Lombardi Maria Stella, Gilliéron Corine, Berkelaar Majoska, Gabay Cem
Division of Rheumatology, Department of Internal Medicine Specialties, University Hospitals of Geneva, Geneva, Switzerland.
Department of Pathology and Immunology, University of Geneva School of Medicine, Geneva, Switzerland.
PLoS One. 2017 Oct 3;12(10):e0185426. doi: 10.1371/journal.pone.0185426. eCollection 2017.
Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-κB activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus represent a potential novel therapy to prevent bone erosions.
破骨细胞是负责骨吸收的大型多核细胞。破骨细胞的过度炎症激活会导致骨侵蚀，这是类风湿性关节炎（RA）等几种疾病的标志。盐诱导激酶（SIK）构成一个激酶亚家族，由三个成员（SIK1、-2和-3）组成。抑制SIK激酶活性可诱导巨噬细胞产生抗炎表型。由于破骨细胞起源于巨噬细胞来源的前体，我们推测SIK在破骨细胞生成中发挥作用。我们使用小鼠巨噬细胞系RAW264.7和骨髓来源的巨噬细胞（BMM）分析了SIK1、-2和-3在破骨细胞分化中的表达和功能。我们发现所有三种SIK都在完全分化的破骨细胞中表达，并且在BMM来源的破骨细胞中，SIK1和SIK3蛋白的表达增加。有趣的是，泛SIK抑制剂HG-9-91-01通过剂量依赖性地降低破骨细胞分化标志物（即组织蛋白酶K、基质金属蛋白酶-9和抗酒石酸酸性磷酸酶）和骨吸收活性，显著抑制破骨细胞生成。对RAW细胞中RANKL激活的信号通路的分析表明，SIK抑制剂不影响RANKL诱导的ERK1/2、JNK、p38或NF-κB激活，但会导致c-Fos和NFATc1蛋白水平显著下调，这两个主要转录因子参与破骨细胞特异性基因的调控。此外，SIK抑制部分增加了蛋白酶体介导的c-Fos降解。通过CRISPR/Cas9方法生成了SIK2和SIK3基因敲除的RAW细胞。SIK2基因敲除以及程度较轻的SIK3基因敲除重现了SIK小分子抑制剂的作用，从而证实了SIK抑制对减少破骨细胞生成作用的特异性。总体而言，我们的结果支持这样一种观点，即SIK信号通路在控制破骨细胞生成的检查点中发挥重要作用。因此，SIK激酶抑制剂可能代表一种预防骨侵蚀的潜在新疗法。
