Osteoclasts originated from hematopoietic stem cells are multi-nucleated cells that can resorb the bone matrix. Receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) signaling pathway is crucial for the differentiation and activation of osteoclasts. In this study, we investigated for the first time whether or not RANKL induced mitogen- and stress-activated kinase 1 (MSK1) phosphorylation at Ser 376. Activation of MSK1 was detected as soon as 5 min after RANKL stimulation and sparsely detected at 30 min after stimulation. RANKL-induced MSK1 phosphorylation occurred in a dose-dependent manner. MSK1 is known as a downstream signaling molecule of cAMP-dependent protein kinase (PKA). Treatment with the PKA inhibitor H89 significantly suppressed c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) induction upon RANKL stimulation. In addition, cAMP response element-binding protein (CREB) phosphorylation was extremely inhibited by H89 treatment. Mitogen-activated protein kinases (MAPKs) have been investigated for induction of MSK1 phosphorylation. Specific signaling pathway inhibitors for p38 and extracellular signal-regulated kinases (ERKs) significantly blocked RANKL-induced MSK1 activation. Finally, as a downstream effector of the p38-MSK1 pathway, c-Fos transcriptional activity was determined. RANKL-mediated elevation of c-Fos transcriptional activity was significantly suppressed by p38 inhibitor. Moreover, a dominant negative form of CREB suppressed activation of NFATc1. In conclusion, RANKL-stimulated MSK1 phosphorylation could play a role in induction of NFATc1 through CREB and c-Fos activation as a downstream molecule of p38, ERK MAPKs, and PKA. Our results support basic information for the development of osteoclast specific inhibitors.

Introduction
 Materials and Methods
  Materials
  Cell culture
  Western blotting
  CCK assay
  Luciferase assay
  Retrovirus infection
  Statistics
 Results
 Discussion
 Acknowledgements
 ORCID
 References

• Jeongim Ha(Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Jung Hye Hwang(1Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Seul Gi Kwon(Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Da Hye Park(Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Tae Wan Kim(Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Deok Gyeong Kang(Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Kyung Hee Kang(Swine Science and Technology Center, Gyeongnam National University of Science & Technology)
• Chul Wook Kim(Swine Science and Technology Center, Gyeongnam National University of Science & Technology) Corresponding author
• Il-Suk Kim(Department of Animal Resource Technology, Gyeongnam National University of Science & Technology)