Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog-Wnt signaling, causing campomelia.

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with ß-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

Mechanical induction of osteoanabolic Wnt1 promotes osteoblast differentiation via Plat.

Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating ß-Catenin. Silencing Wnt1 impairs mechanically induced ß-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/ß-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.

IGF signaling pathway in bone and cartilage development, homeostasis, and disease.

The skeleton plays a fundamental role in the maintenance of organ function and daily activities. The insulin-like growth factor (IGF) family is a group of polypeptide substances with a pronounced role in osteoblast differentiation, bone development, and metabolism. Disturbance of the IGFs and the IGF signaling pathway is inextricably linked with assorted developmental defects, growth irregularities, and jeopardized skeletal structure. Recent findings have illustrated the significance of the action of the IGF signaling pathway via growth factors and receptors and its interactions with dissimilar signaling pathways (Wnt/ß-catenin, BMP, TGF-ß, and Hh/PTH signaling pathways) in promoting the growth, survival, and differentiation of osteoblasts. IGF signaling also exhibits profound influences on cartilage and bone development and skeletal homeostasis via versatile cell-cell interactions in an autocrine, paracrine, and endocrine manner systemically and locally. Our review summarizes the role and regulatory function as well as a potentially integrated gene network of the IGF signaling pathway with other signaling pathways in bone and cartilage development and skeletal homeostasis, which in turn provides an enlightening insight into visualizing bright molecular targets to be eligible for designing effective drugs to handle bone diseases and maladies, such as osteoporosis, osteoarthritis, and dwarfism.

Type 1 collagen: Synthesis, structure and key functions in bone mineralization.

Collagen is a highly abundant protein in the extracellular matrix of humans and mammals, and it plays a critical role in maintaining the body's structural integrity. Type I collagen is the most prevalent collagen type and is essential for the structural integrity of various tissues. It is present in nearly all connective tissues and is the main constituent of the interstitial matrix. Mutations that affect collagen fiber formation, structure, and function can result in various bone pathologies, underscoring the significance of collagen in sustaining healthy bone tissue. Studies on type 1 collagen have revealed that mutations in its encoding gene can lead to diverse bone diseases, such as osteogenesis imperfecta, a disorder characterized by fragile bones that are susceptible to fractures. Knowledge of collagen's molecular structure, synthesis, assembly, and breakdown is vital for comprehending embryonic and foetal development and several aspects of human physiology. In this review, we summarize the structure, molecular biology of type 1 collagen, its biomineralization and pathologies affecting bone.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

KIAA0753 enhances osteoblast differentiation suppressed by diabetes.

Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.

Ap-2ß regulates cranial osteogenic potential via the activation of Wnt/ß-catenin signaling pathway.

The skull is a fundamental bone that protects the development of brain and consists of several bony elements, such as the frontal and parietal bones. Frontal bone exhibited superior in osteogenic potential and regeneration of cranial defects compared to parietal bone. However, how this regional difference is regulated remains largely unknown. In this study, we identified an Ap-2ß transcriptional factor with a higher expression in frontal bone, but its molecular function in osteoblasts needs to be elucidated. We found that Ap-2ß knockdown in preosteoblasts leads to reduced proliferation, increased cell death and impaired differentiation. Through RNA-seq analysis, we found that Ap-2ß influences multiple signaling pathways including the Wnt pathway, and overexpression of Ap-2ß showed increased nuclear ß-catenin and its target genes expressions in osteoblasts. Pharmacological activation of Wnt/ß-catenin signaling using LiCl treatment cannot rescue the reduced luciferase activities of the ß-catenin/TCF/LEF reporter in Ap-2ß knockdown preosteoblasts. Besides, transient expression of Ap-2ß via the lentivirus system could sufficiently rescue the inferior osteogenic potential in parietal osteoblasts, while Ap-2ß knockdown in frontal osteoblasts resulted in reduced osteoblast activity, reduced active ß-catenin and target genes expressions. Taken together, our data demonstrated that Ap-2ß modulates osteoblast proliferation and differentiation through the regulation of Wnt/ß-catenin signaling pathway and plays an important role in regulating regional osteogenic potential in frontal and parietal bone.

Interaction between p21-activated kinase 4 and ß-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity.

Recent studies have confirmed that melatonin and N6-methyladenosine (m6A) modification can influence bone cell differentiation and bone formation. Melatonin can also regulate a variety of biological processes through m6A modification. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) serves as a reader of m6A modification. In this study, we used the hindlimb unloading model as an animal model of bone loss induced by simulated microgravity and used 2D clinorotation to simulate a microgravity environment for cells on the ground. We found that hnRNPA2B1 was downregulated both in vitro and in vivo during simulated microgravity. Further investigations showed that hnRNPA2B1 could promote osteoblast differentiation and that overexpression of hnRNPA2B1 attenuated the suppression of osteoblast differentiation induced by simulated microgravity. We also discovered that melatonin could promote the expression of hnRNPA2B1 under simulated microgravity. Moreover, we found that promotion of osteoblast differentiation by melatonin was partially dependent on hnRNPA2B1. Therefore, this research revealed, for the first time, the role of the melatonin/hnRNPA2B1 axis in osteoblast differentiation under simulated microgravity. Targeting this axis may be a potential protective strategy against microgravity-induced bone loss and osteoporosis.

Erythropoietin regulates osteoclast formation via up-regulating PPARγ expression.

Erythropoietin (EPO), expressed in red blood progenitor cells, primarily regulates erythropoiesis by binding to its receptor. Besides anemia, recent studies have identified new therapeutic indications for EPO that are not connected to red blood cell formation. Elevated EPO levels harm bone homeostasis in adult organisms and are associated with increased osteoclast; however, the underlying molecular mechanisms remain unclear. This study demonstrated that EPO enhanced osteoclast differentiation and bone resorption in vitro. We showed that EPO promoted osteoclast formation by up-regulating PPARγ expression through activating the Jak2/ERK signaling pathway. Consistently, PPARγ antagonists rescued the hyperactivation of osteoclasts due to EPO, while PPARγ agonists reversed the EMP9-mediated decrease in osteoclast differentiation. Further, exposing female mice to EPO for two months led to a decrease in bone mass and increased osteoclast numbers. The present results suggested that EPO promotes osteoclastogenesis by regulating the Jak2/ERK/ PPARγ signaling pathway. From a clinical perspective, the risk of compromised bone health should be considered when using EPO to treat anemia in post-operative patients with intertrochanteric fractures of the femur, as it could significantly impact the patient's recovery and quality of life.

Wnt3a-induced LRP6 phosphorylation enhances osteoblast differentiation to alleviate osteoporosis through activation of mTORC1/ß-catenin signaling.

OBJECTIVE: Osteoporosis (OP) is a common cause of morbidity and mortality in older individuals. The importance of Wnt3a in osteogenic activity and bone tissue homeostasis is well known. Here, we explored the possible molecular mechanism by which Wnt3a mediates the LRP6/mTORC1/ß-catenin axis to regulate osteoblast differentiation in OP. METHODS: OP-related key genes were identified through a bioinformatics analysis. A ROS17/2.8 cell differentiation system for rat osteogenic progenitors and a rat model of senile OP were constructed for in vitro and in vivo mechanism verification. RESULTS: Bioinformatics analysis revealed that LRP6 was poorly expressed in OP and may play a key role in the occurrence of OP by affecting osteoblast differentiation. LRP6 knockdown inhibited osteoblast differentiation in an in vitro model. In addition, Wnt3a promoted osteoblast differentiation by inducing LRP6 phosphorylation. Moreover, LRP6 promoted mTORC1 expression, which indirectly promoted ß-catenin expression, thus promoting osteoblast differentiation. Finally, an in vivo assay revealed that LRP6 inhibition improved OP. CONCLUSION: Our study provides evidence that Wnt3a induces phosphorylation of LRP6 to activate the mTORC1/ß-catenin axis, thus promoting osteoblast differentiation and ultimately improving OP in aged rats.

Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

Update on the Genetics of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous heritable skeletal dysplasia characterized by bone fragility and deformity, growth deficiency, and other secondary connective tissue defects. OI is now understood as a collagen-related disorder caused by defects of genes whose protein products interact with collagen for folding, post-translational modification, processing and trafficking, affecting bone mineralization and osteoblast differentiation. This review provides the latest updates on genetics of OI, including new developments in both dominant and rare OI forms, as well as the signaling pathways involved in OI pathophysiology. There is a special emphasis on discoveries of recessive mutations in TENT5A, MESD, KDELR2 and CCDC134 whose causality of OI types XIX, XX, XXI and XXI, respectively, is now established and expends the complexity of mechanisms underlying OI to overlap LRP5/6 and MAPK/ERK pathways. We also review in detail new discoveries connecting the known OI types to each other, which may underlie an eventual understanding of a final common pathway in OI cellular and bone biology.

BMP7-induced osteoblast differentiation requires hedgehog signaling and involves nuclear mechanisms of gene expression control.

During the morphological changes occurring in osteoblast differentiation, Sonic hedgehog (Shh) plays a crucial role. While some progress has been made in understanding this process, the epigenetic mechanisms governing the expression of Hh signaling members in response to bone morphogenetic protein 7 (BMP7) signaling in osteoblasts remain poorly understood. To delve deeper into this issue, we treated pre-osteoblasts (pObs) with 100 ng/mL of BMP7 for up to 21 days. Initially, we validated the osteogenic phenotype by confirming elevated expression of well-defined gene biomarkers, including Runx2, Osterix, Alkaline Phosphatase (Alp), and bone sialoprotein (Bsp). Simultaneously, Hh signaling-related members Sonic (Shh), Indian (Ihh), and Desert (Dhh) Hedgehog (Hh) exhibited nuanced modulation over the 21 days in vitro period. Subsequently, we evaluated epigenetic markers, and our data revealed a notable change in the CpG methylation profile, considering the methylation/hydroxymethylation ratio. CpG methylation is a reversible process regulated by DNA methyltransferases and demethylases, including Ten-eleven translocation (Tets), which also exhibited changes during the acquisition of the osteogenic phenotype. Specifically, we measured the methylation pattern of Shh-related genes and demonstrated a positive Pearson correlation for GLI Family Zinc Finger 1 (Gli1) and Patched (Ptch1). This data underscores the significance of the epigenetic machinery in modulating the BMP7-induced osteogenic phenotype by influencing the activity of Shh-related genes. In conclusion, this study highlights the positive impact of epigenetic control on the expression of genes related to hedgehog signaling during the morphogenetic changes induced by BMP7 signaling in osteoblasts.

Systematical mutational analysis of teriparatide on anti-osteoporosis activity by alanine scanning.

Osteoporosis is a progressive systemic skeletal disease that decreases bone density and bone quality, making them fragile and easy to break. In spite of effective anti-osteoporosis potency, teriparatide, the first anabolic medications approved for the treatment of osteoporosis, was proven to exhibit various side effects. And the relevant structure-activity relationship (SAR) of teriparatide was in need. In this work, we performed a systematical alanine scanning against teriparatide and synthesized 34 teriparatide derivatives. Their biological activities were evaluated and the importance of each residue for anti-osteoporosis activity was also revealed. A remarkable decrease in activity was observed for alanine replacement of the residue Gly12, His14, Ser17, Arg20 and Leu24, showcasing the important role of these residues in teriparatide on anti-osteoporosis activity. On contrary, when Gly13 and Gln30 were mutated to Ala, the peptide derivatives exhibited the significantly increased activities, demonstrating that these two residues could be readily replaced. Our research expanded the peptide library of teriparatide analogues and presented a potential opportunity for designing the more powerful anti-osteoporosis peptide agents.

Resveratrol modulates the inflammatory response in hPDLSCs via the NRF2/HO-1 and NF-κB pathways and promotes osteogenic differentiation.

OBJECTIVE: The purpose of this study was to investigate resveratrol's specific role as an anti-inflammatory and osteogenic differentiation of hPDLSCs in periodontitis and to reveal the mechanisms involved. BACKGROUND: Numerous studies have shown that inhibiting the inflammatory response of periodontal tissues and promoting the regeneration of alveolar bone are crucial treatments for periodontitis. Resveratrol has been found to have certain anti-inflammatory property. However, the anti-inflammatory mechanism and osteogenic effect of resveratrol in periodontitis are poorly understood. MATERIALS AND METHODS: We constructed an in vitro periodontitis model by LPS stimulation of hPDLSCs and performed WB, RT-qPCR, and immunofluorescence to analyze inflammatory factors and related pathways. In addition, we explored the osteogenic ability of resveratrol in in vitro models. RESULTS: In vitro, resveratrol ameliorated the inflammatory response associated with activation of the NF-κB pathway through activation of the NRF2/HO-1 pathway, characterized by inhibition of p65/p50 nuclear translocation and the proinflammatory cytokines interleukin-1ß levels. Resveratrol also has a positive effect on osteogenic differentiation. CONCLUSIONS: Observations suggest that resveratrol modulates the inflammatory response in hPDLSCs via the NRF2/HO-1 and NF-κB pathways and promotes osteogenic differentiation.

Fibroblasts inhibit osteogenesis by regulating nuclear-cytoplasmic shuttling of YAP in mesenchymal stem cells and secreting DKK1.

BACKGROUND: Fibrous scars frequently form at the sites of bone nonunion when attempts to repair bone fractures have failed. However, the detailed mechanism by which fibroblasts, which are the main components of fibrous scars, impede osteogenesis remains largely unknown. RESULTS: In this study, we found that fibroblasts compete with osteogenesis in both human bone nonunion tissues and BMP2-induced ectopic osteogenesis in a mouse model. Fibroblasts could inhibit the osteoblastic differentiation of mesenchymal stem cells (MSCs) via direct and indirect cell competition. During this process, fibroblasts modulated the nuclear-cytoplasmic shuttling of YAP in MSCs. Knocking down YAP could inhibit osteoblast differentiation of MSCs, while overexpression of nuclear-localized YAP-5SA could reverse the inhibition of osteoblast differentiation of MSCs caused by fibroblasts. Furthermore, fibroblasts secreted DKK1, which further inhibited the formation of calcium nodules during the late stage of osteogenesis but did not affect the early stage of osteogenesis. Thus, fibroblasts could inhibit osteogenesis by regulating YAP localization in MSCs and secreting DKK1. CONCLUSIONS: Our research revealed that fibroblasts could modulate the nuclear-cytoplasmic shuttling of YAP in MSCs, thereby inhibiting their osteoblast differentiation. Fibroblasts could also secrete DKK1, which inhibited calcium nodule formation at the late stage of osteogenesis.

Knockdown of the UL-16 binding protein 1 promotes osteoblast differentiation of human mesenchymal stem cells by activating the SMAD2/3 pathway.

Osteoporosis is caused by the imbalance of osteoblasts and osteoclasts. The regulatory mechanisms of differentially expressed genes (DEGs) in pathogenesis of osteoporosis are of significant and needed to be further investigated. GSE100609 dataset downloaded from Gene Expression Omnibus (GEO) database was used to identified DEGs in osteoporosis patients. KEGG analysis was conducted to demonstrate signaling pathways related to enriched genes. Osteoporosis patients and the human mesenchymal stem cells (hMSCs) were obtained for in vivo and in vitro resaerch. Lentivirus construction and viral infection was used to knockdown genes. mRNA expression and protein expression were detected via qRT-PCR and western blot assay separately. Alkaline phosphatase (ALP) activity detection, alizarin Red S (ARS) staining, and expression of bone morphogenetic protein 2 (BMP2), osteocalcin (OCN) and Osterix were evaluated to determine osteoblast differentiation capacity. UL-16 binding protein 1 (ULBP1) gene was upregulated in osteoporosis and downregulated in differentiated hMSCs. Knockdown of ULBP1 increased ALP activity, mineralization ability evaluated by ARS staining, expression of BMP2, OCN and Osterix in differentiated hMSCs. Furthermore, rescue experiment demonstrated that suppressed ULBP1 boosted osteoblast differentiation by activating TNF-ß signaling pathway. Knockdown of ULBP1 gene could promoted osteoblast differentiation by activating TNF-ß signaling pathway in differentiated hMSCs. ULBP1 may be a the Achilles' heel of osteoporosis, and suppression of ULBP1 could be a promising treatment for osteoporosis.

Loss of BACH1 improves osteogenic differentiation in glucocorticoid-induced hBMSCs through restoring autophagy.

BACKGROUND: Glucocorticoid-induced osteoporosis (GIOP) is the most common type of secondary osteoporosis. Recently, autophagy has been found to be related with the development of various diseases, including osteoporosis and osteoblast differentiation regulations. BTB and CNC homology 1 (BACH1) was a previously confirmed regulator for osteoblast differentiation, but whether it's could involve in glucocorticoid-induced human bone mesenchymal stem cells (hBMSCs) differentiation and autophagy regulation remain not been elucidated. METHODS: hBMSCs were identified by flow cytometry method, and its differentiation ability were measured by ARS staining, oil O red, and Alcian blue staining assays. Gene and proteins were quantified via qRT-PCR and western blot assays, respectively. Autophagy activity was determined using immunofluorescence. ChIP and dual luciferase assay validated the molecular interactions. RESULTS: The data revealed that isolated hBMSCs exhibited positive of CD29/CD44 and negative CD45/CD34. Moreover, BACH1 was abated gradually during osteoblast differentiation of hBMSCs, while dexamethasone (Dex) treatment led to BACH1 upregulation. Loss of BACH1 improved osteoblast differentiation and activated autophagy activity in Dex-challenged hBMSCs. Autophagy-related proteins (ATG3, ATG4, ATG5, ATG7, ATG12) were repressed after Dex treatment, while ATG3, ATG7 and BECN1 could be elevated by BACH1 knockdown, especially ATG7. Moreover, BACH1 could interact ATG7 promoter region to inhibit its transcription. Co-inhibition of ATG7 greatly overturned the protective roles of BACH1 loss on osteoblast differentiation and autophagy in Dex-induced hBMSCs. CONCLUSION: Taken together, our results demonstrated that silencing of BACH1 mitigated Dex-triggered osteogenic differentiation inhibition by transcriptionally activating ATG7-mediated autophagy, suggesting that BACH1 may be a therapeutic target for GIOP treatment.

MicroRNA 98-5p Overexpression Contributes to Delayed Fracture Healing via Targeting BMP-2.

MicroRNAs (miRNAs) are related to the regulation of bone metabolism. Delayed fracture healing (DFH) is a common complication after fracture surgery. The study attempted to examine the role of miR-98-5p and bone morphogenetic protein (BMP)-2 with the onset of DFH. A total of 140 patients with femoral neck fracture were recruited, including 80 cases with normal fracture healing (NFH) and 60 cases with DFH. MC3T3-E1 cells were induced cell differentiation for cell function experiments. Real-time quantitative polymerase chain reaction (RT-qPCR) was carried out to test mRNA levels. Cell proliferation and apoptosis were determined via CCK-8 and flow cytometry assay. Luciferase reporter assay was done to verify the targeted regulatory relationship of miR-98-5p with BMP-2. In comparison with NFH cases, DFH patients owned high levels of serum miR-98-5p and low concentration of BMP-2, and the levels of the two indexes are significantly negatively correlated. Both miR-98-5p and BMP-2 had the ability to predict DFH, while their combined diagnostic value is the highest. BMP-2 was demonstrated to be the target gene of miR-98-5p. Overexpression of BMP-2 reversed the role of miR-98-5p in MC3T3-E1 cell proliferation, apoptosis and differentiation. Increased miR-98-5p and decreased BMP-2 serve as potential biomarkers for the diagnosis of DFH. MiR-98-5p overexpression inhibits osteoblast proliferation and differentiation via targeting BMP-2.