Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway

Abstract

Objectives: To clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC).

Materials and methods: Tooth extraction models of Ctsk knockout mice (Ctsk^-/- ) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK-8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk^-/- JBMMSC was sent to RNA-seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis.

Results: We found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up-regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA-seq results showed that coding genes of key enzymes in glycolysis were significantly up-regulated in Ctsk^-/- JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC's regeneration was blocked subsequently.

Conclusions: Our findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.

Keywords: Cathepsin K; alveolar bone; glycolysis; jaw bone marrow mesenchymal stem cells; osteogenic differentiation.

FIGURE 1

Ctsk deficiency promotes alveolar bone regeneration. Eight‐week‐old Ctsk^−/− mice and their WT littermates were used for tooth extraction. Three mice were sacrificed 3, 7, 10 and 14 d after tooth extraction. A, Representative Micro‐CT scanning images of the extraction socket along the longitudinal direction of the maxillae. B, BV/TV (%), Tb. Th (mm) and Tb. Sp (mm) of trabecular bone in the extraction socket were analysed. C, Representative images of H&E staining paraffin sections. D, Representative images of Masson’s trichrome staining paraffin sections. The statistical analysis was shown: **P < .01

FIGURE 2

Ctsk deficiency accelerates osteoblast activity during the process of alveolar bone filling. A, Representative images of TRAP‐stained paraffin sections. Red arrowheads indicate osteoclasts. B, Number of TRAP‐positive osteoclast per alveolar bone surface was analysed. C, Immunohistochemistry staining of osterix in extraction socket healing process. D, Quantitative analysis of Osx‐positive cells. The statistical analysis was shown: *P < .05, **P < .01

FIGURE 3

CTSK is expressed in JBMMSC and osteoblasts during the process of alveolar bone filling. A, Immunohistochemistry staining of CTSK in the extraction socket filling process. B, CTSK‐positive cells per mm² was assessed. C, Distribution of osteoclasts was analysed by TRAP staining. Red arrowheads indicate osteoclasts. D, Number of TRAP‐positive osteoclast per alveolar bone surface was analysed. E, Immunofluorescent staining of CTSK (green) in extraction socket (7d post‐extraction). CD44 (red) and CD90 (red) were stained as the bone marrow mesenchymal stem cells marker. Nuclei were stained with the DAPI (blue). White arrowheads indicate CTSK⁺/CD90⁺ or CTSK⁺/CD44⁺ cells. F, Immunofluorescent staining of CTSK (green) and Osterix (red) in extraction socket (7d post‐extraction). Nuclei were stained with the DAPI (blue). White arrowheads indicate CTSK⁺/OSX⁺cells. The statistical analysis was shown: **P <.01; ***P <.001. AB, alveolar bone; BC, bone marrow cavity; ES, extraction socket; TB, trabecular bone

FIGURE 4

Endogenous Ctsk deficiency promotes JBMMSC proliferation and osteogenic differentiation. JBMMSC from Ctsk^‐/‐ mice and their WT littermates were cultured. A, Flow cytometry was used to detect the expression of JBMMSC surface markers. B, The expression of endogenous CTSK in JBMMSC and its deficiency in JBMMSC from Ctsk ^‐/‐ mice were confirmed by Western blot. C, CTSK was mainly located in lysosomal in WT JBMMSC by immunofluorescence. D, Influence of Ctsk knockout or inhibition by ODN on proliferation of JBMMSC was assessed by CCK‐8 (* represent WT compared to Ctsk^‐/‐ group; # represent WT compared to ODN group). E, Expressions of osteogenic‐related proteins of ALP and Runx2 were detected by Western blot after osteogenic induction for 7 d, and quantitative analyses were shown in (F). G, Mineralized nodules of JBMMSC were assayed by alizarin red staining after osteogenic induction for 15 d. H, Alizarin red staining was quantified with a spectrophotometer after dissolving by 10% cetylpyridinium chloride. The statistical analysis was shown: *P < .05;**P < .01; ***P < .001; #P < .05; ###P < .001

FIGURE 5

Ctsk deficiency or inhibition promotes glycolysis. JBMMSC from Ctsk^‐/‐ mice and their WT littermates were cultured and differentially expressed genes were selected by RNA‐seq. A, Five differentially expressed genes of key enzymes in glycolysis were detected by RNA‐seq. B, Expression levels of the five differentially expressed genes were confirmed by RT‐qPCR. C, WT and Ctsk^‐/‐JBMMSC were stimulated with 1 μmol/L ODN or 1 μmol/L DMSO for 48 h and then detected extracellular acidification rate by Seahorse. Glucose consumption (D) and lactate production (E) were performed to study the effect of Ctsk deficiency or inhibition on glycolysis. The statistical analysis was shown: *P < .05; **P < .01; ***P < .001

FIGURE 6

C tsk deficiency or inhibition promotes JBMMSC proliferation and osteogenic differentiation by up‐regulating glycolysis. JBMMSC from WT mice were stimulated with 1 μmol/L DMSO, 1 μmol/L ODN, 10 μmol/L 3PO or 1 μmol/L ODN + 10 μmol/L 3PO. A, Blocking glycolysis by 3PO inhibited the effect of ODN on JBMMSC proliferation (* represent WT compared to ODN group; # represent ODN compared to 3PO+ODN group). B, Expressions of osteogenic‐related proteins of ALP and Runx2 were detected by Western blot after osteogenic induction and chemical reagent treatment for 7 d. C, Quantitative analysis of Western blot images. D, Representative images of alizarin red staining of JBMMSC after osteogenic induction and chemical reagent treatment for 15 d and quantitative analyses were shown (E). The statistical analysis was shown: *P < .05; **P < .01; ***P < .001; #P < .05; ###P < .001