SAT0462 Overexpression of Prohibitin-1 Inhibits Rankl-Induced Activation of P38-Elk-1-Sre Signaling Axis Blocking MKK6 Activity
Authors: C.-H. Lee, J.-M. Oh, W.-H. Yoo, S.-H. Beak, J.-J. Choi, S.-W. Choi, J.-T. Yeon, W.-S. Lee, M.-S. Lee
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> 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. <h3>Objectives</h3> In the present study, we investigated the effects of PHB on osteoclast differentiation in vitro and bone resorption ex vivo. <h3>Methods</h3> 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. <h3>Results</h3> 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. <h3>Conclusions</h3> 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. <h3>Disclosure of Interest</h3> None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.2301