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

Abstract

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.

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

“…Expression of SMAD4, which has a major role in regulating osteoblast viability and bone homeostasis 45 , was also significantly increased in hBMSCs treated with XAV-939. In addition, we identified TNF, NFκB, and STAT signaling among the top activated signaling pathways in XAV-939-treated hBMSCs; all are known to play a role in osteoblast differentiation and bone formation 29 .…”

“…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 important for osteoblast and adipocyte differentiation of hMSCs 25 , 27 – 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 .…”

“…Two-fold cutoff and p(corr) < 0.05 (Benjamini–Hochberg multiple testing corrected) were used to define significantly changed transcripts. Pathway and functional annotation analyses were conducted using the Ingenuity Pathway Analysis (Ingenuity Systems, https://www.ingenuity.com/ ) 29 , 71 . upregulated genes ≤ 2 FC (fold change) and corrected p value < 0.05 were chosen for analysis.…”

“…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.…”

Syndecan-1 Facilitates the Human Mesenchymal Stem Cell Osteo-Adipogenic Balance

“…Interestingly, the induction of BMP2 and PTCH expression in undifferentiated SDC-1 KD cultures suggests the BMP and SHH signalling pathways are activated. BMP2 is a predominantly pro-osteogenic factor for hMSCs [44,45] and SHH signalling is also required for hMSC osteogenic differentiation [46,47]. When undifferentiated SDC-1 KD cultures were differentiated, these cultures appeared to have enhanced osteogenic differentiation, with increased von Kossa and Alizarin Red staining.…”