Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells

Background. Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. Methods. A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. Results. BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. Conclusions. We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

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Notch Signaling Inhibition by LY411575 Attenuates Osteoblast Differentiation and Decreased Ectopic Bone Formation Capacity of Human Skeletal (Mesenchymal) Stem Cells

Stem Cells International ◽

10.1155/2019/3041262 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Nihal AlMuraikhi ◽

Dalia Ali ◽

Radhakrishnan Vishnubalaji ◽

Muthurangan Manikandan ◽

Muhammad Atteya ◽

...

Keyword(s):

Gene Expression ◽

Bone Formation ◽

Notch Signaling ◽

Global Gene Expression ◽

Osteoblastic Differentiation ◽

Ectopic Bone Formation ◽

Ectopic Bone ◽

Global Gene Expression Profiling ◽

Mineralized Matrix

Background. Chemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions. Methods. Stem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite–tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo. Results. Among the tested molecules, LY411575, a potent γ-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGFβ, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGFβ1, SPP1, and ERK regulatory networks. Conclusions. We identified γ-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

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3579 The role of CypD/mPTP during osteogenic differentiation - a potential target to prevent bone loss

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.58 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 24-24

Author(s):

Rubens Sautchuk ◽

Brianna H. Shares ◽

Roman A. Eliseev

Keyword(s):

Bone Loss ◽

Bone Formation ◽

Mitochondrial Function ◽

Ectopic Bone Formation ◽

Regulatory Mechanisms ◽

Mrna Levels ◽

Prevent Bone Loss ◽

Ectopic Bone

OBJECTIVES/SPECIFIC AIMS: The study aims to further investigate how cyclophilin D (CypD), the key mPTP opening regulator, affects BMSCs fate and to determine potential regulatory mechanisms involved in CypD regulation during osteogenesis. METHODS/STUDY POPULATION: We evaluated CypD mRNA expression in mouse BMSCs and in osteogenic-like (OL) cells during the course of OB differentiation. CypD protein level was also probed. Moreover, BMSCs had their mPTP activity recorded during osteoinduction. We further analyzed the effect of CypD genetic deletion on osteogenesis in vitro and in vivo. For our in vivo model, we performed the ectopic bone formation assay to asses differences in ossicle formation when CypD KO BMSCs were transplanted compared to wild type littermate BMSCs. In our in vitro model, we transfected OL cells with either CypD gain of function or CypD loss of function vector and measured their osteogenic differentiation potential. Additionally, we treated BMSCs with CypD inhibitor and compare to non-treated BMSCs for mineralization level. To determine potential regulatory mechanisms involved in CypD regulation, we analyzed the CypD gene (Ppif) promoter for potential transcription factor (TF) binding sites and found multiple Smad-binding elements within this promoter. Smads (Smad1, 5, 8) are TFs downstream from Bone Morphogenic Protein (BMP) signaling pathway that transmit cell differentiation signaling, and exert either activating or inhibitory effects on a variety of genes. We also transfect OL cells with Smad1 vector and analyzed for CypD mRNA levels. RESULTS/ANTICIPATED RESULTS: - Our data showed that CypD mRNA levels decreased in both primary cells and OL cells at day 7 and day 14 in osteogenic media. - Osteogenic induction also decreased mPTP activity. - In vivo ectopic bone formation assay showed increased ossicle fo DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest that downregulation of CypD increases OB differentiation due to improved OxPhos activity led by mPTP closure. Our results corroborate reports of CypD downregulation and mPTP closure during neuronal differentiation in developing rat brains as well as in cardiomyocyte differentiation in developing mouse hearts. Our studies also suggest a yet unknown mechanism linking differentiation signaling with mitochondrial function – BMP/Smad mediated downregulation of CypD transcription. As initially mentioned, in a previous study, our lab showed that CypD KO mice present higher mitochondrial function and osteogenicity in aged BMSCs and less osteoporosis burden. Taken together, these results suggest that CypD can be a potential target to prevent bone loss in aging.

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BMPER Enhances Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells

Cellular Physiology and Biochemistry ◽

10.1159/000487969 ◽

2018 ◽

Vol 45 (5) ◽

pp. 1927-1939 ◽

Cited By ~ 6

Author(s):

Fei Xiao ◽

Chuandong Wang ◽

Chenglong Wang ◽

Yuan Gao ◽

Xiaoling Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Formation ◽

Bone Repair ◽

Ectopic Bone Formation ◽

Bmp Signaling ◽

Coupling Process ◽

Ectopic Bone

Background/Aims: During bone repair and remodeling, osteogenesis is coupled with angiogenesis. Bone morphogenetic protein (BMP) antagonists are important modulators of BMP signaling and bone homeostasis. Several investigations have demonstrated that one ‘BMP antagonist’, BMP-binding endothelial cell precursor-derived regulator (BMPER), participates in the regulation of BMP signaling. In this study, we examined the role of BMPER in the osteogenesis-angiogenesis coupling process. Methods: Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were used in this experiment. After overexpressing or silencing BMPER with lentiviruses or siRNA, hBMSCs were stimulated by BMP-2, and osteogenic differentiation activity was detected by alkaline phosphatase and alizarin red staining. VEGF and endostatin release were assessed by ELISA. HUVEC migration was detected by the cell scratch test and transwell migration assay, and in vitro angiogenesis was determined by the tube formation assay. Bone formation was assessed using in vivo femoral monocortical defect and ectopic bone formation models. Results: BMP-2 upregulated BMPER expression. Overexpression of BMPER remarkably enhanced BMP-2-induced osteogenic differentiation, while suppression of BMPER effectively inhibited this process both in vitro and in vivo. In addition, overexpression of BMPER promoted BMP-2-induced VEGF expression in vitro and vascularization in the ectopic bone formation model. Conclusion: BMPER functions as a positive regulator of the osteogenesis-angiogenesis coupling process in hBMSCs, suggesting a novel therapeutic role of BMPER in the regenerative capacity of bone repair.

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Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

Scientific Reports ◽

10.1038/s41598-020-73439-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Nuha Almasoud ◽

Sarah Binhamdan ◽

Ghaydaa Younis ◽

Hanouf Alaskar ◽

Amal Alotaibi ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Expression Profiling ◽

Osteoblast Differentiation ◽

Global Gene Expression ◽

Osteoblastic Differentiation ◽

Global Gene Expression Profiling ◽

Mineralized Matrix

Abstract Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

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Evaluation of a Thiolated Chitosan Scaffold for Local Delivery of BMP-2 for Osteogenic Differentiation and Ectopic Bone Formation

BioMed Research International ◽

10.1155/2013/878930 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 22

Author(s):

In-Ho Bae ◽

Byung-Chul Jeong ◽

Min-Suk Kook ◽

Sun-Hun Kim ◽

Jeong-Tae Koh

Keyword(s):

Bone Formation ◽

Release Kinetics ◽

Ectopic Bone Formation ◽

Bone Morphogenetic Protein 2 ◽

Osteoblastic Cell ◽

Mineral Density ◽

Thiolated Chitosan ◽

Ectopic Bone

Thiolated chitosan (Thio-CS) is a well-established pharmaceutical excipient for drug delivery. However, its use as a scaffold for bone formation has not been investigated. The aim of this study was to evaluate the potential of Thio-CS in bone morphogenetic protein-2 (BMP-2) delivery and bone formation.In vitrostudy showed that BMP-2 interacted with the Thio-CS and did not affect the swelling behavior. The release kinetics of BMP-2 from the Thio-CS was slightly delayed (70%) within 7 days compared with that from collagen gel (Col-gel, 85%), which is widely used in BMP-2 delivery. The BMP-2 released from Thio-CS increased osteoblastic cell differentiation but did not show any cytotoxicity until 21 days. Analysis of thein vivoectopic bone formation at 4 weeks of posttransplantation showed that use of Thio-CS for BMP-2 delivery induced more bone formation to a greater extent (1.8 fold) than that of Col-gel. However, bone mineral density in both bones was equivalent, regardless of Thio-CS or Col-gel carrier. Taken together, Thio-CS system might be useful for delivering osteogenic protein BMP-2 and present a promising bone regeneration strategy.

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Phosphate graphene as an intrinsically osteoinductive scaffold for stem cell-driven bone regeneration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815434116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4855-4860 ◽

Cited By ~ 23

Author(s):

Anne M. Arnold ◽

Brian D. Holt ◽

Leila Daneshmandi ◽

Cato T. Laurencin ◽

Stefanie A. Sydlik

Keyword(s):

Bone Formation ◽

Bone Regeneration ◽

Bone Cells ◽

Standard Of Care ◽

Ectopic Bone Formation ◽

Bone Morphogenetic Protein 2 ◽

Ectopic Bone ◽

Human Bone Morphogenetic Protein

Synthetic, resorbable scaffolds for bone regeneration have potential to transform the clinical standard of care. Here, we demonstrate that functional graphenic materials (FGMs) could serve as an osteoinductive scaffold: recruiting native cells to the site of injury and promoting differentiation into bone cells. By invoking a Lewis acid-catalyzed Arbuzov reaction, we are able to functionalize graphene oxide (GO) to produce phosphate graphenes (PGs) with unprecedented control of functional group density, mechanical properties, and counterion identity. In aqueous environments, PGs release inducerons, including Ca2+ and PO43−. Calcium phosphate graphene (CaPG) intrinsically induces osteogenesis in vitro and in the presence of bone marrow stromal cells (BMSCs), can induce ectopic bone formation in vivo. Additionally, an FGM can be made by noncovalently loading GO with the growth factor recombinant human bone morphogenetic protein 2 (rhBMP-2), producing a scaffold that induces ectopic bone formation with or without BMSCs. The FGMs reported here are intrinsically inductive scaffolds with significant potential to revolutionize the regeneration of bone.

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