Article Text
Abstract
Background Prohibitin-1 (PHB) regulates diverse cellular processes by controlling several signaling pathways and many studies have evaluated its therapeutic potential in various diseases, including cancer, inflammatory bowel disease, diabetes, and obesity.
Objectives In the present study, we investigated the effects of PHB on osteoclast differentiation in vitro and bone resorption ex vivo.
Methods Bone marrow cells were obtained from 5∼8-week-old male ICR mice. BMMs were cultured for 4 days with M-CSF (30 ng/ml) and RANKL (100 ng/ml). To generate osteoclasts from the co-culture of primary osteoblasts and bone marrow cells, primary osteoblasts from newborn ICR mouse calvariae were prepared.BMMs and primary osteoblasts were co-cultured. After 6 days in culture, the cells were fixed and stained for TRAP. Mature osteoclasts derived from retrovirus-transduced BMMs were cultured with RANKL (100 ng/ml) and M-CSF (30 ng/ml). After MNC were observed at 6 days for the presence of resorption pits, the slides were washed with PBS and treated with 5% sodium hypochlorite for 5 min. After being washed with PBS and dried, the plate was photographed under a light microscope. Resorbed areas were quantified using the ImageJ program. Luciferase reporter plasmid was constructed by cloning c-Fos-luc vector into pGL3 basic and serum response element was constructed by inserting two copies of the SRE sequence into the pGL3 basic vector. Full-length human RANK cDNA was amplified from human leukocyte cDNA and cloned into the HindIII-EcoRI site of pcDNA3.1. To measure luciferase activity, human embryonic kidney HEK293T cells were plated in triplicate for 1 day. After 48 h the transfected cells were lysed with lysis buffer and the luciferase activity measured using the dual-luciferase assay system and Wallac EnVision microplate reader.
Results During osteoclastogenesis, induced PHB suppressed osteoclast differentiation in mouse bone marrow macrophage (BMM) cultures stimulated by the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and co-cultures of bone marrow cells and osteoblasts. Over-expression of PHB using retrovirus inhibited the protein expression of c-Fos, nuclear factor of activated T cells (NFAT) c1, and phosphorylation of p38 in BMMs. PHB inhibited constitutively active forms of MKK6 (CA-MKK6) and induced SRE activation and the phosphorylation of Elk-1, but no direct inhibition of Elk-1-SRE was observed. PHB also inhibited the bone resorptive activity of mature osteoclasts in a pit formation assay.
Conclusions Our results demonstrate that, during RANKL-mediated osteoclastogenesis, induced PHB exerts inhibitory effects on osteoclast differentiation via suppression of MKK6-induced activation of the p38-Elk-c-Fos-NFATc1 signaling axis. These findings could be important for treating bone resorption diseases, such as osteoporosis and rheumatoid arthritis.
Disclosure of Interest None declared
DOI 10.1136/annrheumdis-2014-eular.2301