Peripheral blood monocyte-expressed ANXA2 gene is involved in pathogenesis of osteoporosis in humans

Low bone mineral density (BMD) is a risk factor of osteoporosis and has strong genetic determination. Genes influencing BMD and fundamental mechanisms leading to osteoporosis have yet to be fully determined. Peripheral blood monocytes (PBM) are potential osteoclast precursors, which could access to bone resorption surfaces and differentiate into osteoclasts to resorb bone. Herein, we attempted to identify osteoporosis susceptibility gene(s) and characterize their function(s), through an initial proteomics discovery study on PBM in vivo, and multiscale validation studies in vivo and in vitro. Utilizing the quantitative proteomics methodology LC-nano-ESI-MS(E), we discovered that a novel protein, i.e. ANXA2, was up-regulated twofold in PBM in vivo in Caucasians with extremely low BMD (cases) versus those with extremely high BMD (controls) (n = 28, p < 0.05). ANXA2 gene up-regulation in low BMD subjects was replicated at the mRNA level in PBM in vivo in a second and independent case-control sample (n = 80, p < 0.05). At the DNA level, we found that SNPs in the ANXA2 gene were associated with BMD variation in a 3(rd) and independent case-control sample (n = 44, p < 0.05), as well as in a random population sample (n = 997, p < 0.05). The above integrative evidence strongly supports the concept that ANXA2 is involved in the pathogenesis of osteoporosis in humans. Through a follow-up cellular functional study, we found that ANXA2 protein significantly promoted monocyte migration across an endothelial barrier in vitro (p < 0.001). Thus, elevated ANXA2 protein expression level, as detected in low BMD subjects, probably stimulates more PBM migration through the blood vessel walls to bone resorption surfaces in vivo, where they differentiate into higher number of osteoclasts and resorb bone at higher rates, thereby decreasing BMD. In conclusion, this study identified a novel osteoporosis susceptibility gene ANXA2, and suggested a novel pathophysiological mechanism, mediated by ANXA2, for osteoporosis in humans.     

Attenuated Monocyte Apoptosis,a New Mechanism for Osteoporosis Suggested by a Transcriptome-Wide Expression Study of Monocytes

BackgroundOsteoporosis is caused by excessive bone resorption (by osteoclasts) over bone formation (by osteoblasts). Monocytes are important to osteoporosis by serving as progenitors of osteoclasts and produce cytokines for osteoclastogenesis.AimTo identify osteoporosis-related genes, we performed microarray analyses of monocytes using Affymetrix 1.0 ST arrays in 42 (including 16 pre- and 26 postmenopausal) high hip BMD (bone mineral density) vs. 31 (including 15 pre- and 16 postmenopausal) low hip BMD Caucasian female subjects. Here, high vs. low BMD is defined as belonging to top vs. bottom 30% of BMD values in population.MethodDifferential gene expression analysis in high vs. low BMD subjects was conducted in the total cohort as well as pre- and post-menopausal subjects. Focusing on the top differentially expressed genes identified in the total, the pre- and the postmenopausal subjects (with a p <5E-03), we performed replication of the findings in 3 independent datasets of microarray analyses of monocytes (total N = 125).ResultsWe identified (in the 73 subjects) and successfully replicated in all the 3 independent datasets 2 genes, DAXX and PLK3. Interestingly, both genes are apoptosis induction genes and both down-regulated in the low BMD subjects. Moreover, using the top 200 genes identified in the meta-analysis across all of the 4 microarray datasets, GO term enrichment analysis identified a number of terms related to induction of apoptosis, for which the majority of component genes are also down-regulated in the low BMD subjects. Overall, our result may suggest that there might be a decreased apoptosis activity of monocytes in the low BMD subjects.ConclusionOur study for the first time suggested a decreased apoptosis rate (hence an increased survival) of monocytes, an important osteoclastogenic cell, as a novel mechanism for osteoporosis.     

MiR-133a in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis

Background

Osteoporosis mainly occurs in postmenopausal women, which is characterized by low bone mineral density (BMD) due to unbalanced bone resorption by osteoclasts and formation by osteoblasts. Circulating monocytes play important roles in osteoclastogenesis by acting as osteoclast precursors and secreting osteoclastogenic factors, such as IL-1, IL-6 and TNF-α. MicroRNAs (miRNAs) have been implicated as important biomarkers in various diseases. The present study aimed to find significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.

Methodology/Principal Findings

We used ABI TaqMan® miRNA array followed by qRT-PCR validation in circulating monocytes to identify miRNA biomarkers in 10 high and 10 low BMD postmenopausal Caucasian women. MiR-133a was upregulated (P=0.007) in the low compared with the high BMD groups in the array analyses, which was also validated by qRT-PCR (P=0.044). We performed bioinformatic target gene analysis and found three potential osteoclast-related target genes, CXCL11, CXCR3 and SLC39A1. In addition, we performed Pearson correlation analyses between the expression levels of miR-133a and the three potential target genes in the 20 postmenopausal women. We did find negative correlations between miR-133a and all the three genes though not significant.

Conclusions/Significance

This is the first in vivo miRNA expression analysis in human circulating monocytes to identify novel miRNA biomarkers underlying postmenopausal osteoporosis. Our results suggest that miR-133a in circulating monocytes is a potential biomarker for postmenopausal osteoporosis.     

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.     

Generation of novel bone forming cells (monoosteophils) from the cathelicidin-derived peptide LL-37 treated monocytes

Background

Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair.

Methods and Findings

Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK).

Conclusion

Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These cells may have wide ranging implications in the clinic including repair of broken bones and treatment of osteoporosis.     

Recent advances in the genetics of osteoporosis

It has been known for over 20 years that osteoporosis is highly influenced by genetic factors. Bone mineral density (BMD) has also been shown to be highly heritable. Other known risk factors for osteoporotic fractures such as reduced bone quality, femoral neck geometry and bone turnover are now also known to be heritable. Susceptibility to osteoporosis is mediated, in all likelihood, by multiple genes each having small effect. Different approaches are being used currently to identify the many genes responsible. These include linkage studies in man and experimental animals as well as candidate gene studies and alterations in gene expression. Linkage studies have identified multiple quantitative trait loci (QTL) for regulation of BMD and, with twin studies, have indicated that the effects of these loci are partly site-dependent and sex-specific. On the whole, the genes responsible for BMD regulation at these QTL have not yet been isolated. Most studies have used the candidate gene approach. The vitamin D receptor gene (VDR), the collagen type I alpha 1 gene (COLIA1) and estrogen receptor gene (ER) alpha have been most widely investigated and found to play a role in regulating BMD, but the effects are modest and together probably account for less than 5% of the heritable contribution to BMD. Genes may vary in their influence of particular intermediate phenotypes, and we now know that not all genes influencing BMD will be important in fracture. In addition, the study of other diseases such as osteoarthritis and metabolic bone syndromes may prove fruitful in highlighting genes which overlap to osteoporosis as well. As large scale genetic testing becomes more cost-effective, recent findings have illustrated the potential of novel approaches. These include combining large multi-national populations for candidate gene analysis, meta-analyses, DNA pooling studies and gene expression studies.     

Long non-coding RNA-DANCR in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis

Osteoporosis is a common disease characterized by low bone mineral density (BMD) and low trauma fractures, mainly resulting from exceeding bone resorption by osteoclasts over bone formation by osteoblasts. Circulating monocytes are directly involved in osteoclastogenesis, and lncRNAs are believed to be involved in the osteoblast differentiation. However, no study has been conducted to identify the roles of lncRNA in circulating monocytes associated with human osteoporosis. In this study, we found significant upregulation of DANCR in the blood mononuclear cells (MNCs) from low-BMD patients with the qRT-PCR analyses. We further found that DANCR promoted the expression of IL6 and TNF-α at both mRNA level and protein level in MNCs. After deletion of DANCR with siRNAs, the levels of IL6 and TNF-α are decreased in the MNCs from low-BMD postmenopausal women. Moreover, DANCR level was correlated with IL6 and TNF-α in postmenopausal women with low BMD. Furthermore, we found that DANCR-induced IL6 and TNF-α in MNCs had bone-resorbing activity. These results indicate that DANCR is involved in the pathology of osteoporosis and may be as a biomarker for postmenopausal osteoporosis.     

MiR-422a as a Potential Cellular MicroRNA Biomarker for Postmenopausal Osteoporosis

Background

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.

Methodology/Principal Findings

We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression.

Conclusions/Significance

Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.     

Human circulating monocytes can express receptor activator of nuclear factor-kappaB ligand and differentiate into functional osteoclasts without exogenous stimulation

Osteoclast formation from mononuclear precursors is believed to require accessory cells expressing receptor activator of nuclear factor-kappaB ligand (RANKL). We recently identified a human cell population originated from circulating CD14(+) monocytes, called monocyte-derived multipotential cells (MOMCs), which can differentiate into several distinct mesenchymal cells, neuron and endothelial cells. This study was undertaken to examine whether MOMCs can differentiate into functional osteoclasts. MOMCs prepared from peripheral blood of healthy volunteers cultured on fibronectin for 7 days at high density (8 x 10(5) cells cm(-2)), but not at regular density (2 x 10(4) cells cm(-2)), resulted in the appearance of tartrate-resistant acid phosphatase-positive giant multi-nucleated cells forming actin ring without exogenous osteoclastogenic factors. A subset of these cells showed bone resorption capacity on dentine slices and expression of genes for cathepsin K and calcitonin receptor, characteristic of functional osteoclasts. Such osteoclast differentiation was not observed in high-density culture of circulating monocytes, macrophages or dendritic cells, or the high-density culture of MOMCs on type I collagen. Among cells of the monocyte lineage, untreated MOMCs exclusively showed gene and protein expression of RANKL. When osteoprotegerin/IgG1 Fc chimera was added to high-density MOMC cultures, osteoclast formation was completely inhibited by neutralizing the endogenous RANKL. These results indicate that human MOMCs derived from circulating monocytes can express RANKL and differentiate into functional osteoclasts without RANKL-expressing accessory cells.     

Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3 transgenic mice

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT, and TL1A, induces osteoclast formation from monocyte, macrophage, and bone stromal marrow cells. However, the function of DcR3 on bone formation remains largely unknown. To understand the function of DcR3 in bone formation in vivo, transgenic mice overexpressing DcR3 were generated. Bone mineral density (BMD) and bone mineral content (BMC) of total body were significantly lower in DcR3 transgenic mice as compared with wild-type controls. The difference in BMD and BMC between DcR3 transgenic and control mice was confirmed by histomorphometric analysis, which showed a 35.7% decrease in trabecular bone volume in DcR3 transgenic mice in comparison with wild-type controls. The number of osteoclasts increased in DcR3 transgenic mice. In addition, local administration of DcR3 (30 microg/ml, 10 microl, once/day) into the metaphysis of the tibia via the implantation of a needle cannula significantly decreased the BMD, BMC, and bone volume of secondary spongiosa in tibia. Local injection of DcR3 also increased osteoclast numbers around trabecular bone in tibia. Furthermore, coadminstration of soluble tumor necrosis factor receptor inhibitor/Fc chimera (TNFRSF1A) but not osteoprotegerin inhibited the action of DcR3. In addition, in an assay of osteoclast activity on substrate plates, DcR3 significantly increased the resorption activity of mature osteoclasts. Treatment with higher concentrations of DcR3 slightly increased nodule formation and alkaline phosphatase activity of primary cultured osteoblasts. These results indicate that DcR3 may play an important role in osteoporosis or other bone diseases.     

Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation

Objective

The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH) and bone mineral density (BMD) in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells.

Methods

We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA) was used to detect serum FSH, luteinizing hormone (LH), and estradiol (E₂). Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E₂. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL), and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP) staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank), Trap, matrix metalloproteinase-9 (Mmp-9) and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction).

Results

1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner.

Conclusions

The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.     

Polymorphism in vitamin D receptor and osteoprotegerin genes in Egyptian rheumatoid arthritis patients with and without osteoporosis

1α,25-Dihydroxyvitamin D3 upregulates the expression of the receptor activator of nuclear factor kB ligand (RANKL), and downregulates osteoprotegerin (OPG) expression. We tested the effects of polymorphisms in the vitamin D receptor gene (VDR), and OPG gene in rheumatoid arthritis (RA) patients and healthy controls and their relationship to bone mineral density (BMD) and development of osteoporosis. Three hundred and fifty women were evaluated, 200 women having RA and 150 healthy control. The subjects were genotyped for polymorphism at BsmI in VDR and A163G in OPG genes by polymerase chain reaction followed by restriction fragment length polymorphism analysis. BMD was also measured. In A163G, the G allele increased the risk for RA and for the development of osteoporosis. We found a significant association between lower hip (BMD-h) and genotype variants of VDR (BsmI) and OPG A163G in RA patients with osteoporosis. Our results suggested that OPG A163G polymorphism was associated with RA susceptibility and with the development of osteoporosis in these patients. Also, VDR and OPG genes are important candidates for osteoporosis in RA patients.     

RANKL-RANK signaling in osteoclastogenesis and bone disease

Hundreds of millions of people worldwide are affected by bone-related diseases, such as osteoporosis and rheumatoid arthritis. Understanding the molecular mechanisms of bone metabolism is crucial for developing novel drugs for treating such diseases. In particular, genetic experiments showing that the receptor activator of NF-kappaB (RANK), its ligand RANKL, and the decoy receptor OPG are essential, central regulators of osteoclast development and osteoclast function were significant turning points in our understanding of bone diseases. RANKL-RANK signaling activates a variety of downstream signaling pathways required for osteoclast development. Moreover, molecular cross-talk between RANKL-RANK and other ligand-receptor systems fine-tunes bone homeostasis in normal physiology and disease. Designing novel drugs that target RANKL-RANK and their signaling pathways in osteoclasts could potentially revolutionize the treatment of many diseases associated with bone loss such as arthritis, tooth loss, cancer metastases or osteoporosis.     

钙敏感受体基因、白介素6基因与中国人群骨密度相关联

钙敏感受体是钙新陈代谢的一个重要因素,白介素6是参与破骨细胞分化及功能的一种多效细胞因子。因此,钙敏感受体基因和白介素6基因都为骨矿物质代谢的重要候选基因。本研究旨在利用数量性状传递不平衡检测法,检测白介素6基因和钙敏感受体基因与腰椎和髋部骨密度的关联和连锁,以证实它们是否为影响中国人群骨密度的重要候选基因。本研究的样本为来自中国上海的401个中国核心家庭,共1,263个个体,均为汉族。每个核心家庭由父母双亲和至少一个20～45岁的健康绝经前女儿组成。腰椎与髋部的骨密度采用Hologic QDR 2000＋双能X射线扫描仪进行了检测。用PE377测序仪及相关软件分别对白介素6和钙敏感受体基因中的一个CA重复多态微卫星位点进行了基因分型。分析结果表明钙敏感受体基因（CA）12等位基因（P=0．006）及（Ca）18等位基因（P=0．02）与股骨颈骨密度之间存在显著的整体关联。白介素6基因的（CA）18等位基因与整个髋部（P=0．021）、股骨颈（P=0．041）以及转子间区（P=0．029）骨密度之间均存在显著的家庭内关联。白介素6基因（CA）n位点与腰椎BMD之间存在显著的连锁（P=0．001）。本研究结果表明白介素6基因和钙敏感受体基因可能为与中国人群骨密度变异相关联的候选基因。     

Gene variants for osteoporosis and their pleiotropic effects in aging

The prevalence of osteoporosis is raising worldwide as improving conditions of living and treatment of other common diseases continuously increases life expectancy. Thus, osteoporosis affects most women above 80 years of age and, at the age of 50, the lifetime risk of suffering an osteoporosis-related fracture approaches 50% in women and 20% in men. Numerous genetic, hormonal, nutritional and life-style factors contribute to the acquisition and maintenance of bone mass. Among them, genetic variations explain as much as 70% of the variance for bone mineral density (BMD) in the population. Dozens of quantitative trait loci (QTLs) for BMD have been identified by genome screening and linkage approaches in humans and mice, and more than 100 candidate gene polymorphisms tested for association with BMD and/or fracture. Sequence variants in the vitamin D receptor (VDR), collagen 1 alpha 1 chain (Col1A1), estrogen receptor alpha (ESR1), interleukin-6 (IL-6) and LDL receptor-related protein 5 (LRP5) genes were all found to be significantly associated with differences in BMD and/or fracture risk in multiple replication studies. Moreover, some genes, such as VDR and IL-6, were shown to interact with non-genetic factors, i.e. calcium intake and estrogens, to modulate BMD. Since these gene variants have also been associated with other complex disorders, including cancer and coronary heart disease, they may represent common genetic susceptibility factors exerting pleiotropic effects during the aging process.     

Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells

Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.     

Integrin αMβ2 is differently expressed by subsets of human osteoclast precursors and mediates adhesion of classical monocytes to bone

Bone-degrading osteoclasts are formed through fusion of their monocytic precursors. In the population of human peripheral blood monocytes, three distinct subsets have been identified: classical, intermediate and non-classical monocytes. We have previously shown that when the monocyte subsets are cultured on bone, significantly more osteoclasts are formed from classical monocytes than from intermediate or non-classical monocytes. Considering that this difference does not exist when monocyte subsets are cultured on plastic, we hypothesized that classical monocytes adhere better to the bone surface compared to intermediate and non-classical monocytes. To investigate this, the different monocyte subsets were isolated from human peripheral blood and cultured on slices of human bone in the presence of the cytokine M-CSF. We found that classical monocytes adhere better to bone due to a higher expression of the integrin αMβ2 and that their ability to attach to bone is significantly decreased when the integrin is blocked. This suggests that integrin αMβ2 mediates attachment of osteoclast precursors to bone and thereby enables the formation of osteoclasts.     

Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair.