IGF2 modulates the microenvironment for osteoclastogenesis

We previously reported that hypoxic stress enhanced osteoclast differentiation via increasing insulin-like growth factor 2 (IGF2) production. However, the mechanisms underlying IGF2 stimulation remains unknown. In this study, we investigated the molecular mechanisms of osteoclastogenesis by IGF2 treatment. Primary mouse bone marrow cells were cultured with IGF2. Total RNAs were applied to a DNA microarray analysis, and quantitative RT-PCR was then used to confirm the microarray data and clarify which cells expressed the relative genes. The most interesting findings were the upregulations of CXC chemokine ligand 7 (CXCL7) expression in stromal cells and stromal cell-derived factor 1 (SDF1) expression in osteoblastic cells with IGF2 treatment. The addition of exogenous SDF1 to CXCL7 increased the number of osteoclasts and promoted the formation of giant osteoclasts. These results suggest that IGF2 modulates the microenvironment around osteoclast precursor cells. SDF1 together with CXCL7 may promote the formation of giant osteoclasts.     

Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we used human bone marrow cells (BMCs) to investigate the role of hypoxic exposure on human osteoclast (OC) formation in the presence of tumor necrosis factor (TNF). Exposing the BMCs to 3%, 5%, or 10% O₂ in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, consistent with OCs. The addition of TNF under hypoxic conditions generated significantly greater numbers of mature OCs with more nuclei than OCs generated under normoxic conditions. Longer initial hypoxic exposure increased the number of OC precursor cells and facilitated the differentiation of OC precursor cells into multinucleated OCs. Quantitative RT-PCR analysis revealed that RANKL and TNFR1 were expressed at higher levels in non-OC cells from BMCs under hypoxic conditions than under normoxic conditions. Furthermore, to confirm the involvement of TNF-induced signaling, we examined the effects of blocking antibodies against TNFR1 and TNFR2 on OC formation under hypoxic conditions. The TNFR1 antibody was observed to significantly suppress OC formation. These results suggest that hypoxic exposure plays an important role in TNF-induced osteoclastogenesis from human BMCs.     

IGF2 derived from SH-SY5Y neuroblastoma cells induces the osteoclastogenesis of human monocytic precursors

The insulin-like growth factors 2 (IGF2) is a peptide hormone that binds to the insulin-like growth factor 1 receptor (IGF1R) and is abundantly stored in bone. IGF1R is deeply involved in the pathogenesis of many cancers that growth within bone and is also involved in osteoclast biology. Among different cell lines representative of osteolytic tumors, we found a very high expression of IGF2 in SH-SY5Y cells derived from neuroblastoma (NB). We previously showed that NB cells induce an osteolytic process through the Osteoprotegerin/RANKL/RANK and the canonical Wnt pathway system. Here, we hypothesized that NB promotes osteoclastogenesis also via IGF2. First, we demonstrated the presence of IGF1R on the osteoclast basolateral membrane, and we observed a cyclic IGF1R activation along with the differentiation process, also when induced by SH-SY5Y. Moreover, we found that IGF2 mRNA expression in SH-SY5Y cells was further increased when co-cultured with mesenchymal stromal cells, suggesting that IGF2 is important for NB interaction with the bone microenvironment. Finally, the treatment of SH-SY5Y cells with an anti-IGF2 siRNA or the addition of anti-IGF1R molecules impaired NB-induced osteoclastogenesis, even though the chemoattraction of monocytes by NB cells was unaffected. Our findings suggest that in IGF2-producing osteolytic tumors IGF1R is a good candidate for targeted therapies in combination with conventional drugs.     

Endogenous production of tumor necrosis factor by primary cultures of murine calvarial cells: influence on IL-6 production and osteoclast development

We have previously shown that tumor necrosis factor (TNF) and interleukin-1 (IL-1) acted synergistically to stimulate the production of IL-6 by bone marrow stromal and osteoblastic cells; and that an antibody to IL-6 inhibited TNF-induced osteoclast development in murine calvarial cell cultures. Prompted by this evidence, we have now examined whether TNF and/or IL-1 are produced by murine calvarial cells, and whether these cytokines are involved in IL-6 production and osteoclast formation. When cultured under basal conditions, calvarial cells produced TNF and IL-6, and were able to form bone resorbing osteoclasts. A neutralizing antibody against TNF suppressed both basal IL-6 production and the formation of bone resorbing osteoclasts. The anti-TNF antibody also inhibited IL-6 production in response to exogenous IL-1 or parathyroid hormone (PTH). In contrast, a neutralizing anti-IL-1 receptor antibody had no effect on basal, TNF- or PTH-stimulated IL-6 production. These findings suggest that TNF, but not IL-1, is produced by murine bone cells and that endogenous TNF induces the IL-6 production, osteoclast formation, and bone resorption exhibited by these cultures under basal conditions. Furthermore, bone cell-derived TNF amplifies the stimulatory effect of exogenous IL-1 or PTH on IL-6 production by calvarial cells.     

CXCR7 Silencing Attenuates Cell Adaptive Response to Stromal Cell Derived Factor 1α after Hypoxia

Previous studies have shown that chemotactic factor stromal-cell derived factor 1α (SDF1α) promotes cell recovery from hypoxic injury via its main receptor C-X-C chemokine receptor type (CXCR) 4. However, the role of its new receptor CXCR7 on cell repair against hypoxia and cell response to SDF1α remains largely unknown. In this study, neurons induced from hippocampal progenitor cells were pre-conditioned in hypoxia for 4h and subsequently monitored to investigate the function of SDF1α on cell repair after hypoxia. Neurons were assessed for their cell morphology, actin filament polymerization and migration capability. SDF1α protein levels increased significantly 1 h after hypoxia compared to control (P<0.01), and it reached a peak at 24 h after hypoxia. Moreover, addition of SDF1α promoted neurite outgrowth and actin filament polymerization both in normoxic and hypoxic cells compared to untreated cells. Cell migration showed a time-dependent increase with SDF1α stimulation in both groups, and hypoxic cells illustrated a significant augment at 0.5 h, 1 h and 12 h after SDF1α application compared to normoxic cells (P<0.01). CXCR7 expression also increased with time dependence after hypoxia and demonstrated a two-fold upregulation compared to control at 24 h after hypoxia. With CXCR7 silencing, axon elongation and actin filament polymerization induced by SDF1α were inhibited sharply both in normoxic and hypoxic cells. CXCR7 silencing also leads to reduced hypoxic cell migration at 0.5 h, 1 h, 12 h, 24 h and 36 h after SDF1α application (P<0.01), but it failed to reduce normoxic cell migration induced by SDF1α at 0.5 h, 1 h and 12 h (P>0.05). 24 h SDF1α stimulation led to higher ERK1/2 phosphorylation compared to control, and ERK1/2 phosphorylation increased more in hypoxic cells than that in normoxic cells. This study suggested that CXCR7 plays an important role on cell repair processing induced by SDF1α, and CXCR7 silencing attenuates cell adaptive response to acute SDF1α stimulation (≤12 h) after hypoxia.     

MicroRNA-26a Regulates RANKL-Induced Osteoclast Formation

Osteoclasts are unique cells responsible for the resorption of bone matrix. MicroRNAs (miRNAs) are involved in the regulation of a wide range of physiological processes. Here, we examined the role of miR-26a in RANKL-induced osteoclastogenesis. The expression of miR-26a was up-regulated by RANKL at the late stage of osteoclastogenesis. Ectopic expression of an miR-26a mimic in osteoclast precursor cells attenuated osteoclast formation, actin-ring formation, and bone resorption by suppressing the expression of connective tissue growth factor/CCN family 2 (CTGF/CCN2), which can promote osteoclast formation via up-regulation of dendritic cell-specific transmembrane protein (DC-STAMP). On the other hand, overexpression of miR-26a inhibitor enhanced RANKL-induced osteoclast formation and function as well as CTGF expression. In addition, the inhibitory effect of miR-26a on osteoclast formation and function was prevented by treatment with recombinant CTGF. Collectively, our results suggest that miR-26a modulates osteoclast formation and function through the regulation of CTGF.     

NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis

We previously identified functional N-methyl-D-aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and Rank ligand (RankL). Using RT-PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi-quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose-dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western-blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF-kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF-kappa B pathway.     

MCP-1 is induced by receptor activator of nuclear factor-{kappa}B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-kappaB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.     

RANKL induces components of the extrinsic coagulation pathway in osteoclasts

Prothrombin is converted to thrombin by factor Xa in the cell-associated prothrombinase complex. Prothrombin is present in calcified bone matrix and thrombin exerts effects on osteoblasts as well as on bone resorption by osteoclasts.We investigated whether (1) osteoclasts display factor Xa-dependent prothrombinase activity and (2) osteoclasts express critical regulatory components upstream of the prothrombinase complex.The osteoclast differentiation factor RANKL induced formation of multinucleated TRAP positive cells concomitant with induction of prothrombinase activity in cultures of RAW 264.7 cells and bone marrow osteoclast progenitors.Expression analysis of extrinsic coagulation factors revealed that RANKL enhanced protein levels of factor Xa as well as of coagulation factor III (tissue factor). Inhibition assays indicated that factor Xa and tissue factor were involved in the control of prothrombinase activity in RANKL-differentiated osteoclasts, presumably at two stages (1) conversion of prothrombin to thrombin and (2) conversion of factor X to factor Xa, respectively.Activation of the extrinsic coagulation pathway during osteoclast differentiation through induction of tissue factor and factor Xa by a RANKL-dependent pathway indicates a novel role for osteoclasts in converting prothrombin to thrombin.     

Hypoxia Increases Gefitinib-Resistant Lung Cancer Stem Cells through the Activation of Insulin-Like Growth Factor 1 Receptor

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as “gefitinib-resistant persisters” (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1–all genes involved in stemness–were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.     

Thyroid hormone stimulates osteoclast differentiation by a mechanism independent of RANKL-RANK interaction

It is well known that thyroid hormone excess causes bone loss. However, the precise mechanism of bone loss by thyroid hormone still remains unclear. When T(3) was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of indomethacin and IL-6 Ab. T(3) increased the expression of osteoprotegerin (OPG) messenger RNA (mRNA), but not of receptor activator of nuclear factor kappaB ligand (RANKL) in unfractionated bone cells, suggesting that the stimulatory effect of T(3) on osteoclast formation was not mediated by the RANKL/OPG system. We next examined the direct effect of T(3) on osteoclast precursors in the absence of osteoblasts, using hemopoietic blast cells derived from spleen cells. T(3) (1 pM-100 nM) dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors. OPG did not inhibit T(3)-induced osteoclast formation from osteoclast precursor cells. The polymerase chain reaction (PCR) product corresponding in size to the mouse T(3) receptor alpha1 cDNA was detected in osteoclast precursors from mouse hemopoietic blast cells as well as mouse heart and mouse osteoblastic cell line MC3T3-E1 cells, suggesting that T(3) directly stimulated osteoclast-like cell formation from osteoclast precursors in the absence of osteoblasts. Further, T(3) increased the expression of c-Fos mRNA at 15 min and 24 h and Fra-1 mRNA at 2 and 6 h in osteoclast precursors. Consistent with the increased expression of c-Fos mRNA observed by RT-PCR, the activation of c-Fos occurred in osteoclast precursor cells stimulated by T(3), while the activation of neither NF-kappaB nor MAPKs was observed by immunoblot analysis. Antisense oligodeoxynucleotides (as-ODN) complementary to c-Fos mRNA at 1 microM significantly inhibited T(3)-induced osteoclast-like cell formation from osteoclast precursors in the absence of stromal cells while sense-ODN did not affect T(3)-induced osteoclast-like cell formation. These results indicate that T(3) directly stimulates osteoclast differentiation at least in part by up-regulation of c-fos protein in osteoclast precursor cells.     

miR-221-3p targets Ang-2 to inhibit the transformation of HCMECs to tip cells

Postembryonic angiogenesis is mainly induced by various proangiogenic factors derived from the original vascular network. Previous studies have shown that the role of Ang-2 in angiogenesis is controversial. Tip cells play a vanguard role in angiogenesis and exhibit a transdifferentiated phenotype under the action of angiogenic factors. However, whether Ang-2 promotes the transformation of endothelial cells to tip cells remains unknown. Our study found that miR-221-3p was highly expressed in HCMECs cultured for 4 h under hypoxic conditions (1% O₂). Moreover, miR-221-3p overexpression inhibited HCMECs proliferation and tube formation, which may play an important role in hypoxia-induced angiogenesis. By target gene prediction, we further demonstrated that Ang-2 was a downstream target of miR-221-3p and miR-221-3p overexpression inhibited Ang-2 expression in HCMECs under hypoxic conditions. Subsequently, qRT-PCR and western blotting methods were performed to analyse the role of miR-221-3p and Ang-2 on the regulation of tip cell marker genes. MiR-221-3p overexpression inhibited CD34, IGF1R, IGF-2 and VEGFR2 proteins expression while Ang-2 overexpression induced CD34, IGF1R, IGF-2 and VEGFR2 expression in HCMECs under hypoxic conditions. In addition, we further confirmed that Ang-2 played a dominant role in miR-221-3p inhibitors promoting the transformation of HCMECs to tip cells by using Ang-2 shRNA to interfere with miR-221-3p inhibitor-treated HCMECs under hypoxic conditions. Finally, we found that miR-221-3p expression was significantly elevated in both serum and myocardial tissue of AMI rats. Hence, our data showed that miR-221-3p may inhibit angiogenesis after acute myocardial infarction by targeting Ang-2 to inhibit the transformation of HCMECs to tip cells.     

EGF-like ligands stimulate osteoclastogenesis by regulating expression of osteoclast regulatory factors by osteoblasts: implications for osteolytic bone metastases

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.     

Carboxypeptidase E Is a Novel Modulator of RANKL-Induced Osteoclast Differentiation

Osteoclasts are large polykaryons that have the unique capacity to degrade bone and are generated by the differentiation of myeloid lineage progenitors. To identify the genes involved in osteoclast development, we performed microarray analysis, and we found that carboxypeptidase E (CPE), a prohormone processing enzyme, was highly upregulated in osteoclasts compared with their precursors, bone marrow-derived macrophages (BMMs). Here, we demonstrate a novel role for CPE in receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. The overexpression of CPE in BMMs increases the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts and the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are key regulators in osteoclastogenesis. Furthermore, employing CPE knockout mice, we show that CPE deficiency attenuates osteoclast formation. Together, our data suggest that CPE might be an important modulator of RANKL-induced osteoclast differentiation.     

人骨保护素N端片段在大肠杆菌中的表达及其活性分析

骨保护素 (OPG)成熟肽N端D1～D4结构域仅由 2个外显子编码 .以人基因组DNA作为模板 ,采用重叠延伸PCR得到N端D1～D4域编码序列 ,并在其上游引入 2×His密码子序列 ,然后克隆入载体pQE 30进行表达 ,SDS PAGE表明 8×His融合蛋白主要以包涵体形式存在 ,可被抗OPG抗体识别 .变性条件下通过Ni NTA金属螯合亲和层析对表达产物进行纯化后再经梯度透析进行复性 ,采用破骨细胞样细胞 (osteoclast likecell ,OLC)诱导分化实验来检测重组蛋白的生物活性 ,证实单核 巨噬细胞集落刺激因子 (M CSF)和破骨细胞分化因子 (ODF)可协同促进多核OLC的生成 ,但加入重组OPG片段后 ,OLC生成显著减少 .     

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A.

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A-binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1alpha,25 dihydroxyvitamin D(3), prostaglandin E(2), and interleukin-1alpha. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF-kappaB ligand (RANKL). RT-PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony-stimulating factor-dependent bone marrow macrophages (M-BMMPhis). In addition, phosphorylation of Smad2 was observed in M-BMMPhis stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A-enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.