Regulates Osteoblast Proliferation Research Articles

Cu/Gd co-doped hydroxyapatite/poly lactic-co-glycolic acid composites enhance MRI imaging and bone defect regeneration.

Background: The hydroxyapatite (HA)/poly(lactide-co-glycolide) acid (PLGA) composite material is a widely used orthopedic implant due to its excellent biocompatibility and plasticity. Recent advancements in cation doping have expanded its potential biological applications. However, conventional HA/PLGA composites are not visible under X-rays post-implantation and have limited osteogenic induction capabilities. Copper (Cu) is known to regulate osteoblast proliferation and differentiation, while gadolinium (Gd) can significantly enhance the magnetic resonance imaging (MRI) capabilities of materials. Methods: This study aimed to investigate whether incorporating Cu and Gd into an HA/PLGA composite could enhance the osteogenic properties, in vivo bone defect repair, and MRI characteristics. We prepared a Cu/Gd@HA/PLGA composite and assessed its performance. Results: Material characterization confirmed that Cu/Gd@HA retained the morphology and crystal structure of HA. The Cu/Gd@HA/PLGA composite exhibited excellent nuclear magnetic imaging capabilities, porosity, and hydrophilicity, which are conducive to cell adhesion and implant detection. In vitro experiments demonstrated that the Cu/Gd@HA/PLGA composite enhanced the proliferation, differentiation, and adhesion of MC3T3-E1 cells, and upregulated COL-1 and BMP-2 expression at both gene and protein levels. In vivo studies showed that the Cu/Gd@HA/PLGA composite maintained strong T1-weighted MRI signals and significantly improved the bone defect healing rate in rats. Conclusion: These findings indicate that the Cu/Gd@HA/PLGA composites significantly enhance T1-weighted MRI capabilities, promote osteoblast proliferation and differentiation in vitro, and accelerate bone defect healing in vivo.

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Proliferation and differential regulation of osteoblasts cultured on surface-phosphorylated cellulose nanofiber scaffolds

Phosphorus-containing polymers have received much attention for their excellent ability to regulate bone cell differentiation and calcification. Given the increasing concern about environmental issues, it is promising to utilize “green” biomaterials to construct novel cell culture scaffolds for bone tissue engineering. Herein, surface-phosphorylated cellulose nanofibers (P-CNFs) were fabricated as a novel green candidate for osteoblast culture. Compared with native CNF, P-CNFs possessed shorter fiber morphology with tunable phosphate group content (0–1.42 mmol/g). The zeta-potential values of CNFs were enhanced after phosphorylation, resulting in the formation of uniform and stable scaffolds. The cell culture behavior of mouse osteoblast (MC3T3-E1) cells showed a clear phosphate content-dependent cell proliferation. The osteoblast cells adhered well and proliferated efficiently on P-CNF0.78 and P-CNF1.05, with phosphate contents of 0.78 and 1.05 mmol/g, respectively, whereas the cells grown on native CNF substrate formed aggregates due to poor cell attachment and exhibited limited cell proliferation. In addition, the P-CNF substrates with optimal phosphate content provided a favorable cellular microenvironment and significantly promoted osteogenic differentiation and calcification, even in the absence of a differentiation inducer. The bio-based P-CNFs are expected to mimic the bone components and provide a means to regulate osteoblast proliferation and differentiation in bone tissue engineering.

Comprehensive ceRNA network for MACF1 regulates osteoblast proliferation

BackgroundPrevious studies have shown that microtubule actin crosslinking factor 1 (MACF1) can regulate osteoblast proliferation and differentiation through non-coding RNA (ncRNA) in bone-forming osteoblasts. However, the role of MACF1 in targeting the competing endogenous RNA (ceRNA) network to regulate osteoblast differentiation remains poorly understood. Here, we profiled messenger RNA (mRNA), microRNA (miRNA), and long ncRNA (lncRNA) expression in MACF1 knockdown MC3TC‑E1 pre‑osteoblast cells.ResultsIn total, 547 lncRNAs, 107 miRNAs, and 376 mRNAs were differentially expressed. Significantly altered lncRNAs, miRNAs, and mRNAs were primarily found on chromosome 2. A lncRNA-miRNA-mRNA network was constructed using a bioinformatics computational approach. The network indicated that mir-7063 and mir-7646 were the most potent ncRNA regulators and mef2c was the most potent target gene. Pathway enrichment analysis showed that the fluid shear stress and atherosclerosis, p53 signaling, and focal adhesion pathways were highly enriched and contributed to osteoblast proliferation. Importantly, the fluid shear stress and atherosclerosis pathway was co-regulated by lncRNAs and miRNAs. In this pathway, Dusp1 was regulated by AK079370, while Arhgef2 was regulated by mir-5101. Furthermore, Map3k5 was regulated by AK154638 and mir-466q simultaneously. AK003142 and mir-3082-5p as well as Ak141402 and mir-446 m-3p were identified as interacting pairs that regulate target genes.ConclusionThis study revealed the global expression profile of ceRNAs involved in the differentiation of MC3TC‑E1 osteoblasts induced by MACF1 deletion. These results indicate that loss of MACF1 activates a comprehensive ceRNA network to regulate osteoblast proliferation.

Fabrication of piezoelectric Ca-P-Si-doped PVDF scaffold by phase-separation-hydration: Material characterization, in vitro biocompatibility and osteoblast redifferentiation

Native bone is piezoelectric in nature and generates abundant surface charges under mechanical compression, which regulate osteoblast proliferation, differentiation, adhesion, and so on. Poly (vinylidene fluoride) (PVDF) is becoming one of the most popular piezoelectric polymers because of its easy processability and good biocompatibility. Unfortunately, because only the β and γ crystal phases of PVDF have piezoelectricity, post-treatments, for example, polarizing at high temperature, are required to enhance the piezoelectricity of PVDF scaffolds after fabrication. In this study, we reported a phase-separation-hydration method to fabricate a calcium phosphate silicate (CPS)-doped PVDF scaffold. Our method fabricated a better piezoelectric scaffold than native bone without further processing (∼ 3 pC/N vs. 0.7 pC/N). In addition, the scaffold was mechanically compatible (∼ 7 MPa) with the cancellous bone with sufficient porosity (∼ 45%) to facilitate osteoblast infiltration and bone ingrowth. The in vitro biocompatibility analyses proved that the prepared CPS-PVDF scaffold was biocompatible with osteoblast cells and encouraged osteoblast redifferentiation. In conclusion, our results suggest that this CPS-PVDF scaffold is a promising candidate for bone tissue engineering applications.

Fluid shear stress regulates osteoblast proliferation and apoptosis via the lncRNA TUG1/miR-34a/FGFR1 axis.

LncRNAs and microRNAs play critical roles in osteoblast differentiation and bone formation. However, their exact roles in osteoblasts under fluid shear stress (FSS) and the possible mechanisms remain unclear. The aim of this study was to explore whether and how miR‐34a regulates osteoblast proliferation and apoptosis under FSS. In this study, FSS down‐regulated miR‐34a levels of MC3T3‐E1 cells. MiR‐34a up‐regulation attenuated FSS‐induced promotion of proliferation and suppression of apoptosis. Luciferase reporter assay revealed that miR‐34a directly targeted FGFR1. Moreover, miR‐34a regulated osteoblast proliferation and apoptosis via FGFR1. Further, we validated that lncRNA TUG1 acted as a competing endogenous RNA (ceRNA) to interact with miR‐34a and up‐regulate FGFR1 protein expression. Furthermore, lncRNA TUG1 could promote proliferation and inhibit apoptosis. Taken together, our study revealed the key role of the lncRNA TUG1/miR‐34a/FGFR1 axis in FSS‐regulated osteoblast proliferation and apoptosis and may provide potential therapeutic targets for osteoporosis.

Thyroid-stimulating hormone decreases the risk of osteoporosis by regulating osteoblast proliferation and differentiation

BackgroundAs the incidence of secretory osteoporosis has increased, bone loss, osteoporosis and their relationships with thyroid-stimulating hormone (TSH) have received increased attention. In this study, the role of TSH in bone metabolism and its possible underlying mechanisms were investigated.MethodsWe analyzed the serum levels of free triiodothyronine (FT3), free thyroxine (FT4), and TSH and the bone mineral density (BMD) levels of 114 men with normal thyroid function. In addition, osteoblasts from rat calvarial samples were treated with different doses of TSH for different lengths of time. The related gene and protein expression levels were investigated.ResultsA comparison of the BMD between the high-level and low-level serum TSH groups showed that the TSH serum concentration was positively correlated with BMD. TSH at concentrations of 10 mU/mL and 100 mU/mL significantly increased the mRNA levels of ALP, COI1 and Runx2 compared with those of the control (P < 0.05, P < 0.01). Bone morphogenetic protein (BMP)2 activity was enhanced with both increased TSH concentration and increased time. The protein levels of Runx2 and osterix were increased in a dose-dependent manner.ConclusionsThe circulating concentrations of TSH and BMD were positively correlated with normal thyroid function in males. TSH promoted osteoblast proliferation and differentiation in rat primary osteoblasts.

Fluid shear stress-induced down-regulation of microRNA-140-5p promotes osteoblast proliferation by targeting VEGFA via the ERK5 pathway

ABSTRACT Purpose Fluid shear stress (FSS) plays a critical role in osteoblast proliferation. However, the role of miRNA in osteoblast proliferation induced by FSS and the possible molecular mechanisms remain to be defined. The aim of the present study was to investigate whether miR-140-5p regulates osteoblast proliferation under FSS and its molecular mechanism. Materials and methods miR-140-5p expression was measured by qRT-PCR. Western blot was used to measure the expressions of P-ERK1/2, ERK1/2, P-ERK5 and ERK5. The levels of VEGFA, PCNA, CDK4 and Cyclin D1 were identified through qRT-PCR and western blot, respectively. Cell proliferation was detected by CCK-8 assay and EdU labeling assay. Dual-luciferase reporter assay was used to validate the target of miR-140-5p. Results miR-140-5p was significantly down-regulated when MC3T3-E1 cells were exposed to FSS. We then confirmed that up-regulation of miR-140-5p inhibited and down-regulation of miR-140-5p promoted osteoblast proliferation. In addition, FSS promotes osteoblast proliferation via down-regulating miR-140-5p. Luciferase reporter assay demonstrated that VEGFA is a direct target of miR-140-5p. Furthermore, transfection of mimic-140-5p inhibited the up-regulation of VEGFA protein level induced by FSS, suggesting that FSS regulates VEGFA protein expression via miR-140-5p. Further investigations demonstrated that VEGFA could promote osteoblast proliferation. Lastly, we demonstrated that miR-140-5p regulates osteoblast proliferation and ERK5 activation through VEGFA. Conclusions Our study demonstrates that FSS-induced the down-regulation of miR-140-5p promotes osteoblast proliferation through activing VEGFA/ERK5 signaling pathway. These findings may provide a novel mechanism of FSS-induced osteoblast proliferation and offer a new avenue to further investigate osteogenesis induced by mechanical loading.

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation.

Both parathyroid hormone (PTH) and mechanical signals are able to regulate bone growth and regeneration. They also can work synergistically to regulate osteoblast proliferation, but little is known about the mechanisms how PTH and mechanical signals interact with each other during this process. In this study, we investigated responses of MC3T3-E1 osteoblasts to PTH and oscillatory fluid flow. We found that osteoblasts are more sensitive to mechanical signals in the presence of PTH according to ERK1/2 phosphorylation, ATP release, CREB phosphorylation, and cell proliferation. PTH may also reduce the osteoblast refractory period after desensitization due to mechanical signals. We further found that the synergistic responses of osteoblasts to fluid flow or ATP with PTH had similar patterns, suggesting that synergy between fluid flow and PTH may be through the ATP pathway. After we inhibited ATP effects using apyrase in osteoblasts, their synergistic responses to mechanical stimulation and PTH were also inhibited. Additionally, knocking down P2Y2 purinergic receptors can significantly attenuate osteoblast synergistic responses to mechanical stimulation and PTH in terms of ERK1/2 phosphorylation, CREB phosphorylation, and cell proliferation. Thus, our results suggest that PTH enhances mechanosensitivity of osteoblasts via a mechanism involving ATP and P2Y2 purinergic receptors.

Silencing long noncoding RNA colon cancer-associated transcript-1 upregulates microRNA-34a-5p to promote proliferation and differentiation of osteoblasts in osteoporosis.

Long noncoding RNAs (lncRNAs) have been revealed to be related to multiple physiological and pathology processes such as development, carcinogenesis, and osteogenesis. It is reported that lncRNAs might exert function in osteoblast differentiation and bone formation. Here, we determined this study to clarify whether lncRNA CCAT1 could regulate osteoblast proliferation and differentiation in ovariectomized rats with osteoporosis. The osteoporosis models were established by bilateral ovariectomy and treated with CCAT1 siRNAs to discuss the effect of CCAT1 on pathological changes and osteocyte apoptosis in ovariectomized rats with osteoporosis. The osteoblasts from ovariectomized rats were cultured in vitro, which were then treated with CCAT1 siRNAs to explore the role of CCAT1 in osteoblast proliferation and differentiation. Moreover, the relationships among CCAT1, miR-34a-5p, and SMURF2 were confirmed. CCAT1 and SMURF2 were amplified while miR-34a-5p expression was inhibited in bone tissues and osteoblasts of ovariectomized rats with osteoporosis. Inhibited CCAT1 improved pathology and restricted osteocyte apoptosis of bone tissues in ovariectomized rats with osteoporosis in vivo, and also enhanced differentiation, mineralization abilities, and proliferation, and suppressed apoptosis of osteoblasts from ovariectomized rats in vitro through upregulating miR-34a-5p expression. LncRNA CCAT1 could competitively bind with miR-34a-5p to prevent the degradation of its target gene SMURF2. Results of this research suggested that the CCAT1 inhibits the proliferation and differentiation of osteoblasts in rats with osteoporosis by binding to miR-34a-5p, providing novel biomarkers for osteoporosis treatment.

Protein Kinase G2 Is Essential for Skeletal Homeostasis and Adaptation to Mechanical Loading in Male but Not Female Mice.

We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway positively regulates osteoblast proliferation, differentiation, and survival in vitro, and that cGMP-elevating agents have bone-anabolic effects in mice. Here, we generated mice with an osteoblast-specific (OB) knockout (KO) of type 2 PKG (gene name Prkg2) using a Col1a1(2.3 kb)-Cre driver. Compared to wild type (WT) littermates, 8-week-old male OB Prkg2-KO mice had fewer osteoblasts, reduced bone formation rates, and lower trabecular and cortical bone volumes. Female OB Prkg2-KO littermates showed no bone abnormalities, despite the same degree of PKG2 deficiency in bone. Expression of osteoblast differentiation- and Wnt/β-catenin-related genes was lower in primary osteoblasts and bones of male KO but not female KO mice compared to WT littermates. Osteoclast parameters were unaffected in both sexes. Since PKG2 is part of a mechano-sensitive complex in osteoblast membranes, we examined its role during mechanical loading. Cyclical compression of the tibia increased cortical thickness and induced mechanosensitive and Wnt/β-catenin-related genes to a similar extent in male and female WT mice and female OB Prkg2-KO mice, but loading had a minimal effect in male KO mice. We conclude that PKG2 drives bone acquisition and adaptation to mechanical loading via the Wnt/β-catenin pathway in male mice. The striking sexual dimorphism of OB Prkg2-KO mice suggests that current U.S. Food and Drug Administration-approved cGMP-elevating agents may represent novel effective treatment options for male osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Dchs1-Fat4 regulation of osteogenic differentiation in mouse.

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of Yap-Runx2 and Taz-Runx2 complexes is altered in Dchs1/Fat4 mutant osteoblasts. In conclusion, these data identify Dchs1-Fat4 as a signalling pathway in osteoblast differentiation, reveal its crucial role within the early Runx2 progenitors, and identify distinct requirements for Yap and Taz during osteoblast differentiation.

Highly-expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β-catenin and mTOR/HIF1α/VEGF signaling.

The P2X7 receptor, an ATP‐gated ion channel, is critical for cancer cell growth, invasiveness, and angiogenesis. Previous studies indicate that P2X7 regulates osteoblast proliferation and osteodeposition and that high P2X7 expression has a pro‐growth effect in osteosarcoma. However, how it functions in osteosarcoma cell growth and metastasis is not clear. Thus, we elucidated molecular mechanisms of P2X7‐dependent positive regulation of osteosarcoma cell proliferation, invasion, migration, epithelial to mesenchymal transition (EMT), and angiogenesis using in vitro and in vivo models. We confirm that P2X7 is highly‐expressed in human osteosarcoma tumor tissues and HOS/MNNG, MG63, U2OS, SW1353 and SAOS‐2 cell lines. P2X7 receptor stimulation enhanced HOS/MNNG and SAOS‐2 cell proliferation, migration and invasion; but knockdown of P2X7 expression or receptor inhibition had opposite effects. P2X7 positively regulated glycogen content, epithelial to mesenchymal transition and stemness of HOS/MNNG cells. P2X7 activation promoted PI3K/Akt/GSK3β/β‐catenin and mTOR/HIF1α/VEGF signaling, thereby mediating pro‐tumor effects of osteosarcoma cells. Consistent with data from in vitro experiments, systemic administration of P2X7 agonist induced tumor growth, metastasis and tumor‐associated bone destruction in osteosarcoma‐bearing nude mice, whereas a P2X7 antagonist reversed these effects. Thus, the P2X7 receptor participates in regulation of osteosarcoma growth and metastasis and we offer evidence that P2X7 may be a promising therapeutic target for treating osteosarcoma.

Loss of cyclin-dependent kinase 1 impairs bone formation, but does not affect the bone-anabolic effects of parathyroid hormone

Bone mass is maintained by a balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Although recent genetic studies have uncovered various mechanisms that regulate osteoblast differentiation, the molecular basis of osteoblast proliferation remains unclear. Here, using an osteoblast-specific loss-of-function mouse model, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates osteoblast proliferation and differentiation. Quantitative RT-PCR analyses revealed that Cdk1 is highly expressed in bone and is down-regulated upon osteoblast differentiation. We also noted that Cdk1 is dispensable for the bone-anabolic effects of parathyroid hormone (PTH). Cdk1 deletion in osteoblasts led to osteoporosis in adult mice due to low bone formation, but did not affect osteoclast formation in vivo Cdk1 overexpression in osteoblasts promoted proliferation, and conversely, Cdk1 knockdown inhibited osteoblast proliferation and promoted differentiation. Of note, we provide direct evidence that PTH's bone-anabolic effects occur without enhancing osteoblast proliferation in vivo Furthermore, we found that Cdk1 expression in osteoblasts is essential for bone fracture repair. These findings may help reduce the risk of nonunion after bone fracture and identify patients at higher risk for nonresponse to PTH treatment. Collectively, our results indicate that Cdk1 is essential for osteoblast proliferation and that it functions as a molecular switch that shifts osteoblast proliferation to maturation. We therefore conclude that Cdk1 plays an important role in bone formation.

The effects of photobiomodulation therapy on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via miR-503/Wnt3a pathway.

Photobiomodulation therapy (PBMT) has been demonstrated as regulating osteoblast proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in osteoblast, but the role of miRNAs in the PBMT-based promotion of osteoblast proliferation remains unclear. This study aimed to investigate the effects of PBMT treatment (3.75J/cm2) on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via the miR-503/Wnt3a pathway; meanwhile, detect the expressions of miR-503 and Wnt3a after PBMT treatment and the role of miR-503 in regulating Wnt signaling molecules Wnt3a, β-catenin, Runx2, apoptotic proteins caspase-3, and Bcl-2 in vitro. The PBMT parameters were as follows: 808nm continuous wavelength, 0.401W output power, 0.042W/cm2 power density, 9.6cm2 spot size, 36J energy, 3.75J/cm2 energy density, 90s irradiation for three times per 12h, 14.5cm distance of the laser source and the angle of divergence of the laser beam was 7°. In our present study, the target relationship was predicted and verified by bioinformatics analysis and luciferase reporter assays. Gene mRNA and protein expressions were examined by qPCR and western blot analysis. The MTT method was used to evaluate the effect of miR-503 on MC3T3-E1 cells proliferation. And cell apoptosis was examined by flow cytometry. The results showed that PBMT treatment reduced the expression of miR-503 and increased the level of Wnt3a (p < 0.01). Bioinformatics analysis and luciferase reporter assays revealed that Wnt3a was a target of miR-503, and Wnt3a was regulated by miR-503. Furthermore, miR-503 was found to functionally inhibit proliferation and promote apoptosis (p < 0.01). And during this process, Wnt3a, β-catenin, Runx2, and Bcl-2 expressions were significantly inhibited (p < 0.01); however, caspase-3 level was upregulated (p < 0.01). These results suggest that miR-503 plays a role in osteoblast proliferation and apoptosis in response to PBMT, which is potentially amenable to therapeutic manipulation for clinical application.

Fluid shear stress promotes osteoblast proliferation through the NFATc1-ERK5 pathway

ABSTRACTPurpose: Extracellular-regulated kinase 5 (ERK5) is thought to regulate osteoblast proliferation. To further understand how ERK5 signaling regulates osteoblast proliferation induced by fluid shear stress (FSS), we examined some potential signaling targets associated with ERK5 in MC3T3-E1 cells.Methods: MC3T3-E1 cells were treated with XMD8-92 (an ERK5 inhibitor) or Cyclosporin A (CsA, a nuclear factor of activated T cells (NFAT) c1 inhibitor) and/or exposed to 12 dyn/cm2 FSS. Phosphorylated-ERK5 (p-ERK5) and expression levels of NFATc1, ERK5, E2F2, and cyclin E1 were analyzed by western blot. The mRNA levels of genes associated with cell proliferation were analyzed by Polymerase Chain Reaction (PCR) array. Subcellular localization of p-ERK5 and NFATc1 were determined by immunofluorescence. Cell proliferation was evaluated by MTT assay.Results: NFATc1 expression was up-regulated by FSS. XMD8-92 only blocked ERK5 activation; however, CsA decreased NFATc1 and p-ERK5 levels, including after FSS stimulation. Exposure to NFATc1 inhibitor or ERK5 inhibitor resulted in decreased E2F2 and cyclin E1 expression and proliferation by proliferative MC3T3-E1 cells. Furthermore, immunofluorescence results illustrated that NFATc1 induced ERK5 phosphorylation, resulting in p-ERK5 translocation to the nucleus.Conclusions: Our results reveal that NFATc1 acts as an intermediate to promote the phosphorylation of ERK5 induced by FSS. Moreover, activated NFATc1-ERK5 signaling up-regulates the expression of E2F2 and cyclin E1, which promote osteoblast proliferation.

Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation.

Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation.

Phenotypic research on senile osteoporosis caused by SIRT6 deficiency.

Osteoporosis is a serious public bone metabolic disease. However, the mechanisms underlying bone loss combined with ageing, which is known as senile osteoporosis, remains unknown. Here we show the detailed phenotype of this disease caused by SIRT6 knock out (KO) in mice. To the best of our knowledge, this is the first study to reveal that SIRT6 is expressed in both bone marrow stroma cells and bone-related cells in both mouse and human models, which suggests that SIRT6 is an important regulator in bone metabolism. SIRT6-KO mice exhibit a significant decrease in body weight and remarkable dwarfism. The skeleton of the SIRT6-KO mouse is deficient in cartilage and mineralized bone tissue. Moreover, the osteocalcin concentration in blood is lower, which suggests that bone mass is markedly lost. Besides, the tartrate-resistant acid phosphatase 5b (TRAP5b) concentration is much higher, which suggests that bone resorption is overactive. Both trabecular and cortical bones exhibit severe osteopenia, and the bone mineral density is decreased. Moreover, double-labelling analysis shows that bone formation is much slower. To determine whether SIRT6 directly regulates bone metabolism, we cultured primary bone marrow stromal cells for osteogenesis and osteoclastogenesis separately to avoid indirect interference in vivo responses such as inflammation. Taken together, these results show that SIRT6 can directly regulate osteoblast proliferation and differentiation, resulting in attenuation in mineralization. Furthermore, SIRT6 can directly regulate osteoclast differentiation and results in a higher number of small osteoclasts, which may be related to overactive bone resorption.

Sika Deer Antler Collagen Type I-Accelerated Osteogenesis in Bone Marrow Mesenchymal Stem Cells via the Smad Pathway.

Deer antler preparations have been used to strengthen bones for centuries. It is particularly rich in collagen type I. This study aimed to unravel part of the purported bioremedial effect of Sika deer antler collagen type I (SDA-Col I) on bone marrow mesenchymal stem cells. The results suggest that SDA-Col I might be used to promote and regulate osteoblast proliferation and differentiation. SDA-Col I might potentially provide the basis for novel therapeutic strategies in the treatment of bone injury and/or in scaffolds for bone replacement strategies. Finally, isolation of SDA-Col I from deer antler represents a renewable, green, and uncomplicated way to obtain a biomedically valuable therapeutic.