RANKL up-regulates brain-type creatine kinase via poly(ADP-ribose) polymerase-1 during osteoclastogenesis

Abstract

Receptor activator of nuclear factor κB ligand (RANKL) is the key regulator for osteoclast formation and function. During osteoclastogenesis, RANKL-stimulated signals differentially modulate expression of a large number of proteins. Using proteomics approaches, we identified that brain-type cytoplasmic creatine kinase (Ckb) was greatly induced in mature osteoclasts. Ckb has been shown to contribute to osteoclast function. However, the mechanisms of Ckb regulation and the contribution of other isoforms of creatine kinase during RANKL-induced osteoclastogenesis are unknown. We found that Ckb was the predominant isoform of creatine kinase during osteoclastogenesis. Real-time PCR confirmed that RANKL induced ckb mRNA expression by over 40-fold in primary mouse bone marrow macrophages and Raw 264.7 cells. The RANKL-responsive region was identified within the -0.4- to -0.2-kb 5'-flanking region of the ckb gene. Affinity binding purification followed by mass spectrometry analysis revealed that poly(ADP-ribose) polymerase-1 (PARP-1) bound to the -0.4/-0.2-kb fragment that negatively regulated expression of ckb in response to RANKL stimulation. Electrophoretic mobility shift assays with PARP-1-specific antibody located the binding site of PARP-1 to the TTCCCA consensus sequence. The expression of PARP-1 was reduced during RANKL-induced osteoclastogenesis, concurrently with increased expression of Ckb. Consistently, knockdown of PARP-1 by lentivirus-delivered shRNA enhanced ckb mRNA expression. The activity of PARP-1 was determined to be required for its inhibitory effect on the ckb expression. In summary, we have demonstrated that PARP-1 is a negative regulator of the ckb expression. Down-regulation of PARP-1 is responsible for the up-regulation of ckb during RANKL-induced osteoclastogenesis.

FIGURE 1.

Expression of Ckb during RANKL-induced osteoclastogenesis. BMMs were treated with (RANKL) or without (Control) RANKL for 72 h. A, osteoclastogenesis was depicted by TRAP staining. B, protein extracts were subjected to SDS-PAGE analysis followed by Coomassie Blue staining. The arrow indicates the RANKL-up-regulated protein, which was identified as Ckb by mass spectrometry analysis. C, Western blotting analyses of Ckb in BMMs (upper) or Raw 264.7 cells (lower) with or without exposure to RANKL for 72 h. GAPDH was used as a loading control. D, quantitative analysis of Ckb production determined by Western blotting analysis in C. E and F, the mRNA expression of creatine kinase isoforms was determined by RT-PCR (E) and quantitative real-time PCR (F). Representative pictures of three independent experiments are shown. *, p < 0.01.

FIGURE 2.

Identification of RANKL-responsive region on the ckb promoter. A, Raw 264.7 cells were transfected with a luciferase reporter construct driven by a −3.5-kb fragment located at 5′ of the ckb gene. The transfected cells were treated with (solid bars) or without (open bars) RANKL for up to 72 h. A plasmid encoding Renilla luciferase was used to normalize the transfection efficiency. RANKL-induced luciferase activity in the absence of RANKL was set as 1. B, BMMs were exposed to M-CSF and RANKL for up to 72 h. The expression of ckb and osteoclast markers including TRAP, Cath K, and CTR was determined by RT-PCR. Representative results of three independent experiments are shown. C, Raw 264.7 cells were transfected with luciferase reporter constructs containing indicated fragments from the 5′-flanking region of ckb and subsequently treated with or without RANKL for 72 h. Relative luciferase activities are shown. *, p < 0.05; **, p < 0.01. RLU, relative luciferase units; NS, not significant.

FIGURE 3.

Affinity purification of factors that bound to RANKL-responsive region and verification of the binding by PARP-1. A, Coomassie Blue staining of affinity-purified proteins. Nuclear extracts from control and RANKL-treated Raw 264.7 cells were incubated with −0.4/−0.2 kb-biotinylated probe. After being separated with streptavidin-conjugated beads, protein samples were subjected to SDS-PAGE followed by mass spectrometry analysis. The arrow points to PARP-1 identified by mass spectrometry analysis. B, Western blotting analysis of the binding of PARP-1 to the biotinylated DNA probe. Nuclear extracts were used for affinity pulldown assays, and the captured protein-DNA complexes were subjected to Western blot analysis using anti-PARP1 or anti-NFATc1 antibody. C, control; R, RANKL. C, EMSA using wild type (wt) (left) or mutant (mu) −261/−256 bp) ckb probe (right) as described under “Experimental Procedures.” ³²P-labeled probe was incubated with nuclear extracts from control or differentiated Raw 264.7 cells. The competition assays were performed in the presence of a 100-fold molar excess of the unlabeled probes. D, supershift EMSA analysis of PARP-1 DNA complex. Nuclear extracts preincubated with anti-PARP1 serum or control rabbit IgG prior to the addition of the probe were subjected to EMSA. Ab, antibody.

FIGURE 4.

Production of Ckb is inversely associated with expression of PARP-1 during osteoclastogenesis. BMMs were differentiated into osteoclasts with RANKL, and protein samples harvested at different time points were subjected to Western blotting analysis using anti-Ckb, PARP-1, or GAPDH antibody. The arrow and asterisk label different cleaved forms of PARP-1. Representative results from three independent experiments are shown.

FIGURE 5.

PARP-1 is functionally involved in the expression of ckb. A, Raw 264.7 cells and BMMs were cultured for 48 h in the presence of vehicle (dimethyl sulfoxide (DMSO)) or PARP-1-specific inhibitor, PJ34, and expression of ckb was determined by quantitative RT-PCR. B, Western blotting analysis of PARP-1 in BMMs infected with lentiviruses carrying PARP-1 shRNAs. GAPDH was used as a loading control. C, analysis of ckb mRNA by quantitative RT-PCR in BMMs with control or PARP-1 shRNAs. All results presented are the mean ± S.D. of three independent experiments, *, p < 0.05; **, p < 0.01.