Targeting casein kinase 2 and ubiquitin-specific protease 7 to modulate RUNX2-mediated osteogenesis in chronic kidney disease

Abstract

Objective: Chronic Kidney Disease (CKD) frequently leads to Mineral Bone Disorder (MBD), which significantly affects patient quality of life due to bone fragility and metabolic disturbances. This study investigates the role of Casein Kinase 2 (CK2) and Ubiquitin-Specific Protease 7 (USP7) in modulating Runt-related Transcription Factor 2 (RUNX2)-driven osteogenesis in a CKD-MBD mouse model.

Methods: A CKD-MBD mouse model was established using 5/6 nephrectomy. Bioinformatic analysis of CKD-related datasets identified RUNX2 and USP7 as key genes implicated in bone metabolism. In vivo and in vitro experiments were conducted to assess the effects of CK2-mediated phosphorylation and USP7-induced deubiquitination on RUNX2 stability and function. Histomorphometry, Enzyme-Linked Immunosorbent Assay (ELISA), and micro-CT analyses were performed to evaluate bone density, strength, and metabolic markers.

Results: RUNX2 and USP7 were significantly downregulated in CKD-MBD mice. Silencing RUNX2 impaired osteoblast differentiation, reduced bone density, and increased bone turnover, while CK2 overexpression restored RUNX2 activity by phosphorylation, recruiting USP7 to stabilize RUNX2. Enhanced osteoblast differentiation and improved bone metabolism were observed in CKD-MBD mice upon CK2 activation.

Conclusion: CK2 activation promotes RUNX2 phosphorylation and stabilization by USP7, leading to improved osteogenesis and bone metabolism in CKD-MBD. Targeting the CK2/USP7/RUNX2 axis presents a potential therapeutic strategy for managing CKD-related bone disorders.

Keywords: Casein kinase 2; Chronic kidney disease; Mineral bone metabolism disorder; Runt-related transcription factor 2; Ubiquitin-specific protease 7.

Fig. 1

Bioinformatics Analysis to Identify Key Genes Involved in CKD Development. Note: (A) Heatmap of DEGs, where the color scale from blue to orange represents expression values from low to high; (B) PPI network graph of candidate genes, where the color gradient from green to yellow represents Degree values from high to low; (C) Significant downregulation of Runx2 in CKD (Treat, n = 10) compared to control samples (Control, n = 8); (D) Bubble plot of GO and KEGG pathway enrichment analysis for candidate genes, where the size of the circles represents the number of selected genes and the color indicates the significance level of the enrichment analysis; (E) Significant downregulation of Usp7 in CKD (Treat, n = 12) compared to control samples (Control, n = 12)

Fig. 2

In vitro Cell Experimental Validation of the Interaction between USP7 and RUNX2. Note: (A) Immunoprecipitation assay of USP7 binding with RUNX2; (B) RT-qPCR and Western Blot analysis of mRNA and protein levels of USP7 and RUNX2 in HEK293T cells in sh-NC and sh-USP7 groups; (C) Evaluation of the protein half-life of RUNX2; (D) In vitro deubiquitination experiment measuring the impact of USP2 on the ubiquitination levels of RUNX2; (E) Western Blot analysis of the protein levels of RUNX2 in HEK293T cells in different groups, where “ns” indicates no significant difference between the groups (P > 0.05), and * denotes a significant difference between the groups (P < 0.05); all cellular experiments were performed in triplicate

Fig. 3

In vitro Cell Experimental Validation of CK2 and RUNX2/USP7 Interaction. Note: (A) Western Blot analysis of the protein expression levels of CK2 and RUNX2 in various groups of MC3T3-E1 cells; (B) Western Blot analysis of the protein expression levels of RUNX2 in different groups of MC3T3-E1 cells; (C) RT-qPCR analysis of the relative expression levels of osteoblast marker genes (ALP, Collagen-1, and Osteocalcin) in different groups of MC3T3-E1 cells; (D) Immunoprecipitation experiment to detect the interaction between RUNX2 and CK2; (E) Phosphorylation kinase assay assessing the regulatory role of CK2 in the phosphorylation of RUNX2; (F) Ubiquitination experiment measuring the ubiquitination levels of RUNX2 in various groups of cells after treatment with MG132; (G) In vitro cell experimental validation of the mutual regulation among CK2, USP7, and RUNX2. * indicates a significant difference between the groups (P < 0.05), and all cellular experiments were conducted in triplicate

Fig. 4

Bone Formation and Bone Metabolism State in CKD-MBD Mice. Note: (A) Histomorphometric analysis of bone tissue in the two groups of mice, including erosion perimeter/bone perimeter (E.Pm/B.Pm), osteoclast number (Oc.N/BS), osteoclast perimeter/bone perimeter (Obs/BS), and Bone Formation rate (BFR/Ob), BFR/BS, MS/BS, and MAR; (B) ELISA measurement of serum PTH, ALP, and FGF23 levels in the two groups of mice; (C) Western blot analysis of the expression of RUNX2 protein in bone tissue of the two groups of mice; (D) RT-qPCR measurement of RUNX2 RNA expression levels. * indicates comparisons between the two groups, where *P < 0.05 denotes significance. Each group consisted of 6 mice

Fig. 5

Impact of RUNX2 Silencing on Bone Turnover Rate, Bone Density, Bone Strength, and Osteoclastogenesis in CKD-MBD Mice. Note: (A) RT-qPCR to detect RUNX2 expression levels in various tissues; (B) ELISA assessment of expression levels of PINP and CTx in the serum of each group of mice; (C) Micro-CT 3D scanning (200 μm) of trabecular and cortical bone regions in each group of mice; (D) Quantitative evaluation of bone parameters from the Micro-CT 3D scanning results, including BV/TV, Tb. N (mm^− 1), Tb. Th (mm), Tb. Sp, mm, Ct. Ar (mm²), and Ct. Th (mm²) (* indicates comparison with sh-NC group, P < 0.05); (E) H&E staining of the tibial bone tissue in each group of mice (200 μm); (F) Quantitative histomorphological evaluation of the MASSON stained tissues. * denotes significance in comparisons between the two groups (P < 0.05). Each group comprised 6 mice

Fig. 6

Impact of CK2 Silencing on RUNX2 Expression and Bone Turnover Rate, Bone Density, and Bone Strength in Mice. Note: (A) RT-qPCR to detect RUNX2 expression levels in various tissues; (B) Western blot analysis of CK2 and RUNX2 protein levels in bone tissue of each group of mice; (C) ELISA assessment of PINP and CTx expression levels in the serum of each group of mice; (D) Micro-CT 3D scanning (200 μm) of trabecular and cortical bone regions in each group of mice; (E) Quantitative evaluation of bone parameters from the Micro-CT 3D scanning results, including BV/TV, Tb. N (mm^− 1), Tb. Th (mm), Tb. Sp, mm, Ct. Ar (mm²), and Ct. Th (mm²); (F) H&E staining of tibial bone tissue in each group of mice (200 μm); (G) Quantitative histomorphological assessment of the MASSON stained tissues. *indicates significance in comparisons between the two groups (P < 0.05), and ** denotes significance at P < 0.01. Each group consisted of 6 mice

Fig. 7

The Impact of CK2 Regulating RUNX2 on Bone Turnover Rate, Bone Density, and Bone Strength in CKD-MBD Mice. Note: (A) RT-qPCR to detect RUNX2 expression levels in various tissues; (B) Western blot analysis of CK2 and RUNX2 protein levels in bone tissue of each group of mice; (C) ELISA assessment of PINP and CTx expression levels in the serum of each group of mice; (D) Micro-CT 3D scanning (200 μm) of trabecular and cortical bone regions in each group of mice; (E) Quantitative evaluation of bone parameters from the Micro-CT 3D scanning results, including BV/TV, Tb. N (mm^− 1), Tb. Th (mm), Tb. Sp, mm, Ct. Ar (mm²), and Ct. Th (mm²); (F) H&E staining of tibial bone tissue in each group of mice (200 μm); (G) Quantitative histomorphological assessment of the MASSON stained tissues. * indicates significance in comparisons between the two groups (P < 0.05). Each group comprised 6 mice.=

Fig. 8

Molecular Mechanism Diagram of CK2 Phosphorylation Modifications on RUNX2 Affecting MBD in CKD Mice