Figure 7.

Effect of Saa3 on maturation and activity of osteoclasts. Saa3 up-regulates osteoclast-specific genes, increases osteoclastic fusion, and triggers their bone-resorbing activity. Macrophage-like, preosteoclastic RAW264.7 cells treated with MCSF (30 ng/ml) and RANKL (10 ng/ml) for 3 d significantly up-regulate osteoclast marker genes: Acp5 (A), Ctsk (B), and Calcr (C). Remarkably, cotreatment with M/R and recSaa3 significantly further increases the expression of these osteoclast-specific genes (A–C). Sole treatment with recSaa3 shows a tendency to increase the mRNA levels of Acp5, Ctsk, and Calcr (A–C). D) Treatment of RAW264.7 cells with M/R for 10 d leads to TRAP-positive cells. Cotreatment of these cells with recSaa3 doubles the TRAP-positive osteoclast-like cells. E) Treatment of primary mouse monocytes/macrophages with M/R and with or without recSaa3 for 12 d. Combined treatment significantly increases the number of multinuclear cells, counting 3–5 nuclei per cell and counting over 5 nuclei per cell (E). Calvarial explants from 4- to 5-d-old mice were treated for 12 d with recSaa3, and mineral content was measured with Alizarin Red staining. A significantly decreased mineral content in recSaa3-treated samples is observed, suggesting increased osteoclastic activity in these samples (F). 18S rRNA is a reference gene for RT-qPCR analysis. Values are represented as the mean ± sd. M/R-treated cells are set to 1 in (A)–(C), and control (Ctrl) or treated probes are referred to as fold change to M/R. In (E) and (F), untreated control cells are set to 1, and recSaa3-treated cells/calvaria are referred to as fold change to Ctrl. For all graphs, n = 3 except for (F), n = 10. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.