Eri Kawashita

Plasminogen/Plasmin Modulates Bone Metabolism by Regulating the Osteoblast and Osteoclast Function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg−/− mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg−/− OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg−/− mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg−/− OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg−/− mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

Abnormal production of macrophage inflammatory protein‐1α by microglial cell lines derived from neonatal brains of Sandhoff disease model mice

Sandhoff disease (SD) is a lysosomal β‐hexosaminidase deficiency involving excessive accumulation of undegraded substrates, including terminal N‐acetylglucosamine‐oligosaccharides and GM2 ganglioside, and progressive neurodegeneration. Our previous study demonstrated remarkable induction of macrophage inflammatory factor‐1α (MIP‐1α) in microglia in the brains of SD model mice as a putative pathogenic factor for SD via microglia‐mediated neuroinflammation. In this study, we established microglial cell lines (WT‐ and SD‐Mg) from wild‐type and SD mice, and first demonstrated the enhanced production of MIP‐1α in SD‐Mg. Inhibitors of protein kinase C (PKC) and Akt reduced the production of MIP‐1α by SD‐Mg. Elevated activation of Akt and partial translocation of PKC isozymes (α, βI, βII, and δ) from the cytoplasm to the membrane in SD‐Mg were also revealed by means of immunoblotting. Furthermore, it was demonstrated that intracellular extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and phospholipase C (PLC), but not phosphoinositide 3‐kinase, should contribute to the induction of MIP‐1α in SD‐Mg, and that PLC could independently regulate the activation of both PKC and Akt. We proposed here that the deregulated activation of PLC should cause the enhanced MIP‐1α production via plural signaling pathways mediated by PKC and Akt, followed by extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase, in SD‐Mg.

Urokinase-type plasminogen activator receptor is associated with the development of adipose tissue

Urokinase-type plasminogen activator receptor (uPAR) plays a role in cellular responses which include cellular adhesion, differentiation, proliferation and migration. The aim of this study was to clarify the role of uPAR on the development of adipose tissue. To clarify the role of uPAR on adipogenesis, we examined the effect of uPAR overexpression and uPAR deficiency on the adipocyte differentiation. Adipocyte differentiation was induced by incubation of 3T3-L1 cells with differentiation media containing insulin, dexamethasone, and 1-methyl-3-isobutyl-xanthin. uPAR overexpression by transfection of uPAR expression vector induced adipocyte differentiation. In addition, we examined the difference in adipocyte differentiation of mesenchymal stem cells from wild-type mice and uPAR knockout (uPAR-/-) mice. The uPAR deficiency attenuated differentiation media-induced adipocyte differentiation. Moreover, we found that the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway attenuated uPAR overexpression-induced adipocyte differentiation, and uPAR overexpression induced the activation of Akt. We also found that an increase of the adipose tissue mass in uPAR-/- mice was less than that observed in wild-type mice. The present results suggest that uPAR plays a pivotal role in the development of adipose tissue through PI3K/Akt pathway.

uPA Attenuated LPS-induced Inflammatory Osteoclastogenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases.

ASB14780, an Orally Active Inhibitor of Group IVA Phospholipase A2, Is a Pharmacotherapeutic Candidate for Nonalcoholic Fatty Liver Disease

We have previously shown that high-fat cholesterol diet (HFCD)–induced fatty liver and carbon tetrachloride (CCl4)–induced hepatic fibrosis are reduced in mice deficient in group IVA phospholipase A2 (IVA-PLA2), which plays a role in inflammation. We herein demonstrate the beneficial effects of ASB14780 (3-[1-(4-phenoxyphenyl)-3-(2-phenylethyl)-1H-indol-5-yl]propanoic acid 2-amino-2-(hydroxymethyl)propane-1,3-diol salt), an orally active IVA-PLA2 inhibitor, on the development of fatty liver and hepatic fibrosis in mice. The daily coadministration of ASB14780 markedly ameliorated liver injury and hepatic fibrosis following 6 weeks of treatment with CCl4. ASB14780 markedly attenuated the CCl4-induced expression of smooth muscle α-actin (α-SMA) protein and the mRNA expression of collagen 1a2, α-SMA, and transforming growth factor-β1 in the liver, and inhibited the expression of monocyte/macrophage markers, CD11b and monocyte chemotactic protein-1, while preventing the recruitment of monocytes/macrophages to the liver. Importantly, ASB14780 also reduced the development of fibrosis even in matured hepatic fibrosis. Additionally, ASB14780 also reduced HFCD-induced lipid deposition not only in the liver, but also in already established fatty liver. Furthermore, treatment with ASB14780 suppressed the HFCD-induced expression of lipogenic mRNAs. The present findings suggest that an IVA-PLA2 inhibitor, such as ASB14780, could be useful for the treatment of nonalcoholic fatty liver diseases, including fatty liver and hepatic fibrosis.

α2AP mediated myofibroblast formation and the development of renal fibrosis in unilateral ureteral obstruction

Renal fibrosis is the final common pathway of a wide variety of chronic kidney diseases. Myofibroblast formation via the differentiation of from tissue-resident fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs), and epithelial-to-mesenchymal transition (EMT) is known to play a pivotal role in the development of renal fibrosis. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated the role of alpha 2-antiplasmin (α2AP) in myofibroblast formation and the development of renal fibrosis. We observed the development of renal fibrosis using unilateral ureteral obstruction (UUO). α2AP had accumulated in the UUO-induced obstructed kidneys and α2AP deficiency attenuated UUO-induced renal fibrosis in mice. The degree of myofibroblast formation in the obstructed kidneys of α2AP−/− mice was less than that in α2AP+/+ mice. In vitro, α2AP induced myofibroblast formation in renal tubular epithelial cells (RTECs), renal fibrosblasts, and bone marrow-derived mesenchymal stem cells (MSCs). α2AP also induced the production of TGF-β, which is known to be a key regulator of myofibroblast formation and fibrosis. α2AP-induced the TGF-β production was significantly reduced by SP600125, c-Jun N-terminal kinase (JNK) specific inhibitor. Our findings suggest that α2AP induces myofibroblast formation in the obstructed kidneys, and mediates the development of renal fibrosis.

Prostaglandin E2 reverses aberrant production of an inflammatory chemokine by microglia from Sandhoff disease model mice through the cAMP-PKA pathway.

Background Sandhoff disease (SD) is a neurodegenerative lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including terminal GlcNAc-oligosaccharides and GM2 ganglioside. Microglia-mediated neuroinflammation contributes to the pathogenesis and progression of SD. Our previous study demonstrated that MIP-1α, a putative pathogenic factor for SD, is up-regulated in microglial cells derived from SD model mice (SD-Mg) through activation of Akt and JNK. Methodology/Principal Findings In this study, we first demonstrated that prostaglandin E2 (PGE2), which is one of the lipid mediators derived from arachidonic acid and is known to suppress activation of microglia, reduced the aberrant MIP-1α production by SD-Mg to the same level as by WT-Mg. PGE2 also attenuated the activation of Akt and JNK. The inhibition of MIP-1α production and the activation of Akt and JNK occurred through the EP2 and 4/cAMP/PKA signaling pathway in the murine microglia derived from SD model mice. Conclusions/Significance We propose that PGE2 plays a role as a negative regulator of MIP-1α production in the pathogenesis of SD, and that PGE2-EP2 and 4/cAMP/PKA signaling could be a target pathway for therapy for SD.

Group IVA phospholipase A(2) deficiency prevents CCl4-induced hepatic cell death through the enhancement of autophagy.

Group IVA phospholipase A2 (IVA-PLA2), which generates arachidonate, plays a role in inflammation. IVA-PLA2-deficiency reduced hepatotoxicity and hepatocyte cell death in mice that received a single dose of carbon tetrachloride (CCl4) without any inhibitory effects on CCl4-induced lipid peroxidation. An immunoblot analysis of extracts from wild-type mouse- and IVA-PLA2 KO mouse-derived primary hepatocytes that transiently expressed microtubule-associated protein 1 light chain 3B (LC3) revealed a higher amount of LC3-II, a typical index of autophagosome formation, in IVA-PLA2-deficient cells, suggesting the enhancement of constitutive autophagy. IVA-PLA2 may promote CCl4-induced cell death through the suppression of constitutive autophagy in hepatocytes.

Enhancement by Uridine Diphosphate of Macrophage Inflammatory Protein-1 Alpha Production in Microglia Derived from Sandhoff Disease Model Mice

Sandhoff disease (SD) is a lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including GM2 ganglioside, and progressive neurodegeneration. Macrophage inflammatory protein-1α (MIP-1α) is a crucial factor for microglia-mediated neuroinflammation in the onset or progression of SD. However, the transmitter-mediated production of MIP-1α in SD is still poorly understood.Extracellular nucleotides, including uridine diphosphate (UDP), leaked by either injured or damaged neuronal cells activate microglia to trigger chemotaxis, phagocytosis, macropinocytosis, and cytokine production.In this study, we demonstrated that UDP enhanced the production of MIP-1α by microglia derived from SD mice (SD-Mg), but not that from wild-type mice (WT-Mg). The UDP-induced MIP-1α production was mediated by the activation of P2Y6 receptor, ERK, and JNK. We also found the amount of dimeric P2Y6 receptor protein to have increased in SD-Mg in comparison to WT-Mg. In addition, we demonstrated that the disruption of lipid rafts enhanced the effect of UDP on MIP-1α production and the disordered maintenance of the lipid rafts in SD-Mg. Thus, the accumulation of undegraded substrates might cause the enhanced effect of UDP in SD-Mg through the increased expression of the dimeric P2Y6 receptors and the disordered maintenance of the lipid rafts. These findings provide new insights into the pathogenic mechanism and therapeutic strategies for SD.

CD62-mediated activation of platelets in cerebral white matter lesions in patients with cognitive decline.

BACKGROUND Vascular dementia is related to intracranial arteriosclerosis associated with deep white matter lesions (DWMLs). DWMLs have been linked to thrombogenesis due to sustained platelet activation; therefore, an accurate hematological marker is needed. This study was done to evaluate the usefulness of a new method to examine the function of activated platelets in order to assess DWMLs associated with cognitive decline. METHODS A total of 143 individuals (70.4 ± 6.1 years old) who underwent hospital-based health screening using head MRI were evaluated. DWLs were evaluated on T2-weighted and FLAIR images by semi-quantitatively grading them from Grade 0 (none) to Grade 3 (severe) using the Fazekas classification. Cognitive function was evaluated using the MMSE and the word fluency test. Platelet activation was assessed using fluorescence-labeled anti-human platelet monoclonal antibodies and semi-quantitatively determining PAC-1- and CD62P-positive rates by flow cytometry. RESULTS Significant increases in hypertension and CD62P levels were observed with increasing DWML grade (2.6% in Group 0, 3.1% in Group 1, 4.1% in Group 2, and 5.0% in Group 3). CD62P levels were defined as elevated when they were above the mean+2SD of the Grade 0 group, and the odds ratio of the Grade 2+3 group was 3.03. A significant negative correlation was observed between CD62P levels and word fluency tests or the MMSE score. CONCLUSION Elevations in CD62P levels, which reflect platelet function activation, were associated with white matter lesions accompanied by a decline in cognitive function. CD62P levels may be useful as a sensitive clinical marker for the early detection of DWMLs with cognitive decline.