The Erk‐Mediated Phosphorylation of Non‐Muscle Caldesmon Alters Osteoclastogenesis in RANKL‐Induced Raw264.7 Cells

Abstract

The RANKL-RANK signaling pathway is the key event for osteoclastogenesis accompanied by the podosome formation for cell adhesion or extracellular matrix remodeling. Recently, we showed that non-muscle caldesmon (l-CaD) is a key constituent of podosome in RANKL-induced osteoclasts (OCs). Using different decoy peptides mimicking the Pak- or Erk-mediated phosphorylation at the C-terminal end of l-CaD along with full-length mutants of l-CaD also suggested that the l-CaD phosphorylation may involve in the control of osteoclastogenesis. In this study, we used the Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) to examine the altering effects of the Erk signaling pathway leading to changes of l-CaD phosphorylation on the RANKL-induced TRAP activity and cell-cell fusion in osteoclastogenesis. Our study showed that administrations of t-BHQ concentrations of 1, 10, 25, and 50 microM for 30 min or 24 hr before RANKL induction caused increases of l-CaD phosphorylation by 1.13, 1.14, 1.82, and 1.88 folds, respectively, in concomitant with the increased number of small size of differentiated OCs by 2-, 3-, 4-, and 4-folds, respectively, but the decreased fusion index by 0.66, 0.5, 0.3, and 0.3 in a dose dependent manner. However, complete inhibition of Erk phosphorylation with the concomitant 50~60% decrease of l-CaD phosphorylation by treatment with 10 microM PD0325901 for 30 min or 24 hr before RANKL induction completely abolished the OC differentiation. Results obtained here suggest that the Erk-activated l-CaD phosphorylation is required for RANKL-induced OC differentiation, however the dynamic control of l-CaD unphosphorylation/phosphorylation is important for the control of cell-cell fusion in osteoclastogenesis. Support or Funding Information This study is supported by Ministry of Science and Technology of Taiwan Government ( MOST-106-2320-B-005-003-MY2) RANKL-induced formation of actin ring around the cell periphery in differentiated OCs stained with total l-CaD/F-actin (top panel, A), phosphorylated p-l-CaD/F-actin (bottom, A), and vinculin/F-actin (B), and resolved by confocal immunofluorescence imaging. Actin ring structure is composed of the core (labeled with l-CaD, presumbably unphosphorylated) and the peripheral rim (labeled with vinculin, as well as phosphorylated p-l-CaD). Treatments with t-BHQ concentrations of 1, 10, 25, and 50 microM for 24 hr increased l-CaD phosphorylation. Treatments with t-BHQ concentrations of 1, 10, 25, and 50 microM for 24 hr before RANKL induction increased the number of small size of differentiated OCs, but decreased the fusion index in a dose dependent manner. Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) were used for 30 min and 24 hr to examine alterations of Erk and l-CaD phosphorylation. Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) were used for 30 min and 24 hr to examine the altering effects on RANKL-induced TRAP activity and cell-cell fusion in osteoclastogenesis. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.