Signaling In Osteoblasts Research Articles

2003 - ACUTE MYELOID LEUKEMIA CELLS SEIZE SEROTONIN SIGNALING IN OSTEOBLASTS TO ENGRAFT AND PROLIFERATE

Patients with myelodysplasia or acute myeloid leukemia (AML) show decreased osteoblast numbers, and this is reproduced in mouse models of acute leukemia. Manipulating the osteoblast pool directly affects leukemia progression: osteoblast ablation increases leukemia burden, while maintenance of osteoblast numbers decreases it and prolongs survival. Here we show that the protective effect of osteoblasts against leukemia does not rely on a certain threshold of osteoblast numbers but rather requires a specific signaling pathway. Treatment of leukemic mice with parathyroid hormone, which increases osteoblast numbers, had no effect in leukemia progression. In contrast, maintaining the osteoblast pool in leukemic mice by decreasing the synthesis of gut-derived serotonin, reduced AML burden and prolonged survival. Using the human-AML MLL-AF9 model, we examined whether deletion of one of the main serotonin receptors expressed in osteoblasts affects leukemia progression. Global deletion of Htr1b in mice prevented leukemia. While removal of Htr1b in LepR+ positive mesenchymal stem cells had no effect in leukemia progression, its deletion in Col1a1-expressing osteoblast precursors significantly prevented lethality in the mutant mice. In order to dissect the crosstalk between leukemia-cells and osteoblasts, we co-cultured primary-human osteoblasts and AML cells. RNAseq analysis identified a signature of chemoattractants and pro-inflammatory cytokines secreted by osteoblasts upon exposure to AML cells, suggesting that the protective mechanism exerted by osteoblasts activates the anti-tumor immune response against leukemia. In summary, our data suggest that AML cells activate serotonin signaling in osteoblasts to promote their engraftment and expansion. Targeting this pathway may render the niche hostile to leukemia by enhancing the anti-tumor immune response elicited by osteoblasts.

Influence of single nucleotide polymorphisms (SNPs) in genetic susceptibility towards periprosthetic osteolysis.

Wear debris-induced inflammatory osteolysis remains a significant limiting factor for implant replacement surgeries. Hence, a comprehensive understanding of the complex network of cellular and molecular signals leading to these inflammatory responses is required. Both macrophages and monocytes have a critical role in the instigation of the inflammatory reaction to wear debris but differ in the extent to which they induce cytokine expression in patients. Lately, single nucleotide polymorphisms (SNPs) have been associated with genetic susceptibility among individual patients with implant failure. Studies have shown that SNPs in key pro-inflammatory cytokines and their receptors are associated with osteolytic susceptibility. Likewise, SNPs within several genes involved in the regulation of bone turnover have also been found to be associated with wear debris induced osteolysis. It is presumed that SNP variance might play a decisive role in the activation and signaling of macrophages, osteoblasts, chondrocytes, fibroblasts and other cells involved in inflammatory bone loss. Understanding the extent to which SNPs exist among genes that are responsible for inflammatory bone loss may provide potential targets for developing future therapeutic interventions. Herein, we attempt to summarize the various susceptible genes with possible SNP variance that could contribute to the severity of periprosthetic osteolysis in patients with implants.

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice.

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1-3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.

A Differential Hypofunctionality of G\u03b1i Proteins Occurs in Adolescent Idiopathic Scoliosis and Correlates with the Risk of Disease Progression

Adolescent idiopathic scoliosis is the most prevalent spine deformity and the molecular mechanisms underlying its pathophysiology remain poorly understood. We have previously found a differential impairment of melatonin receptor signaling in AIS osteoblasts allowing the classification of patients into three biological endophenotypes or functional groups (FG1, FG2 and FG3). Here, we provide evidence that the defect characterizing each endophenotype lies at the level of Gαi proteins leading to a systemic and generalized differential impairment of Gi-coupled receptor signaling. The three Gαi isoforms exhibited a selective serine phosphorylation patterns for each AIS endophenotype resulting in a differential reduction in Gαi protein activity as determined by cellular dielectric spectroscopy and small interfering RNA methods. We found that one endophenotype (FG2) with phosphorylated Gαi1 and Gαi2 was consistently associated with a significantly high risk of spinal deformity progression when compared to the other two endophenotypes (FG1 and FG3). We further demonstrated that each endophenotype is conserved among affected family members. This study expands our understanding of the mechanism underlying the Gi-coupled receptor signaling dysfunction occurring in AIS and provides the first evidence for its hereditary nature. Collectively, our findings offers a new perspective on Gαi hypofunctionality in a human disease by revealing specific serine phosphorylation signatures of Gαi isoforms that may facilitate the identification of AIS patients at risk of spinal deformity progression.

CCN3 Facilitates Runx2 and Osterix Expression by Inhibiting miR-608 through PI3K/Akt Signaling in Osteoblasts.

CCN3, otherwise known as the nephroblastoma overexpressed (NOV) protein, is a cysteine-rich protein that belongs to the CCN family and regulates several cellular functions. Osteoblasts are major bone-forming cells that undergo proliferation, mineralization, renewal, and repair during the bone formation process. We have previously reported that CCN3 increases bone morphogenetic protein 4 (BMP-4) production and bone mineralization in osteoblasts, although the role of CCN3 remains unclear with regard to osteogenic transcription factors (runt-related transcription factor 2 (Runx2) and osterix). Here, we used alizarin red-S and alkaline phosphatase staining to show that CCN3 enhances osteoblast differentiation. Stimulation of osteoblasts with CCN3 increases expression of osteogenic factors such as BMPs, Runx2, and osterix. Moreover, we found that the inhibition of miR-608 expression is involved in the effects of CCN3 and that incubation of osteoblasts with CCN3 promotes focal adhesion kinase (FAK) and Akt phosphorylation. Our results indicate that CCN3 promotes the expression of Runx2 and osterix in osteoblasts by inhibiting miR-608 expression via the FAK and Akt signaling pathways.

Connexin43 enhances Wnt and PGE2-dependent activation of β-catenin in osteoblasts.

Connexin43 is an important modulator of many signaling pathways in bone. β-Catenin, a key regulator of the osteoblast differentiation and function, is among the pathways downstream of connexin43-dependent intercellular communication. There are striking overlaps between the functions of these two proteins in bone cells. However, differential effects of connexin43 on β-catenin activity have been reported. Here, we examined how connexin43 influenced both Wnt-dependent and Wnt-independent activation of β-catenin in osteoblasts in vitro. Our data show that loss of connexin43 in primary osteoblasts or connexin43 overexpression in UMR106 cells regulated active β-catenin and phospho-Akt levels, with loss of connexin43 inhibiting and connexin43 overexpression increasing the levels of active β-catenin and phospho-Akt. Increasing connexin43 expression synergistically enhanced Wnt3a-dependent activation of β-catenin protein and β-catenin transcriptional activity, as well as Wnt-independent activation of β-catenin by prostaglandin E2 (PGE2). Finally, we show that the activation of β-catenin by PGE2 required signaling through the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β) pathway, as the PI3K inhibitor, LY-294002, disrupted the synergy between connexin43 and PGE2. These data show that connexin43 regulates Akt and β-catenin activity and synergistically enhances both Wnt-dependent and Wnt-independent β-catenin signaling in osteoblasts.

Geniposide attenuates cadmium‑induced oxidative stress injury via Nrf2 signaling in osteoblasts.

Geniposide, as a type of iridoid glycoside, has antioxidative capacity. However, the mechanism underlying the effect of geniposide in cadmium (Cd)-induced osteoblast injury remains only partly elucidated. In the present study, Cell Counting Kit-8 (CCK-8) was used to determine MC-3T3-E1 cell viability. Flow cytometry was used to determine the rate of apoptosis and levels of reactive oxygen species (ROS). Oxidative stress-related factors were assessed using enzyme-linked immunosorbent method (ELISA). Quantitative real-time polymerase chain reaction (qPCR) and western blotting were used to evaluate apoptosis- and bone formation-related genes and nuclear factor erythroid 2-related factor (Nrf2) signaling. It was demonstrated that geniposide increased the viability of the Cd-treated MC-3T3-E1 cells. Geniposide decreased apoptosis and ROS accumulation compared to these parameters in the Cd group. Geniposide attenuated oxidative stress-related factors, malondialdehyde and lactate dehydrogenase and increased antioxidant key enzyme superoxidase dismutase (SOD). The expression levels of Bax, Bcl-2 and survivin were modulated by geniposide. Additionally, the mRNA and protein expression of the receptor activator of NF-κB ligand (RANKL) and osterix were significantly increased, while osteoprotegerin was decreased by geniposide treatment compared to the Cd groups. Geniposide also enhanced Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) expression. The present study identified a potential agent for the treatment of Cd-induced osteoblast injury.

Collagen XIII-derived ectodomain regulates bone angiogenesis and intracortical remodeling

Osteoporosis is the most common degenerative bone disease that occurs when the balance of bone production and resorption is perturbed. Loss of bone mass or alteration in its quality leads to significant weakening of the bones and subsequently to higher fracture risk. Collagen XIII (ColXIII) is a conserved transmembrane protein expressed in many mesenchymal tissues. Here we show that ColXIII is a regulator of bone remodeling niche. In this study, we found that ColXIII expression is significantly upregulated in osteoporotic patients. In view of that, we studied bone homeostasis in ColXIII-overexpressing mice (Col13a1oe) up to 72 weeks of age and observed a cortical bone overgrowth followed by a drastic bone loss, together with increased bone vascularization. Moreover, our results demonstrate that the ColXIII-derived ectodomain enhances angiogenesis through β1-integrins and the JNK pathway. Consequently, these data suggest that ColXIII has a role in age-dependent cortical bone deterioration with possible implications for osteoporosis and fracture risk.

Disruption of glucocorticoid signalling in osteoblasts attenuates age-related surgically induced osteoarthritis

Aging is a major risk factor for osteoarthritis (OA). Skeletal expression and activity of the glucocorticoid-activating enzyme 11β-hydroxysteroid-dehydrogenase type 1 increases progressively with age in humans and rodents. Here we investigated the role of endogenous osteocytic and osteoblastic glucocorticoid (GC) signalling in the development of osteoarthritic bone and cartilage damage in mice. We utilized transgenic (tg) mice in which glucocorticoid signalling is disrupted in osteoblasts and osteocytes via overexpression of the glucocorticoid-inactivating enzyme, 11β-hydroxysteroid-dehydrogenase type 2. Osteoarthritis was induced in 10- and 22-week-old male transgenic mice (tg-OA, n=6/group) and their wildtype littermates (WT-OA, n=7-8/group) by surgical destabilization ofthemedial meniscus (DMM). Sham-operated mice served as controls (WT- & tg-Sham, n=3-5 and 6-8/group at 10- and 22-weeks of age, respectively). Sixteen weeks after DMM surgery, mice developed features of cartilage degradation, subchondral bone sclerosis and osteophyte formation. These changes did not differ between WT and tg mice when OA was induced at 10-weeks of age. However, when OA was induced at 22-weeks of age, cartilage erosion was significantly attenuated in tg-OA mice compared to WT-OA littermates. Similarly, subchondral bone volume (-5.2%, 95% confidence intervals (CI) -9.1 to-1.2%, P=0.014) and osteophyte size (-4.0mm2, 95% CI -7.5 to-0.5mm2, P=0.029) were significantly reduced in tg-OA compared to WT-OA mice. Glucocorticoid signalling in cells of the osteoblast lineage promotes the development of surgically-induced osteoarthritis in older, but not younger, male mice. These data implicate osteoblasts and osteocytes in the progression of DMM-OA, via a glucocorticoid-dependent and age-related pathway.

Addition of an oligoglutamate domain to bone morphogenic protein 2 confers binding to hydroxyapatite materials and induces osteoblastic signaling.

Nonautologous bone grafts have limited osteoinductive potential and thus there is substantial interest in reconstituting these graft materials with osteogenic factors such as bone morphogenic protein 2 (BMP2). However, one limitation of this approach is that BMP2 is typically weakly bound to the graft, which can lead to side effects associated with BMP2 dissemination. In the current study we added a hydroxyapatite (HA)-binding domain onto BMP2 to increase coupling to the graft surface. A sequence consisting of eight glutamate residues (E8) was inserted into the C-terminus of BMP2, and the recombinant protein (rBMP2-E8) was expressed in E. coli. Compared with rBMP2, rBMP2-E8 displayed markedly enhanced binding to HA disks and was better retained on the disks following exposure to vigorous wash steps. Furthermore, rBMP2-E8 was purified using a heparin column, and evaluated for its capacity to stimulate osteoblastic cell signaling. Treatment of SAOS2 cells with rBMP2-E8 induced SMAD 1/5 activation, confirming that the protein retains activity. Collectively these results suggest that the E8 domain serves as an effective tool for improving rBMP2 coupling to graft materials. The increased retention of rBMP2-E8 on the graft surface is expected to prolong BMP2’s osteoinductive activity within the graft site, while simultaneously reducing off-target effects.

Novel Role for Claudin-11 in the Regulation of Osteoblasts via Modulation of ADAM10-Mediated Notch Signaling.

The claudin (Cldn) family comprises 27 members of 20 to 34 kDa transmembrane tight junction proteins. In addition to Cldns' established canonical role as barriers controlling paracellular flow of molecules, a distinct noncanonical role for them as mediators of cell signaling is now emerging. In our studies evaluating Cldn family expression levels during osteoblast differentiation, Cldn-11 showed the largest increase (60-fold). Immunohistochemistry studies revealed high Cldn-11 expression in trabecular (Tb) bone lining cells. Micro-CT analysis of femurs and vertebrae of Cldn-11 knock-out (KO) mice at 12 weeks of age exhibited a 40% (p < 0.01) reduction in Tb bone volume adjusted for tissue volume compared with control mice, a change caused by significant reductions in Tb number and thickness and increase in Tb separation. Histomorphometry and serum biomarker studies revealed that reduced bone formation, not increased resorption, is the cause for reduced Tb bone volume in the Cldn-11 KO mice. Cldn-11 KO osteoblasts expressed reduced ALP and BSP, whereas Cldn-11 overexpression in MC3T3-E1 cells increased expression of ALP and BSP. Mechanistically, Cldn-11 interacted with tetraspanin (Tspan)3 in osteoblasts, and Tspan3 knockdown reduced osteoblast differentiation. Because members of the Tspan family regulate cell functions via Notch signaling, we evaluated whether Cldn-11/Tspan3 regulates Notch signaling in osteoblasts. Accordingly, Notch targets Hey1 and Hey2 were significantly upregulated in Cldn-11 overexpressing cultures but downregulated in both Cldn-11 KO and Tspan3 knockdown osteoblasts. Because ADAM10 has been shown to interact with Tspan family members to regulate Notch signaling, we evaluated whether Cldn-11 regulates ADAM10 expression. Cldn-11 overexpressing cells express more mature ADAM10, and an ADAM10 inhibitor blocked the Cldn-11 effect on osteoblast differentiation. Based on these data, we propose Cldn-11 as a novel component of an osteoblast cell surface protein complex, comprising Tspan3 and ADAM10, which regulates Notch signaling and cell differentiation. © 2019 American Society for Bone and Mineral Research.

New explanation for autosomal dominant high bone mass: Mutation of low-density lipoprotein receptor-related protein 6.

LRP5 encodes low-density lipoprotein receptor-related protein 5 (LRP5). When LRP5 with a Frizzled receptor join on the surface of an osteoblast and bind a member of the Wnt family of ligands, canonical Wnt/β-catenin signaling occurs and increases bone formation. Eleven heterozygous gain-of-function missense mutations within LRP5 are known to prevent the LRP5 inhibitory ligands sclerostin and dickkopf1 from attaching to LRP5's first β-propeller, and thereby explain the rare autosomal dominant (AD) skeletal disorder "high bone mass" (HBM). LRP6 is a cognate co-receptor of LRP5 and similarly controls Wnt signaling in osteoblasts, yet the consequences of increased LRP6-mediated signaling remain unknown. We investigated two multi-generational American families manifesting the clinical and routine laboratory features of LRP5 HBM but without an LRP5 defect and instead carrying a heterozygous LRP6 missense mutation that would alter the first β-propeller of LRP6. In Family 1 LRP6 c.602C>T, p.A201V was homologous to LRP5 HBM mutation c.641C>T, p.A214V, and in Family 2 LRP6 c.553A>C, p.N185H was homologous to LRP5 HBM mutation c.593A>G, p.N198S but predicting a different residue at the identical amino acid position. In both families the LRP6 mutation co-segregated with striking generalized osteosclerosis and hyperostosis. Clinical features shared by the seven LRP6 HBM family members and ten LRP5 HBM patients included a broad jaw, torus palatinus, teeth encased in bone and, reportedly, resistance to fracturing and inability to float in water. For both HBM disorders, all affected individuals were taller than average for Americans (Ps < 0.005), but with similar mean height Z-scores (P = 0.7606) and indistinguishable radiographic skeletal features. Absence of adult maxillary lateral incisors was reported by some LRP6 HBM individuals. In contrast, our 16 patients with AD osteopetrosis [i.e., Albers-Schönberg disease (A-SD)] had an unremarkable mean height Z-score (P = 0.9401) lower than for either HBM group (Ps < 0.05). DXA mean BMD Z-scores in LRP6 HBM versus LRP5 HBM were somewhat higher at the lumbar spine (+7.8 vs +6.5, respectively; P = 0.0403), but no different at the total hip (+7.9 vs +7.7, respectively; P = 0.7905). Among the three diagnostic groups, only the LRP6 HBM DXA BMD values at the spine seemed to increase with subject age (R = +0.7183, P = 0.0448). Total hip BMD Z-scores were not significantly different among the three disorders (Ps > 0.05), and showed no age effect (Ps > 0.1). HR-pQCT available only for LRP6 HBM revealed indistinct corticomedullary boundaries, high distal forearm and tibial total volumetric BMD, and finite element analysis predicted marked fracture resistance. Hence, we have discovered mutations of LRP6 that cause a dento-osseous disorder indistinguishable without mutation analysis from LRP5 HBM. LRP6 HBM seems associated with generally good health, providing some reassurance for the development of anabolic treatments aimed to enhance LRP5/LRP6-mediated osteogenesis.

Allium fistulosum (Welsh onion) and Portulaca oleracea increase longitudinal bone growth in weanling rats possibly by promoting TGF-β and IGF-1 signaling

Abstract Longitudinal bone growth mainly occurs during adolescence and can be enhanced by growth hormones. We hypothesized that water extracts from roots of Allium fistulosum L. (Welsh onion, WO; 15 or 45 mg/kg bw/day) and Portulaca oleracea L. (PO; 15 or 45 mg/kg bw/day) can stimulate longitudinal bone growth in weanling male rats for 4 weeks. Femur and tibia lengths were higher in the WO-H and PO-H groups than in the control. The proliferative and hypertrophic zones of the growth plate were greater in the legs of WO-H and PO-H rats than in the control; a similar increase was observed in the positive-control (growth hormone, 20 μg/kg bw/day). WO-H and PO-H treatments were found to potentiate insulin-like growth factor-1 (IGF-1) signaling (pIRS-2 → pAkt) in MG-63 osteoblasts compared to control. WO-H and PO increased cell proliferation possibly by activating transforming growth factor (TGF)-β signaling via elevated BMP-2 and Smad4 expression in MG-63 cells. In conclusion, WO and PO are potential to promote the proliferation of the leg growth plate similar to growth hormone.

TMCO1-mediated Ca2+ leak underlies osteoblast functions via CaMKII signaling

Transmembrane and coiled-coil domains 1 (TMCO1) is a recently identified Ca2+ leak channel in the endoplasmic reticulum. TMCO1 dysfunction in humans is associated with dysmorphism, mental retardation, glaucoma and the occurrence of cancer. Here we show an essential role of TMCO1 in osteogenesis mediated by local Ca2+/CaMKII signaling in osteoblasts. TMCO1 levels were significantly decreased in bone from both osteoporosis patients and bone-loss mouse models. Tmco1−/− mice exhibited loss of bone mass and altered microarchitecture characteristic of osteoporosis. In the absence of TMCO1, decreased HDAC4 phosphorylation resulted in nuclear enrichment of HADC4, which leads to deacetylation and degradation of RUNX2, the master regulator of osteogenesis. We further demonstrate that TMCO1-mediated Ca2+ leak provides local Ca2+ signals to activate the CaMKII-HDAC4-RUNX2 signaling axis. The establishment of TMCO1 as a pivotal player in osteogenesis uncovers a novel potential therapeutic target for ameliorating osteoporosis.

Systemic administration of low-dose naltrexone increases bone mass due to blockade of opioid growth factor receptor signaling in mice osteoblasts

AimsOpioid receptor blockers such as naloxone and naltrexone have been suggested to have a bone mass-increasing effect. However, the mechanisms at play have not been clarified. We examined the effects of naltrexone on osteoblasts and determined the expression of opioid growth factor receptor (OGFR) in osteoblasts. Naltrexone blocks the OGFR and other canonical opioid receptors. Thus, we designed experiments to clarify the effects of naltrexone on bone tissue by examining the physiological role of OGFR signaling in osteoblasts and the changes in bone structure after naltrexone systemic administration in mice. Main methodsWe used mouse osteoblast-like cell line MC3T3-E1 for in vitro experiments. We cultured MC3T3-E1 cells in the presence of the OGFR agonist met-enkephalin (met-enk). Then, we measured cell proliferation activity and analyzed the expression levels of cell proliferation-related genes. For our in vivo experiments, we administered naltrexone intraperitoneally to mice daily for 28 days and administered the animals in the control group equivalent volumes of saline. After sacrificing the mice, we performed micro-computed tomography and bone morphology analyses. Key findingsMet-enk suppressed cell proliferation in MC3T3-E1 cells. Moreover, Low dose naltrexone administration significantly increased their femoral bone mass, bone formation ratio, and osteoblast number/bone surface values when comparing the values for the same variables in the control group. SignificanceOur results suggest that naltrexone increases bone mass due to osteoblast number increments caused by the OGFR signaling block. Opioid receptor blockers have potential as therapeutic agents for osteoporosis as well as opioid antagonists.

Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A2A receptors (A2ARs) and Wingless/Integrated-β-catenin pathways. Cytosolic β-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular β-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) β-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular β-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 μM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 β-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular β-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, β-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

Activation of cancer-related and mitogen-activated protein kinase signaling pathways in human mature osteoblasts isolated from patients with type 2 diabetes

Diabetes mellitus is a disease of glucose metabolism, and it adversely affects bone metabolism and increases the risk of cancer development. Previously, we reported a method for the direct isolation of human mature osteoblasts and indicated that osteoblasts were associated with type 2 diabetes mellitus-related signaling pathways. In addition, a recent report suggested that osteoblasts are involved in glucose metabolism. Thus, we sought to examine the effects of diabetes on osteoblast signaling in vivo. We recruited eight patients with type 2 diabetes and eight non-diabetic individuals. We isolated human mature osteoblasts from the resected femoral heads during orthopaedic surgery and extracted their RNA. We compared the gene expression between the two groups by RNA microarray and pathway analyses. Microarray analysis showed significant differences in 885 of 19,463 genes between the two groups (p < 0.05), and pathway analysis revealed that pathways related to cancer and the mitogen-activated protein kinase signaling pathway were significantly activated in the diabetes group (p < 0.01). These preliminary findings suggest that diabetes affects intracellular signaling in human mature osteoblasts and that osteoblasts might not only play a key role in the regulation of bone and glucose metabolism, but might also be related to cancer metabolism. We plan to conduct further studies to examine signaling in diabetic osteoblasts and to further investigate the genes and pathways identified here.

Sostdc1: A soluble BMP and Wnt antagonist that is induced by the interaction between myeloma cells and osteoblast lineage cells.

Multiple myeloma (MM) is characterised by destructive lytic bone disease, caused by induction of bone resorption and impaired bone formation. Our understanding of the molecular mechanisms responsible for osteoblast suppression, are limited. Using the 5T2MM murine model of MM we have previously shown that suppression of the activity of a known inhibitor of bone formation Dikkopf-1 (Dkk1) prevents the development of lytic bone disease. Here we have demonstrated that another potential inhibitor of bone formation, sclerostin domain containing 1 (Sostdc1) is expressed at low levels in MM and osteoblast lineage cells when these cells are grown separately in cell culture but its expression is significantly induced in both cell types when these cells are in contact. The distribution of Sostdc1 staining in bones infiltrated with 5TGM1 myeloma cells in vivo suggested its presence in both myeloma and osteoblast lineage populations when in close proximity. We have also shown that recombinant Sostdc1 inhibits both bone morphogenic proteins (BMP2 and 7) and Wnt signalling in primary osteoblasts and suppresses differentiation of these cells. Together, these findings suggest that Sostdc1 expression in 5TGM1-infiltrated bones as a result of the interaction between myeloma and osteoblast lineage populations, could result in suppression of osteoblast differentiation.

Abrogated glucocorticoid signalling in osteoblasts prevents diet-induced obesity, insulin resistance and bone loss

Abrogated glucocorticoid signalling in osteoblasts prevents diet-induced obesity, insulin resistance and bone loss

Free fatty acid receptor 4-β-arrestin 2 pathway mediates the effects of different classes of unsaturated fatty acids in osteoclasts and osteoblasts

Bone is a dynamic tissue that is constantly remodelled by bone resorbing osteoclasts and bone forming osteoblasts, respectively. A breakdown in the remodelling process underlies several bone diseases such as osteoporosis. Unsaturated fatty acids (UFAs) have been shown to have beneficial effects on bone health. However, the mechanism of action of UFAs in bone remains unclear. Free fatty acid receptor 4 (FFAR4) is expressed in bone cells and preferentially binds ω−3 and ω−7 UFAs. Therefore, we sought to determine if FFAR4 influenced the action of different classes of UFAs in bone cells. FFAR4 and potential signalling pathways, β-arrestin 2 (βarr2) and Gαq, were silenced in RAW264.7 murine macrophages (pre-osteoclasts) and MC3T3-E1 murine pre-osteoblasts. Cell differentiation, activation of signalling pathways and expression of regulatory genes were evaluated. The ω−3 UFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω−7 UFA, palmitoleic acid (PLA), were shown to require the FFAR4/βarr2 signalling pathway to inhibit osteoclast differentiation in RAW264.7 murine macrophages. The ω−6 UFA, arachidonic acid, and the ω−9 UFA, oleic acid (OA), were shown to inhibit osteoclast formation but did not use FFAR4. DHA, EPA, PLA and OA enhanced osteoblast signalling through the FFAR4/βarr2 signalling axis. This study reveals that FFAR4/βarr2 signalling may mediate the bone protective effects of different classes of UFAs in osteoclasts and osteoblasts.