Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.     

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.     

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone.     

Up-regulation of proteinase-activated receptor 1 expression in astrocytes during HIV encephalitis

Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide. Activated cells expressed significantly increased levels of IL-1 beta, inducible NO synthase, and PAR-1 mRNA. Moreover, supernatants of these same cells were neurotoxic, which was inhibited by an N-methyl-D-aspartate receptor antagonist. Striatal implantation of the PAR-1-activating peptide significantly induced brain inflammation and neurobehavioral deficits in mice compared with mice implanted with the control peptide or saline. Since HIV-related neurological disease is predicated on brain inflammation and neuronal injury, the expression of PAR-1 in HIV encephalitis (HIVE) was investigated. Immunohistochemical analysis revealed that PAR-1 and (pro)-thrombin protein expression was low in control brains, but intense immunoreactivity was observed on astrocytes in HIVE brains. Similarly, PAR-1 and thrombin mRNA levels were significantly increased in HIVE brains compared with control and multiple sclerosis brains. These data indicated that activation and up-regulation of PAR-1 probably contribute to brain inflammation and neuronal damage during HIV-1 infection, thus providing new therapeutic targets for the treatment of HIV-related neurodegeneration.     

Effect of different growth factors on human osteoblasts activities: a possible application in bone regeneration for tissue engineering

Cultured human primary osteoblasts reproduce the phenotypic differentiation and maturation of cells in vivo. We have investigated the influence of three isoforms of transforming growth factor beta (TGF-beta1, TGF-beta2 and TGF-beta3), three fibroblast growth factors (FGF-2, FGF-4 and FGF-6) and the active metabolite of Vitamin D [1,25-(OH)(2)D3] on proliferation, alkaline phosphatase activity and mineralization of human osteoblasts during a period of 24 days of culture. TGF-beta isoforms and three FGFs examined have been proved to be inducers of osteoblasts proliferation (higher extent for TGF-beta and FGF-2) and inhibitors of alkaline phosphatase activity and osteoblasts mineralization. Combination of these growth factors with the active form of Vitamin D induced osteodifferentiation. In fact Vitamin D showed an additive effect on alkaline phosphatase activity and calcium content, induced by FGF-2 and TGF-beta in human osteoblast. These results highlight the potential of proliferating cytokines' combination with mineralizing agents for in vitro bone growth induction in bone tissue engineering.     

Nitric oxide mediates 17beta-estradiol-stimulated human and rodent osteoblast proliferation and differentiation

Oestradiol can stimulate osteoblast activity. Osteoblast function is thought to be regulated by nitric oxide (NO). We hypothesised that the effect of 17beta-oestradiol (17beta-E(2)) on osteoblast activity is mediated by NO. This hypothesis was tested using osteoblasts isolated from human trabecular bone, calvariae of rats, endothelial NO synthase (eNOS) gene-deficient mice, and their wild-type counterparts. Our results show that 17beta-E(2) dose-dependently stimulated proliferation and differentiation of primary human, rat and wild-typeosteoblasts. The presence of N(G)-monomethyl-l-arginine (10(-3) M), an inhibitor of NOS activity, blocked the 17beta-E(2)-(10(-7) M)-induced increases in thymidine incorporation (P < 0.01), alkaline phosphatase activity (P < 0.01) and bone nodule formation (P < 0.01) of wild-type, human and rat osteoblasts, respectively. Moreover, 17beta-E(2) did not induce a response in eNOS gene-deficient osteoblasts. 17beta-E(2) also increased total eNOS enzyme expression in rat osteoblasts. These findings indicate 17beta-E(2) modulates osteoblast function by NO-dependent mechanisms mediated via the eNOS isoform.     

Bone morphogenetic protein receptor IB signaling mediates apoptosis independently of differentiation in osteoblastic cells

Bone morphogenetic protein-2 (BMP-2) is an important regulator of osteoblast differentiation. However, the regulation of osteoblast apoptosis by BMP signaling remains poorly understood. Here we examined the role of type I BMP receptor (BMP-RI) in osteoblast apoptosis promoted by BMP-2. Despite undetectable BMP-RIB expression in OHS4 cells, BMP-2 or BMP-2 overexpression increased osteoblast differentiation similarly as in SaOS2 cells which express BMP-RIB, as shown by alkaline phosphatase and CBFA1/RUNX2 expression. In contrast to SaOS2 cells, however, BMP-2 or BMP-2 overexpression did not increase caspase-9 and caspases-3, -6, and -7 activity and DNA fragmentation in OHS4 cells. Consistently, BMP-2 increased protein kinase C (PKC) activity, and PKC inhibition suppressed BMP-2-induced caspase activity in SaOS2 but not in OHS4 cells that lack BMP-RIB. A dominant negative BMP-RIB inhibited BMP-2-induced caspase activity, whereas wild-type BMP-RIB promoted caspase activity induced by BMP-2 in SaOS2 and MC3T3-E1 cells. Wild-type BMP-RIB rescued the apoptotic response to BMP-2, and a constitutively active BMP-RIB restored the apoptotic signal in OHS4 cells, supporting an essential role for BMP-RIB in osteoblast apoptosis. We also assessed whether BMP-2-induced apoptosis occurred independently of osteoblast differentiation. General inhibition of caspases did not abolish BMP-2-induced alkaline phosphatase and CBFA1/RUNX2 expression in SaOS2 cells. Furthermore, broad caspases inhibition increased matrix mineralization but did not reverse the BMP-2 effect on mineralization in MC3T3-E1 cells. These results indicate that BMP-2-induced apoptosis was mediated by BMP-RIB in osteoblasts and occurred independently of BMP-2-induced osteoblast differentiation, which provides additional insights into the dual mechanism of BMP-2 action on osteoblast fate.     

高浓度地塞米松通过下调LMP-1表达抑制大鼠成骨细胞的分化

为探讨地塞米松（dexamethasone,DEX)抑制成骨细胞分化的机制,观察了不同浓度DEX对体外培养大鼠成骨细胞的碱性磷酸酶活性,骨钙素（osteocalcin,OC)合成,I型胶原蛋白表达的影响。并用RT-PCR方法检测了成骨细胞中LIM矿化蛋白1mRNA的表达量,结果显示：低浓度（10^-9mol/L）的DEX能增强碱性磷酸酶的活性、OC的分泌和I型胶原蛋白的表达;而高浓度（10^-7mol/L)的DEX对它们则起抑制作用,并下调成骨细胞正调节因子LMP-1mRNA的表达,上述结果表明,低浓度的DEX促进成骨细胞的分化;高浓度的DEX则抑制成骨细胞的分化,其抑制作用可能是通过下调LMP-1mRNA的表达而实现的。     

Cytotoxicity study of high gold content Degutan surfaces of various degrees of roughness with fibroblasts (BALB 3T3) and osteoblasts (hFOB 1.19)]

The cytotoxicity of Degutan surfaces with different degrees of roughness, and the effect of surface structures on osteoblast proliferation and differentiation, was investigated with standardised cell culture systems. Fibroblast cell lines (BALB/3T3) and osteoblast cell lines (hFOB 1.19) were used. The number and variability of the cells were determined for assessment of proliferation and alkaline phosphatase activity, collagen I and osteocalcin production were used as parameters for differentiation. In the early phase, the largest numbers of cells and greatest proliferation were measured on polished Degutan surfaces. In the late phase, however, larger numbers of cells and a greater degree of proliferation were to be seen on sandblasted and sandblasted/heat-treated Degutan surfaces. No differences were found for collagen I, osteocalcin production or alkaline phosphatase activity. Neither the osteoblasts nor the fibroblasts revealed a toxic effect of Degutan. The results for osteoblast differentiation correlate with recent studies on identical structured titanium surfaces. In view of the immeasurable amount of ion release, Degutan may be considered an ideal model for an inert material surface.     

Thrombin, a survival factor for cultured myoblasts

Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors. PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes. The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion. Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis. Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin. Neither a PAR-1- nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression. Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h. Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor. These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.     

Protease-activated receptors in the musculoskeletal system

Protease-activated receptors (PARs) mediate cellular responses to a subset of extracellular proteases, including blood coagulation factors and proteases produced by inflammatory cells. Cells in bone, cartilage and muscle exhibit cell type-specific expression patterns and functional responses for the different PARs. Activators of PAR-1 include thrombin, and activators of PAR-2 include trypsin and tryptase; PARs-3 and -4 are also receptors for thrombin. Thrombin stimulates PAR-1-mediated proliferative responses in osteoblasts, chondrocytes and myoblasts, and in developing muscle, PAR-1 activation by thrombin appears to mediate activity-dependent polyneuronal synapse reduction. In bone, activation of PAR-2 leads to inhibition of osteoblast-mediated osteoclast differentiation induced by hormones or cytokines, and in muscle, PAR-2 activation leads to stimulation of myoblast proliferation. Although there is some evidence for a role for PARs expressed by cells of the musculoskeletal system at specific stages of development, their major role appears to be in protecting the tissues from the destructive effects of inflammation and promoting regeneration. This review discusses the regulation of cell function in the musculoskeletal system by receptor-mediated responses to proteases. Expression patterns of PARs, the circumstances in which PAR activators are likely to be present, functional responses of PAR activation, and responses to thrombin for which receptors have not yet been identified are considered.     

The PAR-1-activating peptide facilitates pepsinogen secretion in rats

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.     

Influence of Novel Nano-Mesoporous Bioactive Glass on the Regulation of IGF-II Gene Expression in Osteoblasts

To investigate whether the mesoporous bioactive glass (MBG) exerts any in vitro bioactivity on rat osteoblasts and the potential regulatory mechanism of this bioactivity. Rat osteoblasts were incubated in the presence and absence (control) of ionic dissolution product of MBG in minimal essential medium. The osteoblast proliferation and differentiation were measured using MTT and alkaline phosphatase methods. The IGF-II mRNA expression in osteoblasts was measured by RT-PCR. IGF-II protein and IGFBP were measured by ELISA. The level of alkaline phosphatase activity was increased to 125% of control. Expression of IGF-II mRNA was increased to 125% of control. There was a 175 and 237% increase in the concentration of unbound IGF-II protein and IGFBP, respectively, in the conditioned media of treated osteoblasts. The osteoblast proliferation was 92% of control. The ionic dissolution product of MBG was able to promote the differentiation of osteoblasts, probably by inducing IGF-II expression at both mRNA and protein level.     

Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4

It is commonly accepted that thrombin exerts its proinflammatory properties through the activation of proteinase-activated receptor (PAR)-1, although two other thrombin receptors have been discovered: PAR-3 and PAR-4. In this study, we have investigated the mechanisms and the receptors involved in thrombin-induced leukocyte/endothelial cell interactions by using selective agonists and antagonists of thrombin receptors in an in vivo intravital microscopy system. Topical addition of selective PAR-1 agonists to rat mesenteric venules failed to reproduce the increased leukocyte rolling and adhesion observed after thrombin topical addition. When added together with the selective PAR-1 antagonist RWJ-56110, thrombin was still able to provoke increased leukocyte rolling and adherence. The thrombin-induced leukocyte rolling and adherence was not affected by pretreatment of rats with an anti-platelet serum. Selective PAR-4-activating peptide was able to reproduce the effects of thrombin on leukocyte rolling and adhesion. Intraperitoneal injection of PAR-4-activating peptide also caused a significant increase in leukocyte migration into the peritoneal cavity. In rat tissues, PAR-4 expression was detected both on endothelium and isolated leukocytes. Taken together, these results showed that in rat mesenteric venules, thrombin exerts proinflammatory properties inducing leukocyte rolling and adherence, by a mechanism independent of PAR-1 activation or platelet activation. However, PAR-4 activation either on endothelial cells or on leukocytes might be responsible for the thrombin-induced effects. These findings suggest that PAR-4 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence and recruitment, and that in addition to PAR-1, PAR-4 could be involved in proinflammatory properties of thrombin.     

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.