Role of chromatin modulator Dpy30 in osteoclast differentiation and function

Abstract

Osteoclasts are the principal bone resorption cells crucial for homeostatic bone remodeling and pathological bone destruction. Increasing data demonstrate a vital role of histone methylation in osteoclastogenesis. As an integral core subunit of H3K4 methyltransferases, Dpy30 is notal as a key chromatin regulator for cell growth and differentiation and stem cell fate determination, particularly in the hematopoietic system. However, its role in osteoclastogenesis is currently unknown. Herein, we generated Dpy30^F/F; LysM-Cre^+/+ mice, which deletes Dpy30 in myeloid cells, to characterize its involvement in osteoclast differentiation and function. Dpy30^F/F; LysM-Cre^+/+ mice showed increased bone mass, evident by impaired osteoclastogenesis and defective osteoclast activity, but no alteration of osteoblast numbers and bone formation. Additionally, our ex vivo analysis showed that the loss of Dpy30 significantly impedes osteoclast differentiation and suppresses osteoclast-related gene expression. Moreover, Dpy30 deficiency significantly decreased the enrichment of H3K4me3 on the promoter region of NFATc1. Thus, we revealed a novel role for Dpy30 in osteoclastogenesis through epigenetic mechanisms, and that it could potentially be a therapeutic target for bone destruction diseases.

Keywords: Bone resorption; Dpy30; H3K4 methylation; Osteoclast.

Fig. 1.

Dpy30 expression during RANKL-induced osteoclastogenesis. 12-week-old wild type bone marrow macrophages (BMMs) were exposed to RANKL (100 ng/mL) for 3 days. (A) Dpy30 and Ctsk gene expression determined by qPCR (n=3). Data are mean ± SD.*P < 0.05; **P < 0.01 relative to D0. (B) Western blotting for Dpy30, H3, H3K4me1, H3Kme2 and H4K4me3.

Fig. 2.

Dpy30 deficient mice resulted in increased bone mass. (A) Representative μCT images of femurs from Ctrl and Dpy30^CKO mice (12-week-old). (B) Bone morphometric analysis and quantification from A (n=6); (C) Representative μCT images of femurs from Ctrl and Dpy30^CKO mice (22-week-old). (D) Bone morphometric analysis and quantification from C (n=7); Data are mean ± SD. *P < 0.05; **P < 0.01. BV/TV, bone volume per tissue volume; Tb.N, trabecular bone number; Tb.Th, trabecular bone thickness; Tb.Sp, trabecular bone space; BMD, bone mineral density; Ct.Th, cortical thickness. Scale bar: 100 μm.

Fig. 3.

Dpy30 deficient mice showed decreased osteoclasts cell numbers. (A) Representative Goldner’s Trichrome staining images of femoral static histomorphometric analysis. Scale bar: 100 μm. (B) Quantification of femoral static histomorphometric analysis from A (n=7). (C) Representative fluorescence-labeled images of femoral dynamic histomorphometric analysis. Scale bar: 100 μm. (D) Quantification of femoral dynamic histomorphometric analysis from C (n=7). (E) Representative TRAP staining image of femurs. Scale bar: 50 μm. (F) Quantification of N.Oc/BS from E (n=6). (G) Serum analysis of CTX-1 (n = 10). N.Oc/BS, number of osteoclast per bone surface; N.Ob/BS, number of osteoblast per bone surface; OcS/BS, osteoclast surface per bone surface; ObS/BS, osteoblast surface per bone surface. MS/BS, mineralizing surface; MAR, mineral apposition rate; BFR/BS, bone form rate per bone surface; BFR/BV, bone form rate per bone volume. Data are mean ± SD. **P < 0.01.

Fig. 4.

Dpy30 deficiency inhibits osteoclast formation and function. (A) Flow cytometry plot of osteoclast precursor population from bone marrow of 12-week-old Ctrl and Dpy30^CKO mice. (B) Percentage of osteoclast precursor population as aforementioned (n = 6). (C) TRAP staining image of mature osteoclast from 12-week-old Ctrl and Dpy30^CKO mice BMMs stimulated with RANKL (10 ng/ml or 100 ng/ml) at day 3. Scale bar: 50 μm. (D) Quantification of TRAP⁺ cell number in C (n=7). (E) F-actin ring fluorescence staining image of mature osteoclasts at day 3. Scale bar: 100 μm. (F) Quantification of F-actin ring⁺ number in E (n=7). (G) Bone resorption area by WGA staining in the cultures of mature osteoclasts at day 8. Scale bar: 100 μm. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Dpy30 downregulates the expression of osteoclast-related genes at the transcription and protein level. (A) qPCR analysis of mRNA expression of Dpy30, NFATc1, Ctsk, Trap, Car2, Atp6v0d2 and Oscar during osteoclastogenesis using BMMs from the 12-week-old Ctrl and Dpy30^CKO mice stimulated with RANKL (100 ng/ml) at different days (n=3). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Western blotting of Dpy30, NFATc1 and Ctsk expression of cells as aforementioned.

Fig. 6.

Dpy30 deficiency inhibits H3K4 methylation at the NFATc1 promoter. (A) Total protein levels and phosphorylation of MAPKs and NF-ĸB were assessed in Ctrl and DPY30^CKO BMMs stimulated by RANKL (100 ng/ml) for 0, 5, 15 or 30 minutes. (B) Western blotting of H3, H3K4me1, H3K4me2 and H3K4me3 in Ctrl and Dpy30^CKO BMMs stimulated with RANKL (100 ng/ml) at different days. (C) Ctrl and Dpy30^CKO BMMs were stimulated by RANKL (100 ng/ml) for 1 day. The enrichment of H3K4me3 on the NFATc1 promoter region was evaluated by ChIP assay (n=3). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7.. Schematic diagram of the role of Dpy30 in osteoclastogenesis.

Dpy30 regulates H3K4me3 on the promoter of transcription factor NFATc1 and thus regulates RANKL-induced osteoclastogenesis.