertility in male mice

金沢大学がん研究所がん分子細胞制御The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. © 2008 Springer Science+Business Media B.V

Gene Knockout and Metabolome Analysis of Carnitine/Organic Cation Transporter OCTN1

金沢大学医薬保健研究域薬学系Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1. Methods: We first constructed octn1 gene knockout (octn1-/-) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined. Results: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1-/- mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1-/- mice. The octn1-/- mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn\u27s disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis. Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases. © 2010 Springer Science+Business Media, LLC

Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.In Press / 2013-03-18公開予定

Glycoprotein Hyposialylation Gives Rise to a Nephrotic-Like Syndrome That Is Prevented by Sialic Acid Administration in GNE V572L Point-Mutant Mice

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparent myopathies or motor dysfunctions. However, they had a short lifespan and exhibited renal impairment with massive albuminuria. Histological analysis showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte foot process morphologies in the kidneys. Glycan analysis using several lectins revealed glomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin, which is one of important molecules for the glomerular filtration barrier. Administering Neu5Ac to the mutant mice from embryonic stages significantly suppressed the albuminuria and renal pathology, and partially recovered the glomerular glycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observed in these mutant mice resulted from impaired glomerular filtration due to the hyposialylation of podocyte glycoproteins, including podocalyxin. Furthermore, it was possible to prevent the nephrotic-like disease in these mice by beginning Neu5Ac treatment during gestation