Marzaioli Viviana, McMorrow Jason P, Angerer Hannes, Gilmore Alyssa, Crean Daniel, Zocco Davide, Rooney Peadar, Veale Douglas, Fearon Ursula, Gogarty Martina, McEvoy Alice N, Stradner Martin H, Murphy Evelyn P
Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
Arthritis Rheum. 2012 Oct;64(10):3290-301. doi: 10.1002/art.34554.
To elucidate histamine receptor-mediated signaling pathways, transcriptional events, and target gene expression in human cartilage.
Histamine modulation of cartilage destruction was assessed by Safranin O staining and proteoglycan release. H(1) , H(2) , H(3) , and H(4) histamine receptor-dependent regulation of transcription factors (nuclear receptor 4A1 [NR4A1], NR4A2, and NR4A3), RANKL, and osteoprotegerin (OPG) messenger RNA (mRNA) levels were measured in primary and SW-1353 chondrocyte cells using quantitative polymerase chain reaction and selective histamine receptor antagonists. Soluble RANKL and OPG protein levels were determined using enzyme-linked immunosorbent assays. NR4A protein levels and transactivity were evaluated by Western blot analysis, immunocytochemistry, and luciferase reporter assays. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A short hairpin RNA.
Primary human chondrocyte cells expressed differential steady-state levels of H(1) -H(4) histamine receptor mRNA. In combination with tumor necrosis factor α, histamine significantly promoted cartilage proteoglycan depletion and release. Histamine modulated the expression of NR4A1-3 orphan receptors in primary and immortalized human chondrocyte cells in a time- and concentration-dependent manner. Histamine selectively signaled through H(1) and H(2) histamine receptors in chondrocytes to modulate RANKL and NR4A2 expression. The temporal effects of histamine on NR4A2 gene transcription were reduced in cells pretreated with inhibitors directed against protein kinase A, MAPK, and NF-κB signaling pathways. Histamine modulated the expression of RANKL with modest effects on OPG levels, leading to increased RANKL:OPG mRNA and protein ratios. Stable knockdown of NR4A1-3 expression resulted in reduced endogenous OPG levels and the loss of histamine-dependent regulation of RANKL expression.
Our findings indicate that histamine, via H(1) and H(2) histamine receptors, contributes to joint disease by enhancing the ratio of RANKL to OPG expression through altered NR4A activity in human chondrocyte cells.
阐明组胺受体介导的信号通路、转录事件以及人类软骨中的靶基因表达。
通过番红O染色和蛋白聚糖释放评估组胺对软骨破坏的调节作用。使用定量聚合酶链反应和选择性组胺受体拮抗剂，在原代和SW - 1353软骨细胞中测量转录因子（核受体4A1 [NR4A1]、NR4A2和NR4A3）、核因子κB受体活化因子配体（RANKL）和骨保护素（OPG）信使核糖核酸（mRNA）水平的H(1)、H(2)、H(3)和H(4)组胺受体依赖性调节。使用酶联免疫吸附测定法测定可溶性RANKL和OPG蛋白水平。通过蛋白质印迹分析、免疫细胞化学和荧光素酶报告基因测定评估NR4A蛋白水平和转录活性。通过慢病毒转导NR4A短发夹RNA实现NR4A1 - 3的稳定缺失。
原代人软骨细胞表达不同稳态水平的H(1) - H(4)组胺受体mRNA。与肿瘤坏死因子α联合时，组胺显著促进软骨蛋白聚糖的消耗和释放。组胺以时间和浓度依赖性方式调节原代和永生化人软骨细胞中NR4A1 - 3孤儿受体的表达。组胺在软骨细胞中通过H(1)和H(2)组胺受体选择性地发出信号，以调节RANKL和NR4A2的表达。在用针对蛋白激酶A、丝裂原活化蛋白激酶（MAPK）和核因子κB信号通路的抑制剂预处理的细胞中，组胺对NR4A2基因转录的时间效应降低。组胺调节RANKL的表达，对OPG水平影响较小，导致RANKL:OPG mRNA和蛋白比率增加。NR4A1 - 3表达的稳定敲低导致内源性OPG水平降低以及组胺依赖性RANKL表达调节的丧失。
我们的研究结果表明，组胺通过H(1)和H(2)组胺受体，通过改变人软骨细胞中的NR4A活性，提高RANKL与OPG表达的比率，从而导致关节疾病。
