The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

Abstract

Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

Adseverin plays a role in osteoclast differentiation and periodontal disease‐mediated bone loss

“…Although it is very similar in structure to the ubiquitously expressed gelsolin (Ads contains 6 gelsolin‐like domains), the tissue distribution of Ads is mainly limited to the kidney and intestine (11, 12). Notably, although our recent research shows that Ads regulates OCG in cultured cells (13), there have been no detailed analyses of the regulation of expression of Ads in bone. Gelsolin and Ads promote actin filament depolymerization by severing actin filaments in a calcium‐dependent manner while remaining bound to the barbed end of the actin filament (14).…”

“…Similar to gelsolin, the severing activity of adseverin is regulated by phosphatidylinositol 4,5‐bis‐phosphate (PIP2) (15), but despite their seemingly similar roles in regulating actin filament structure, recent data suggest that these 2 proteins may play unique roles in osteoclast differentiation. Only Ads is required in the osteoclastogenic phase, whereas gelsolin is required in the bone resorption phase (13).…”

“…Although 2 separate microarray analyses studies showed that Ads is up‐regulated during OCG (13) and in mature osteoclasts (16), little is known about the role of Ads in actin filament turnover and OCG. Here we investigated the role of Ads in the regulation of the osteoclast actin cytoskeleton and the consequences of Ads deficiency on osteoclast fusion and function.…”

Bioaggregate Inhibits Osteoclast Differentiation, Fusion, and Bone Resorption In Vitro

“…For OCG, 5 Â 10 4 RAW264.7 cells (passages 5-15; ATCC, Manassas, VA) were seeded into 6-well culture dishes and cultured with 60 ng/mL soluble recombinant RANKL (sRANKL) (R&D Systems, Minneapolis, MN) for 4 days as described previously (18). At day 2.5 of the OCG induction, cell-cell fusion among the RAW264.7 cells began to occur.…”

“…Western blotting was performed as described previously (18). The nuclear and cytoplasmic proteins were extracted using the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific Pierce, Rockford, IL) according to the manufacturer's instructions.…”