CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65

Abstract The E3 ubiquitin ligase complex CDC20‐activated anaphase‐promoting complex/Cyclosome (APC/CCDC20) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co‐activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well‐known cell cycle‐related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA‐binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11‐dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle‐independent function of CDC20, establishes APC11CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone‐related diseases.

A-D Western blot analyses (A) and qRT-PCR (B-D) of the expression of CDC20 and osteogenic marker RUNX2, OCN. Cells were cultured in osteogenic medium for 7 and 14 days (n = 6). E, F The knockout efficiency of Cdc20 (E) and the expression of osteogenic marker Runx2 (F) in BMSCs of Sp7-Cre;Cdc20 f/f and Cdc20 f/f mice determined by qRT-PCR (n = 5). G Representative images of light and fluorescence of lentivirus infected NC and CDC20sh hBMSCs. Scale bar: 500 lm.

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The knockdown efficiency of CDC20 in NC and CDC20sh hBMSCs determined by qRT-PCR (n = 5). I, J The expression of RUNX2 in NC and CDC20sh hBMSCs after 7 days osteogenic differentiation determined by qRT-PCR (I) and Western blot analyses (J) (n = 5). K Western blot analyses of Myc-CDC20, Myc-CDC20 171-499 fragment (containing WD40 domain), Myc-CDC20 1-170 fragment (lacking WD40 domain) plasmids expression in HEK293T cells. L Western blot analyses of the degradation of the substrate Cyclin B1 under the overexpression of truncated fragments of CDC20.
Data information: Data are displayed as mean AE SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by a two-tailed unpaired Student's t-test or one-way ANOVA followed by a Tukey's post hoc test and defined as ***P < 0.001. A-D The expression of CDC20 (A) and NF-jB pathway downstream genes IL-8, IL-6, and ICAM1 (B-D) of NC and CDC20si HEK293T cells after TNF-a stimulation determined by qRT-PCR (n = 6). E Western blot analyses of the degradation of endogenous p65 protein in NC and CDC20si HEK293T cells. F Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. HEK293T cells were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 lM MG132 (the proteasome inhibitor) for 6 h before collected. G, H Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in hBMSCs. I, J

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Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in mBMSCs. K The co-localization of CDC20 and p65 in hBMSCs. Scale bar: 20 lm.
Data information: Data are displayed as mean AE SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey's post hoc test and defined as ***P < 0.001.  The knockdown efficiency of APC11 in hBMSCs was determined by qRT-PCR (n = 6).
Data information: Data are displayed as mean AE SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by a Tukey's post hoc test and defined as ***P < 0.001.  Figure EV5. CDC20 regulated osteogenic differentiation of BMSCs in a p65-dependent manner, related to Fig 8. A Fluorescent staining of lentiviruses injected from tail intravenously. B, C The efficiency of p65 knockdown determined by qRT-PCR (B) and Western blot (C) of BMSCs in Sp7-Cre;Cdc20 f/f mice (n = 6). D, E The ALP staining (D) and ALP quantification (E) of control and CDC20 knockdown hBMSCs after 7 days osteogenic differentiation treated with negative control or p65si RNA (n = 5). F, G The ALP staining (F) and ALP quantification (G) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation treated with BAY 11-7,082 (n = 6).

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The expression of RUNX2 in BMSCs from Cdc20 f/f mice and Sp7-Cre;Cdc20 f/f mice after 7 days osteogenic differentiation treated with negative control or p65si, determined by qRT-PCR (n = 5).
Data information: Data are displayed as mean AE SD and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA followed by independent two-tailed Student's t-tests or a Tukey's post hoc test and defined as *P < 0.05, **P < 0.01, ***P < 0.001.