Sclerostin Expression Research Articles

Unraveling the osteocyte in CKD-MBD post–renal transplantation

Changes in indices of mineral metabolism, bone protein expression, and bone turnover were assessed between pre- and post-renal transplant bone biopsies obtained 12 months apart. Circulating sclerostin and fibroblast growth factor 23 (FGF-23) levels decreased, and a low bone turnover state was highly prevalent on follow-up. In contrast, bone sclerostin expression increased, whereas FGF-23 bone expression was unchanged/decreased. These findings underscore the limitations of circulating biomarkers and the critical role of bone biopsy to understand osteocyte biology in chronic kidney disease-mineral bone disorder.

Effects of sclerostin on lipopolysaccharide-induced inflammatory phenotype in human odontoblasts and dental pulp cells.

Previously we have demonstrated that sclerostin inhibits stress-induced odontogenic differentiation of odontoblasts and accelerates senescence of dental pulp cells (DPCs) Odontoblasts and DPCs are main functioning cells for inflammation resistance and tissue regeneration in dentine-pulp complex. Sclerostin is relevant for systemic inflammation and chronic periodontitis processes, but its effects on dental pulp inflammation remains unclear. In this study, we found that sclerostin expression of odontoblasts was elevated in lipopolysaccharide-induced inflammatory environment, and exogenous sclerostin increased the production of pro-inflammatory cytokines in inflamed odontoblasts. Furthermore, sclerostin activated the NF-κB signaling pathway in inflamed odontoblasts and the NF-κB inhibitor reversed the exaggerative effects of sclerostin on the pro-inflammatory cytokines production. Additionally, sclerostin promoted adhesion and migration of inflamed DPCs, while inhibiting odontoblastic differentiation of inflamed DPCs. Sclerostin also might enhance pulpal angiogenesis. Taken together, it can therefore be inferred that sclerostin is upregulated in inflamed odontoblasts under pulpal inflammatory condition to enhance inflammatory responses in dentine-pulp complex and impair reparative dentinogenesis. This indicates that sclerostin inhibition might be a therapeutic target for anti-inflammation and pro-regeneration during dental pulp inflammation.

Experimental periodontitis in Msx2 mutant mice induces alveolar bone necrosis.

Msx2 homeoprotein is a key transcription factor of dental and periodontal tissue formation and is involved in many molecular pathways controlling mineralized tissue homeostasis such as Wnt/sclerostin pathway. This study evaluated the effect of Msx2-null mutation during experimental periodontitis in mice. Experimental periodontitis was induced for 30 days in wild-type and Msx2 knock-in Swiss mice using Porphyromonas gingivalis infected ligatures. In knock-in mice, Msx2 gene was replaced by n-LacZ gene encoding β-galactosidase. Periodontal tissue response was assessed by histomorphometry, tartrate-resistant acid phosphatase histoenzymology, β-galactosidase, sclerostin immunochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling assay. Expression of Msx2 gene expression was also evaluated in human gingival biopsies using RT-qPCR. During experimental periodontitis, osteonecrosis area and osteoclast number were significantly elevated in knock-in mice compared with wild-type mice. Epithelial downgrowth and bone loss was similar. Sclerostin expression in osteocytes appeared to be reduced during periodontitis in knock-in mice. Msx2 expression was detected in healthy and inflamed human gingival tissues. These data indicated that Msx2 pathway influenced periodontal tissue response to experimental periodontitis and appeared to be a protective factor against alveolar bone osteonecrosis. As shown in other inflammatory processes such as atherothrombosis, genes initially characterized in early development could also play an important role in human periodontal pathogenesis.

Pathophysiological characteristics and gene transcriptional profiling of bone microstructure in a low calcium diet fed laying hens

Calcium depletion is a valuable non-invasive tool for studying skeletal system disorders. A low calcium diet (LCD) was used to examine the pathophysiological characteristics and molecular mechanisms of osteoporotic damage in caged laying hens. Sixty 64-wk-old laying hens were randomly housed in single-bird cages, and the cages were divided into 2 treatments: fed a regular calcium diet (RCD, 3.69%) or a LCD (1.56%) for 8 wk. The diet-induced changes of serum bone remodeling indicators, bone strength, microstructure of the distal femur, and the gene expression profiling of keel bone were measured. Compared to RCD hens, LCD hens had higher activity of serum alkaline phosphatase and tartrate resistant acid phosphatase but lower serum calcium concentrations with reduced tibial and femoral mass, width, and strength (P < 0.05). In addition, LCD hens had greater densities of osteoclasts, osteoblasts, connective tissue cells, and osteoid in the trabecular bone (P < 0.05). The transcriptome analysis revealed that 563 unigenes were differentially expressed in keel bone between LCD and RCD hens. The Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that these differentially expressed genes were mainly associated with the osteoporosis-related signaling pathways involved in the biological functions of the bone cellular and extracellular structural modulations. The real-time PCR analysis further confirmed that the LCD enhanced the mRNA expression of collagen type 1 alpha 2, integrin-binding sialoprotein and periostin, but inhibited sclerostin expression. These findings indicate that LCD hens have a higher bone turnover and micro-architectural damage compared to RCD hens. The results further evidence dietary supplement of calcium is a critical nutrient strategy for improving hen skeletal health.

Periostin-modified bone marrow mesenchymal stem cells from osteoporotic rats promote alveolar bone regeneration.

Bone regeneration is impaired in patients with osteoporosis. Previous studies have shown that periostin (Postn) shows great potential in bone regeneration treatments. However, the role of Postn in bone marrow mesenchymal stem cells (BMMSCs) remains to be elucidated. In this study, we isolated BMMSCs from ovariectomized rats (OVX-BMMSCs) and normal rats. Then, the expression levels of Postn and osteogenesis in OVX-BMMSCs were detected by alizarin red and alkaline phosphatase substrate staining, qPCR, and western blotting. We found that the levels of Postn in OVX-BMMSCs were significantly reduced. Furthermore, Postn overexpression in OVX-BMMSCs using recombinant lentivirus could improve the expression of alkaline phosphatase, runt-related transcription factor 2, and osteocalcin and reduce the expression of sclerostin. Besides, micro-computed tomography analysis, hematoxylin-eosin, and Masson's staining showed that the healing of the alveolar bone defect in osteoporotic rats could be promoted using Postn-modified OVX-BMMSC sheets. In conclusion, Postn-modified OVX-BMMSCs might restore the osteogenic capacity and promote alveolar bone regeneration, which may serve as a new therapeutic approach for bone regeneration in osteoporosis.

Hypoxia, hypoxia-inducible transcription factors and oxygen-sensing prolyl hydroxylases in bone development and homeostasis.

To summarize the role of hypoxia signaling in skeletal cells. Hypoxia occurs at several stages during bone development. Skeletal cells, like chondrocytes and osteoblasts, respond to this challenge by stabilizing the hypoxia inducible transcription factor HIF, which induces the expression of angiogenic factors and promotes glycolysis. The increased delivery of oxygen and nutrients, together with metabolic adaptations, prevent chondrocyte cell death in the growth plate and promote bone formation by osteoblasts. However, excessive HIF levels have to be avoided during bone development as the resulting metabolic maladaptations cause skeletal dysplasia. Recent studies show that HIF also targets other genes to increase bone mass: it decreases osteoclastogenesis by increasing osteoprotegerin expression and represses sclerostin expression by epigenetic mechanisms, resulting in increased bone formation and decreased resorption. Moreover, increased HIF signaling in osteolineage cells promotes primary and metastatic breast tumor growth, and induces erythropoietin (EPO) production, resulting in polycythemia. Finally, HIF can directly or indirectly through increasing EPO levels, induce the expression and processing of FGF23 and may thereby affect mineral homeostasis and vitamin D metabolism. HIF signaling in skeletal cells not only affects their behavior but also influences erythropoiesis and possibly mineral homeostasis.

Effect of TNF-α-Induced Sclerostin on Osteocytes during Orthodontic Tooth Movement.

Osteocytes are abundant cells in bone, which contribute to bone maintenance. Osteocytes express receptor activator of nuclear factor kappa-B ligand (RANKL) and regulate osteoclast formation. Orthodontic tooth movement (OTM) occurs by osteoclast resorption of alveolar bone. Osteocyte-derived RANKL is critical in bone resorption during OTM. Additionally, tumor necrosis factor-α (TNF-α) is important in osteoclastogenesis during OTM. Sclerostin has been reported to enhance RANKL expression in the MLO-Y4 osteocyte-like cell line. This study investigated the effect of TNF-α on sclerostin expression in osteocytes during OTM. In vitro analysis of primary osteocytes, which were isolated from DMP1-Topaz mice by sorting the Topaz variant of GFP-positive cells, revealed that SOST mRNA expression was increased when osteocytes were cultured with TNF-α and that RANKL mRNA expression was increased when osteocytes were cultured with sclerostin. Moreover, the number of TRAP-positive cells was increased in osteocytes and osteoclast precursors cocultured with sclerostin. In vivo analysis of mouse calvariae that had been subcutaneously injected with phosphate-buffered saline (PBS) or TNF-α revealed that the number of TRAP-positive cells and the percentage of sclerostin-positive osteocytes were higher in the TNF-α group than in the PBS group. Furthermore, the level of SOST mRNA was increased by TNF-α. As an OTM model, a Ni-Ti closed-coil spring connecting the upper incisors and upper-left first molar was placed to move the first molar to the mesial direction in wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFRsKO) mice. After 6 days of OTM, the percentage of sclerostin-positive osteocytes on the compression side of the first molar in TNFRsKO mice was lower than that in WT mice. In this study, TNF-α increased sclerostin expression in osteocytes, and sclerostin enhanced RANKL expression in osteocytes. Thus, TNF-α may play an important role in sclerostin expression in osteocytes and enhance osteoclast formation during OTM.

Mechanisms of palmitate-induced lipotoxicity in osteocytes.

Lipotoxicity is defined as cellular toxicity observed in the presence of an abnormal accumulation of fat and adipocyte-derived factors in non-fat tissues. Palmitic acid (PA), an abundant fatty acid in the bone marrow and particularly in osteoporotic bones, affects osteoblastogenesis and osteoblast function, decreasing their survival through induction of apoptosis and dysfunctional autophagy. In this study, we hypothesized that PA also has a lipotoxic effect on osteocytes in vitro. Initially, we tested the effect of PA on osteocyte-derived factors DKK1, sclerostin and RANKL. Then, we tested whether PA affects survival and causes apoptosis in osteocytes. Subsequently, we investigated the effect of PA on autophagy by detecting the membrane component LC3-II (Western blot) and staining it and lysosomes with Lysotracker Red dye. PA decreases RANKL, DKK1 and sclerostin expression in osteocytes. In addition, we found that PA induces apoptosis and reduces osteocyte survival. PA also caused autophagy failure identified by a significant increase in LC3-II and a reduced number of autophagosomes/lysosomes in the cytoplasm. In addition to the effects of PA on RANKL, DKK1 and sclerostin expression, which could have significant deleterious impact on bone cell coupling and bone turnover, PA also induced apoptosis and reduced autophagy in osteocytes. Considering that apoptosis and cell dysfunction are two common changes occurring in the osteocytes of osteoporotic bone, our findings suggest that PA could play a role in the pathogenesis of the disease. Suppression of these effects could bring new potential targets for therapeutic interventions in the future.

Regulation of sclerostin in glucocorticoid-induced osteoporosis (GIO) in mice and humans.

Glucocorticoids (GC) are used for the treatment of inflammatory diseases, including various forms of arthritis. However, their use is limited, amongst others, by adverse effects on bone. The Wnt and bone formation inhibitor sclerostin was recently implicated in the pathogenesis of GC-induced osteoporosis. However, data are ambiguous. The aim of this study was to assess the regulation of sclerostin by GC using several mouse models with high GC levels and two independent cohorts of patients treated with GC. Male 24-week-old C57BL/6 and 18-week-old DBA/1 mice exposed to GC and 12-week-old mice with endogenous hypercortisolism displayed reduced bone formation as indicated by reduced levels of P1NP and increased serum sclerostin levels. The expression of sclerostin in femoral bone tissue and GC-treated bone marrow stromal cells, however, was not consistently altered. In contrast, GC dose- and time-dependently suppressed sclerostin at mRNA and protein levels in human mesenchymal stromal cells, and this effect was GC receptor dependent. In line with the human cell culture data, patients with rheumatoid arthritis (RA, n = 101) and polymyalgia rheumatica (PMR, n = 21) who were exposed to GC had lower serum levels of sclerostin than healthy age- and sex-matched controls (−40%, P < 0.01 and −26.5%, P < 0.001, respectively). In summary, sclerostin appears to be differentially regulated by GC in mice and humans as it is suppressed by GCs in humans but is not consistently altered in mice. Further studies are required to delineate the differences between GC regulation of sclerostin in mice and humans and assess whether sclerostin mediates GC-induced osteoporosis in humans.

Mendelian Randomization Analysis Reveals a Causal Influence of Circulating Sclerostin Levels on Bone Mineral Density and Fractures.

ABSTRACTIn bone, sclerostin is mainly osteocyte‐derived and plays an important local role in adaptive responses to mechanical loading. Whether circulating levels of sclerostin also play a functional role is currently unclear, which we aimed to examine by two‐sample Mendelian randomization (MR). A genetic instrument for circulating sclerostin, derived from a genomewide association study (GWAS) meta‐analysis of serum sclerostin in 10,584 European‐descent individuals, was examined in relation to femoral neck bone mineral density (BMD; n = 32,744) in GEFOS and estimated bone mineral density (eBMD) by heel ultrasound (n = 426,824) and fracture risk (n = 426,795) in UK Biobank. Our GWAS identified two novel serum sclerostin loci, B4GALNT3 (standard deviation [SD]) change in sclerostin per A allele (β = 0.20, p = 4.6 × 10−49) and GALNT1 (β = 0.11 per G allele, p = 4.4 × 10−11). B4GALNT3 is an N‐acetyl‐galactosaminyltransferase, adding a terminal LacdiNAc disaccharide to target glycocoproteins, found to be predominantly expressed in kidney, whereas GALNT1 is an enzyme causing mucin‐type O‐linked glycosylation. Using these two single‐nucleotide polymorphisms (SNPs) as genetic instruments, MR revealed an inverse causal relationship between serum sclerostin and femoral neck BMD (β = –0.12, 95% confidence interval [CI] –0.20 to –0.05) and eBMD (β = –0.12, 95% CI –0.14 to –0.10), and a positive relationship with fracture risk (β = 0.11, 95% CI 0.01 to 0.21). Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower eBMD, and greater B4GALNT3 expression in arterial tissue (probability >99%). Our findings suggest that higher sclerostin levels are causally related to lower BMD and greater fracture risk. Hence, strategies for reducing circulating sclerostin, for example by targeting glycosylation enzymes as suggested by our GWAS results, may prove valuable in treating osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc.

1983-P: WNT Pathway and Bone Fragility in Postmenopausal Women with Type 2 Diabetes

Fragility fractures are major complications in type 2 diabetes (T2D), but mechanisms underlying bone fragility are still poorly clarified. Bone mineral density (BMD) underestimates T2D fracture incidence, since a normal or even higher BMD is associated with a greater fracture risk than healthy population. We hypothesized that bone fragility in T2D results from poor bone microarchitecture and bone quality caused by WNT signaling impairment and lower bone formation. We aimed to evaluate the association between bone gene expression of WNT pathway and features of bone microarchitecture and strength. We enrolled T2D (n=13) and nondiabetic (controls, n=75) postmenopausal women undergoing standard hip arthroplasty. Serum and proximal femur samples from recruited subjects were collected at the moment of surgical procedure. Gene expression of sclerostin (SOST), WNT5a, WNT10b, SFRP5, GSK3β, osteocalcin and RUNX2 was analyzed by real-time PCR. Total-body, lumbar and femoral BMD were assessed by bone densitometry. A trabecular core from bone samples was analyzed by Micro X-ray computed tomography and by compression tests. Age (75.23 ± 8.47 vs. 73.24 ± 5.77) and BMI (29.92 ± 5.38 vs. 27.68 ± 5.62 Kg/m2) did not differ between T2D and controls respectively. Glycated hemoglobin was 6.48 ± 1.7 mmol/mol. We found that T2D subjects had no differences in BMD and in bone microarchitecture parameters compared to controls. Neither bone turnover nor WNT serum markers differed among groups. SOST gene expression was increased (p=0.003) whereas RUNX2 expression was decreased in T2D vs. controls (p=0.017), independently by BMI and age. Osteocalcin and other WNT related genes expression did not differ among groups. Compression tests did not show impairment in bone strength in T2D vs. controls. In conclusion, although a good glycemic control and no alterations in bone density, microarchitecture and strength, we found an increase in SOST and a decrease in RUNX2 gene expression, implying a lower bone formation in T2D. Disclosure A. Piccoli: None. F. Cannata: None. F. Russo: None. V.L. Greto: None. C. Isgrò: None. G. Leanza: None. R. Strollo: None. C. Massaroni: None. C. Pedone: None. S. Silvestri: None. G. Vadalà: None. V. Denaro: None. T. Bisogno: None. R. Papalia: None. P. Pozzilli: None. M. Maccarrone: None. N. Napoli: None. Funding Campus Bio-Medico University

Rapamycin attenuates articular cartilage degeneration by inhibiting β-catenin in a murine model of osteoarthritis

ABSTRACTPurpose: To investigate whether systemic injection of rapamycin attenuates articular cartilage degeneration by inhibiting β-catenin in a murine model of osteoarthritis (OA).Materials and methods: Ten-week-old male C57BL/6j wild-type (WT) mice and SOST-knockout (SOST−/−) mice were randomized to a sham group, a vehicle-treated group, and a rapamycin-treated group. Mice in the vehicle-treated group underwent destabilizing of the medial meniscus (DMM) in the right knee, and were then treated with vehicle. Mice in the rapamycin treatment group underwent DMM and were treated with rapamycin. Safranin O-Fast green staining and Osteoarthritis Research Society International (OARSI) modified Mankin score were used to evaluate the histopathological features of the articular cartilage in the knee. The expression of light chain 3 (LC3) was evaluated by immunofluorescence, whereas the expression of ATG5, matrix metallopeptidase 13 (MMP-13), vascular endothelial growth factor (VEGF), sclerostin, and β-catenin were evaluated by immunohistochemistry. TUNEL staining was used to determine apoptosis of chondrocytes.Results: In vehicle-treated mice when compared with mice in the sham group, the OARSI scores, expression of MMP-13, VEGF, sclerostin, β-catenin, and chondrocyte apoptosis were significantly increased, whereas the expression of LC3 and ATG5 were significantly decreased. A systemic injection of rapamycin activated chondrocyte autophagy, which increased the expression of LC3 and ATG-5, and reduced OARSI scores, the expression of β-catenin, MMP-13, and VEGF, and chondrocyte apoptosis in rapamycin treated mice when compared with vehicle-treated mice.Conclusions: Systemic injection of rapamycin attenuated articular cartilage degeneration by inhibiting β-catenin in a murine model of OA.

FGF-2 targets sclerostin in bone and myostatin in skeletal muscle to mitigate the deleterious effects of glucocorticoid on musculoskeletal degradation

AimMyokines are associated with regulation of bone and muscle mass. However, limited information is available regarding the impact of myokines on glucocorticoid (GC) mediated adverse effects on the musculoskeletal system. This study investigates the role of myokine fibroblast growth factor-2 (FGF-2) in regulating GC-induced deleterious effects on bone and skeletal muscle. MethodsPrimary osteoblast cells and C2C12 myoblast cell line were treated with FGF-2 and then exposed to dexamethasone (GC). FGF-2 mediated attenuation of the inhibitory effect of GC on osteoblast and myoblast differentiation and muscle atrophy was assessed through quantitative PCR and western blot analysis. Further, FGF-2 was administered subcutaneously to dexamethasone treated mice to collect bone and skeletal muscle tissue for in vivo analysis of bone microarchitecture, mechanical strength, histomorphometry and for histological alterations in treated tissue samples. Key findingsFGF-2 abrogated the dexamethasone induced inhibitory effect on osteoblast differentiation by modulating BMP-2 pathway and inhibiting Wnt antagonist sclerostin. Further, dexamethasone induced atrophy in C2C12 cells was mitigated by FGF-2 as evident from down regulation of atrogenes expression. FGF-2 prevented GC-induced impairment of mineral density, biomechanical strength, trabecular bone volume, cortical thickness and bone formation rate in mice. Additionally, skeletal muscle tissue from GC treated mice displayed weak myostatin immunostaining and reduced expression of atrogenes following FGF-2 treatment. SignificanceFGF-2 mitigated GC induced effects through inhibition of sclerostin and myostatin expression in bone and muscle respectively. Taken together, this study exhibited the role of exogenous FGF-2 in sustaining osteoblastogenesis and inhibiting muscle atrophy in presence of glucocorticoid.

Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Nuclear protein 1 (NUPR1) is a multifunctional stress-induced protein involved in regulating tumorigenesis, apoptosis, and autophagy. Bone homeostasis is maintained by bone-resorbing osteoclasts and bone-forming osteoblasts and osteocytes. We aimed to determine the role of NUPR1 in bone metabolism. Using microcomputed tomography, we found that mice lacking Nupr1 exhibited increased bone volume. Histologic analysis showed that Nupr1 deficiency decreased osteoclast numbers but increased osteoblast numbers and osteoid formation. In vitro culture of bone marrow macrophages showed that receptor activator of NF-κB ligand-induced osteoclastogenesis was down-regulated in Nupr1-deficient mice. In contrast, primary osteoblasts from Nupr1-deficient mice revealed that proliferation of osteoblasts and expression of bone matrix proteins were markedly enhanced. In addition, expression of autophagy-related genes, formation of autophagosomes, and cell survival were up-regulated in Nupr1-deficient osteoblasts. In contract, deletion of Nupr1 reduced the formation of osteocyte cellular projection, which is an indicator of mature osteocytes. Importantly, we found that the expression of sclerostin (Sost), an inhibitor of bone formation, was down-regulated in the osteoblasts and osteocytes of Nupr1-deficient mice. Conversely, Nupr1 overexpression enhanced Sost expression in primary osteoblasts. Collectively, these results indicate that Nupr1 deficiency increases bone volume by attenuating production of Sost and osteoclastogenesis and enhancing differentiation of osteoblasts.-Shiraki, M., Xu, X., Iovanna, J. L., Kukita, T., Hirata, H., Kamohara, A., Kubota, Y., Miyamoto, H., Mawatari, M., Kukita, A. Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

Breast cancer bone metastases often cause a debilitating non-curable condition with osteolytic lesions, muscle weakness and a high mortality. Current treatment comprises chemotherapy, irradiation, surgery and anti-resorptive drugs that restrict but do not revert bone destruction. In metastatic breast cancer cells, we determined the expression of sclerostin, a soluble Wnt inhibitor that represses osteoblast differentiation and bone formation. In mice with breast cancer bone metastases, pharmacological inhibition of sclerostin using an anti-sclerostin antibody (Scl-Ab) reduced metastases without tumor cell dissemination to other distant sites. Sclerostin inhibition prevented the cancer-induced bone destruction by augmenting osteoblast-mediated bone formation and reducing osteoclast-dependent bone resorption. During advanced disease, NF-κB and p38 signaling was increased in muscles in a TGF-β1-dependent manner, causing muscle fiber atrophy, muscle weakness and tissue regeneration with an increase in Pax7-positive satellite cells. Scl-Ab treatment restored NF-κB and p38 signaling, the abundance of Pax7-positive cells and ultimately muscle function. These effects improved the overall health condition and expanded the life span of cancer-bearing mice. Together, these results demonstrate that pharmacological inhibition of sclerostin reduces bone metastatic burden and muscle weakness with a prolongation of the survival time. This might provide novel options for treating musculoskeletal complications in breast cancer patients. .

Effect of Collagen Cross-Link Deficiency on Incorporation of Grafted Bone.

Bone matrix collagen, is one of the major contributors to bone quality. No studies have examined how bone quality affects the results of bone transplantation. Collagen cross-links (CCL) are the key factor in collagen properties. The purpose was to investigate the influences of CCL for both grafted bone and recipient site bone on the success of bone augmentation. Four-week-old male Wister rats (n = 54) were divided into control and test groups. Control and test groups equally sub-divided into donors and recipients. An additional six rats were used to characterize bone at day zero. Test groups received 0.2% beta-aminoproperionitrile (BAPN) for 4 weeks as CCL inhibitor. Animals were further divided into donor and recipient groups. The transplanted bone chips integrated with host bone by 25% more in CCL-deficient animals compared to control. However, no difference in cortical thickness among all conditions. CCL-deficient transplanted bone did not show any extra signs of osteocyte apoptosis, while sclerostin expression was comparable to that in control. The host periosteum of CCL-deficient animals showed higher cellular activity, as well as higher bone quantity and osteoclast activity. Collagen cross-links deficiency in host bone might accelerate the incorporation of grafted bone. effect. Incorporation of the bone grafts appears to depend mainly on host condition rather than graft condition.

Osteocyte TSC1 promotes sclerostin secretion to restrain osteogenesis in mice.

Osteocytes secrete the glycoprotein sclerostin to inhibit bone formation by osteoblasts, but how sclerostin production is regulated in osteocytes remains unclear. Here, we show that tuberous sclerosis complex 1 (TSC1) in osteocytes promotes sclerostin secretion through inhibition of mechanistic target of rapamycin complex 1 (mTORC1) and downregulation of Sirt1. We generated mice with DMP1-Cre-directed Tsc1 gene deletion (Tsc1 CKO) to constitutively activate mTORC1 in osteocytes. Although osteocyte TSC1 disruption increased RANKL expression and osteoclast formation, it markedly reduced sclerostin production in bone, resulting in severe osteosclerosis with enhanced bone formation in mice. Knockdown of TSC1 activated mTORC1 and decreased sclerostin, while rapamycin inhibited mTORC1 and increased sclerostin mRNA and protein expression levels in MLO-Y4 osteocyte-like cells. Furthermore, mechanical loading activated mTORC1 and prevented sclerostin expression in osteocytes. Mechanistically, TSC1 promotes sclerostin production and prevents osteogenesis through inhibition of mTORC1 and downregulation of Sirt1, a repressor of the sclerostin gene Sost. Our findings reveal a role of TSC1/mTORC1 signalling in the regulation of osteocyte sclerostin secretion and bone formation in response to mechanical loading in vitro. Targeting TSC1 represents a potential strategy to increase osteogenesis and prevent bone loss-related diseases.

Sclerostin inhibits odontogenic differentiation of human pulp-derived odontoblast-like cells under mechanical stress.

Sclerotic dentin is a natural self-protective barrier beneath non-carious cervical lesions (NCCLs), which are mainly induced by mechanical stress. Sclerostin is a mechanosensory protein and serves as an inhibitor of dentinogenesis. However, its function on mechanotransduction in dentine-pulp complex has not been elucidated yet. In this study, decreased sclerostin expression was detected in odontoblasts beneath NCCL-affected sclerotic dentin. Then human pulp-derived odontoblast-like cells (hOBs) were subjected to mechanical strain (MS) in vitro: the results showed that MS-induced upregulation of odontogenic differentiation markers (dentin sialophosphoprotein, osteopontin, osteocalcin, and runt-related transcription factor 2) in hOBs with downregulated sclerostin expression, and this inductive differentiation was attenuated when sclerostin was overexpressed. Additionally, MS activated ERK1/2 pathway and ERK1/2 inhibition restored MS-induced downregulation of sclerostin. Proteasome inhibitor MG132 could also rescue MS-induced decrease of sclerostin. Furthermore, MS suppressed STAT3 pathway, which could be reversed by sclerostin overexpression. STAT3 inhibition was shown to ameliorate the reduction of odontogenic markers induced by sclerostin overexpression. Taken together, we conclude that MS downregulates sclerostin expression via the ERK1/2 and proteasome signaling pathways to promote odontogenic differentiation of hOBs through the STAT3 signaling pathway. It can therefore be inferred that under mechanical stress, sclerostin inhibition promotes reactive dentin formation by enhancing odontogenic differentiation of odontoblasts, which might be one of potential forming mechanisms of sclerotic dentin beneath NCCLs.

Blueberry juice protects osteocytes and bone precursor cells against oxidative stress partly through SIRT1

Oxidative stress and abnormal osteocyte apoptosis are often related to dysregulation of bone turnover and chronic bone loss, and so fruit and vegetables with high antioxidant potential may play an important role in the prevention and/or management of osteoporosis. Osteocytes are the main regulators of bone remodelling. For the first time, we demonstrate here that blueberry juice (BJ), obtained from Vaccinium myrtillus, rich in polyphenols, shows antioxidant and antiosteoclastogenic properties in MLO‐Y4 osteocytes. We report that BJ prevents oxidative stress‐induced apoptosis and reverses the increase in receptor activator of nuclear factor κB ligand and sclerostin expression, crucial factors for osteoclast activation and bone resorption. BJ is also able to prevent oxidative stress‐induced cell cytotoxicity in bone marrow mesenchymal stromal cells (MSCs), which are considered to be an important tool for cell therapy in bone disorders. No significant difference in preventing these events was observed between BJ and blueberry dry extract containing equal amounts of total soluble polyphenols. We have also shown that blueberry acts as both an antioxidant and an activator of sirtuin type 1, a class III histone deacetylase involved in cell death regulation and considered a molecular target for blocking bone resorption without affecting osteoclast survival. Overall, these novel data obtained in osteocytes and MSCs may help us clarify the mechanisms by which blueberry counteracts oxidative stress‐induced damage in bone remodelling and osteogenesis at the cellular and molecular level. Our findings are consistent with the reported beneficial effects of blueberry on bone tissue reported in animal studies, which suggest that blueberry may be a useful supplement for the prevention and/or management of osteoporosis and osteogenic process.

Increased Osteoblastic Activity Suppressed Proliferation of Multiple Myeloma Plasma Cells

In vitro experimental study. To investigate the impact of increased osteoblastic activity on the proliferation and survival of multiple myeloma (MM) plasma cells in vitro SUMMARY OF BACKGROUND DATA.: MM is one of representative hematologic malignancies that cause skeletal-related events (SREs) and dysregulation of bone remodeling is known as a key pathomechanism of disease progression and skeletal-related events. However, decreased proliferation of MM at fracture sites is frequently noted in clinical situations regardless of systemic disease activity. Co-culture under various conditions was used to investigate effects of increased osteoblastic activity on survival and proliferation of MM plasma cells. MM plasma cells were cultured in culture media (control) and co-cultured with human mesenchymal stem cells (hMSCs, group I), osteoblasts (OBs) induced from hMSCs (group II) or bone morphogenic protein-2 (BMP-2, group III). Proliferation measured as extracellular signal-regulated kinase (ERK) and immunoglobulin G (Ig G) expression and apoptosis measured as fluorescence-activated cell sorting (FACS) with annexin V method, caspase-3, and stat-3 expression were assessed for cultured MM plasma cells, along with expression of sclerostin. After 72 hours of co-culture, group II and III showed decreased ERK expression compared with controls. Lower Ig G expression was also noted for groups II and III compared with controls. Group I did not show significantly decreased Ig G and ERK expression compared with controls. Expressions of caspase-3 in groups II and III were higher than controls. Co-culture with hMSCs showed decreased caspase-3 expression compared with control. FACS with annexin V showed higher apoptosis in groups II and III. Sclerostin expression was also decreased in osteoblastic conditions compared with the control and hMSCs co-culture condition. Collectively, our data suggest that increased osteoblastic conditions may provide not only prevention of SREs but also anti-tumor effects on MM cells in the bone marrow environment. N/A.