Inhibition of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) stimulates osteoblastogenesis by potentiating bone morphogenetic protein 2 (BMP2) responses

Abstract

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA-PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA-PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP2) response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast-specific conditional Prkdc-knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA-PKcs negatively regulates osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.

1. INTRODUCTION

DNA-dependent protein kinase (DNA-PK) is a multiprotein complex that plays a critical role in DNA repair, cell cycle control, and transcription regulation (Goodwin & Knudsen, 2014). The core component of DNA-PK is represented by the catalytic subunit, DNA-PKcs, which is a serine/threonine kinase encoded by the protein kinase, DNA-activated, catalytic (Prkdc) gene and belongs to the phosphatidylinositol-3 kinase-related kinase subfamily (Collis, DeWeese, Jeggo, & Parker, 2005). DNA-PKcs is a major component of the DNA repair/V(D)J recombination machinery and class switching recombination in lymphocytes (Gao et al., 1998). As a result, absence of DNA-PKcs results in severe combined immunodeficient mice and is associated with an increase in sensitivity to DNA-damaging agents (Gustafsson, Abramenkovs, & Stenerlow, 2014). Emerging evidence links DNA-PKcs to functions beyond double-strand breaks (DSBs) repair, such as cell cycle progression (K. J. Lee et al., 2011; Watanabe et al., 2003), signal transduction (Xu, Fang, Zhu, Zhu, & Zhou, 2014), and transcriptional regulation (Goodwin & Knudsen, 2014).

There is evidence suggesting that DNA-PKcs activates Protein Kinase B/AKT to stimulate osteoblastogenesis in response to low dose irradiation (Xu et al., 2014). Similarly, an inhibitor of the 26S proteasome was shown to increase DNA-PKcs amounts and reduce radiation-induced apoptosis of cultured osteoblastic cells (Chandra et al., 2018). But whether DNA-PKcs plays a role to control osteoblastogenesis in the absence of ionizing radiation remains to be determined.

DNA-PKcs is very abundant in mammalian cells (Hartley et al., 1995), suggesting it has biological functions beyond those associated with non-homologous end joining. This broad expression may preclude long-term inhibition of the enzyme, but it is conceivable that a potential safe therapeutic window for transient inhibition of DNA-PKcs activity in short-term local bone-related applications could be identified.

Here we show that downregulation of DNA-PKcs expression or pharmacological inhibition of its activity caused cell cycle blockage, significantly increased osteoblast differentiation and extracellular matrix formation, and potentiated cell responsiveness to the bone morphogenetic protein 2 (BMP2). In vivo pharmacological DNA-PKcs inhibition and osteoblast-specific knockout of Prkdc enhanced bone biomechanical properties. Taken together, these results support DNA-PKcs as a potential pharmacological target for bone regeneration.

2. MATERIALS and METHODS

2.1. Cell culture –

All cell lines were cultured in a humidified 5% CO₂ incubator at 37°C and maintained with a cocktail of 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Life Technologies, Grand Island, NY). The calvaria-derived pre-osteoblast cell line MC3T3-E1 (subclone 4) (D. Wang et al., 1999) (generously provided by Dr. Renny T. Franceschi, University of Michigan, Ann Arbor, MI; RRID: CVCL_5440) were maintained in αMEM (alpha minimum essential media) supplemented with 10% fetal bovine serum (FBS) (Gibco). Patient-derived human adipose-derived mesenchymal stromal cells (AMSCs) were harvested from the perivascular stromal fraction of patients undergoing elective lipo-aspiration. Patients were consented and the study was approved by the Mayo Clinic institutional review board (IRB# 07–008842). The cells presented here were extensively characterized in previous studies (Dudakovic et al., 2014; Dudakovic, Camilleri, Lewallen, et al., 2015). AMSCs were maintained in advanced MEM medium containing 5% PLTMax (a clinical grade commercial platelet lysate product; Mill Creek Life Sciences, Rochester, MN), 2mM Glutamax, 2 U/ml heparin (hospital pharmacy). C2C12 mouse myoblast cells (ATCC Cat# CRL-1772, RRID: CVCL_0188) and human embryonic kidney 293 (HEK293T) cells (American Type Culture Collection, Manassas, VA; ATCC Cat# CRL-3216, RRID: CVCL_0063) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% FBS.

2.2. Osteogenic differentiation and treatment –

Cells were plated in complete αMEM medium (Gibco) and grown until confluency. To induce differentiation, the culture medium was freshly supplemented with 50 μg/ml ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich). NU7441 (1 μM; Selleckchem, Houston, TX), mimosine (200 μM; Sigma-Aldrich) or vehicle (DMSO) (Sigma-Aldrich) were added to the media. Cells were harvested for total RNA or protein extraction at the indicated time points. Where indicated, 300 ng/ml recombinant human bone morphogenetic protein 2 (rhBMP2, Sigma-Aldrich) or vehicle (H₂O) was added for 48 h prior to RNA isolation and for 2 h prior to protein extraction. For the AMSCs, the culture medium was replaced with osteogenic medium (containing human osteogenic supplement) and treated with NU7441 or DMSO.

2.3. Western blot analysis –

Whole cell extracts were isolated using radioimmune precipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich). Protein concentrations were determined by the Bradford assay kit (ThermoFisher Scientific) and equal amounts of cell lysates were subjected to SDS-PAGE gel electrophoresis and Western blot analysis using standard protocols. Primary antibodies were used as follows: rabbit anti-DNA-PKcs (1:1000, Abcam Cat# 3922–1, RRID: AB_10896006); rabbit anti-phosphorylated-SMAD1 (Ser463/465)/SMAD5 (Ser463/465) (pSMAD1/5) (1:1000, Cell Signaling Technology Cat# 9516, RRID: AB_491015), rabbit anti-SMAD1 (1:1000, Cell Signaling Technology Cat# 6944, RRID: AB_10858882). Loading control proteins were α-Tubulin (1:5000) (Sigma-Aldrich Cat# T6074, RRID:AB_477582) or GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) (1:1000, Cell Signaling Technology Cat# 2118, RRID: AB_561053). HRP-conjugated secondary antibodies and dilutions were: goat anti-mouse IgG (1:10 000, Santa Cruz, Dallas, TX) and goat anti-rabbit IgG (1:10 000, Santa Cruz).

2.4. Total RNA Extraction, reverse transcription, and quantitative PCR –

Total RNA was extracted from cells with TRIzol reagent (Invitrogen, ThermoFisher Scientific) according to the manufacturer’s protocol. One (1) μg total RNA was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific Applied Biosystems). cDNA was used for RT-qPCR carried out in a 7500 real-time PCR system (ThermoFisher Scientific Applied Biosystems) using TaqMan universal PCR master mix and gene-specific TaqMan probes (Table 4). Gene expression in AMSCs was quantified using quantitative PCR with QuantiTect SYBR Green PCR Kit (Qiagen, Mississauga, ON) and the CFX384 Real-Time System (BioRad) (Mayo Clinic). All experiments were performed in triplicate and transcript levels were normalized to control genes using the comparative CT method (Livak & Schmittgen, 2001).

Table 4.

List of TaqMan probes

Gene	Probe ID
Actb	Mm0007939_s1
Alpl	Mm00475834_m1
B2M	Mm00437762_m1
Bglap	AIWR1XJ
Col1a1	Mm00801666_g1
Dlx5	Mm00438430_m1
Gapdh	Mm03302249_g1
Ibsp	Mm00492555_m1
Id3	Mm01188138_g1
Opn	Mm0043767_m1
Prkdc	Mm01342967_m1
Smad1	Mm00484721_m1
Smad5	Mm01341687_g1
Smad6	Mm000484738_m1
Sp7	Mm04209856_m1

2.5. Alizarin red S and von Kossa staining –

Extracellular matrix calcification was estimated using Alizarin Red S (Puchtler, Meloan, & Terry, 1969) or von Kossa staining. Cells were fixed with 4% formaldehyde for 15 min at room temperature, rinsed in deionized water, and then stained in Alizarin red for 20 min at room temperature. Stained cells were extensively washed with deionized water. For quantification, cells were incubated with 800 μl of a 10% acetic acid solution for 30 min with gentle shaking. Then, 500 μl of the mixture was added to 100 μl NH₄OH and optical density was read on a spectrophotometer at a wavelength of 450 nm. For von Kossa staining, the cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 5% AgNO3 (Sigma-Aldrich) for 5 min at room temperature.

2.6. Short hairpin RNA (shRNA) Gene Silencing –

the shRNAs vector against mouse Prkdc (TRCN0000194985 [shPrkdc#4]) was obtained from Sigma-Aldrich. shRNAs pLKO.1 lentiviral plasmids were used as non-targeting shRNA control (SHC002). Lentiviral particles and infection was carried out by co-transfecting HEK293T cells with 2.5 μg of pMD2.G (viral envelope encoding vector, a gift from Dr. Didier Trono; Addgene plasmid #12259, RRID: Addgene_12259) and 7.5 μg psPAX2 (viral packaging vector, a gift from Dr. Didier Trono; Addgene plasmid #12260, RRID: Addgene_12260) together with 10 μg of the different constructs using the calcium phosphate method (Chen & Okayama, 1987). To generate stable cell lines, the media containing viral particles was filtered using a 0.45 μm filter (Millipore, Billerica, MA) and lentiviruses were transduced into MC3T3-E1 cells in the presence of 4 μg/ml polybrene (Sigma-Aldrich) 48 h post-infection. Control cells were transfected with empty pTRIPZ vector. Twenty-four (24) h post-infection, infected cells were selected with 5 μg/mL puromycin (Sigma-Aldrich) for 72 h and positive clones were established.

2.7. RNA-seq –

MC3T3-E1 cells were grown in αMEM (Gibco) supplemented with 10% FBS (Gibco) to confluency, followed by a treatment with either NU7441 (1 μM) (Selleckchem) or DMSO (Sigma) for 16h. Cells were harvested, and total RNA was isolated using mRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Triplicates from each of the three independent experiments were pooled together, quantified, and assessed by RNA-Seq as described with modifications (Dudakovic et al., 2014). RNA libraries were prepared according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina). Briefly, poly-A mRNA was purified from total RNA using oligo dT magnetic beads. Purified mRNA was fragmented at 95°C for 8 minutes, eluted from the beads and primed for first strand cDNA synthesis. RNA fragments were copied into first strand cDNA using SuperScript III reverse transcriptase and random primers (Invitrogen). Next, second strand cDNA synthesis was performed using DNA polymerase I and RNase H. The double-stranded cDNA was purified using a single AMPure XP bead (Agencourt) clean-up step. The cDNA ends were repaired and phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide kinase followed by a single AMPure XP bead clean-up. Blunt-ended cDNAs were modified to include a single 3’ adenylate (A) residue using Klenow exo- (3’ to 5’ exo minus). Paired-end DNA adaptors (Illumina) with a single “T” base overhang at the 3’ end were immediately ligated to the ‘A tailed’ cDNA population. Unique indexes, included in the standard TruSeq Kits (12-Set A and 12-Set B) were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. The resulting constructs were purified by two consecutive AMPure XP bead clean-up steps. The adapter-modified DNA fragments were enriched by 12 cycles of PCR using primers included in the Illumina Sample Prep Kit. The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip. A final quantification, using Qubit fluorometry (Invitrogen), confirmed sample concentrations.

Libraries were loaded onto flow cells at concentrations of 8–10 pM to generate cluster densities of 700,000/mm² following the standard protocol for the Illumina cBot and cBot Paired end cluster kit version 3. Flow cells were sequenced as 51 × 2 paired end reads on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and HCS v2.0.12 data collection software. Base-calling was performed using Illumina’s RTA version 1.17.21.3 (RRID: SCR_014332). The RNA-Seq data were analyzed using the standard RNA-Seq workflow by Mayo Bioinformatics Core called MAPRSeq v.1.2.1 (Kalari et al., 2014), which includes alignment with TopHat 2.0.6 (RRID: SCR_013035) (Kim et al., 2013) and quantification of gene expression using the HTSeq software (RRID: SCR_005514) (Anders, Pyl, & Huber, 2015). Normalized gene counts were also obtained from MAPRSeq where expression for each gene were normalized to 1 million reads and corrected for gene length (Fragments Per Kilobase pair per Million mapped reads, FPKM). RNA-Seq data were deposited in the Gene Expression Omnibus of the National Institute for Biotechnology Information (GSE141686).

FPKM values were determined from triplicates from each of the three independent biological replicates. Secondary mRNA-Seq was then analyzed as previously described (Camilleri et al., 2016; Dudakovic et al., 2014; Dudakovic et al., 2016; Dudakovic, Camilleri, Xu, et al., 2015). Briefly, non-coding RNAs were removed and then several filters were applied to compare the expression of vehicle- and NU7441-treated MC3T3-E1 cells. First, genes with a robust expression (FPKM > 0.3) were included in the analysis. Then, genes were selected based on a 1.4-fold-change between the two conditions. Finally, genes with a Student’s t-test p-value of less than 0.05 were selected. Database for Annotation, Visualization, and Integration Discovery (DAVID, RRID: SCR_001881) bioinformatics Resources v6 was used to determine biological pathways that are affected by NU7441 treatment.

2.8. Affymetrix array –

Total RNA was isolated from MC3T3-E1 cells differentiated in osteogenic medium for 14 days in the presence of NU7441 (1 μM) or DMSO (3 biological replicates per condition). Total RNA was purified with RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s guidelines. The steps from RNA amplification and hybridization to raw data preparation were performed by the McGill University Genome Quebec Innovation Center using the Affymetrix Mouse Genome 2.0 Gene ST array platform (Affymetrix, Santa Clara, CA). CEL files containing probe intensity data were analyzed using FlexArray software (version 1.6.2). Briefly, raw data were normalized using Affymetrix power tools (APT). Significant differences between the two groups was detected using the Cyber-T test (p-value ≤ 0.05), and the Benjamini-Hochberg false discovery rate (FDR) adjustment of p-values was applied to correct for multiple testing. Genes with an FDR corrected p-value ≤ 0.05 and fold change ≥ 1.2 or ≤ 0.7, were considered significantly differentially expressed. Gene Ontology (Gene Ontology Consortium, RRID: SCR_017505) (Ashburner et al., 2000) and Kyoto Encyclopedia of Genes and Genomes (KEGG, RRID: SCR_012773) pathway enrichment analysis (Kanehisa & Goto, 2000) were performed using Enrichr (Kuleshov et al., 2016). GO terms with FDR corrected p-value ≤ 0.05 were considered enriched. Affymetrix array data were deposited in the Gene Expression Omnibus of the National Institute for Biotechnology Information (GSE151332).

2.9. Pharmacological inhibition of DNA-PKcs at steady-state in vivo –

Animal use protocol No. 7436 was reviewed and approved by the McGill Institutional Animal Care and Use Committee and followed the guidelines of the Canadian Council on Animal Care. Sixteen (16)-week-old female wild-type C57BL/6 mice (10 mice/group) were injected intraperitoneally with a single daily dose of 10 mg/kg NU7441 (Zhao et al., 2006) or the vehicle (DMSO/40% polyethylene glycol 400 [PEG400]/PBS) for 21 days. Body weight were recorded twice per week for each mouse.

2.10. Three-point bending test –

Mouse femurs were gently covered with PBS-soaked gauze and stored at −20°C until ready for testing. Mechanical properties were assessed using an Instron model 5943 single column table frame machine (Instron, Norwood, MA) as previously described (Martineau et al., 2018). Raw output used for comparison was stiffness (N/mm), maximum load (N), and Young’s modulus (MPa/mm/mm).

2.11. Osteoblast Prkdc-deficient mice –

The Prkdc floxed mice (Prkdc^fl/fl) have been described before (Mishra et al., 2015). Prkdc ^fl/fl were crossed with Osterix-Cre (Osx-Cre) transgenic mice (Rodda & McMahon, 2006). Mice with Prkdc deletion under the Osx promoter Cre driver were generated (Osx-Cre;Prkdc^fl/fl; designated as Prkdc_Ob^−/−). These conditional knockout mice were compared with littermates with a control genotype Prkdc^+/fl or Prkdc^fl/fl, or with heterozygous conditional knockout genotype, Osx-Cre;Prkdc^+/fl. Mice genotyping was conducted by PCR using tail genomic DNA with the PKEx81f (50-CTGGAGCCTATGTGCTAATGTACAG-30) and PKEx82r (5’-CTGTTTCTGTACGGTTAGCTCG GCTG-3’) primers that flank the LoxP sites of the targeting construct (Mishra et al., 2015).

2.12. Micro-computed tomography (μCT) –

Femurs were collected and cleaned of soft tissue, fixed overnight in 4% paraformaldehyde at 4°C, washed with PBS, then dehydrated in 50% ethanol and stored in 70% ethanol until ready for testing. They were analyzed using a SkyScan 1272 high-resolution μCT (Bruker Micro-CT, Kontich, Belgium). Two regions of the mouse femur were evaluated: the secondary spongiosa in the distal metaphysis and the cortical bone in the mid-diaphysis. Scans were acquired at an image pixel size of 6 μm using a voltage of 52 kV, a current of 192 μA, a 0.25 mm aluminum filter, an angular step of 0.4 degrees, an integration time of 2250 ms, an averaging frame of 3 and a 2K resolution. The scans were reconstructed using the SkyScan NRecon Program (Bruker). Reconstructions were performed using a ring artifact reduction value of 8, a smoothing value of 1 and a beam-hardening correction coefficient of 30%. For evaluation of the femur mid-diaphyseal cortical bone, a region of 0.5 mm in length (~90 μCT slices per specimen) at the midshaft was used for the analysis. For evaluation of the secondary spongiosa in the distal metaphysis of the femur, a region of interest including only trabecular bone beginning 1 mm proximal to the growth plate and extending proximally for 2 mm was included in the analysis using SkyScan CTan (Bruker). The manual contouring approach on a slice-by-slice basis was used to delineate the trabecular region of femur, whereas the automated approach was used for the cortical region. The images were thresholded using two global threshold values, followed by a despeckling filter. For trabecular bone regions, we assessed the bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), trabecular number (Tb.N, mm−1), and connectivity. For cortical bone at the femoral midshaft, we measured the cortical bone volume density (BV/TV, %), the cortical thickness (mm), the endosteum perimeter (mm) and mean polar moment of inertia (mm⁴).

2.13. Statistical analysis –

Treatment groups within experiments were performed in triplicate. Error bars on all graphs represent standard error of the mean (SEM). Each experiment was repeated at least three times. Statistical analysis was performed using PRISM 6 (GraphPad, San Diego, CA) to determine significance among treatment groups. Student’s t-test (two-tailed) was utilized for pair-wise comparisons, and ANOVA followed by the Tukey-Kramer post-hoc test was performed for group comparisons. A probability (p) value lower than 0.05 was accepted as significant.

2.14. Data availability –

The RNA-Seq data (Accession number: GSE141686) and the Affymetrix array data (Accession number: GSE151332) were deposited in the Gene Expression Omnibus database of the National Institute for Biotechnology Information.

3. RESULTS

3.1. DNA-PKcs inhibition enhances osteogenic differentiation of MC3T3-E1 cells

We cultured MC3T3-E1 osteoblastic cells in differentiation medium for up to 28 days. DNA-PKcs protein levels remained constant throughout the differentiation cycle (Fig. 1). To test the potential role of DNA-PKcs in osteogenic differentiation, cells were cultured in the presence or absence of 1 μM of the potent and specific DNA-PKcs inhibitor, NU7441 (Leahy et al., 2004). Treatment with NU7441 significantly enhanced the expression of early (Sp7, Alpl, Col1a1, Ibsp) and late (Opn, Bglap) osteoblastic differentiation markers at day 7 and/or day 14 (Fig. 2A). Mineralization was also enhanced by NU7441 (Fig. 2B, C). Similar results were obtained when Prkdc was knocked down by shRNA (Fig. 3A–C). Thus both pharmacological and genetic DNA-PKcs inhibition increased osteoblast differentiation and mineralization.

Figure 1. Expression of DNA-PKcs in osteoblasts.

Western blot analysis of DNA-PKcs expression throughout differentiation of MC3T3-E1 cells. Alpha-tubulin was used as a loading control.

Figure 2. The DNA-PKcs inhibitor NU7441 induces gene expression and matrix mineralization in MC3T3-E1 osteoblastic cells.

Confluent MC3T3-E1 cells were cultured in osteogenic medium with NU7441 (1 μM) or DMSO for the times indicated. (A) Total RNA was isolated from cells at day 7 and 14 of differentiation, and expression of Sp7 (Osterix), Alpl (alkaline phosphatase), Col1a1 (α1 chain of type I collagen), Ibsp (bone sialoprotein), Opn (osteopontin), and Bglap (osteocalcin) was analyzed by RT-qPCR. Values are normalized to Gapdh and expressed relative to vehicle treatment on day 7, which was arbitrarily ascribed a value of 1. (B) The calcium content was visualized by Alizarin Red S (upper) and von Kossa (lower) staining at the indicated differentiation time points. (C) Alizarin red concentration was quantified spectrophotometrically using image analysis software after extraction of the staining from samples. Data are presented as mean ± SEM. Statistical analysis used one-way ANOVA test with post-hoc analysis; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

Figure 3. Prkdc knockdown induces differentiation marker gene expression in MC3T3-E1 cells.

(A) Prkdc knockdown was determined by measuring protein levels (α-Tubulin is used as loading control). (B) MC3T3-E1 cells were infected with either control or Prkdc-expressing lentiviruses. Cells were cultured in osteogenic medium for the times indicated. Total RNA was prepared, and gene expression for Alpl, Col1a1, Ibsp, Bglap, and Opn was analyzed by RT-qPCR. Values are normalized to Gapdh and expressed relative to vehicle treatment on day 7, which was arbitrarily ascribed a value of 1. (C) The calcium content was visualized by Alizarin Red S (upper) and von Kossa (lower) staining. Data are presented as mean ± SEM. Statistical analysis used one-way ANOVA test with post-hoc analysis; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

3.2. Inhibition of DNA-PKcs by NU7441 induces osteogenic differentiation of human adipose-derived mesenchymal stromal cells

To establish that the observed stimulatory effect of NU7441 on osteogenic differentiation was not restricted to the mouse MC3T3-E1 cell line, we tested NU7441 on patient-derived human adipose mesenchymal stromal cells (AMSCs). AMSCs have the capacity to differentiate into osteogenic, chondrogenic, cardiac, and adipogenic tissues depending on the applied stimulus (Ullah, Subbarao, & Rho, 2015). AMSCs were grown in inductive medium for osteogenic differentiation in the presence of different concentrations of NU7441 (0.125 to 1 μM) over a course of 7 days. The viability of AMSCs was not significantly compromised by NU7441 (data not shown). NU7441 up-regulated mRNA levels of ALPL and COL1A1 in a dose-dependent manner with the strongest effect observed following NU7441 treatment at 1 μM for 7 days (Fig. 4A). In addition, NU7441 treatment induced the expression of ID3 (inhibitor of DNA binding 3) and RUNX2 (runt-related transcription factor 2) at day 3 and 7 of differentiation, respectively (Fig. 4B). Pharmacological inhibition of DNA-PKcs in AMSCs increased mineralization (Fig. 4C, D). These results show that NU7441 stimulated osteoblastic differentiation of human mesenchymal stem cells and confirm that the observed effects were not restricted to established murine cell lines.

Figure 4. Inhibition of DNA-PKcs induces osteogenesis of human AMSCs.

(A) Human adipose-derived mesenchymal stromal cells (AMSCs) were grown in osteogenic medium and treated with various concentrations of NU7441 (0.125 to 1 μM) or vehicle (DMSO). ALPL and COL1A1 transcript abundance was determined by RT-qPCR. (B) ID3 and RUNX2 expression was determined by RT-qPCR. Values are normalized to Gapdh and expressed relative to vehicle treatment, which was arbitrarily ascribed a value of 1. (C) The calcium content was visualized by Alizarin Red S staining at day 8 of differentiation. (D) Mineralized area was quantified using Image J. Data are presented as mean ± SEM. Statistical analysis using one-way ANOVA test with post-hoc analysis (panel A) or unpaired two tailed t-test (B, D); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

3.3. Inhibition of DNA-PKcs by NU7441 blocks cell cycle

To further examine the effects of DNA-PKcs inhibition on osteoblastic cells, we compared gene expression profiles in confluent MC3T3-E1 cells treated with NU7441 or vehicle for 16h, using high-throughput RNA-Seq analysis. Results show that 488 genes were down-regulated and 77 genes were up-regulated after NU7441 treatment with an average expression greater than 0.3 FPKM, fold change greater than 1.4-fold; and p ≤ 0.05 (Fig. 5A). Functional annotation analysis using DAVID (Huang da, Sherman, & Lempicki, 2009a, 2009b) (v6.7) indicates that the up-regulated genes are associated with biological processes such as ‘positive regulation of transcription, DNA-templated’ and ‘positive regulation phosphatidylinositol 3-kinase signaling’, which aligns with DNA-PKcs. The primary gene ontology category for the down-regulated genes is cell cycle control (n=147 of 488 total; enrichment score 75.31). Most of these genes are involved in mitosis, cell division and cell cycle (Fig. 5B).

Figure 5. DNA-PKcs inhibitor induces cell cycle block.

(A) Bar graph showing the differentially expressed genes (fold change >1.4, FDR <0.05) determined by RNA-Seq from confluent MC3T3-E1 cells treated with NU7441 (1 μM) or vehicle control (DMSO) for 16h. (B) Biological processes of the differentially expressed genes using DAVID.

We tested whether the osteogenic effect of NU7441 treatment were solely a consequence of a cell cycle block. MC3T3-E1 cells were grown in osteogenic medium and treated with the cell cycle inhibitor mimosine (San Martin et al., 2009), in parallel to treatment with NU7441 for up to 7 days of differentiation. In contrast to the NU7441 treatment, we found that mRNA expression levels of Sp7, Alpl, Col1a1, Ibsp, and Bglap were significantly decreased in the presence of mimosine compared to control (Fig. 6). In the same experiment we observed the stimulatory effect of NU7441 treatment on the same target genes (Fig. 6). Together, we interpret these data to mean that pharmacological inhibition of DNA-PKcs by NU7441 induces cell cycle block, but that other mechanisms stimulate osteogenic differentiation downstream of the mitotic arrest.

Figure 6. Cell cycle inhibition does not induce differentiation of MC3T3-E1 cells.

Confluent MC3T3-E1 cells were cultured in osteoblast differentiation medium with NU7441 (1 μM), mimosine (200 μM) or vehicle (DMSO) for up to seven days. Total RNA was isolated from cells at day 3 and 7 of differentiation, and expression of Sp7, Alpl, Col1a1, Ibsp, and Bglap was analyzed by RT-qPCR. Values are normalized to Gapdh and expressed relative to vehicle treatment on day 3, which was arbitrarily ascribed a value of 1. Data are presented as mean ± SEM. Statistical analysis used one-way ANOVA test with post-hoc analysis; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

3.4. Identification of genes differentially regulated by longer-term treatment with NU7441

In order to identify pathways affected by long term inhibition of DNA-PKcs, we performed a whole genome gene expression profiling using the Affymetrix mouse GeneChip system (MoGen2.0 gene ST arrays). MC3T3-E1 cells grown in osteogenic medium were treated with vehicle or NU7441 for 14 days. Isolated RNA was subjected to microarray analysis. We found 49 genes whose expression was responsive to NU7441 treatment (p< 0.05; FDR < 0.05; fold), of which 19 were up-regulated over 1.2-fold and 28 were down-regulated to less than 0.7 compared to the vehicle-treated control (Table 1). We confirmed these data by RT-qPCR on biological triplicates; the expression pattern matched closely for the genes from the microarray data that we tested (data not shown). Analysis of the genes up-regulated by NU7441 treatment showed enrichment in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathways (Fig. 7A). GO analysis (Ashburner et al., 2000; The Gene Ontology, 2019) of differentially expressed genes performed using the Enrichr web-based tools demonstrated that these genes associate with distinct biological processes, including ‘skeletal system morphogenesis’, ‘skeletal system development’, ‘bone development’ and ‘embryonic skeletal system development’ (Fig. 7B). Differentially expressed genes include the previously identified NU7441 targets, Alpl, Sp7, Bglap, and Ibsp (Table 1).

Table 1.

Differentially expressed genes following 14 days of treatment of MC3T3-E1 cells with NU7441

Upregulated genes		Downregulated genes
Gene	Fold change	Gene	Fold change
Txnip	2.18	Gpr35	0.70
Oaslg	2.03	Sh3bp2	0.69
Pthlr	1.75	Tnfrsf12a	0.69
Ccl5	1.61	Timp3	0.69
Bglap	1.56	Pvr	0.69
Tnfrsf19	1.51	Tfgbi	0.68
Id3	1.49	Ddah1	0.68
Kazaldl	1.45	Spry4	0.68
Alpl	1.46	Ccl2	0.68
Sp7	1.45	Grem1	0.68
Ibsp	1.43	Il18rap	0.67
Crlfl	143	Sparcl1	0.65
Igf2	1.43	Ero1l	0.65
Gpr116	1.41	Gadd45a	0.63
Cyp39a1	1.39	Il33	0.63
Ehd3	1.31	Gdpd1	0.62
Thbs1	1.25	Clic5	0.61
Dlx5	1.22	Dhrs9	0.58
Prelp	1.21	March11	0.56
		Ptgs2	0.56
		Serpina3n	0.56
		Lum	0.56
		Cst6	0.55
		Dpt	0.53
		Fabp4	0.50
		Il1rl1	0.48
		Atp1b1	0.46
		Ch25h	0.44

mRNA from MC3T3-E1 cells was harvested following treatment with 1 μM NU7441 for 14 days and analyzed using GeneChip MoGen2.0 gene ST arrays. Nineteen (19) genes were upregulated over 1.2-fold, and 28 genes were downregulated less than 0.7 (p<0.05; FDR<0.05; fold).

Figure 7. Gene ontology and KEGG pathway enrichment analysis of microarray data.

(A) KEGG enriched pathways of the up-regulated gene following NU7441 treatment. (B) Biological process category of the significantly up- and down-regulated genes in differentiating MC3T3-E1 cells treated with NU7441 for 14 days.

Another enriched pathway is the transforming growth factor-beta (TGF-β) signaling pathway (Fig. 7A), that also includes the Bone Morphogenetic Proteins (BMPs). Two direct targets of BMP2, Id3, which mediates BMP2-induced osteoblast differentiation of mesenchymal stem cells (Peng et al., 2004) and Dlx5 (Distal-Less Homeobox 5), a skeletal tissue-specific and BMP-signaling-specific transcription factor expressed in later stages of osteoblast differentiation (Lee et al., 2003), were upregulated (Table 1). In parallel, Dpt (dermatopontin) and Grem1 (gremlin 1), two inhibitors of BMP2 signaling activity (Behnam, Murray, & Brochmann, 2006), were downregulated (Table 1). This suggested that inhibition of DNA-PKcs activity may potentiate BMP2 responses in osteoblasts.

3.5. Prkdc knockdown potentiates BMP2 effects in C2C12 cells

The mouse C2C12 multipotent mesenchymal precursor cell line is known for its ability to undergo osteoblastic differentiation upon treatment with BMP2 (Katagiri et al., 1994). To examine the effect of DNA-PKcs inhibition or knockdown on response to BMP2 stimulation, we knocked down Prkdc in C2C12 cells (Fig. 8A). Prkdc knockdown was confirmed by RT-qPCR (Fig. 8B) and Western blot analysis (Fig. 8C). We studied the effect of Prkdc knockdown in confluent C2C12 cultures stimulated with BMP2 for 48h, evaluating BMP2 responses as Smad1/5 phosphorylation and direct target gene expression. Prkdc knockdown further enhanced the expression of Sp7, Alpl, Id3 and Bglap in C2C12 cells in response to BMP2 stimulation compared to BMP2 alone (Fig. 8D). Similarly, BMP2-induced phosphorylation of SMAD1/5 was further increased in the absence of DNA-PKcs (Fig. 8E). These results suggest that silencing of DNA-PKcs expression stimulates the ability of BMP2 to induce differentiation of C2C12 myoblasts into the osteoblast lineage.

Figure 8. Prkdc knockdown potentiates BMP2 signaling in C2C12 cells.

(A) Schematic representing the experimental design. (B, C) Gene and protein knockdown efficiency with scrambled shRNA (Scr) and shRNA against DNA-PKcs (#4) quantified by RT-qPCR (B) and immunoblotting (C) in C2C12 cells. α-Tubulin is shown as a loading control. (D) Prkdc-deficient C2C12 cells were treated at confluency with 300 ng/ml BMP2 or vehicle (H₂O) for 48h, and RT-qPCR was performed for Sp7, Alpl, Id3, and Bglap. Values are normalized to Gapdh and expressed relative to vehicle treatment in scr shRNA-transfected cells, which was arbitrarily ascribed a value of 1. (E) Prkdc-deficient C2C12 cells were treated at confluency with 300 ng/ml BMP2 or vehicle (H₂O) for 2h. Whole cell extracts were generated, followed by immunoblotting, and α-Tubulin is provided as a loading control. Data are presented as mean ± SEM. Statistical analysis used unpaired two tailed t-test (B) or one-way ANOVA with post-hoc analysis; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

3.6. Inhibition of DNA-PKcs by NU7441 potentiates BMP2 effects in MC3T3-E1 cells and AMSCs

Confluent MC3T3-E1 were treated with NU7441 for 5 days in differentiation medium followed by BMP2 stimulation on day 5 for 48h (Fig. 9A, top). BMP2 regulates osteogenic differentiation via target genes including Id3, Sp7, Runx2 and Dlx5 (Lee et al., 2003; Matsubara et al., 2008). NU7441 increased the mRNA levels of Alpl, Col1a1, Bglap and the BMP downstream genes Id3, Dlx5, and Sp7 which were further enhanced by the sequential treatment with BMP2 (Fig. 9A). SMAD1/5 phosphorylation was increased in the presence of NU7441 at day 7 of differentiation and further enhanced in the presence of both NU7441 and BMP2 (Fig. 9B). As expected, NU7441 did not affect Smad1 and Smad5 mRNA (data not shown) or protein levels (Fig. 9B), establishing that NU7441 affects BMP2 signaling specifically at the level of protein phosphorylation, perhaps via endogenous paracrine activation of BMP receptors.

Figure 9. DNA-PKcs inhibition potentiates BMP2 effects in MC3T3-E1 cells and AMSCs.

(A) MC3T3-E1 cells cultured in osteogenic medium were treated for 5 days with NU7441 (1 μM) or vehicle (DMSO) prior to stimulation with 200 ng/ml BMP2 or vehicle (H₂O) for 48h. Total RNA was harvested and RT-qPCR for Sp7, Alpl, Id3, Col1a1, Bglap and Dlx-5 was performed. Values are normalized to Gapdh and expressed relative to vehicle treatment in the absence of BMP2, which was arbitrarily ascribed a value of 1. (B) Confluent MC3T3-E1 cells cultured in osteogenic medium were treated for 7 days with NU7441 (1 μM) or vehicle (DMSO) and stimulated with 200 ng/ml BMP2 or vehicle (H₂O) for 1h. Whole cell extracts were generated, followed by immunoblotting for phospho- and total SMAD1/5. GAPDH was used as a loading control. (C) AMSCs cultured in osteogenic medium were treated for 5 days with NU7441 (1 μM) or DMSO prior to stimulation with 200 ng/ml BMP2 or H₂O for 48h, and ID3 expression was analyzed by RT-qPCR. Data are presented as mean ± SEM. Statistical analysis used one-way ANOVA test with post-hoc analysis; *, p ≤ 0.05; **, p ≤ 0.01; ***, p≤0.001; ****, p ≤ 0.0001.

The stimulatory effect of NU7441 on AMSCs osteogenic differentiation was also enhanced by treatment with BMP2. ID3 expression was enhanced by BMP2 treatment but this effect was enhanced in NU7441-treated cells (Fig. 9C). Together, these data show that inhibition of DNA-PKcs enhances the osteogenic effects of BMP2 in osteoblast precursors as well as in committed osteoblasts.

3.7. Inhibition of DNA-PKcs in vivo increases bone biomechanical properties

Based on the osteogenic potential of NU7441 in vitro, we also assessed its effect on bone homeostasis in vivo. Sixteen (16)-week-old female C57BL/6 mice were intraperitoneally injected with NU7441 at a dose of 10 mg/kg/day or vehicle for three weeks (Fig. 10A). The mean body weights between the cohorts remained statistically similar (p > 0.9; t test) (Fig. 10B), showing that the treatment was well tolerated. All mice survived the 21-day-treatment and no abnormal appearance or behavior was observed. The clinical chemistry and hematological analysis did not reveal any treatment-related toxic effect (data not shown). No significant differences were observed in trabecular and cortical bone parameters in the femoral neck or distal radius of the NU7441-treated group compared to the vehicle controls by micro-CT analysis (Table 2). Interestingly, in the three-point bending test of the femoral shaft, NU7441 treatment for three weeks resulted in a significantly higher stiffness and maximum load compared to the vehicle group (Fig. 10C). These results show that short-term treatment with an inhibitor of DNA-PKcs is well tolerated and positively impacts bone tissue. They support the potential use of these compounds in bone regenerative medicine.

Figure 10. NU7441 enhances bone biomechanical properties in vivo.

(A) Schematic representing the experimental design. Female C57BL/6 mice of 16 weeks of age (n=10) were treated daily for 3 weeks with 10 mg/kg of NU7441 or vehicle (DMSO/40% PEG400/PBS) via intraperitoneal injection. (B) Body weight was recorded weekly. (C) Femurs were collected and their biomechanical parameters were measured using three-point bending tests. Data are presented as mean ± SEM. Statistical analysis used unpaired two tailed t-test; *, p ≤ 0.05; **, p ≤ 0.01 versus vehicle.

Table 2.

Effect of NU7441 on cortical and trabecular bone parameters

	DMSO (n=10)	NU7441 (n=7)
Cortical thickness (mm)	0.18 ± 0.002	0.18 ± 0.002
Cortical BV/TV (%)	93.95 ± 0.06	93.97 ± 0.06
Cross-sectional area (mm2)	0.87 ± 0.015	0.88 ± 0.015
Medullary area (mm2)	0.05 ± 0.001	0.05 ± 0.0005
Trabecular BV/TV (%)	3.07 ± 0.19	3.25 ± 0.24
Trabecular thickness (mm)	0.04 ± 0.001	0.04 ± 0.001
Trabecular separation (mm)	0.35 ± 0.01	0.36 ± 0.01
Trabecular number (mm−1)	0.81 ± 0.04	0.82 ± 0.07

Nineteen-week old female mice were treated once daily with 10 mg/kg of NU7441 for 21 days.

3.8. Phenotype of osteoblast Prkdc-deficient mice

We examined the role of DNA-PKcs in bone formation and homeostasis by conditionally deleting Prkdc in osteoblasts using the Osx-Cre driver strain. Heterozygous Prkdc^+/fl mice were mated with heterozygous Osx-Cre;Prkdc^+/flox to generate mutant Osx-Cre;Prkdc^fl/fl (Prkdc_Ob^−/−) conditional knockout offspring and their control littermates. The offspring were born at the expected Mendelian ratios. The ability of the Cre recombinase to specifically excise the floxed Prkdc allele was assessed by RT-qPCR using the RNA extracted from calvarial osteoblasts of wild-type and Prkdc_Ob^−/− mice. Prkdc expression was reduced by around 35% in the Prkdc_Ob^−/− mouse compared to Osx-Cre;Prkdc^+/+ (+/+,Cre; Fig. 11A), demonstrating poor efficacy of the Cre driver with the Prkdc floxed allele.

Figure 11. Phenotype of osteoblast-specific Prkdc-deficient mice.

(A) Expression of Prkdc in calvarial osteoblasts of wild type (+/+,Cre) and mutant Prkdc_Ob^−/− mice using RT-qPCR. (B) Comparison of body weight between genotypes. (C) Femurs were collected and stiffness (Young’s Modulus) was measured using three-point bending tests. Data are presented as mean ± SEM. Statistical analysis used unpaired two tailed t-test (A) or one-way ANOVA with post-hoc analysis; *, p ≤ 0.05; **, p ≤ 0.01.

Prkdc_Ob^−/− mice had no obvious phenotype in terms of gross appearance and size and were fertile (not shown). Prkdc_Ob^−/− mice and their littermates had similar body weight (Fig. 11B). Since floxed allele excision was low, it is not surprising that we observed no significant difference in trabecular and cortical bone parameters in femurs from 6-week-old male Prkdc_Ob^−/− mice compared with littermate controls using μCT analysis (Table 3). However, three-point bending testing indicated that stiffness (Young’s modulus) was significantly increased by 30% in Prkdc_Ob^−/− compared to control littermates (Fig 11C), a phenotypic manifestation that strongly suggests that the biomechanical changes measured following NU7441 treatment of wild-type mice (Fig. 10C) were caused by DNA-PKcs inhibition.

Table 3.

Trabecular and cortical parameters in Prkdc_Ob^−/− mice and control littermates

	+/+,Cre	+/−,Cre	PrkdcOb−/−
Trabecular BV/TV (%)	9.06 ± 1.23	6.73 ± 0.97	7.47 ± 1.5
Trabecular thickness (mm)	0.038 ± 0.001	0.03 ± 0.004	0.04 ± 0.001
Trabecular separation (mm)	0.29 ± 0.02	0.24 ± 0.04	0.31 ± 0.02
Trabecular number (mm−1)	2.31 ± 0.24	1.93 ± 0.15	1.96 ± 0.31
Connectivity	2586 ± 218	1979 ± 33	2063 ± 276
Cortical BV/TV (%)	36.2 ± 0.74	34.97 ± 0.93	34.66 ± 0.44
Cortical thickness (mm)	0.11 ± 0.003	0.11 ± 0.003	0.11 ± 0.004
Endosteum perimeter (mm)	3.37 ± 0.12	3.48 ± 0.09	3.33 ± 0.09
Mean polar moment of inertia (mm4)	0.05 ± 0.006	0.06 ± 0.005	0.05 ± 0.005

Values are mean ± SE, n=8 mice per group. No statistically significant differences between groups by one-way ANOVA. +/+,Cre, Osx-Cre;Prkdc^+/+; +/−,Cre, Osx-Cre;Prkdc^+/fl; Prkdc_Ob^−/−, Osx-Cre;Prkdc^fl/fl.

4. DISCUSSION

We explored a potential role for DNA-PKcs in osteoblastogenesis and bone homeostasis. Genetic or pharmacologic inhibition of DNA-PKcs increased osteoblast differentiation and extracellular matrix formation in vitro, while it ameliorated the biomechanical properties of bones in vivo. We conclude that under steady-state conditions, DNA-PKcs acts to negatively regulate osteoblast differentiation.

Our results contrast with the sparse literature dealing with DNA-PKcs and osteoblastogenesis. Two studies have examined the function of DNA-PKcs in the context of low-dose irradiation of osteoblasts (Xu et al., 2014) or focal radiation-induced osteoporosis (Chandra et al., 2018). In both cases it was suggested that DNA-PKcs exerts positive effects on osteoblasts, either by increasing expression of differentiation markers downstream from signaling through protein kinase B/AKT (Xu et al., 2014) or by enhancing survival following irradiation (Chandra et al., 2018). DNA-PK acts as a molecular sensor for DNA damage and an activator of DNA break repair to preserve genome integrity (Callen et al., 2009). Therefore it is not surprising that under low dose conditions it could compensate for the effects of radiation-induced damage and allow osteoblastogenesis to carry forward. The broad high-level expression of DNA-PKcs in many cell types however clearly suggests that it may have roles beyond its DNA break repair functions, for example, its role in transcriptional regulation (Goodwin & Knudsen, 2014).

Inhibition of DNA-PKcs at confluency results in a decrease in the expression of cell cycle genes. While it is generally accepted that DNA-PK has little to no role in p53 activation and cell cycle arrest in response to DNA damage (S. Wang et al., 2000), DNA-PKcs activity is required for chromosome segregation and normal cell cycle progression through mitosis (K. J. Lee et al., 2011). Cells stop proliferating in order to begin and maintain differentiation (Strehl, Schumacher, de Vries, & Minuth, 2002), but our results using the cell cycle inhibitor mimosine (San Martin et al., 2009) show that cell cycle block is not sufficient to induce osteoblastic differentiation. It is worth mentioning that inhibition of Enhancer of Zeste homolog 2 (Ezh2), a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (Dudakovic et al., 2018; Dudakovic et al., 2016), as well as Runx2 activation (Galindo et al., 2005; Pratap et al., 2003) in each case are cytostatic while promoting osteogenesis. The new findings with DNA-PKcs corroborate the general concept that induction of quiescence and onset of osteoblast maturation may be functionally coupled, and that DNA-PKcs may play an important role in this functional coupling in the osteoblast lineage.

We showed that long term pharmacological or genetic DNA-PKcs inhibition enhances the expression of differentiation markers including Sp7, Alpl, Col1a1, and Bglap as well as calcium deposition in murine MC3T3-E1 cells, and prompts the osteogenic capacity of human AMSCs. The effect of DNA-PKcs inhibition on osteoblast differentiation occurred in the absence of exogenous damage. Gene activating and suppressing roles for DNA-PKcs have been previously shown (Barlev et al., 1998; Kuhn, Gottlieb, Jackson, & Grummt, 1995; Okazaki, Nishimori, Ogata, & Fujita, 2003; Wong et al., 2009) without being restricted to damage response (Goodwin & Knudsen, 2014).

At later time points, DNA-PKcs inhibition increased SMAD1/5 phosphorylation in both myoblasts and osteoblasts, the expression of BMP2 downstream genes in osteoblasts, and potentiated BMP2-induced osteoblastic differentiation. The mechanism leading to increased SMAD1/5 phosphorylation in the presence of NU7441 but in the absence of exogenous BMP2 treatment remains to be determined, but may involve paracrine BMP signaling that is endogenously produced by precursor cells. The transcriptional activity of activated SMADs is influenced by histone modifications such as the H3K27 trimethylation mark (Morikawa, Koinuma, Miyazono, & Heldin, 2013) which could contribute to the observed potentiation of BMP2 signals.

Because of its significant osteoinductive properties, recombinant human BMP2 is FDA-approved for use in orthopaedic applications to enhance bone repair in the treatment of non-union fractures, spinal surgeries and oral maxillofacial procedures (de Freitas et al., 2013; Simmonds et al., 2013; Wei et al., 2012). Despite the significant positive effects of BMP2, its clinical use is limited due to several drawbacks including rapid degradation, high costs, safety and efficacy concerns, and need for high doses (Carreira et al., 2014; Crandall et al., 2013; Ehnert et al., 2012; Fu et al., 2013; Moatz & Tortolani, 2013; Oryan, Alidadi, Moshiri, & Bigham-Sadegh, 2014; Rodgers, Marascalchi, Grobelny, Smith, & Samadani, 2013). Strategies to enhance the safety and positive osteoinductive effects of BMP2 in bone regenerative medicine would have significant benefits. Pharmacological inhibition of DNA-PKcs may offer a promising therapeutic tool to increase BMP signaling activity in combination therapy. The DNA-PKcs inhibitor NU7441 was originally developed as an anticancer agent based on its ability to potentiate cell death mediated by chemotherapy- and radiation therapy-induced DSBs (Gavande et al., 2016). It may be argued that blocking DNA-PKcs activity may be hazardous to health. Knocking out any of the three components of DNA-PK in mice causes severe combined immunodeficiency and premature aging (Errami et al., 1996; Park et al., 2017; van der Burg et al., 2009). However, DNA-PKcs heterozygous mice did not have this defect (Ghonim et al., 2015). In addition, numerous studies have indicated the beneficial effect of DNA-PKcs inhibition in the context of chemotherapy-induced DNA damage to reduce therapeutic resistance in cancer therapy (Gavande et al., 2016). Like most inhibitor drugs, NU7441 only partially inhibits DNA-PKcs kinase activity at tolerated doses. The residual DNA-PKcs activity may be sufficient to protect against naturally occurring DNA damage, offering a potential therapeutic window. For example, it has been shown that moderately decreasing DNA-PKcs activity protects against diet-induced obesity and insulin resistance, and against mitochondrial decline, and improves physical fitness of aged mice (Park et al., 2017). Here, we observed that daily NU7441 treatment of adult female mice for 3 weeks showed promising efficacy as it significantly induced bone biomechanical properties compared with the vehicle group. However, no significant changes were observed in cortical bone parameters under those conditions. One interpretation of this data is that inhibition of DNA-PKcs could improve bone matrix properties through inducing the expression of matrix proteins such as type I collagen, bone sialoprotein, and alkaline phosphatase, without impacting the total amount of bone formed.

Additional insight will be provided by studying the phenotype of osteoblast-specific Prkdc-deficient mice in which excision of the floxed allele is more efficient than we have been able to accomplish to date. These experiments are in progress in our laboratory. At any rate, the incomplete penetrance of the Prkdc mutation that we report herein, which can be equated to partial inhibition achieved by treatment with the inhibitor drug NU7441, yielded a similar phenotype of improved biomechanical properties of the mutant bones.

In conclusion, our results identified DNA-PKcs as a potential therapeutic target in bone regeneration and treatment of skeletal diseases. Future studies with in vivo models where DNA-PKcs expression is maximally inhibited in osteoblasts will be essential to fully appreciate its clinical importance and relevance.