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. 2019 Nov 21:2019:3435901.
doi: 10.1155/2019/3435901. eCollection 2019.

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

Nihal AlMuraikhi  1 , Nuha Almasoud  1 , Sarah Binhamdan  1 , Ghaydaa Younis  1 , Dalia Ali  2 , Muthurangan Manikandan  1 , Radhakrishnan Vishnubalaji  3 , Muhammad Atteya  1   4 , Abdulaziz Siyal  1 , Musaad Alfayez  1 , Abdullah Aldahmash  1 , Moustapha Kassem  1   2   5 , Nehad M Alajez  3
Affiliations

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

Nihal AlMuraikhi et al. Stem Cells Int. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of BMS-833923 treatment on the osteoblast differentiation of human bone marrow skeletal (mesenchymal) stem cells (hMSCs). (a) Representative alkaline phosphatase (ALP) staining of BMS-833923-treated hMSCs (3.0 μM) on day 10 postosteoblastic differentiation compared to DMSO-treated control cells. Magnification: 10x. (b) Quantification of ALP activity in BMS-833923-treated hMSCs (3.0 μM) on day 10 postosteoblastic differentiation compared to DMSO-treated control cells. (c) Assay for cell viability using alamarBlue assay BMS-833923-treated hMSCs (3.0 μM) on day 10 postosteoblastic differentiation compared to DMSO-treated control cells. Data are presented as mean ± SEM (n = 16). DMSO: dimethyl sulfoxide.
Figure 2
Figure 2
Effects of BMS-833923 treatment on human bone marrow skeletal (mesenchymal) stem cell (hMSC) functions in vitro. hMSCs were induced to osteoblast differentiation in the presence of BMS-833923 (3.0 μM) or vehicle (DMSO), and osteoblast differentiation was determined by cytochemical staining with Alizarin red for an in vitro formed mineralized matrix (a) and expression of osteoblast-specific genes by quantitative RT-PCR (b). Magnification: 10x. Data are presented as mean ± SEM, n = 6. ∗P < 0.05; ∗∗∗P < 0.0005. (c) Expression of GLI1 and PCTH1 in hMSCs treated with BMS-833923 (3.0 μM) for 24 hours and measured using qRT-PCR, n = 6. Abbreviations: ALP—alkaline phosphatase; COL1A1—collagen Type I Alpha 1; ON—osteonectin; DMSO—dimethyl sulfoxide; GLI1—GLI Family Zinc Finger 1; PTCH1—patched 1.
Figure 3
Figure 3
BMS-833923 exerts significant effects on multiple signaling pathways during osteoblast differentiation of human bone marrow skeletal (mesenchymal) stem cells (hMSCs). hMSCs were induced to osteoblast differentiation in the presence of BMS-833923 (3.0 μM) or vehicle (DMSO). (a) Heat map and unsupervised hierarchical clustering performed on differentially expressed genes during osteoblastic differentiation. (b) Pie chart illustrating the distribution of selected intracellular signaling pathways enriched in the downregulated genes identified in BMS-833923-treated hMSCs compared to DMSO-treated control cells. (c) Validation of a selected panel of downregulated genes in BMS-833923-treated hMSCs compared to DMSO-treated control using qRT-PCR. Gene expression was normalized to β-actin. Data are presented as mean fold change ± SEM (n = 6), ∗∗∗P < 0.0001.
Figure 4
Figure 4
Bioinformatic analysis of signaling networks regulated in BMS-833923-treated human bone marrow skeletal (mesenchymal) stem cells (hMSCs). (a) Disease and function heat map depicting activation (red) or inhibition (blue) of the indicated functional and disease categories identified in the downregulated transcripts in BMS-833923-treated hMSCs. (b) Heat map illustrating the tissue development functional category. (c) Illustration of the TNF and (d) NFκB networks with predicted activated state based on transcriptome data. Figure legend illustrates the interaction between molecules within the network. (e) Illustration of the connective tissue disorders, organismal injury and abnormalities, and skeletal and muscular disorder network.
Figure 5
Figure 5
BMS-833923 reduces in vivo ectopic bone formation. BMS-833923-treated human bone marrow skeletal (mesenchymal) stem cells (hMSCs) and vehicle-treated control cells were implanted with hydroxyl apatite/tricalcium phosphate (HA/TCP) subcutaneously into immune deficient mice. The histology of in vivo bone formation was examined in H&E- (a) and Sirius red- (b) stained sections. Black arrows in (a) refer to formed bone. In Sirius red-stained slides (b), red color identifies collagen tissue staining. Magnification: 10x (first row; scale bar = 200 μm) and 20x (second row; scale bar = 100 μm). n = 3 implants/treatment. ∗∗P < 0.001; ∗∗∗P < 0.0001. H&E: hematoxylin and eosin.
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