The Role of Filamin A in Osteoclastogenesis

Osteoclasts are bone-resorbing cells important physiologically for bone development, remodeling, and calcium regulation. They are formed by fusion of monocytes but the mechanisms involved are unclear. Migration of monocytes toward each other likely precedes critical fusion events. Cellular migration involves actin cytoskeleton reorganization, with F-actin assembly at the leading edge of migrating cells driving locomotion. Filamin A (FLNa) crosslinks F-actin and is crucial for actin remodeling and cellular migration. Thus, FLNa may be important in osteoclastogenesis (OCG) by regulating monocyte migration. Objective: To investigate the role of FLNa in regulating monocyte migration and subsequent OCG. Methods: Bone marrow from WT and FLNa-null SV129-Black6 mice was flushed from tibia and femurs with alpha-MEM and a monocyte-rich cell suspension was recovered after centrifugation over Ficoll-Paque-PLUS. Monocyte chemotaxis was evaluated using Boyden chambers with 5um membrane pores against M-CSF and RANKL. OCG was induced by adding M-CSF (20ng/mL) and RANKL (100ng/mL) to 1x10⁶ cells in glass chamber slides for 6 days. Osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP). Osteoclast activity was evaluated by plating cells on osteologic discs and performing von Kossa staining for resorption pits made. Results: FLNa-null monocytes had a severe migration defect against M-CSF and RANKL (see Table 1). They generated less osteoclasts with ³3 nuclei (3.3 ±1.7/FOV) compared to WT (14.8±3.1/FOV, p<0.01). % of nuclei in multinucleated TRAP⁺osteoclasts to total nuclei in TRAP⁺cells per FOV was 50.3±3.5% for WT but only 24.0±0.3% for FLNa-null cells (p<0.05). FLNa-null osteoclasts were able to initiate formation of similar numbers of resorption pits as WT (135.3±50.6 and 170.3±37.0, respectively; p>0.05), however total resorption area/FOV and avg area/pit were significantly higher in WT than FLNa-null osteoclasts (299.1±28.6 vs. 76.9±27.7, respectively, and 1.9±0.3 vs. 0.6±0.02, respectively; both p<0.05). Conclusion: FLNa is important in osteoclastogenesis likely through its regulation of monocyte migration.

Table 1. Monocyte migration toward chemoattractants M-CSF and RANKL. (Results are nuclei counted per FOV expressed as mean±SEM, n=3)
	M-CSF				RANKL
[ ] ng/mL	0	20	50	100	0	100	500
WT	18.1±1.9	38.1±4.2*	38.5±4.9*	40.3±4.8*	46.2±12.3	78.2±8.0*	103.7±9.1*
FLNa-null	15.3±2.0	16.2±2.4*	14.4±1.4*	15.1±2.4*	32.0±7.1	35.3±4.6*	43.4±2.5*
*p<0.05 for WT vs. FLNa-null at each chemoattractant concentration used