A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.


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The experimental results were analyzed using GraphPad Prism (ver.8.3.0). All flow cytometry data were analyzed on FlowJo Software. IPA (ver.01-10) was used for pathway analysis from mass spectrometry data. For RNA sequencing, reads are aligned to mouse genome GRCm38 by using STAR aligner (ver.2.3.0e) and mapped reads were indexed using SAMTools (ver.1.9). Gene counts were obtained by HTSeq-Count (ver.0.11.2) to sorted bam files. DESeq2 (ver. 1.4.5) was employed for differential gene expression analysis from RNA sequencing data and functional enrichment analysis was performed with DAVID (ver. 6.7).
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Generally, sample sizes were calculated on the assumption that a 30% difference in the parameters measured would be considered biologically significant with an estimate of sigma of 10-20% of the expected mean. Alpha and Beta were set to the standard values of .05 and 0.8, respectively.
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All experiments were individually at least 2-3 times in order to confirm reproducibility and findings were consistent.
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Yes, microCT analysis, histology and histomorphometry were performed by individuals (Yeon-Suk Yang, Ren Xu, Na Li) who were blinded to the nature of the mice under analysis (both what specific mouse strains or treatment groups were in the experiment and whether any individual mouse belonged to control versus experimental groups). For other experiments, no blinding was employed as the researcher performing the treatment was also responsible for the analysis.

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Cell line source(s) Cell lines (C3H10T1/2 and HEK293) were purchased from ATCC. Human bone marrow-derived stromal cells (hBMSCs) were purchased from Cyagen Biosciences.

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nature research | reporting summary
October 2018

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We note that only histology data (H&E, immunohistochemistry and immunofluorescence for expression of type 1 collagen, RUNX2, CSNK2 and HAUSP) is currently provided for the analysis of the human heterotopic ossification (HO) tissues. The individuals included seven patients (3 male and 4 females, previously healthy, nonsmoking individuals; age ranging from 29 to 67 years) with pain or decreased hip movement.

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Patients with diagnosis of heterotopic ossification underwent exclusion surgery. After surgery, heterotopic bone samples for this study were stored and analyzed in Yonsei University Severance Hospital, Korea.

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The samples were obtained from human patients in Yonsei University Severance Hospital, Korea under institutional review board approval (IRB No.4-2017-1223.
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For human skeletal stem cell preparation, human bone marrow aspirate (BMA) purchased from StemExpress (BMEDT010F) was incubated for 10 min at room temperature with BD Pharm Lyse hypotonic lysis buffer (BD Biosciences, 555899) for RBC lysis. Cells were washed with cold FACS buffer twice. For mouse skeletal stem cell preparation, E17.5 Csnk2bfl/fl and Csnk2bPrx1 embryonic limbs were dissociated by mechanical and enzymatic digestion (1mg/ml of Collagenase P (Roche, 11213857), 2 mg/ml of Dispase II (Roche, 10165859001), 1 mg/ml of Hyaluronidase (Sigma, H3506) and 10000 unit/ml of DNase I (Roche, 4716728001)) for 1 hour at 37°C under gentle agitation. After digestion, cells were passed through 40 um cell strainer and washed with cold PBS (pH 7.2) containing 0.5% BSA (Fraction V) and 1 mM EDTA. Cell population abundance In general, flow cytometry was limited to analysis and not sorting. It was not feasible to run a purity check on the post-sort population of cells.