FGFR2-Cbl interaction in lipid rafts triggers attenuation of PI3K/Akt signaling and osteoblast survival

Fibroblast growth factor receptor (FGFR) signaling plays an important role in skeletogenesis. The molecular mechanisms triggered by activated FGFR in bone forming cells are however not fully understood. In this study, we identify a role for phosphatidylinositol 3-kinase (PI3K) signaling in cell apoptosis induced by FGFR2 activation in osteoblasts. We show that FGFR2 activation leads to decrease PI3K protein levels, resulting in attenuation of PI3K signaling in human osteoblasts. Biochemical and molecular analyses revealed that the attenuated PI3K signaling induced by FGFR2 activation is due to increased Cbl-PI3K molecular interaction mediated by the Cbl Y731 residue, which results in increased PI3K ubiquitination and proteasome degradation. Biochemical and immunocytochemical analyses showed that FGFR2 and Cbl interact in raft micro-domains at the plasma membrane. FGFR2 activation increases FGFR2 and Cbl recruitment in micro-domains, resulting in increased molecular interactions. Consistently, functional analyses showed that the attenuation of PI3K/Akt signaling triggered by FGFR2 activation results in increased osteoblast apoptosis. These results identify a functional molecular mechanism by which activated FGFR2 recruits Cbl in raft micro-domains to trigger PI3K ubiquitination and proteasome degradation, and reveal a novel role for PI3K/Akt attenuation in the control of osteoblast survival by FGFR2 signaling.     

高糖环境LIF介导STAT3/SOCS3信号通路参与成骨细胞分化机制研究

目的研究高糖环境下白血病抑制因子(leukemia inhibitory factor,LIF)介导STAT3/SOCS3信号通路对成骨细胞分化的影响及其作用机制。方法利用ELISA检测2型糖尿病患者血清白细胞介素6(interleukin-6,IL-6)家族中LIF及炎性因子的水平;通过RT-PCR、Western blot检测体外高糖环境下成骨分化标志物ALP、RUNX2、OCN、OPN以及细胞因子信号抑制因子3(suppressors of cytokine signaling,SOCS3)的m RNA和蛋白表达情况。结果 2型糖尿病患者血清炎性因子和LIF的水平明显升高。高糖或LIF显著降低了成骨分化标志物ALP、RUNX2、OCN和OPN的m RNA和蛋白水平,同时上调了SOCS 3和PSTAT3的表达。加入50 nmol/L或100 nmol/L STAT 3抑制剂JSI-124可改善成骨分化标志物的表达下调。结论 LIF通过介导STAT3/SOCS3信号通路参与高糖诱导下的成骨细胞分化。本研究为高糖导致的成骨障碍奠定了分子生物学理论基础并对靶点药物的研发提供了实验依据。     

线粒体途径相关信号通路对成骨细胞凋亡的作用

骨质疏松症是由于多种原因导致的骨质量和骨密度下降,骨微结构遭到破坏,造成骨脆性增加,从而容易发生骨折的一种全身性代谢性骨病变。其中,成骨细胞是促进骨形成的主要功能细胞,是骨质疏松发生的关键细胞,其功能退变和异常凋亡将会引起骨量丢失,诱导骨质疏松发生。因此,探究成骨细胞的凋亡机制对预防和治疗骨质疏松症具有重要意义。其中,线粒体途径是成骨细胞凋亡的主要途径,是由多基因参与、多通路相互作用而形成的复杂过程。因此,本文综述了线粒体途径的凋亡过程及其众多相关信号通路:PI3k/Akt信号通路、MAPK信号通路、Ca~(2+)/CaM信号通路以及其他信号通路和因子(Keap1/Nrf2信号通路、PHB、FoxO家族、cAMP/CREB),同时还归纳了这些信号通路在成骨细胞经线粒体途径凋亡的过程中发挥的促进或者抑制作用,以及他们在成骨细胞凋亡中的一些相互作用。这为进一步探究成骨细胞的凋亡机制和骨质疏松的发病机制提供了可靠依据,也为更深入研究安全、有效的抗骨质疏松药物提供了一些可能的作用靶点(如:Akt、ERK、JNK、p38、Ca~(2+)、PHB、FoxO3a、CREB、Keap1/Nrf2等)。     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。     

PlexinA2 mediates osteoblast differentiation via regulation of Runx2

The imbalance between bone-resorbing osteoclasts and bone-forming osteoblasts often leads to bone destructive diseases such as osteoporosis. In contrast to the development of several antiresorptive agents for osteoporosis therapy, discovery of anabolic drugs has been difficult because of an insufficient understanding of the complex mechanism of bone formation. In a microarray analysis with mouse preosteoblast cells, we found that PlexinA2 (PlxnA2), a molecule previously known to mediate axon guidance in neural development, was upregulated by the osteogenic factor BMP2. PlxnA2-specific siRNA decreased Runx2 expression, osteoblast differentiation, and mineralization. Runx2 overexpression restored osteoblastic differentiation of PlxnA2-knockdown cells. PlxnA2 was associated with both type 1 and 2 BMP receptors, and BMP2 increased the interaction between PlxnA2 and type 1 receptors. PlxnA2 also affected Smad and Akt signaling pathways downstream of BMP2. Taken together, the results of our study reveal that PlxnA2 has a pro-osteogenic function by modulating BMP2 signaling. Therefore, PlxnA2 may be a useful target for development of bone anabolic therapeutics.     

Raji-MG63细胞共培养体系中心肌营养蛋白样细胞因子1(CLCF1)对RANKL/OPG比值及成骨细胞分化的影响

目的 探讨Raji-MG63细胞共培养体系中,心肌营养蛋白样细胞因子1(cardiotrophin-like cytokine factor 1,CLCF1)基因对骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RAN...     

Prostaglandin D2 induces apoptosis of human osteoclasts through ERK1/2 and Akt signaling pathways

In a recent study we have shown that prostaglandin D₂ (PGD₂) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cell (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD₂-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinositol3-kinase (PI3K) and NF-κB/IκB kinase-2 (IKK2) prior to the treatments of PGD₂ as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-κB signaling pathways. Treatments with both PGD₂ and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased β-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandin production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phosphorylations, and reduced the phosphorylation of β-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD₂ and a CRTH2 agonist. Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD₂, whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K inhibitor treatment. The DP receptor was not implicated in any of the parameters evaluated. Treatment of OCs with PGD₂ as well as its receptor agonists and antagonists did not alter the phosphorylation of RelA/p65 (Ser536). Moreover, the caspase-3 activity was not altered in OCs treated with a selective IKK2/NF-κB inhibitor. In conclusion, endogenous or exogenous PGD₂ induces CRTH2-dependent apoptosis in human differentiated OCs; β-arrestin-1, ERK1/2, and Akt, but not IKK2/NF-κB are probably implicated in the signaling pathways of this receptor in the model studied.     

EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone

Previous reports indicate that ephrinB2 expression by osteoblasts is stimulated by parathyroid hormone (PTH) and its related protein (PTHrP) and that ephrinB2/EphB4 signaling between osteoblasts and osteoclasts stimulates osteoblast differentiation while inhibiting osteoclast differentiation. To determine the role of the ephrinB2/EphB4 interaction in the skeleton, we used a specific inhibitor, soluble EphB4 (sEphB4), in vitro and in vivo. sEphB4 treatment of cultured osteoblasts specifically inhibited EphB4 and ephrinB2 phosphorylation and reduced mRNA levels of late markers of osteoblast/osteocyte differentiation (osteocalcin, dentin matrix protein‐1 [DMP‐1], sclerostin, matrix‐extracellular phosphoglycoprotein [MEPE]), while substantially increasing RANKL. sEphB4 treatment in vivo in the presence and absence of PTH increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1α1, and PTH receptor [PTHR1]), but despite a substantial increase in osteoblast numbers, there was no significant change in bone formation rate or in late markers of osteoblast/osteocyte differentiation. Rather, in the presence of PTH, sEphB4 treatment significantly increased osteoclast formation, an effect that prevented the anabolic effect of PTH, causing instead a decrease in trabecular number. This enhancement of osteoclastogenesis by sEphB4 was reproduced in vitro but only in the presence of osteoblasts. These data indicate that ephrinB2/EphB4 signaling within the osteoblast lineage is required for late stages of osteoblast differentiation and, further, restricts the ability of osteoblasts to support osteoclast formation, at least in part by limiting RANKL production. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis. © 2013 American Society for Bone and Mineral Research.     

Emodin Regulates Bone Remodeling by Inhibiting Osteoclastogenesis and Stimulating Osteoblast Formation

Bone remodeling, a physiological process in which new bone is formed by osteoblasts and the preexisting bone matrix is resorbed by osteoclasts, is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this process can cause various pathological conditions, including osteoporosis. Emodin, a naturally occurring anthraquinone derivative found in Asian herbal medicines, has numerous beneficial pharmacologic effects, including anticancer and antidiabetic activities. However, the effect of emodin on the regulation of osteoblast and osteoclast activity has not yet been investigated. We show here that emodin is a potential target for osteoporosis therapeutics, as treatment with this agent enhances osteoblast differentiation and bone growth and suppresses osteoclast differentiation and bone resorption. In this study, emodin suppressed receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced osteoclast differentiation of bone marrow macrophages (BMMs) and the bone‐resorbing activity of mature osteoclasts by inhibiting RANKL‐induced NF‐κB, c‐Fos, and NFATc1 expression. Emodin also increased ALP, Alizarin Red‐mineralization activity, and the expression of osteoblastogenic gene markers, such as Runx2, osteocalcin (OCN), and ALP in mouse calvarial primary osteoblasts, as well as activated the p38‐Runx2 pathway, which enhanced osteoblast differentiation. Moreover, mice treated with emodin showed marked attenuation of lipopolysaccharide (LPS)‐induced bone erosion and increased bone‐forming activity in a mouse calvarial bone formation model based on micro‐computed tomography and histologic analysis of femurs. Our findings reveal a novel function for emodin in bone remodeling, and highlight its potential for use as a therapeutic agent in the treatment of osteoporosis that promotes bone anabolic activity and inhibits osteoclast differentiation. © 2014 American Society for Bone and Mineral Research.     

PTEN regulates matrix synthesis in adult human chondrocytes under oxidative stress

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified as an important tumor suppressor gene. PTEN functions as a negative regulator of phosphoinositol‐3‐kinase (PI3K)‐Akt and MEK/ERK signaling. The PI3K‐Akt pathway is critical for cell survival, differentiation, and matrix synthesis. Oxidative stress is considered a critical factor in the onset and progression of osteoarthritis (OA). Therefore, we investigated the function of PTEN in OA chondrocytes under oxidative stress. Chondrocytes were treated with insulin‐like growth factor‐1 (IGF‐1) and/or tert‐butyl hydroperoxide (tBHP), which causes oxidative stress. The expression levels of type2 collagen (Col2a1) and aggrecan were analyzed by real‐time PCR, and phosphorylation of Akt and ERK1/2 was analyzed by Western blotting. Chondrocytes were treated with PTEN‐specific small interfering RNA (siRNA), as well as IGF‐1 and/or tBHP. PTEN and IGF‐1 expressions in OA chondrocytes were increased. The downregulation of PTEN expression increased the expression levels of Col2a1 and aggrecan, and increased proteoglycan synthesis under oxidative stress. Oxidative stress decreased the phosphorylation of Akt and increased that of ERK1/2. The downregulation of PTEN expression increased Akt phosphorylation, but did not increase that of ERK 1/2. Our results suggest that PTEN regulates matrix synthesis via the PI3K‐Akt pathway under oxidative stress. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:231–237, 2014.     

Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKCdelta and STAT3.

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCdelta and STAT3, offering new lines of therapeutic investigations. INTRODUCTION: Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation. MATERIALS AND METHODS: Proliferation was analyzed by 3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated. RESULTS: OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCdelta inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction. CONCLUSIONS: These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.     

Downregulation of Wnt signaling by increased expression of Dickkopf-1 and -2 is a prerequisite for late-stage osteoblast differentiation of KS483 cells.

We examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation. It has been shown that Wnt signaling in mature osteoblasts needs to be downregulated to enable the formation of a mineralized matrix. Using RNA interference, we showed that this is, at least in part, accomplished by upregulation of the Wnt antagonists Dickkopf-1 and -2. INTRODUCTION: The role of Wnt signaling in the initiation of osteoblast differentiation has been well studied. However, the role during late-stage differentiation is less clear. We have examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation. MATERIALS AND METHODS: We treated murine bone marrow and mesenchymal stem cell-like KS483 cells with either LiCl or Wnt3A during several stages of osteoblast differentiation. In addition, we generated stable KS483 cell lines silencing either the Wnt antagonist Dkk-1 or -2 RESULTS: Activation of Wnt signaling by LiCl inhibits the formation of a mineralized bone matrix in both cell types. Whereas undifferentiated KS483 cells respond to Wnt3A by inducing nuclear beta-catenin translocation, differentiated cells do not. This is at least in part accomplished by upregulated expression of Dkk-1 and -2 during osteoblast differentiation. Using RNA interference, we showed that Dkk-1 plays a crucial role in blunting the BMP-induced alkaline phosphatase (ALP) response and in the transition of an ALP+ osteoblast in a mineralizing cell. In contrast, Dkk-2 plays a role in osteoblast proliferation and the initiation of osteoblast differentiation. CONCLUSIONS: Our data suggest that Wnt signaling in maturing osteoblasts needs to be downregulated to enable the formation of a mineralized bone matrix. Furthermore, they suggest that Dkk-1 and Dkk-2 may have distinct functions in osteoblast differentiation.