The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14(+) human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK.

Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), and -benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC = 0.3 μM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α; ) mRNA expression without affecting, , or oncostatin M () expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss.

BACKGROUND We recently reported that the signaling pathway downstream RANK in RANKL-induced osteoclastogenesis is inhibited by cysteine proteinase inhibitors (CPIs) (Strålberg et al., FASEB J, 2013). Here, we demonstrate that osteoclastogenesis stimulated by LPS E.coli in RANKL-primed mouse bone marrow macropages (BMM) is inhibited by CPIs cystatin C, E-64, and Z-RLVG-CHN 2. The inhibitory effect is associated with decreased gene and protein expression, and an active uptake of fluorescent tagged cystatin C dependent on LPS-stimulation. Osteoclast formation was inhibited by all three CPIs in cathepsin K deficient mice. Also, the cathepsin K inhibitor L-006235 did not inhibit osteoclast formation. LPS-stimulated osteoclast formation in skull bones of adult mouse was inhibited by E-64. CONCLUSIONS All together, these data show that: ü Cysteine proteinase inhibitors are important in LPS-induced osteoclastogenesis both in vitro and in vivo ü Osteoclast progenitors take up Cystatin C by a process facilitated by RLprimed LPS-stimulation ü inhibition of osteoclast formation by cysteine proteinase inhibitors is not explained by inhibition of cathepsin K Disclosure of Interest: None Declared RESULTS Printed by A) RL-primed LPS-induced osteoclast differentiation is inhibited by CPIs Cystatin C, E-64, and Z-RLVG-CHN 2 B) CPIs inhibit LPS-induced mRNA and protein expression of Cfos and Nfatc1 C) CPIs inhibit LPS-induced mRNA and protein expression of Acp5, Calcr, and Ctsk E) LPS potentiates the intracellular uptake of CysC in RL-primed osteoclast progenitors F) The cathepsin K inhibitor L-006235 does not inhibit osteoclast formation G) LPS-induced osteoclast formation in skull bones is inhibited by E-64 in vivo

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Background and objectivesThe authors have previously reported that the cysteine proteinase inhibitor (CPI) cystatin C (CysC) reduces osteoclast formation induced by signal pathways activated by either PTH receptor, vitamin D receptor or gp130 in crude mouse bone marrow cultures (ref). In the present study, the authors have investigated if CysC, E-64 (fungal CPI) and the tetrapeptidyl derivative Z-RLVG-CHN 2 (representing Arg 8 -Leu 9 -Val 10 -Gly 11 of the aminoterminal end of CysC) can inhibit osteoclast formation using purifi ed mouse and human osteoclast progenitors stimulated by either RANKL or LPS Escherichia coli. Results All three inhibitors concentration-dependently (IC 50 CysC=0.3 μM, E-64=3μM, Z-RLVG-CHN 2 =0.3 μM) inhibited RANKL induced osteoclast formation in mouse bone marrow macrophage (BMM) cultures; similar observations were made using human peripheral blood CD14 + progenitors. These data were based upon (1) counting the number of TRAP + osteoclasts in cultures on plastic or (2) on bone, (3) assessing actin-ring expressing cells and (4) by analysing pit formation and release of CTX when progenitor cells were cultured on bone. The effect was induced early during differentiation as demonstrated by withdrawal and addition of CysC at different time points, by showing that a variety of RANKL induced osteoclastic genes (Calcr, Acp5, Ctsk, Integrin b3, were downregulated and that BMM was maintained at a macrophage stage (capacity to phagocytose and increased Irf-8). The inhibitory effect was associated with decreased mRNA and protein expression of c-Fos and Nfatc1, and with decreased activation of NF-κB. Inhibition was also associated with decreased mRNA and protein expression of