Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Crosstalk Between the Neuroendocrine System and Bone Homeostasis.

The homeostasis of bone microenvironment is the foundation of bone health and comprises 2 concerted events: bone formation by osteoblasts and bone resorption by osteoclasts. In the early 21st century, leptin, an adipocytes-derived hormone, was found to affect bone homeostasis through hypothalamic relay and the sympathetic nervous system, involving neurotransmitters like serotonin and norepinephrine. This discovery has provided a new perspective regarding the synergistic effects of endocrine and nervous systems on skeletal homeostasis. Since then, more studies have been conducted, gradually uncovering the complex neuroendocrine regulation underlying bone homeostasis. Intriguingly, bone is also considered as an endocrine organ that can produce regulatory factors that in turn exert effects on neuroendocrine activities. After decades of exploration into bone regulation mechanisms, separate bioactive factors have been extensively investigated, whereas few studies have systematically shown a global view of bone homeostasis regulation. Therefore, we summarized the previously studied regulatory patterns from the nervous system and endocrine system to bone. This review will provide readers with a panoramic view of the intimate relationship between the neuroendocrine system and bone, compensating for the current understanding of the regulation patterns of bone homeostasis, and probably developing new therapeutic strategies for its related disorders.

A Brief Review of Bone Cell Function and Importance.

This review focuses on understanding the macroscopic and microscopic characteristics of bone tissue and reviews current knowledge of its physiology. It explores how these features intricately collaborate to maintain the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which plays a pivotal role in shaping not only our physical framework but also overall health. In this work, a comprehensive exploration of microscopic and macroscopic features of bone tissue is presented.

Mechanical stretching determines the orientation of osteoblast migration and cell division.

Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts.

Modulating osteoclasts with nanoparticles: A path for osteoporosis management?

Osteoclasts are the cells responsible for the bone resorption process during bone remodeling. In a healthy situation, this process results from an equilibrium between new matrix formation by osteoblast and matrix resorption by osteoclast. Osteoporosis (OP) is a systemic bone disease characterized by a decreased bone mass density and alterations in bone microarchitecture, increasing fracture predisposition. Despite the variety of available therapies for OP management there is a growing gap in its treatment associated to the low patients' adherence owing to concerns related with long-term efficacy or safety. This makes the development of new and safe treatments necessary. Among the newly developed strategies, the use of synthetic and natural nanoparticles to modulate osteoclasts differentiation, activity, apoptosis or crosstalk with osteoblasts have arisen. Synthetic nanoparticles exert their therapeutic effect either by loading antiresorptive drugs or including molecules for osteoclasts gene regulation. Moreover, this control over osteoclasts can be improved by their targeting to bone extracellular matrix or osteoclast membranes. Furthermore, natural nanoparticles, also known as extracellular vesicles, have been identified to play a key role in bone homeostasis. Consequently, these systems have been widely studied to control osteoblasts and osteoclasts under variable environments. Additionally, the ability to bioengineer extracellular vesicles has allowed to obtain biomimetic systems with desirable characteristics as drug carriers for osteoclasts. The analyzed information reveals the possibility of modulating osteoclasts by different mechanisms through nanoparticles decreasing bone resorption. These findings suggest that controlling osteoclast activity using nanoparticles has the potential to improve osteoporosis management. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Cyclopamine targeting hedgehog modulates nuclear control of the osteoblast activity.

It is known that cellular events underlying the processes of bone maintenance, remodeling, and repair have their basis in the embryonic production of bone. Shh signaling is widely described developing important morphogenetic control in bone by modifying the activity of osteoblast. Furthermore, identifying whether it is associated with the modulation of nuclear control is very important to be the basis for further applications. Experimentally, osteoblasts were exposed with cyclopamine (CICLOP) considering up to 1 day and 7 days, here considered an acute and chronic responses respectively. Firstly, we have validated the osteogenic model in vitro by exposing the osteoblasts to classical differentiating solution up to 7 days to allow the analysis of alkaline phosphatase and mineralization. Conversely, our data shows that differentiating osteoblasts present higher activity of inflammasome-related genes, while Shh signaling members were lower, suggesting a negative feedback between them. Thereafter, to better know about the role of Shh signaling on this manner, functional assays using CICLOP (5 µM) were performed and the data validates the previously hypothesis that Shh represses inflammasome related genes activities. Altogether, our data supports the anti-inflammatory effect of Shh signaling by suppressing Tnfα, Tgfß and inflammasome related genes during osteoblast differentiation, and this comprehension might support the understanding the molecular and cellular mechanisms related in bone regeneration by reporting molecular-related osteoblast differentiation.

Mitochondrial ß-oxidation of adipose-derived fatty acids by osteoblasts fuels parathyroid hormone-induced bone formation.

The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone (PTH) treatment, a strategy used to reduce fracture risk, bone formation is preceded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain ß-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regimen. PTH increased the release of fatty acids from adipocytes and ß-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone's anabolic effect. Collectively, these data suggest that PTH's anabolic actions require coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation.

Evidence of the static magnetic field effects on bone-related diseases and bone cells.

Static magnetic fields (SMFs), magnetic fields with constant intensity and orientation, have been extensively studied in the field of bone biology both fundamentally and clinically as a non-invasive physical factor. A large number of animal experiments and clinical studies have shown that SMFs have effective therapeutic effects on bone-related diseases such as non-healing fractures, bone non-union of bone implants, osteoporosis and osteoarthritis. The maintenance of bone health in adults depends on the basic functions of bone cells, such as bone formation by osteoblasts and bone resorption by osteoclasts. Numerous studies have revealed that SMFs can regulate the proliferation, differentiation, and function of bone tissue cells, including bone marrow mesenchymal stem cells (BMSCs), osteoblasts, bone marrow monocytes (BMMs), osteoclasts, and osteocytes. In this paper, the effects of SMFs on bone-related diseases and bone tissue cells are reviewed from both in vivo studies and in vitro studies, and the possible mechanisms are analyzed. In addition, some challenges that need to be further addressed in the research of SMF and bone are also discussed.

How Do Extracellular Vesicles Play a Key Role in the Maintenance of Bone Homeostasis and Regeneration? A Comprehensive Review of Literature.

The maintenance of bone homeostasis includes both bone resorption by osteoclasts and bone formation by osteoblasts. These two processes are in dynamic balance to maintain a constant amount of bone for accomplishing its critical functions in daily life. Multiple cell type communications are involved in these two complex and continuous processes. In recent decades, an increasing number of studies have shown that osteogenic and osteoclastic extracellular vesicles play crucial roles in regulating bone homeostasis through paracrine, autosecretory and endocrine signaling. Elucidating the functional roles of extracellular vesicles in the maintenance of bone homeostasis may contribute to the design of new strategies for bone regeneration. Hence, we review the recent understandings of the classification, production process, extraction methods, structure, contents, functions and applications of extracellular vesicles in bone homeostasis. We highlight the contents of various bone-derived extracellular vesicles and their interactions with different cells in the bone microenvironment during bone homeostasis. We also summarize the recent advances in EV-loaded biomaterial scaffolds for bone regeneration and repair.

Fluid shear stress-induced down-regulation of miR-146a-5p inhibits osteoblast apoptosis via targeting SMAD4.

Fluid shear stress (FSS) plays an important role in osteoblast apoptosis. However, the role of miRNA in osteoblast apoptosis under FSS and possible molecular mechanisms remain unknown. Our aim of the study was to explore whether miR-146a-5p regulates osteoblast apoptosis under FSS and its molecular mechanisms. FSS could down-regulate the expression of miR-146a-5p in MC3T3-E1 cells. We confirm that up-regulation of miR-146a-5p promotes osteoblasts apoptosis and down-regulation of miR-146a-5p inhibits osteoblasts apoptosis. We further demonstrated that FSS inhibits osteoblast apoptosis by down-regulated miR-146a-5p. Dual-luciferase reporter assay validated that SMAD4 is a direct target gene of miR-146a-5p. In addition, mimic-146a-5p suppressed FSS-induced up-regulation of SMAD4 protein levels, which suggests that FSS elevated SMAD4 protein expression levels via regulation miR-146a-5p. Further investigations showed that SMAD4 could inhibit osteoblast apoptosis. We demonstrated that miR-146a-5p regulates osteoblast apoptosis via targeting SMAD4. Taken together, our present study showed that FSS-induced down-regulation miR-146a-5p inhibits osteoblast apoptosis via target SMAD4. These findings may provide novel mechanisms for FSS to inhibit osteoblast apoptosis, and also may provide a potential therapeutic target for osteoporosis.

Aging Relevant Metabolite Itaconate Inhibits Inflammatory Bone Loss.

Progressive bone loss during aging makes osteoporosis one of the most common and life impacting conditions in geriatric populations. The bone homeostasis is maintained through persistent remodeling mediated by bone-forming osteoblast and bone-resorbing osteoclast. Inflammaging, a condition characterized by increased pro-inflammatory markers in the blood and other tissues during aging, has been reported to be associated with skeletal stem/progenitor cell dysfunction, which will result in impaired bone formation. However, the role of age-related inflammation and metabolites in regulation of osteoclast remains largely unknown. In the present study, we observed dichotomous phenotypes of anti-inflammatory metabolite itaconate in responding to inflammaging. Itaconate is upregulated in macrophages during aging but has less reactivity in responding to RANKL stimulation in aged macrophages. We confirmed the inhibitory effect of itaconate in regulating osteoclast differentiation and activation, and further verified the rescue role of itaconate in lipopolysaccharides induced inflammatory bone loss animal model. Our findings revealed that itaconate is a crucial regulatory metabolite during inflammaging that inhibits osteoclast to maintain bone homeostasis.

Titanium alkalinization improves response of osteoblasts to zoledronic acid.

This investigation is aimed to determine the effect of the modification of titanium surface with NaOH on the metabolism of osteoblasts treated with zoledronic acid (ZA). Machined and NaOH-treated titanium disks were used. Surfaces were characterized by scanning electron microscopy, confocal microscopy, and x-ray photoelectron spectroscopy (XPS) analysis. Human osteoblasts were seeded onto the disks. After 24 h, cells were treated with ZA at 5 µM for 7 days. At this point, cell viability, collagen synthesis, total protein production, alkaline phosphatase activity, and mineral nodule deposition were assessed. The results of surface roughness were descriptively and statistically analyzed (t-Student), while the XPS results were qualitatively described. Cell metabolism data were analyzed by the analysis of variance two-way and Tukey tests at a 5% significance level. The results demonstrated that NaOH-treatment increased surface roughness (p < .05) and confirmed the presence of sodium titanate and a pH switch on the NaOH-treated disks. This modification also resulted in higher cell viability, collagen synthesis, total protein production, and alkaline phosphatase by osteoblasts when compared to cells seeded onto machined disks (p < 0.05). In the presence of ZA, all cellular metabolism and differentiation parameters were significantly reduced for cells seeded on both surfaces (p < 0.05); however, the cells seeded onto modified surfaces showed higher values for these parameters, except for mineral nodule deposition (p < 0.05). NaOH modification improved cell adhesion and metabolism of osteogenic cells even in the presence of ZA. The surface modification of titanium with NaOH solution may be an interesting strategy to improve metabolism and differentiation of osteoblasts and accelerate osseointegration process, mainly for tissues exposed to ZA.

Acacia arabica (Lam.) Willd. On osteoblastogenesis, osteoblast proliferation, osteoclastic activity, and bone calcium mineralization.

OBJECTIVES: Since ancient times Acacia arabica (Lam.) Willd. (AA) consumed for the bone and muscle related disorder like the bone fracture, rheumatoid arthritis, and bone loss. To study the effects of the aqueous (AAA) and ethanolic extract (AAE) of AA on osteoblast proliferation and differentiation, osteoclastic activity and bone matrix mineralization using in vitro primary bone-marrow cultures. METHODS: Effect of AAA and AAE was estimated using four in vitro assays. Primary bone marrow cell culture, isolated from rat femur bone, was used for all the assays. Cell growth and viability were assessed by standard colorimetric assays like MTT assay. The differentiation of mesenchymal stem cells into osteoblastic lineage was evaluated by the measuring the levels of the osteoblast-specific marker, alkaline phosphatase. Antiosteoclastic action and matrix mineralization were measured using TRAP assay and Alizarin red-s staining assay, respectively. RESULTS: It indicates that AAA causes more increase in osteoblast differentiation and a reduction in osteoclast activity as compared to AAE. In osteoblast proliferation assay, AAA was found to promote more cell proliferation as compared to AAE. Higher concentrations of AAA significantly increased mineralization of bone-like matrix. CONCLUSIONS: The extracts of AA have a significant positive influence on osteogenesis and they inhibit osteoclastogenesis. Hence, these extracts have the potential to be developed as a therapy for osteoporosis.

The Role of NRF2 in Bone Metabolism - Friend or Foe?

Bone metabolism is closely related to oxidative stress. As one of the core regulatory factors of oxidative stress, NRF2 itself and its regulation of oxidative stress are both involved in bone metabolism. NRF2 plays an important and controversial role in the regulation of bone homeostasis in osteoblasts, osteoclasts and other bone cells. The role of NRF2 in bone is complex and affected by several factors, such as its expression levels, age, sex, the presence of various physiological and pathological conditions, as well as its interaction with certains transcription factors that maintain the normal physiological function of the bone tissue. The properties of NRF2 agonists have protective effects on the survival of osteogenic cells, including osteoblasts, osteocytes and stem cells. Activation of NRF2 directly inhibits osteoclast differentiation by resisting oxidative stress. The effects of NRF2 inhibition and hyperactivation on animal skeleton are still controversial, the majority of the studies suggest that the presence of NRF2 is indispensable for the acquisition and maintenance of bone mass, as well as the protection of bone mass under various stress conditions. More studies show that hyperactivation of NRF2 may cause damage to bone formation, while moderate activation of NRF2 promotes increased bone mass. In addition, the effects of NRF2 on the bone phenotype are characterized by sexual dimorphism. The efficacy of NRF2-activated drugs for bone protection and maintenance has been verified in a large number of in vivo and in vitro studies. Additional research on the role of NRF2 in bone metabolism will provide novel targets for the etiology and treatment of osteoporosis.

Bone mass and adaptation to mechanical loading are sexually dimorphic in adult osteoblast-specific ERα knockout mice.

Estrogen receptor-alpha (ERα) regulates bone mass and is implicated in bone tissue's response to mechanical loading. The effects of ERα deletion in mice depend on sex, anatomical location, and the cellular stage at which ERα is removed. Few studies have investigated the effect of age on the role of ERα in skeletal maintenance and functional adaptation. We previously demonstrated that bone mass and adaptation to loading were altered in growing 10-week-old female and male mice lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO). Here our goal was to determine the effects of ERα and mechanical loading in skeletally-mature adult mice. We subjected 26-week-old skeletally-mature adult pOC-ERαKO and littermate control (LC) mice of both sexes to two weeks of in vivo cyclic tibial loading. ERα deletion in male mice did not alter bone mass or the response to loading. Adult female pOC-ERαKO mice had reduced cancellous and cortical bone mass and increased adaptation to high-magnitude mechanical loading compared to LC mice. Thus, ERα deletion from mature osteoblasts reduced the bone mass and increased the mechanoadaptation of adult female but not male mice. Additionally, compared to our previous work in young mice, adult female mice had greatly reduced mechanoadaptation and adult male mice retained most of their mechanoadaptation with age.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Melatonin enhances osteoblastogenesis of senescent bone marrow stromal cells through NSD2-mediated chromatin remodelling.

BACKGROUND: Aging-associated osteoporosis is frequently seen in the elderly in clinic, but efficient managements are limited because of unclear nosogenesis. The current study aims to investigate the role of melatonin on senescent bone marrow stromal cells (BMSCs) and the underlying regulating mechanism. METHODS: Melatonin levels were tested by ELISA. Gene expression profiles were performed by RNA-sequencing, enrichment of H3K36me2 on gene promoters was analyzed by Chromatin Immunoprecipitation Sequencing (ChIP-seq), and chromatin accessibility was determined by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). Osteogenesis of BMSCs in vitro was measured by Alizarin Red and Alkaline Phosphatase staining, and in vivo effects of melatonin was assessed by histological staining and micro computed tomography (micro-CT) scan. Correlation of NSD2 expression and severity of senile osteoporosis patients were analyzed by Pearson correlation. RESULTS: Melatonin levels were decreased during aging in human bone marrow, accompanied by downregulation of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2) expression in the senescent BMSCs. Melatonin stimulated the expression of NSD2 through MT1/2-mediated signaling pathways, resulting in the rebalancing of H3K36me2 and H3K27me3 modifications to increase chromatin accessibility of the osteogenic genes, runt-related transcription factor 2 (RUNX2) and bone gamma-carboxyglutamate protein (BGLAP). Melatonin promoted osteogenesis of BMSCs in vitro, and alleviates osteoporosis progression in the aging mice. In clinic, severity of senile osteoporosis (SOP) was negatively correlated with melatonin level in bone marrow, as well as NSD2 expression in BMSCs. Similarly, melatonin remarkably enhanced osteogenic differentiation of BMSCs derived from SOP patients in vitro. CONCLUSIONS: Collectively, our study dissects previously unreported mechanistic insights into the epigenetic regulating machinery of melatonin in meliorating osteogenic differentiation of senescent BMSC, and provides evidence for application of melatonin in preventing aging-associated bone loss.

Cytoskeletal Protein 4.1G Is Essential for the Primary Ciliogenesis and Osteoblast Differentiation in Bone Formation.

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.

Thyroid-Stimulating Hormone Favors Runx2-Mediated Matrix Mineralization in HOS and SaOS2 Cells: An In Vitro and In Silico Approach.

Osteoporosis is a skeletal disease that is both systemic and silent characterized by an unbalanced activity of bone remodeling leading to bone loss. Rising evidences demonstrate that thyroid stimulating hormone (TSH) has an important role in the regulation on the metabolism of bone. However, TSH regulation on human osteoblast essential transcriptional factors has not been identified. Current study examined the role of TSH on human osteoblastic Runx2 expression and their functional genes by in vitro and in slico analysis. Human osteoblast like (HOS and SaoS-2) cells were cultured with DMEM and treated with hTSH at the concentration of 0.01 ng/mL and 10 ng/mL. After treatment, osteoblastic Runx2 and IGF-1R beta expression were studied using RT-PCR and western blot analysis. TSH treatment induced osteoblastic essential transcriptional factor, Runx2 in HOS and SaOS2 cells on 48 h duration and elevated the expression of IGF-IR ß gene and Protein in SaoS-2 cells. TSH also promotes Runx2 responsive genes such as ALP, Collagen and osteocalcin in SaOS2 cells on day 2 to day 14 of 10 ng/mL of treatment and favors' matrix mineralization matrix in these cells. In addition, TSH facilitated human osteoblastic cells to mineralize their matrix confirmed by day 21 of alizarin red calcium staining. In silico study was performed to check CREB and ELK1 interaction with Runx2. Results of in silico analysis showed that TSH mediated signalling molecules such as CREB and ELK1 showed interaction with Runx2 which involve in osteobalstic gene expression and differentiation. Present findings confirm that TSH promotes Runx2 expression, osteoblastic responsive genes and bone matrix formation.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.