Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

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.

Deubiquitinase USP17 Regulates Osteoblast Differentiation by Increasing Osterix Protein Stability.

Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme, specifically known as ubiquitin-specific protease 17, which plays a critical role in regulating protein stability and cellular signaling pathways. However, the role of USP17 during osteoblast differentiation has not been elusive. In this study, we initially investigated whether USP17 could regulate the differentiation of osteoblasts. Moreover, USP17 overexpression experiments were conducted to assess the impact on osteoblast differentiation induced by bone morphogenetic protein 4 (BMP4). The positive effect was confirmed through alkaline phosphatase (ALP) expression and activity studies since ALP is a representative marker of osteoblast differentiation. To confirm this effect, Usp17 knockdown was performed, and its impact on BMP4-induced osteoblast differentiation was examined. As expected, knockdown of Usp17 led to the suppression of both ALP expression and activity. Mechanistically, it was observed that USP17 interacted with Osterix (Osx), which is a key transcription factor involved in osteoblast differentiation. Furthermore, overexpression of USP17 led to an increase in Osx protein levels. Thus, to investigate whether this effect was due to the intrinsic function of USP17 in deubiquitination, protein stabilization experiments and ubiquitination analysis were conducted. An increase in Osx protein levels was attributed to an enhancement in protein stabilization via USP17-mediated deubiquitination. In conclusion, USP17 participates in the deubiquitination of Osx, contributing to its protein stabilization, and ultimately promoting the differentiation of osteoblasts.

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.

YY2 Promotes Osteoblast Differentiation by Upregulating Osterix Transcriptional Activity.

Yin Yang 2 (YY2) is a paralog of YY1, a well-known multifunctional transcription factor containing a C-terminal zinc finger domain. Although the role of YY1 in various biological processes, such as the cell cycle, cell differentiation and tissue development, is well established, the function of YY2 has not been fully determined. In this study, we investigated the functional role of YY2 during osteoblast differentiation. YY2 overexpression and knockdown increased and decreased osteoblast differentiation, respectively, in BMP4-induced C2C12 cells. Mechanistically, YY2 overexpression increased the mRNA and protein levels of Osterix (Osx), whereas YY2 knockdown had the opposite effect. To investigate whether YY2 regulates Osx transcription, the effect of YY2 overexpression and knockdown on Osx promoter activity was evaluated. YY2 overexpression significantly increased Osx promoter activity in a dose-dependent manner, whereas YY2 knockdown had the opposite effect. Furthermore, vectors containing deletion and point mutations were constructed to specify the regulation site. Both the Y1 and Y2 sites were responsible for YY2-mediated Osx promoter activation. These results indicate that YY2 is a positive regulator of osteoblast differentiation that functions by upregulating the promoter activity of Osx, a representative osteogenic transcription factor in C2C12 cells.

Sp7 Transgenic Mice with a Markedly Impaired Lacunocanalicular Network Induced Sost and Reduced Bone Mass by Unloading.

The relationship of lacunocanalicular network structure and mechanoresponse has not been well studied. The lacunocanalicular structures differed in the compression and tension sides, in the regions, and in genders in wild-type femoral cortical bone. The overexpression of Sp7 in osteoblasts resulted in thin and porous cortical bone with increased osteoclasts and apoptotic osteocytes, and the number of canaliculi was half of that in the wild-type mice, leading to a markedly impaired lacunocanalicular network. To investigate the response to unloading, we performed tail suspension. Unloading reduced trabecular and cortical bone in the Sp7 transgenic mice due to reduced bone formation. Sost-positive osteocytes increased by unloading on the compression side, but not on the tension side of cortical bone in the wild-type femurs. However, these differential responses were lost in the Sp7 transgenic femurs. Serum Sost increased in the Sp7 transgenic mice, but not in the wild-type mice. Unloading reduced the Col1a1 and Bglap/Bglap2 expression in the Sp7 transgenic mice but not the wild-type mice. Thus, Sp7 transgenic mice with the impaired lacunocanalicular network induced Sost expression by unloading but lost the differential regulation in the compression and tension sides, and the mice failed to restore bone formation during unloading, implicating the relationship of lacunocanalicular network structure and the regulation of bone formation in mechanoresponse.

Osteoblasts pattern endothelium and somatosensory axons during zebrafish caudal fin organogenesis.

Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones and endothelium. In adult zebrafish, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development we show that the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.

A neomorphic variant in SP7 alters sequence specificity and causes a high-turnover bone disorder.

SP7/Osterix is a transcription factor critical for osteoblast maturation and bone formation. Homozygous loss-of-function mutations in SP7 cause osteogenesis imperfecta type XII, but neomorphic (gain-of-new-function) mutations of SP7 have not been reported in humans. Here we describe a de novo dominant neomorphic missense variant (c.926 C > G:p.S309W) in SP7 in a patient with craniosynostosis, cranial hyperostosis, and long bone fragility. Histomorphometry shows increased osteoblasts but decreased bone mineralization. Mice with the corresponding variant also show a complex skeletal phenotype distinct from that of Sp7-null mice. The mutation alters the binding specificity of SP7 from AT-rich motifs to a GC-consensus sequence (typical of other SP family members) and produces an aberrant gene expression profile, including increased expression of Col1a1 and endogenous Sp7, but decreased expression of genes involved in matrix mineralization. Our study identifies a pathogenic mechanism in which a mutation in a transcription factor shifts DNA binding specificity and provides important in vivo evidence that the affinity of SP7 for AT-rich motifs, unique among SP proteins, is critical for normal osteoblast differentiation.

REV-ERBs negatively regulate mineralization of the cementoblasts.

OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) are dysregulated in periodontitis development and involved in osteogenesis. The current study was aimed at investigating the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms. METHODS: Firstly, the level of ANRIL was tested by qPCR. Then, PDLCs were treated with a mineralizing solution to induce osteogenic differentiation. ALP activity was measured, and protein levels of BMP2, Osterix, and OCN were measured by Western blot. A target of ANRIL was verified using dual-luciferase reporter assay. miR-7 level was measured by qPCR, and the signals of the NF-κB pathway were tested by Western blot. RESULTS: ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were increased to show that PDLCs were differentiated. ANRIL level was increased in differential PDLCs, in which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7. CONCLUSIONS: Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

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.

Zfhx4 regulates endochondral ossification as the transcriptional platform of Osterix in mice.

Endochondral ossification is regulated by transcription factors that include SRY-box transcription factor 9, runt-related protein 2 (Runx2), and Osterix. However, the sequential and harmonious regulation of the multiple steps of endochondral ossification is unclear. This study identified zinc finger homeodomain 4 (Zfhx4) as a crucial transcriptional partner of Osterix. We found that Zfhx4 was highly expressed in cartilage and that Zfhx4 deficient mice had reduced expression of matrix metallopeptidase 13 and inhibited calcification of cartilage matrices. These phenotypes were very similar to impaired chondrogenesis in Osterix deficient mice. Coimmunoprecipitation and immunofluorescence indicated a physical interaction between Zfhx4 and Osterix. Notably, Zfhx4 and Osterix double mutant mice showed more severe phenotype than Zfhx4 deficient mice. Additionally, Zfhx4 interacted with Runx2 that functions upstream of Osterix. Our findings suggest that Zfhx4 coordinates the transcriptional network of Osterix and, consequently, endochondral ossification.

Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin.

Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases.

Sp6/Epiprofin is a master regulator in the developing tooth.

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.

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.

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.

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.