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.
Highlights
Osteoclasts are multinucleated bone-resorbing cells that play a crucial role in physiological bone remodeling, as well as in pathological bone resorption such as osteoporosis and periodontal disease [1, 2]
We determined the expression of other creatine kinase isoforms and their regulation during RANKL-induced osteoclastogenesis
The function of Ckb in regulating osteoclast bone resorption activity in mice has been reported, which may be related to decrease in regeneration of ATP that is required for cytoskeleton reorganization and bone resorption [12, 13]
Summary
Cytoplasmic creatine kinase muscular isoform; Ckb, cytoplasmic creatine kinase brain isoform; Ckmt, creatine kinase mitochondrial isoform; RANKL, receptor activator of NF-B ligand; PARP, poly(ADP-ribose) polymerase; NFAT, nuclear factor of activated T-cells; TRAP, tartrate-resistant acidic phosphatase; Cath K, cathepsin K; CTR, calcitonin receptor; BMM, bone marrow macrophage. RANKL-induced NF-B, NFATc1, and AP-1 signaling pathways play essential roles in activating the expression of osteoclast-specific genes, such as tartrate-resistant acidic phosphatase (TRAP), cathepsin K (Cath K), and calcitonin receptor (CTR), promoting osteoclast differentiation and function. With the use of a luciferase reporter system and electrophoretic mobility shift assays, the PARP-1 binding domain was identified at 5Ј of the ckb gene. These studies provide important insights into molecular regulation of Ckb and its function during RANKL-induced osteoclastogenesis
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