Strålberg Fredrik, Kassem Ali, Kasprzykowski Franciszek, Abrahamson Magnus, Grubb Anders, Lindholm Catharina, Lerner Ulf H
Department of Molecular Periodontology, Umeå University, Umeå, Sweden.
Institute of Chemistry, University of Gdansk, Gdansk, Poland.
J Leukoc Biol. 2017 May;101(5):1233-1243. doi: 10.1189/jlb.3A1016-433R. Epub 2017 Feb 14.
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.
炎症诱导的骨破坏是许多炎性骨骼疾病的主要治疗靶点。本研究旨在探讨半胱氨酸蛋白酶抑制剂胱抑素C、真菌半胱氨酸蛋白酶抑制剂（E-64）和苄氧羰基-精氨酰-亮氨酰-缬氨酰-甘氨酰-重氮甲烷乙酸盐（Z-RLVG-CHN）是否能抑制脂多糖（LPS）诱导的破骨细胞形成。分离小鼠骨髓巨噬细胞（BMMs），用核因子κB受体激活剂配体（RANKL）预处理24小时，然后在有或无抑制剂的情况下用LPS刺激。成年小鼠局部注射LPS，然后用E-64治疗，并通过组织蛋白酶K多核细胞数量评估破骨细胞形成。胱抑素C以时间和浓度依赖性方式抑制LPS诱导的破骨细胞形成（半数抑制浓度[IC] = 0.3 μM）。该作用与抗酒石酸酸性磷酸酶（TRAP）、组织蛋白酶K以及破骨细胞生成转录因子c-Fos和活化T细胞核因子c1（NFATc1）的mRNA和蛋白表达降低有关。胱抑素C也抑制LPS诱导的骨切片上破骨细胞的形成，导致骨陷窝形成减少和骨基质蛋白释放减少。用E-64和Z-RLVG-CHN获得了类似的数据。胱抑素C在LPS刺激的BMMs中被内化，但在未刺激的BMMs中未被内化。LPS诱导的破骨细胞形成依赖于肿瘤坏死因子-α（TNF-α），这3种抑制剂消除了LPS诱导的肿瘤坏死因子超家族2（编码TNF-α的基因；）mRNA表达，而不影响白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）或抑瘤素M（OSM）的表达。E-64抑制局部LPS刺激后颅骨中破骨细胞的形成。得出的结论是，半胱氨酸蛋白酶抑制剂通过抑制TNF-α表达在体内和体外有效抑制LPS诱导的破骨细胞形成。靶向半胱氨酸蛋白酶可能代表一种预防炎性骨丢失的新治疗方式。
