Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

The Cell-Penetrating Peptide GV1001 Enhances Bone Formation via Pin1-Mediated Augmentation of Runx2 and Osterix Stability.

Peptide-based drug development is a promising direction due to its excellent biological activity, minimal immunogenicity, high in vivo stability, and efficient tissue penetrability. GV1001, an amphiphilic peptide, has proven effective as an anti-cancer vaccine, but its effect on osteoblast differentiation is unknown. To identify proteins interacting with GV1001, biotin-conjugated GV1001 was constructed and confirmed by mass spectrometry. Proteomic analyses were performed to determine GV1001's interaction with osteogenic proteins. GV1001 was highly associated with peptidyl-prolyl isomerase A and co-immunoprecipitation assays revealed that GV1001 bound to peptidyl-prolyl cis-trans isomerase 1 (Pin1). GV1001 significantly increased alkaline phosphatase (ALP) activity, bone nodule formation, and the expression of osteogenic gene markers. GV1001-induced osteogenic activity was enhanced by Pin1 overexpression and abolished by Pin1 knockdown. GV1001 increased the protein stability and transcriptional activity of Runx2 and Osterix. Importantly, GV1001 administration enhanced bone mass density in the OVX mouse model, as verified by µCT analysis. GV1001 demonstrated protective effects against bone loss in OVX mice by upregulating osteogenic differentiation via the Pin1-mediated protein stabilization of Runx2 and Osterix. GV1001 could be a potential candidate with anabolic effects for the prevention and treatment of osteoporosis.

[TNF-α regulated SHED osteogenic differentiation through ERK1/2-Runx2 signaling pathway].

PURPOSE: To investigate the effect of TNF-α on osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED), and to analyze the changes of ERK1/2-Runx2 signaling pathway in the regulation process. METHODS: SHED cells were isolated and cultured from normal deciduous permanent teeth of healthy children aged 6-8 years old, and the third passage of SHED cells were taken and divided into control group (osteogenic inducer culture), observation group (osteogenic inducer and TNF-α co-culture) and agonist group (osteogenic inducer, TNF-α and ERK pathway agonist co-culture). The osteogenic differentiation was determined by alizarin red staining. The protein expression levels of Osterix, OPN, ERK1/2, pERK1/2 and Runx2 in SHED cells were determined by Western blot. The expressions of Osterix, OPN, ERK1/2, pERK1/2 and Runx2 mRNA were detected by qRT-PCR. Statistical analysis was performed with SPSS 26.0 software package. RESULTS: Comparison of osteogenic differentiation ability of the three groups of cells showed that red-brown mineralized nodules were observed in the three groups of cells. Compared among the three groups, the control group had the most mineralized nodules, followed by the activation group, and the observation group had the least mineralized nodules. Compared with the control group, the expression levels of Osterix and OPN protein and mRNA in the observation group and the agonist group were significantly decreased, while the expression levels of Osterix and OPN protein and mRNA in the agonist group were significantly higher than those in the observation group. There was no significant difference in the expression levels of ERK1/2 protein and mRNA among the three groups, while the expression levels of pERK1/2 and Runx2 protein and mRNA in the observation group and the agonist group were significantly higher than those in the control group, and the expression levels of pERK1/2 and Runx2 protein and mRNA in the agonist group were significantly higher than those in the observation group. CONCLUSIONS: TNF-α can inhibit osteogenic differentiation of SHED cells, which may be related to the inhibition of ERK1/2-Runx2 signaling pathway.

The adjunctive use of antimicrobial photodynamic therapy, light-emitting-diode photobiomodulation and ozone therapy in regenerative treatment of stage III/IV grade C periodontitis: a randomized controlled clinical trial.

OBJECTIVES: To assess the short-term efficacy of multiple sessions of antimicrobial photodynamic therapy (aPDT), light-emitting-diode (LED) photobiomodulation, and topical ozone therapy applications following surgical regenerative treatments on clinical parameters, patient-centered outcomes, and mRNA expression levels of VEGF, IL-6, RunX2, Nell-1, and osterix in gingival crevicular fluid samples in patients with stage III/IV, grade C periodontitis. MATERIALS AND METHODS: Forty-eight systemically healthy patients were assigned into four groups to receive adjunctive modalities with regenerative periodontal surgical treatment. A 970 ± 15 nm diode laser plus indocyanine-green for aPDT group, a 626 nm LED for photobiomodulation group, and topical gaseous ozone were applied at 0, 1, 3, and 7 postoperative days and compared to control group. The clinical periodontal parameters, early wound healing index (EHI), and postoperative patients' morbidity were evaluated. The mRNA levels of biomarkers were assessed by real-time polymerase chain reaction. RESULTS: No significant difference in the clinical parameters except gingival recession (GR) was identified among the groups. For group-by-time interactions, plaque index (PI) and probing pocket depths (PD) showed significant differences (p = 0.034; p = 0.022). In sites with initial PD > 7 mm, significant differences were observed between control and photobiomodulation groups in PD (p = 0.011), between control and aPDT, and control and photobiomodulation groups in CAL at 6-month follow-up (p = 0.007; p = 0.022). The relative osterix mRNA levels showed a statistically significant difference among the treatment groups (p = 0.014). CONCLUSIONS: The additional applications of aPDT and LED after regenerative treatment of stage III/IV grade C periodontitis exhibited a more pronounced beneficial effect on clinical outcomes in deep periodontal pockets.

Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women.

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

IFT80 promotes early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation as the essential functional component of primary cilia. The effects of IFT80 on early bone healing of extraction sockets have not been well studied. To investigate whether deletion of Ift80 in alveolar bone-derived mesenchymal stem cells (aBMSCs) affected socket bone healing, we generated a mouse model of specific knockout of Ift80 in Prx1 mesenchymal lineage cells (Prx1Cre;IFT80f/f). Our results demonstrated that deletion of IFT80 in Prx1 lineage cells decreased the trabecular bone volume, ALP-positive osteoblastic activity, TRAP-positive osteoclastic activity, and OSX-/COL I-/OCN-positive areas in tooth extraction sockets of Prx1Cre; IFT80f/f mice compared with IFT80f/f littermates. Furthermore, aBMSCs from Prx1Cre; IFT80f/f mice showed significantly decreased osteogenic markers and downregulated migration and proliferation capacity. Importantly, the overexpression of TAZ recovered significantly the expressions of osteogenic markers and migration capacity of aBMSCs. Lastly, the local administration of lentivirus for TAZ enhanced the expression of RUNX2 and OSX and promoted early bone healing of extraction sockets from Prx1Cre; IFT80f/f mice. Thus, IFT80 promotes osteogenesis and early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Carbonate apatite increases gene expression of osterix and bone morphogenetic protein 2 in the alveolar ridge after socket grafting.

PURPOSE: After tooth extraction, preservation of the alveolar ridge by socket grafting attenuates bone resorption. Runt-related transcription factor 2 (RUNX2) and SP7/Osterix (OSX) are transcription factors playing an important role in osteoblast differentiation. The purpose of this study was to evaluate the effects of carbonate apatite (CO3Ap) on osteoblast-related gene and protein expression after socket grafting. METHODS: Alveolar bone and new bone after CO3Ap grafting were collected at the time of implant placement. Levels of mRNA for RUNX2, SP7/OSX, bone morphogenetic protein 2 (BMP2), BMP7 and platelet derived growth factor B were determined by real-time PCR. Immunostaining was performed using antibodies against RUNX2, SP7/OSX, vimentin and cytokeratin. To evaluate bone resorption rates, cone-beam CT (CBCT) imaging was performed after socket grafting and before implant placement. RESULTS: CBCT imaging showed that the average degree of bone resorption at the CO3Ap graft site was 7.15 ± 3.79%. At the graft sites, levels of SP7/OSX and BMP2 mRNA were significantly increased. Replacement of CO3Ap with osteoid was evident histologically, and in the osteoid osteoblast-like cells were stained for SP7/OSX and vimentin. CONCLUSION: These results show that gene expression of both SP7/OSX and BMP2 can be induced by CO3Ap, suggesting that increased expression of SP7/OSX and vimentin may be involved in the BMP pathway.

The histone acetyltransferase Mof regulates Runx2 and Osterix for osteoblast differentiation.

Osteoblast differentiation is regulated by various transcription factors, signaling molecules, and posttranslational modifiers. The histone acetyltransferase Mof (Kat8) is involved in distinct physiological processes. However, the exact role of Mof in osteoblast differentiation and growth remains unknown. Herein, we demonstrated that Mof expression with histone H4K16 acetylation increased during osteoblast differentiation. Inhibition of Mof by siRNA knockdown or small molecule inhibitor, MG149 which is a potent histone acetyltransferase inhibitor, reduced the expression level and transactivation potential of osteogenic key markers, Runx2 and Osterix, thus inhibiting osteoblast differentiation. Besides, Mof overexpression also enhanced the protein levels of Runx2 and Osterix. Mof could directly bind the promoter region of Runx2/Osterix to potentiate their mRNA levels, possibly through Mof-mediated H4K16ac to facilitate the activation of transcriptional programs. Importantly, Mof physically interacts with Runx2/Osterix for the stimulation of osteoblast differentiation. Yet, Mof knockdown showed indistinguishable effect on cell proliferation or apoptosis in MSCs and preosteoblast cells. Taken together, our results uncover Mof functioning as a novel regulator of osteoblast differentiation via the promotional effects on Runx2/Osterix and rationalize Mof as a potential therapeutic target, like possible application of inhibitor MG149 for the treatment of osteosarcoma or developing specific Mof activator to ameliorate osteoporosis.

circRNA422 enhanced osteogenic differentiation of bone marrow mesenchymal stem cells during early osseointegration through the SP7/LRP5 axis.

Circular RNAs (circRNAs) play an important role in biological activities, especially in regulating osteogenic differentiation of stem cells. However, no studies have reported the role of circRNAs in early osseointegration. Here we identified a new circRNA, circRNA422, from rat bone marrow mesenchymal stem cells (BMSCs) cultured on sandblasted, large-grit, acid-etched titanium surfaces. The results showed that circRNA422 significantly enhanced osteogenic differentiation of BMSCs with increased expression levels of alkaline phosphatase, the SP7 transcription factor (SP7/osterix), and lipoprotein receptor-related protein 5 (LRP5). Silencing of circRNA422 had opposite effects. There were two SP7 binding sites on the LRP5 promoter, indicating a direct regulatory relationship between SP7 and LRP5. circRNA422 could regulate early osseointegration in in vivo experiments. These findings revealed an important function of circRNA422 during early osseointegration. Therefore, circRNA422 may be a potential therapeutic target for enhancing implant osseointegration.

Inactivation of Ihh in Sp7-Expressing Cells Inhibits Osteoblast Proliferation, Differentiation, and Bone Formation, Resulting in a Dwarfism Phenotype with Severe Skeletal Dysplasia in Mice.

Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.

Klotho in Osx+-mesenchymal progenitors exerts pro-osteogenic and anti-inflammatory effects during mandibular alveolar bone formation and repair.

Maxillofacial bone defects are commonly seen in clinical practice. A clearer understanding of the regulatory network directing maxillofacial bone formation will promote the development of novel therapeutic approaches for bone regeneration. The fibroblast growth factor (FGF) signalling pathway is critical for the development of maxillofacial bone. Klotho, a type I transmembrane protein, is an important components of FGF receptor complexes. Recent studies have reported the presence of Klotho expression in bone. However, the role of Klotho in cranioskeletal development and repair remains unknown. Here, we use a genetic strategy to report that deletion of Klotho in Osx-positive mesenchymal progenitors leads to a significant reduction in osteogenesis under physiological and pathological conditions. Klotho-deficient mensenchymal progenitors also suppress osteoclastogenesis in vitro and in vivo. Under conditions of inflammation and trauma-induced bone loss, we find that Klotho exerts an inhibitory function on inflammation-induced TNFR signaling by attenuating Rankl expression. More importantly, we show for the first time that Klotho is present in human alveolar bone, with a distinct expression pattern under both normal and pathological conditions. In summary, our results identify the mechanism whereby Klotho expressed in Osx+-mensenchymal progenitors controls osteoblast differentiation and osteoclastogenesis during mandibular alveolar bone formation and repair. Klotho-mediated signaling is an important component of alveolar bone remodeling and regeneration. It may also be a target for future therapeutics.

Long Noncoding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 Promotes the Osteoblast Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Targeting the microRNA-96/Osterix Axis.

OBJECTIVES: To investigate whether and how the long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) sponges microRNA-96 (miR-96) to achieve the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). METHODS: Protein levels were detected by Western blot. Mineralized bone matrix formation was studied by alizarin red staining. Metastasis-associated lung adenocarcinoma transcript 1, miR-96, and osteogenesis-related Messenger RNA expression was assessed by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). The interactions between miR-96 and osterix (Osx), MALAT1, and miR-96 were determined by luciferase reporter assay. RESULTS: The expression of MALAT1 was upregulated whereas that of miR-96 was downregulated in osteogenic hBMSCs. In addition, the expression of MALAT1 significantly decreased whereas that of miR-96 increased in the hBMSCs of osteoporosis (OP) patients. qRT-PCR and alizarin red staining assays showed that MALAT1 silencing or miR-96 overexpression inhibits hBMSC osteogenic differentiation and vice versa. overexpression of miR-96 reversed the promotive effect of MALAT1 on the osteogenic differentiation of hBMSCs. Dual luciferase report assay verified that miR-96 is a regulatory target of MALAT1 and that Osx is a gene target of miR-96. CONCLUSIONS: Taken together, the results demonstrate that MALAT1 promotes the osteogenic differentiation of hBMSCs by regulating the miR-96/Osx axis. Our study provides novel mechanistic insights into the critical role of lncRNA MALAT1 as a microRNA sponge in OP patients and sheds new light on lncRNA-directed diagnostics and therapeutics in OP.

The effects of Omarigliptin on promoting osteoblastic differentiation.

Osteoporosis significantly impacts the normal life of the elderly and is reported to be closely related to dysfunction of osteoblastic differentiation. Runt-related transcription factor-2 (Runx2) is a critical transcriptional factor involved in the regulation of osteoblast differentiation. Omarigliptin is a novel dipeptidyl peptidase-4 (DDP-4) inhibitor and this study proposes to probe into its possible therapeutic function against Osteoporosis by investigating its impacts on osteoblastic differentiation. Osteogenic medium was used to induce osteoblastic differentiation in MC3T3­E1 cells, and was verified by the increased alkaline phosphatase (ALP) activity, enhanced mineralization, and promoted expression level of osteoblastic differentiation-related factors, including bone morphogenetic protein-2 (BMP-2), ALP, osteocalcin (Ocn), collagen type I alpha 1 (Col1a1), Collagen Type I alpha 2 (Col1a2), Runx2, osterix (Sp7), fibroblast growth factor receptor 2 (Fgfr2), and fibroblast growth factor receptor 3 (Fgfr3), accompanied by the activation of the p38 and Akt pathways. After treatment with Omarigliptin, the ALP activity and mineralization were further promoted, accompanied by the further upregulation of osteoblastic differentiation-related factors, and activation of the p38 and Akt pathways. Lastly, Omarigliptin-induced osteoblastic differentiation, promoted ALP activity, and increased expression levels of Sp7, Fgfr2, Fgfr3, BMP-2, Ocn, ALP, Col1a1, and Col1a2, in the osteogenic medium- cultured MC3T3­E1 cells were dramatically abolished by the knockdown of Runx2. Taken together, our data reveal that Omarigliptin promoted osteoblastic differentiation by regulating Runx2.

Skeletal Deformities in Osterix-Cre;Tgfbr2f/f Mice May Cause Postnatal Death.

Transforming growth factor ß (TGFß) signaling plays an important role in skeletal development. We previously demonstrated that the loss of TGFß receptor II (Tgfbr2) in Osterix-Cre-expressing mesenchyme results in defects in bones and teeth due to reduced proliferation and differentiation in pre-osteoblasts and pre-odontoblasts. These Osterix-Cre;Tgfbr2f/f mice typically die within approximately four weeks for unknown reasons. To investigate the cause of death, we performed extensive pathological analysis on Osterix-Cre- (Cre-), Osterix-Cre+;Tgfbr2f/wt (HET), and Osterix-Cre+;Tgfbr2f/f (CKO) mice. We also crossed Osterix-Cre mice with the ROSA26mTmG reporter line to identify potential off-target Cre expression. The findings recapitulated published skeletal and tooth abnormalities and revealed previously unreported osteochondral dysplasia throughout both the appendicular and axial skeletons in the CKO mice, including the calvaria. Alterations to the nasal area and teeth suggest a potentially reduced capacity to sense and process food, while off-target Cre expression in the gastrointestinal tract may indicate an inability to absorb nutrients. Additionally, altered nasal passages and unexplained changes in diaphragmatic muscle support the possibility of hypoxia. We conclude that these mice likely died due to a combination of breathing difficulties, malnutrition, and starvation resulting primarily from skeletal deformities that decreased their ability to sense, gather, and process food.

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.

miR-125a-5p Regulates Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells under Oxidative Stress.

Adipose-derived mesenchymal stem cells (ADSCs) are a well-recognized multilineage stem cell with vital clinical feasibility for tissue regeneration. Extensive evidence indicates that oxidative stress and microRNAs (miRNAs/miRs) play an important role in the osteoinduction of adipose-derived mesenchymal stem cells. In this study, we investigated the mechanism of miR-125a-5p in regulating the osteogenesis of human adipose-derived mesenchymal stem cells (hADSCs) under oxidative stress. The expression of miR-125a-5p lessened gradually during the osteogenic differentiation of hADSCs. Relative to the negative group, the expression levels of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osterix in the miR-125a-5p group were marked lower than those in the miR-125a-5p inhibitor group. The levels of p16, p21, p53, miR-125a-5p, and ROS during osteoinduction of hADSCs were assessed in vitro under oxidative stress and were observed to be upregulated. Further experiments showed that oxidative stress and miR-125a-5p together suppressed the expression of VEGF during osteogenic differentiation of hADSCs and that the inhibition of miR-125a-5p reversed the effect of oxidative stress. In short, our study indicated that miR-125a-5p is induced under oxidative stress and inhibits the expression of VEGF, leading to the reduction of osteogenic differentiation of hADSCs. Our outcomes showed that miR-125a-5p could be a potential clinical target for bone repairing.

7,8-Dihydroxyflavone modulates bone formation and resorption and ameliorates ovariectomy-induced osteoporosis.

Imbalances in bone formation and resorption cause osteoporosis. Mounting evidence supports that brain-derived neurotrophic factor (BDNF) implicates in this process. 7,8-Dihydroxyflavone (7,8-DHF), a plant-derived small molecular TrkB agonist, mimics the functions of BDNF. We show that both BDNF and 7,8-DHF promoted the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells. These effects might be attributed to the activation of the Wnt/ß-catenin signaling pathway as the expression of cyclin D1, phosphorylated-glycogen synthase kinase-3ß (p-GSK3ß), ß-catenin, Runx2, Osterix, and osteoprotegerin (OPG) was all significantly up-regulated. Knockdown of ß-catenin restrained the up-regulation of Runx2 and Osterix stimulated by 7,8-DHF. In particular, blocking TrkB by its specific inhibitor K252a suppressed 7,8-DHF-induced osteoblastic proliferation, differentiation, and expression of osteoblastogenic genes. Moreover, BDNF and 7,8-DHF repressed osteoclastic differentiation of RAW264.7 cells. The transcription factor c-fos and osteoclastic genes such as tartrate-resistant acid phosphatase (TRAP), matrix metalloprotein-9 (MMP-9), Adamts5 were inhibited by 7,8-DHF. More importantly, 7,8-DHF attenuated bone loss, improved trabecular microarchitecture, tibial biomechanical properties, and bone biochemical indexes in an ovariectomy (OVX) rat model. The current work highlights the dual regulatory effects that 7,8-DHF exerts on bone remodeling.

C-C chemokine receptor type 6 modulates the biological function of osteoblastogenesis by altering the expression levels of Osterix and OPG/RANKL.

Circulating inflammatory factors affect osteoblast and osteoclast formation and activity in osteoporosis. Estrogen affects the migration of Th17 cells via the C-C chemokine receptor type 6 (CCR6) and C-C chemokine ligand 20 (CCL20) signaling pathways to modulate bone metabolism; however, it is unclear whether and how CCR6 modulates bone homeostasis. In the present study, CCR6 knockout (CCR6-/-) mice were selected to investigate the effects of CCR6 in the regulation of homeostasis of osteoblasts and osteoclasts. Primary osteoblasts were isolated from the calvarium of newborn CCR6-/- or wild-type mice, followed by osteoblastic differentiation culture in vitro. CCR6 deletion reduced osteoblast activity in terms of alkaline phosphatase (ALP) activity and inhibited osteoblast mineralization according to the results of Alizarin Red S staining, whereas it did not affect the proliferation of osteoblasts. CCR6 deletion inhibited Osterix mRNA expression in osteoblasts during the late stage of mineralization in vitro, while it did not affect mRNA expression levels of runt-related transcription factor 2 (Runx2) and Collagen-1. The ratio of osteoprotegerin (OPG) /receptor activator of nuclear factor κ-Β ligand (RANKL) mRNA level in osteoblasts was decreased by CCR6 deficiency in the culture treated with 1,25(OH)2D3/PGE2, while there was no effect observed in the normal culture environment. The results provide novel insights, such as that CCR6 deletion suppresses osteoblast differentiation by downregulating the expression levels of the transcription factor Osterix, and indirectly promotes osteoclast production by increasing transcription of RANKL. This may be one of the mechanisms via which CCR6 deletion regulates bone metabolism.

Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence.

The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-ß signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-ß signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-ß signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.