IL1ß and TNFα promote RANKL-dependent adseverin expression and osteoclastogenesis.

Adseverin is an actin-binding protein involved in osteoclastogenesis, but its role in inflammation-induced bone loss is not well-defined. Here, we examined whether IL1ß and TNFα regulate adseverin expression to control osteoclastogenesis in mouse primary monocytes and RAW264.7 cells. Adseverin was colocalized with subcortical actin filaments and was enriched in the fusopods of fusing cells. In precursor cells, adseverin overexpression boosted the formation of RANKL-induced multinucleated cells. Both IL1ß and TNFα enhanced RANKL-dependent TRAcP activity by 1.6-fold and multinucleated cell formation (cells with ≥3 nuclei) by 2.6- and 3.3-fold, respectively. However, IL1ß and TNFα did not enhance osteoclast formation in adseverin-knockdown cells. RANKL-dependent adseverin expression in bone marrow cells was increased by both IL1ß (5.4-fold) and TNFα (3.3-fold). Luciferase assays demonstrated that this expression involved transcriptional regulation of the adseverin promoter. Activation of the promoter was restricted to a 1118 bp sequence containing an NF-κB binding site, upstream of the transcription start site. TNFα also promoted RANKL-induced osteoclast precursor cell migration. We conclude that IL1ß and TNFα enhance RANKL-dependent expression of adseverin, which contributes to fusion processes in osteoclastogenesis.

Role of actin filaments in fusopod formation and osteoclastogenesis.

Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 µm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.