Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

Abstract

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-␣ demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-␣ displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-␣ and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or ␣v reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin ␣v␤3, Src, cortactin, etc.) involved in the maturation process.Osteoclasts, the multinucleated and terminally differentiated giant cells, are involved in bone resorption. The adhesion of osteoclasts to the bone during bone resorption leads to the formation of the clear zone, an actin-rich ring-like adhesion zone circumscribing an area of bone resorption. Formation of a clear zone or sealing zone (also known as the sealing ring) has been considered to be a marker of osteoclast activation and is fundamental to the process of osteoclast bone resorption. Sealing ring formation is mediated by the dynamics of the actin cytoskeleton. Distinct pathways and signaling molecules have been shown to play roles in the organization of the sealing ring during bone resorption. Our previous observations in gelsolin null (Gsn Ϫ/Ϫ ) 2 osteoclasts demonstrated that deficiency of this protein blocks podosome assembly and motility. However, the cells still exhibit sealing ring and matrix resorption (1). Therefore, Gsn Ϫ/Ϫ osteoclasts are capable of resorbing bone, but the resorbed areas are small due to the absence of podosomes and the resulting hypomotile nature of osteoclasts (1). Observations in Gsn Ϫ/Ϫ osteoclasts also suggest that the organization of the sealing ring presumably reflect changes in the role of actinbinding proteins. Spatial configurations of actin fil...

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

“…We assessed the expression of p140Cap in OCs but could not unequivocally detect its mRNA or protein. In contrast, cortactin is highly expressed in mature OCs (80) and localizes to individual podosomes and in the podosome belt (8,10,52,53,81,82). In OCs, EB1 and cortactin belong to a similar molecular complex, as shown by immunoprecipitation, even though a direct interaction between the proteins cannot be ascertained.…”

LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L‐plastin

“…In previous studies, it was found that L-plastin deficient OCs exhibited normal differentiation and peripheral podosomes, but these cells showed significant decreases in the formation of the adhesive contact with the bone surface (16) . It has also been demonstrated that phosphorylation of L-plastin at serine residue 5, after a variety of stimuli, lead to increased stability of actin-based OC sealing rings and podosomes that are critical for osteoclast adhesion, and the early process of bone resorption (16) .…”

“…L-plastin is phosphorylated on serine 5 and serine 7 in hematopoietic cells in vivo in response to signals triggering the activation of the immune response, cell migration and proliferation (16-18) . While in vivo and in vitro phosphorylation of serine 5 by cAMP-dependent protein kinase A (PKA) is well documented, it is still unknown which kinase(s) phosphorylate serine 7 (17,18) .…”

L-Plastin promotes podosome longevity and supports macrophage motility

“…Podosomes are small, circular, adhesion-based accumulations of actin-binding and signaling proteins linked to F-actin found predominantly in cells of myeloid origin, including monocytes, macrophages, dendritic cells and osteoclasts (Williams and Ridley, 2000; Linder and Wiesner, 2015). LPL has been previously shown to be a component of macrophage podosomes, but the function of LPL in assembling or maintaining this actin-based structure has been explored in only a few reports (Messier et al, 1993; Babb et al, 1997; Evans et al, 2003; Ma et al, 2010; De Clercq et al, 2013). Tight temporal correlation of LPL with F-actin as podosomes formed, disassembled, and reformed in the IC-21 macrophage cell line suggested that LPL provided structural support to podosome-associated actin (Evans et al, 2003).…”