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

Abstract

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.

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

“…Small molecule inhibitors targeting intracellular signaling pathways have been employed as chemical tools to determine the molecular mechanisms controlling stem cell proliferation and differentiation 25,30 . Our group has employed this chemical biology approach to identify a number of molecular targets and pathways that are www.nature.com/scientificreports/ www.nature.com/scientificreports/ important for osteoblast and adipocyte differentiation of hMSCs 25,[27][28][29]31,34,35 . In the current study, we identified XAV-939 small molecule inhibitor, during small molecule library functional screen, as an enhancer of osteoblast differentiation of hMSCs 25 .…”

“…Human skeletal (mesenchymal) stem cells (hMSCs) are multipotent stem cells that have the potential to proliferate and differentiate into various cell types including bone-forming osteoblasts 23 , 24 . The osteoblastic differentiation of hMSCs involves various signaling pathways including Tankyrase 4 , JAK-STAT signaling 25 , Wnt/β-catenin 26 , TGFβ 27 , Notch signaling 28 , and Hedgehog signaling 29 . However, the relative contribution of these signaling pathways on osteoblastic differentiation remained to be determined.…”

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

“…Ethical approval for all animal experiments was obtained from the Animal Care Committee of King Saud University. In vivo experiments were performed as previously described [19]. In brief, cells were trypsinized to a single-cell suspension and resuspended in culture medium with/without the small molecule inhibitor, BMS-833923.…”

Influence of the TGF-β Superfamily on Osteoclasts/Osteoblasts Balance in Physiological and Pathological Bone Conditions

“…The role played by Notch in both osteoclastogenesis, as well as osteoblast differentiation, remains controversial due to discrepancy in the results obtained by several studies due to the experimental design, cell source, and operating conditions [ 311 , 313 , 314 , 315 ].…”