C/EBPα regulates osteoclast lineage commitment

Abstract

Despite recent insights gained from the effects of targeted deletion of the Finkel-Biskis-Jinkins osteosarcoma oncogene (c-fos), Spleen focus-forming virus (SFFV) proviral integration 1 (PU.1), microphthalmia-associated transcription factor, NF-κB, and nuclear factor of activated cells cytoplasmic 1 (NFATc1) transcription factor genes, the mechanism underlying transcription factors specifying osteoclast (OC) lineage commitment from monocyte/macrophage remains unclear. To characterize the mechanism by which transcription factors regulate OC lineage commitment, we mapped the critical cis-regulatory element in the promoter of cathepsin K (Ctsk), which is expressed specifically in OCs, and found that CCAAT/enhancer binding protein α (C/EBPα) is the critical cis-regulatory element binding protein. Our results indicate that C/EBPα is highly expressed in pre- OCs and OCs. The combined presence of macrophage colony-stimulating factor and receptor activator of NF-κB ligand significantly induces high C/EBPα expression. Furthermore, C/EBPα(-/-) newborn mice exhibited impaired osteoclastogenesis, and a severe osteopetrotic phenotype, but unaffected monocyte/macrophage development. Impaired osteoclastogenesis of C/EBPα(-/-) mouse bone marrow cells can be rescued by c-fos overexpression. Ectopic expression of C/EBPα in mouse bone marrow cells and monocyte/macrophage cells, in the absence of receptor activator of NF-κB ligand, induces expression of receptor activator of NF-κB, c-fos, Nfatc1, and Ctsk, and it reprograms monocyte/macrophage cells to OC-like cells. Our results demonstrate that C/EBPα directly up-regulates c-fos expression. C/EBPα(+/-) mice exhibit an increase in bone density compared with C/EBPα(+/+) controls. These discoveries establish C/EBPα as the key transcriptional regulator of OC lineage commitment, providing a unique therapeutic target for diseases of excessive bone resorption, such as osteoporosis and arthritis.

Fig. 1.

Identification of Ctsk CCRE and CCRE DNA binding protein as C/EBPα. (A) Activity of deletion mutants of the pCCAT construct was measured in RANKL-induced RAW64.7 cells. Schematic representation of each reporter construct is shown. Values are relative to the activity obtained with the largest promoter fragment (pCCAT-1474). CAT activities are normalized with the cotransfected β-gal activity. Experimental data are reported as mean ± SD of triplicate independent samples (n = 5, repeated three times). (B and C) Summary of site-specific mutagenesis of the Ctsk promoter binding site from −116 to −34 and characterization of the CCRE as a C/EBPα site. (D) Gel mobility shift experiment using a ³²P-labeled −53 to −30 WT Ctsk promoter oligonucleotide probe in nuclear extracts prepared from RANKL-induced RAW264.7 cells, with and without 100-fold molar excess of unlabeled WT or mutant oligonucleotides as described on the left. NP, nuclear protein. (E) DNase I footprint assays identify a binding site between −34 to −51 in the −137 to −31 mouse Ctsk promoter. The protected region is indicated by a shaded box, which contains a C/EBPα site. DNase I digestion of the naked DNA incubated with (lane 1) and without (lane 2) RANKL-induced RAW264.7 cell nuclear extracts is shown. G + A Maxam–Gilbert reaction of the −137 to −31 promoter fragment is shown in lane 3.

Fig. 2.

C/EBPα is expressed in pre-OCs and OCs, and it is induced by RANKL. (A) Northern blot hybridization using a C/EBPα-specific probe in RANKL-induced and uninduced C/EBPα^+/+ MBM, GCTs of bone, hSCs, rat osteoblast (ROB), a human macrophage cell line (U-937 cells), and C/EBPα^+/+ mouse tissues. (B) Time-course Western blot analysis of C/EBPα expression in C/EBPα^+/+ MBM cultured with M-CSF (20 ng/mL) alone for 2 d and then stimulated with M-CSF (10 ng/mL)/RANKL (10 ng/mL) for 0–120 h. (C) TRAP staining and immunostaining for Ctsk, C/EBPα, C/EBPβ, and C/EBPδ in GCTs of bone and C/EBPα^+/+ tibiae sections. Normal serum is shown as a control. (D) Immunofluorescence staining of C/EBPα (green) and Ctsk (red) in C/EBPα^+/+ tibiae indicates significant overlap, visualized as a yellow area in the merged image (n > 9). Inset is the magnified image of the boxed area.

Fig. 3.

Increased bone mass and defective osteoclastogenesis in C/EBPα^−/− mice in vivo and in vitro. (A) Radiographic analysis of femora and tibiae indicates osteopetrosis in C/EBPα^−/− newborn mice compared with C/EBPα^+/+ newborn mice (n = 5, repeated three times). (B) Three-dimensional microcomputed tomography images of C/EBPα^+/+ and C/EBPα^−/− newborn mice femora (n = 5, repeated three times). (C) Quantification of bone volume/tissue volume and bone mineral density of C/EBPα^+/+ and C/EBPα^−/− newborn mice femora. *P < 0.05; ***P < 0.001. (D) Histological sections of C/EBPα^−/− newborn mice tibiae have an extended growth plates (arrows) and a dramatic reduction in TRAP⁺ OCs compared with C/EBPα^+/+ controls (n > 50). Boxed areas (Upper) are magnified (Lower). The histological sections of the same area of C/EBPα^−/− newborn mice also show dramatically decreased TRAP⁺ OCs. (E) TRAP stain of RANKL-induced C/EBPα^+/+ and C/EBPα^−/− MBM. (F) TRAP stain of cocultures with MOBs and MBM obtained from C/EBPα^−/− and C/EBPα^+/+ mice.

Fig. 4.

C/EBPα KO reduces the expression of key OC regulators and marker genes. Western blot (A) and quantification of expression (B) of PU.1, Ctsk, c-Fos, and Nfatc1 in C/EBPα^+/+ and C/EBPα^−/− MBM cells cultured with M-CSF (20 ng/mL) alone for 2 d and then stimulated with M-CSF (10 ng/mL)/RANKL (10 ng/mL) for 5 d to generate OC-like cells. *P < 0.05; **P < 0.01. (C) Time-course Western blot analysis of IκBα and p38 and their phosphorylated forms (p-IκBα and p-p38) in C/EBPα^+/+ and C/EBPα^−/− MBM cells cultured with M-CSF (20 ng/mL) alone for 2 d and then stimulated with M-CSF (10 ng/mL)/RANKL (10 ng/mL) for 0–60 min.

Fig. 5.

C/EBPα^−/− mice exhibit a dramatic increase in trabecular bone number, and C/EBPα^−/− impaired osteoclastogenesis is rescued by ectopic expression of c-fos. (A) Compared with C/EBPα^+/+ controls, C/EBPα^−/− tibiae have extended growth plates (arrows) and a dramatic increase in mineralized tissue (bright blue regions), which indicates osteopetrosis (n = 3, repeated three times). (B) Histomorphometric analysis of tibiae from C/EBPα^+/+ and C/EBPα^−/− mice. *P < 0.05; **P < 0.01. N.S., not significant. (C and D) Impaired OC differentiation in C/EBPα^−/− MBM cells cultured with M-CSF/RANKL can be rescued by ectopic expression of c-fos using pBMN–c-fos compared with control virus (pBMN-GFP). ***P < 0.001.

Fig. 6.

C/EBPα induces osteoclastogenesis in the absence of RANKL and activates the c-fos promoter. (A) Western blot and quantification of forced expression of C/EBPα in C/EBPα^+/+ MBM stimulated with 20 ng/mL M-CSF for 24 h and then transduced with pBMN-C/EBPα for 96 h. Compared with the control group, C/EBPα expression increased significantly 96 h after cells were transduced with pBMN-C/EBPα. (B) GFP expression and TRAP expression in C/EBPα^+/+ MBM in which C/EBPα is overexpressed (pBMN-C/EBPα) compared with the control (pBMN-GFP) after 96 h of stimulation with M-CSF (20 ng/mL) alone. (C) qPCR analysis in MBM cultured in M-CSF alone and transduced with pBMN-C/EBPα (+) or with the vector control pBMN-GFP for 96 h. *P < 0.05; **P < 0.01; ***P < 0.001. (D) ChIP analysis of C/EBPα binding to the c-fosA, c-fosB, or upstream promoter region [c-fos (−5 Kb) as a negative control] in MBM stimulated with RANKL/M-CSF to generate mature OCs. (E) Consensus sequence alignment of putative C/EBPα binding sites of mouse, rat, and human c-fos promoter regions. C/EBPα activates c-Fos expression. (F) Truncated c-fos promoters were induced by C/EBPα. Luciferase reporter assays with truncated c-fos promoters indicated the presence of two C/EBPα binding sites (GL3B, control vector).

Fig. 7.

Ectopic expression of C/EBPα reprograms the monocyte/macrophage cell line to OC-like cells in the absence of RANKL. (A) Western blot of C/EBPα expression in RAW264.7 cells (not stimulated with M-CSF/RANKL) transfected with pcDNA3.1-C/EBPα (+) or with the vector control pcDNA3.1 (−). (B) TRAP and Ctsk immunostaining in uninduced RAW264.7 cells in which C/EBPα is overexpressed [pcDNA3.1-C/EBPα (+)] compared with the control [pcDNA3.1 (−)]. Semi-qPCR (C) and qPCR (D) analysis (25 cycles, Hprt served as a loading control) of genes important for monocytes/macrophages and OCs in uninduced RAW264.7 cells transfected with pcDNA3.1-C/EBPα (+) or with the vector control pcDNA3.1 (−).*P < 0.05; **P < 0.01.