Oncostatin M enhances osteoprotegerin synthesis but reduces macrophage colony­stimulating factor synthesis in bFGF­stimulated osteoblast­like cells.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase.

Tramadol is a useful analgesic which acts as a serotonin and noradrenaline reuptake inhibitor in addition to µ-opioid receptor agonist. Cytoplasmic serotonin modulates the small GTPase activity through serotonylation, which is closely related to the human platelet activation. We recently reported that the combination of subthreshold collagen and CXCL12 synergistically activates human platelets. We herein investigated the effect and the mechanism of tramadol on the synergistic effect. Tramadol attenuated the synergistically stimulated platelet aggregation (300 µM of tramadol, 64.3% decrease, p<0.05). Not morphine or reboxetine, but duloxetine, fluvoxamine and sertraline attenuated the synergistic effect of the combination on the platelet aggregation (30 µM of fluvoxamine, 67.3% decrease, p<0.05; 30 µM of sertraline, 67.8% decrease, p<0.05). The geranylgeranyltransferase inhibitor GGTI-286 attenuated the aggregation of synergistically stimulated platelet (50 µM of GGTI-286, 80.8% decrease, p<0.05), in which GTP-binding Rac was increased. The Rac1-GEF interaction inhibitor NSC23766 suppressed the platelet activation and the phosphorylation of p38 MAPK and HSP27 induced by the combination of collagen and CXCL12. Tramadol and fluvoxamine almost completely attenuated the levels of GTP-binding Rac and the phosphorylation of both p38 MAPK and HSP27 stimulated by the combination. Suppression of the platelet aggregation after the duloxetine administration was observed in 2 of 5 patients in pain clinic. These results suggest that tramadol negatively regulates the combination of subthreshold collagen and CXCL12-induced platelet activation via Rac upstream of p38 MAPK.

Thrombopoietin and collagen in low doses cooperatively induce human platelet activation.

Aim: In acute medicine, we occasionally treat life-threatening conditions such as sepsis and trauma, which cause severe thrombocytopenia. Serum thrombopoietin levels have been reported to increase under the condition of thrombocytopenia related to severity. Collagen is a crucial activator of platelets, and Rho family members, such as Rho/Rho-kinase and Rac, play roles as active molecules involved in the intracellular signaling pathways in platelet activation. The present study aimed to elucidate the effects of thrombopoietin (TPO) on subthreshold low-dose collagen-stimulated human platelets in terms of Rho/Rho-kinase and Rac. Methods: Platelet-rich plasma donated from healthy volunteers was stimulated by the subthreshold low-dose of collagen after pretreatment with TPO and/or NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction, or Y27632, an inhibitor of Rho-kinase. Platelet aggregation was measured using an aggregometer based on laser-scattering methods. Proteins involved in intracellular signaling were analyzed using western blotting, and the secretion of platelet-derived growth factor-AB from activated platelets was determined using an enzyme-linked immunosorbent assay. Results: Under the existence of TPO, the low dose of collagen remarkably elicited the aggregation and platelet-derived growth factor-AB secretion of platelets, which were suppressed by NSC23766 and Y27632. The combination of TPO and collagen considerably induced a transient increase of guanosine triphosphate (GTP)-binding Rac and GTP-binding Rho followed by an increase of phosphorylated cofilin, a Rho-kinase substrate. Conclusion: These results strongly suggest that TPO and collagen in low doses cooperatively potentiate human platelet activation through both Rac and Rho/Rho-kinase mediated pathways.

Oncostatin M reduces the synthesis of macrophage-colony stimulating factor stimulated by TGF-ß via suppression of p44/p42 MAP kinase and JNK in osteoblasts.

Bone fracture is an important trauma frequently encountered into emergency medicine as well as orthopedics reflecting an aging society. Oncostatin M, an inflammatory cytokine produced by osteal macrophages, has been considered to play a crucial role in fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts is essential in osteoclastgenesis, and the secretion is stimulated by transforming growth factor-ß (TGF-ß). The aim of this study is to elucidate the effects of oncostatin M on the TGF-ß-induced M-CSF synthesis in osteoblast-like MC3T3-E1 cells and the underlying mechanisms. Oncostatin M attenuated the TGF-ß-stimulated M-CSF release and the mRNA expressions. SMAD3 inhibitor SIS3, p38 MAP kinase inhibitor SB203580, MEK1/2 inhibitor PD98059, and SAPK/JNK inhibitor SP600125 significantly suppressed the M-CSF release. Oncostatin M suppressed the TGF-ß-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK, but failed to affect the phosphorylation of SMAD3 and p38 MAP kinase. Oncostatin M attenuated the TGF-ß-stimulated vascular endothelial growth factor (VEGF) release and the TGF-ß-induced mRNA expressions of VEGF. These results strongly suggest that oncostatin M downregulates TGF-ß signaling upstream of p44/p42 MAP kinase and SAPK/JNK, but not SMAD 2/3 and p38 MAP kinase, in osteoblasts, leading to the attenuation of M-CSF synthesis. Our findings might provide a new therapeutic strategy for the acceleration of fracture healing process.

Amyloid ß protein negatively regulates human platelet activation induced by thrombin receptor-activating protein.

Amyloid ß protein deposition in cerebral vessels, a characteristic of Alzheimer's disease, is a risk factor for intracerebral hemorrhage. Amyloid ß protein directly modulates human platelet function; however, the exact mechanism of action is unclear. Therefore, we investigated the effects of amyloid ß protein on human platelet activation using an aggregometer with laser scattering. Amyloid ß protein decreased platelet aggregation induced by thrombin receptor-activating protein, but not by collagen and ADP. Amyloid ß protein also suppressed platelet aggregation induced by SCP0237 and A3227. Platelet-derived growth factor-AB secretion and phosphorylated-heat shock protein 27 release by thrombin receptor-activating protein were inhibited by amyloid ß protein. Additionally, thrombin receptor-activating protein-induced phosphorylation of JNK and p38 MAP kinase was reduced by amyloid ß protein. Collectively, our results strongly suggest that amyloid ß protein negatively regulates protease-activated receptor-elicited human platelet activation. These findings may indicate a cause of intracerebral hemorrhage due to amyloid ß protein.