[Mechanisms of bone formation by primary cilia].

Primary cilia are immotile cilia assembled from the centriole-derived basal body, and they protrude on the cell surface in almost all cell types during the cell cycle G0 phase. Due to the diffusion barrier at the ciliary base, cilia harbor selective G protein-coupled receptors, growth factor receptors, and ion channels on their membrane. Thus, cilia act as sensory organelles, regulating the proliferation and differentiation of the cells and promoting the formation and maturation of various organs including bone, brain, and kidney. It has been unveiled that malformation and dysregulation of cilia cause organ dysplasia, so-called ciliopathy, thus research on primary cilia has become active during the past 20 years. Research on the roles of cilia in bone formation and its regulatory mechanisms have also progressed. It is widely recognized that cilia of preosteoblasts receive hedgehog and promote differentiation of the cells to osteoblasts, resulting in the formation of skulls and long bones. Recently, it has been shown that a membrane-associated protein 4.1G is important in ciliogenesis, hedgehog signaling, and osteoblast differentiation in neonatal bone formation. In this review, we would like to summarize the roles of primary cilia in bone formation and their regulatory mechanisms including the contribution of 4.1G.

MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1.

The primary cilium undergoes cell cycle-dependent assembly and disassembly. Dysregulated ciliary dynamics are associated with several pathological conditions called ciliopathies. Previous studies showed that the localization of phosphorylated Tctex-1 at Thr94 (T94) at the ciliary base critically regulates ciliary resorption by accelerating actin remodeling and ciliary pocket membrane endocytosis. Here, we show that microtubule-associated serine/threonine kinase family member 4 (MAST4) is localized at the primary cilium. Suppressing MAST4 blocks serum-induced ciliary resorption, and overexpressing MAST4 accelerates ciliary resorption. Tctex-1 binds to the kinase domain of MAST4, in which the R503 and D504 residues are key to MAST4-mediated ciliary resorption. The ciliary resorption and the ciliary base localization of phospho-(T94)Tctex-1 are blocked by the knockdown of MAST4 or the expression of the catalytic-inactive site-directed MAST4 mutants. Moreover, MAST4 is required for Cdc42 activation and Rab5-mediated periciliary membrane endocytosis during ciliary resorption. These results support that MAST4 is a novel kinase that regulates ciliary resorption by modulating the ciliary base localization of phospho-(T94)Tctex-1. MAST4 is a potential new target for treating ciliopathies causally by ciliary resorption defects.

Extracellular Vesicles Derived from 3T3-L1 Adipocytes Enhance Procoagulant Activity.

Obesity is associated with the risk of venous thromboembolism. Thrombi are constantly formed via the coagulation cascade and degraded by the fibrinolytic system, so they tend to form in obese individuals. Adipocytes are involved in thrombus formation in obesity, but it is not clear whether bioactive factors from adipocytes directly initiate or enhance coagulation and thrombosis. In this study, we confirmed that adipocyte-derived extracellular vesicles (ADEVs) enhance procoagulant activity in vitro. ADEVs prepared from the culture supernatant of mature 3T3-L1 adipocytes shortened plasma clotting times. Moreover, the effect of ADEVs on clotting time was weakened when using plasma lacking factors of the extrinsic pathway, but not the intrinsic pathway. ADEVs contain tissue factors and phosphatidylserine, which are involved in the extrinsic pathway, and blockade of these molecules diminished the effects of ADEVs on plasma clotting time. Additionally, the effect of ADEVs on plasma clotting time was further enhanced when cells were stimulated with the proinflammatory cytokine tumor necrosis factor-α. Thus, ADEVs may be a factor in thrombus formation in obesity.

Exposure to Stearate Activates the IRE1α/XBP-1 Pathway in 3T3-L1 Adipocytes.

In the endoplasmic reticulum (ER), accumulation of abnormal proteins with malformed higher-order structures activates signaling pathways (inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP-1) pathway, protein kinase RNA-activated-like endoplasmic reticulum kinase (PERK)/CCAAT/enhancer binding protein-homologous protein (CHOP) pathway and activating transcription factor 6α (ATF6α) pathway) that result in a cellular response suppressing the production of abnormal proteins or inducing apoptosis. These responses are collectively known as the unfolded protein response (UPR). Recently, it has been suggested that the UPR induced by saturated fatty acids in hepatocytes and pancreatic ß cells is involved in the development of metabolic diseases such as diabetes. The effect of palmitate, a saturated fatty acid, on the UPR has also been investigated in adipocytes, which are associated with the development of metabolic disorders, but the results were inconclusive. Therefore, as the major saturated fatty acids present in the daily diet are palmitate and stearate, we examined the effects of these saturated fatty acids on UPR in adipocytes. Here, we show that saturated fatty acids caused limited activation of the UPR in adipocytes. Exposure to stearate for several hours elevated the ratio of spliced XBP-1 mRNA, and this effect was stronger than that of palmitate. Moreover, the phosphorylation level of IRE1α, upstream of XBP-1 and expression levels of its downstream targets such as DNAJB9 and Pdia6 were elevated in 3T3-L1 adipocytes exposed to stearate. On the other hand, stearate did not affect the phosphorylation of PERK, its activation of CHOP, or the cleavage of ATF6α. Thus, in adipocytes, exposure to stearate activates the UPR via the IRE1α/XBP-1 pathway, but not the PERK/CHOP and ATF6α pathway.

Elaidate, a trans fatty acid, suppresses insulin signaling for glucose uptake in a manner distinct from that of stearate.

The dietary intake of elaidate (elaidic acid), a trans-fatty acid, is associated with the development of various diseases. Since elaidate is a C18 unsaturated fatty acid with a steric structure similar to that of a C18 saturated fatty acid (stearate), we previously revealed that insulin-dependent glucose uptake was impaired in adipocytes exposed to elaidate prior to and during differentiation similar to stearate. However, it is still unknown whether the mechanism of impairment of insulin-dependent glucose uptake due to elaidate is similar to that of stearate. Here, we indicate that persistent exposure to elaidate has particular effects on insulin signaling and GLUT4 dynamics. Insulin-induced accumulation of Akt at the plasma membrane (PM) and elevations of phosphorylated Akt and AS160 levels in whole cells were suppressed in adipocytes persistently exposed to 50 µM elaidate. Interestingly, persistent exposure to the same concentration of stearate has no effect on the phosphorylated Akt and AS160 levels. When cells were exposed to these fatty acids, elaidate suppressed insulin-induced fusion, but not translocation, of GLUT4 storage vesicles in the PM, whereas stearate did not suppress the fusion and translocation of GLUT4 storage, indicating that elaidate has suppressive effects on the accumulation of Akt and fusion of GLUT4 storage vesicles and that both elaidate and stearate vary in the mechanisms by which they impair insulin-dependent glucose uptake.

Elevated Expression of Vascular Adhesion Molecule-1, Plasminogen Activator Inhibitor-1, Cyclooxygenase-2, and Thrombomodulin in Human Umbilical Vein Endothelial Cells from Hospitalized Gestational Diabetes Mellitus Patients.

Protein expression in human umbilical vein endothelial cells (HUVECs) is a useful indicator of maternal condition and the intrauterine environment during pregnancy. Therefore, we investigated protein expression in HUVECs obtained from patients with gestational diabetes mellitus (GDM). HUVECs were prepared from the umbilical cords of GDM patients and controls who underwent planned cesarean section between 2013 and 2014 at Teikyo University Hospital (Tokyo, Japan). There were no differences in blood glucose levels between the GDM patients and controls at admission. However, pre-pregnancy body mass index (BMI) was higher in GDM patients, although the changes in gestational BMI were smaller during hospitalization. To evaluate the state of the endothelium, we examined the protein expression levels of vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, thrombomodulin (TM), endothelial nitric oxide synthase, plasminogen activator inhibitor-1 (PAI-1), cyclooxygenase-2 (COX-2), and VE-cadherin, which are altered by various factors in endothelial tissue. VCAM-1, PAI-1, and COX-2 expression was higher in HUVECs from patients with GDM than the controls. Because the pre-pregnancy BMI was higher in GDM patients, we examined the relationship between BMI and protein expression. However, the expression levels of these proteins were not correlated with pre-pregnancy BMI and were higher in HUVECs from BMI-matched GDM patients than from BMI-matched controls. Intriguingly, TM expression was also higher in HUVECs from BMI-matched GDM patients. Thus, expression of VCAM-1, PAI-1, COX-2, and TM may reflect certain factors in the intrauterine environment that are altered in hospitalized GDM patients with controlled body weight.

Activation of PPARγ at an Early Stage of Differentiation Enhances Adipocyte Differentiation of MEFs Derived from Type II Diabetic TSOD Mice and Alters Lipid Droplet Morphology.

Type 2 diabetic Tsumura, Suzuki, obese, diabetes (TSOD) mice gradually gain weight as compared to corresponding Tsumura, Suzuki, non-obesity (TSNO) control mice, and develop insulin resistance. Although development of type 2 diabetes mellitus is associated with dysfunction of adipocytes, little is known about the properties of adipocytes from TSOD mice. Therefore, we attempted to remove intracorporeal factors and elucidate inherent properties of adipocytes of TSOD mice using adipocytes differentiated from mouse embryonic fibroblasts (MEFs) in vitro. Here, we show that MEFs of TSOD have low potency for differentiation into adipocytes. The percentage of Oil red O-stained cells and levels of adipogenic markers in cells differentiated from MEFs of TSOD are lower than those in cells differentiated from MEFs of TSNO. We further show that treatment with an agonist of peroxisome proliferator-activated receptor-γ (PPARγ) (rosiglitazone) at an early stage of differentiation increases the percentage of Oil red O-stained cells in TSOD-MEFs differentiated into adipocytes. Moreover, the lipid droplet size in those adipocytes is larger than that in the adipocytes differentiated from MEFs of TSNO. Although persistent treatment of MEFs of TSOD with rosiglitazone during differentiation increases the percentage of Oil red O-stained cells, the lipid droplet size in adipocytes treated as such does not reach the size of those treated in early stage only. Thus, activation of PPARγ by its agonist at an early stage of differentiation compensates for the low potency toward adipogenic differentiation of, and accelerates formation of enlarged lipid droplets in adipocytes derived from, MEFs of TSOD mice.

Regulatory Functions of Phospholipase A2.

Phospholipase A2 (PLA2) plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction. PLA2 provides precursors for generation of eicosanoids, such as prostaglandins (PGs) and leukotrienes (LTs), when the cleaved fatty acid is arachidonic acid, platelet-activating factor (PAF) when the sn-1 position of the phosphatidylcholine contains an alkyl ether linkage and some bioactive lysophospholipids, such as lysophosphatidic acid (lysoPA). As overproduction of these lipid mediators causes inflammation and tissue disorders, it is extremely important to understand the mechanisms regulating the expression and functions of PLA2. Recent advances in molecular and cellular biology have enabled us to understand the molecular nature, possible function, and regulation of a variety of PLA2 isozymes. Mammalian tissues and cells generally contain more than one enzyme, each of which is regulated independently and exerts distinct functions. Here we classify mammalian PLA2s into three large groups, namely, secretory (sPLA2), cytosolic (cPLA2), and Ca2+-independent PLA2s, on the basis of their enzymatic properties and structures and focus on the general undestanding of the possible regulatory functions of each PLA2 isozyme. In particular, the roles of type II sPLA2 and cPLA2 in lipid mediator generation are discussed.

Positions of Hydroxyl Groups in Chrysin are Critical for Inhibiting Plasminogen Activator Inhibitor-1 Release from Human Umbilical Vein Endothelial Cells.

Chrysin suppresses the TNFα-induced increase in the secretion of plasma plasminogen activator inhibitor 1 (PAl-1), a risk factor for thrombotic diseases, from human umbilical vein endothelial cells (HUVECs). The present study aimed to determine the association between the location of the hydroxyl groups in chrysin.to levels of-PAI-1. in the medium of HUVEC stimulated with TNFα. We cultured HUVEC for 3 h in medium containing chrysin or various flavonoids and then stimulated them with TNFα (10 ng/mL) for 12 h. Levels of PAI-1 antigen measured using ELISA showed that chrysin significantly inhibited the PAl- I increase with an IC50 of 15.6 µM. The flavones, galangin, baicalein, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone and quercetin did not significantly inhibit the PAI- increase. Apigenin and luteolin were cytotoxic and thus their ability to inhibit PAI production could not be evaluated. Chrysin also inhibited PAI- mRNA expression whereas the other compounds did not. Hydroxyl groups located in the A-5 and A-7 positions were essential for the inhibitoryactivity, which along with cytotoxicity, was significantly influenced by adding a third hydroxyl group.

Effects of a diet containing Brazilian propolis on lipopolysaccharide-induced increases in plasma plasminogen activator inhibitor-1 levels in mice.

BACKGROUND: Brazilian propolis has many biological activities including the ability to help prevent thrombotic diseases, but this particular effect has not been proven. Plasma levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, increase under inflammatory conditions such as infection, obesity and atherosclerosis and such elevated levels predispose individuals to a risk of developing thrombotic diseases. AIM: This study aimed to determine the effects of a diet containing Brazilian propolis on lipopolysaccharide (LPS)-induced increases in plasma PAI-1 levels. MATERIALS AND METHODS: Mice were fed with a diet containing 0.5% (w/w) Brazilian propolis for 8 weeks. Thereafter, the mice were subcutaneously injected with saline containing 0.015 mg/kg of LPS and sacrificed 4 h later. RESULTS: Orally administered Brazilian propolis significantly suppressed the LPS-induced increase in PAI-1 antigen and its activity in mouse plasma. CONCLUSION: This study indicated that Brazilian propolis contains natural products that can decrease thrombotic tendencies in mice.

Differentiation with elaidate tends to impair insulin-dependent glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes.

Development of type 2 diabetes mellitus and insulin resistance is associated with a quality of dietary fatty acids such as saturated and unsaturated fatty acids. Dietary fatty acids also include transform of unsaturated fatty acids and intake of transform of oleate (elaidate) is associated with cardiovascular disease. However, little is known about the roles of elaidate in insulin responsiveness. We show here that elaidate impairs insulin-dependent glucose uptake in adipocytes. Differentiation with 10 µM elaidate, which is close to physiological plasma concentration, reduces insulin-dependent glucose uptake. Furthermore, insulin-dependent GLUT4 translocation is disturbed in adipocytes differentiated with elaidate. In addition, analysis of lipolysis and gene expression shows that deteriorative effects of elaidate on insulin responsiveness are limited but not general. Thus, our findings reveal that differentiation with elaidate tends to affect insulin-dependent glucose uptake through alternation of GLUT4 translocation from cytosol to the plasma membrane.

Screening for hemostatic activities of popular Chinese medicinal herbs in vitro.

AIMS: This study aimed to identify new hemostyptics by assessing the coagulation enhancing activity of 114 Chinese herbal extracts in vitro. METHODS: Herbs were boiled in water for 30 min, filtered and then lyophilized filtrates (10 mg/mL) were dissolved in water. Coagulation was assayed as prothrombin time (PT). Plasma diluted in saline was incubated with each extract for 5 min and then PT reagent was added, followed by CaCl2 solution and the time taken to form clots was measured. Extracts that decreased coagulation time were regarded as containing active compounds. The abilities of extracts to activate Factor XII were assessed and the activated form of factor XII (XIIa) was resolved by SDS-PAGE and visualized by silver staining. RESULTS: Coagulation time was obviously shortened by extracts of Alpinia Rhizome, Areca, Artemisia Leaf, Cassia Bark, Danshen Root, Ephedra Herb, Epimedium Herb, Forsythia Fruit, Great Burdock Achene, Moutan Bark, Perilla Herb, Red Paeony Root, Schizonepeta Spike, Senticosus Rhizome, Sweet Annie, Uncaria Thorn and Zanthoxylum Peel. Factor XII was obviously activated by extracts of Artemisia Leaf and Great Burdock Achene, and slightly by Perilla herb. CONCLUSION: Some popular Chinese medicinal herbs have potential as hemostatic agents and could thus be develope as new strategies for the treatment and prevention of bleeding.

Blood coagulation and metabolic profiles in middle-aged male and female ob/ob mice.

Obese and diabetic states in humans are associated with an increased incidence of thrombotic diseases caused by various coagulation abnormalities. Genetically obese ob/ob mice produce metabolic abnormalities similar to those associated with type 2 diabetes. However, little is known about their coagulation features or sex differences. The present study aimed to determine the effects of obese and diabetic complications on blood coagulation and vascular diseases by exploring correlations between blood coagulation and metabolic profiles in middle-aged male and female ob/ob mice. Plasma levels of plasminogen activator inhibitor 1 (PAI-1) were significantly increased, whereas those that of platelet factor-4 (PF-4) was slightly, but significantly increased in male and female ob/ob mice compared with lean counterparts. Prothrombin time (PT) was significantly shortened in female ob/ob mice and activated partial thrombin time (APTT) significantly differed between male and female ob/ob mice. Plasma levels of antithrombin (AT) were significantly increased in male and female ob/ob mice. None of the other coagulation and fibrinolytic factors examined significantly differed between ob/ob mice and lean counterparts. On the contrary, factors such as body weight and cholesterol levels significantly differed between ob/ob and lean mice, whereas glucose, fructosamine and insulin levels significantly differed only in one sex of each strain. These results provided fundamental information about blood coagulation and metabolic features for exploring the function of altered blood coagulation states in ob/ob mice.

Acidic sialidase activity is aberrant in obese and diabetic mice.

Mammalian sialidases (NEU1, NEU2, NEU3 and NEU4) that remove sialic acids from glycoconjugates have been implicated in diverse cellular functions. Human sialidases are involved in the development of various disease states such as cancer, diabetes and arteriosclerosis. Unregulated acidic sialidase NEU1 activity is associated with the pathogenesis of lysosomal storage disorder (LSD) sialidosis, abnormal immune responses and cancer progression. Obesity is closely related to several chronic diseases such as diabetes, cardiovascular diseases, hyperlipidemia or hypertension that are associated with metabolic syndrome. We examined fluctuations in mRNA levels and sialidase activities of NEU1 in two strains of obese and diabetic mice to assess the involvement of NEU1 in obesity. The activity of NEU1 was preferentially higher in epididymal fat and lower in the livers of two strains of obese and diabetic mice. Fluctuations in NEU1 activity might be associated with the pathological status of these tissues in obesity.

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production.

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.

Experimental study on the hemostatc activity of Pollen Typhae: a traditional folk medicine used by external and oral application.

Pollen Typhae is the traditional Chinese herbal medicine widely used to treat the hemorrhagic diseases both by external and oral application. The present study examines the hemostatic properties and its components of Pollen Typhae. Pollen extract significantly reduced prothrombin time (PT), activated partial prothrombin time (APTT) and recalcification time. Pollen extract directly activated factor XII in the coagulation cascade. Acidic polysaccharide in the pollen that adsorbed to the diethylaminoethyl (DEAE) column was the causative agent of factor XII activation. These results suggested that an electronegative charge attributed to an acidic polysaccharide in the pollen extract contributed to the hemostatic activity. We then examined the hemostatic activity of administered pollen extract in the mouse tail bleeding model. Tail bleeding was significantly decreased after oral administration of the pollen extract, whereas the acidic polysaccharide fraction did not affect the duration of tail bleeding. These results suggest that the oral anticoagulant effect of Pollen Typhae is attributed to compounds other than acidic polysaccharides. We concluded that the activation of the intrinsic coagulation pathway by the acidic polysaccharide contributes to the external hemostatic property of Pollen Typhae, and the action of components such as flavonoids that possess anticoagulant activity are causative agent when orally administered.