Experimental Rheumatology, National Institute for Health Research–Leeds Musculoskeletal Biomedical Research Unit, Leeds Institute of Molecular Medicine, Leeds, West Yorkshire, UK.
Ann Rheum Dis. 2012 Dec;71(12):2035-43. doi: 10.1136/annrheumdis-2011-201197. Epub 2012 Jun 7.
To investigate convergence of endoplasmic reticulum stress pathways and enhanced reactive oxygen species (ROS) production, due to intracellular retention of mutant tumour necrosis factor receptor 1 (TNFR1), as a disease mechanism in TNFR-associated periodic syndrome (TRAPS).
Peripheral blood mononuclear cells from patients with TRAPS (n=16) and healthy controls (HC) (n=22) were studied alongside HEK293T cells expressing wild type-TNFR1 or TRAPS-associated mutations. Unfolded protein response (UPR)-associated proteins (protein kinase-like endoplasmic reticulum kinase, PERK), phosphorylated-PERK (p-PERK), phosphorylated inositol-requiring enzyme 1α (p-IRE1α) and spliced X-box binding protein 1 (sXBP1)) were measured by flow cytometry. XBP1 splicing and UPR-associated transcript expression were assessed by reverse transcription PCR/quantitative real-time PCR. ROS levels were measured using CM-H(2)DCFDA and MitoSOX Red in patients' monocytes or HEK293T cells by flow cytometry.
Mutant TNFR1-expressing HEK293T cells had increased TNFR1 expression associated with intracellular aggregation. TRAPS patients had increased sXBP1 transcripts (p<0.01) compared with HC. Raised p-PERK protein was seen, indicative of an UPR, but other UPR-associated transcripts were normal. Increased ROS levels were observed in TRAPS monocytes compared with HCs (p<0.02); these increased further upon IL-6 stimulation (p<0.01). Lipopolysaccharide-stimulated peripheral blood mononuclear cells of patients with TRAPS, but not HCs, demonstrated increased sXBP1 levels (p<0.01), which were reduced by antioxidant treatment (p<0.05).
Patients with TRAPS have evidence of increased sXBP1 and PERK expression but without other signs of classical UPR, and also with high ROS generation that may contribute to the pro-inflammatory state associated with TRAPS. The authors propose a non-traditional XBP1 pathway with enhanced sXBP1 as a novel disease-contributing mechanism in TRAPS.
研究内质网应激途径的趋同以及活性氧（ROS）产生的增强，这是由于突变肿瘤坏死因子受体 1（TNFR1）在细胞内的滞留，这是肿瘤坏死因子相关周期性综合征（TRAPS）的一种疾病机制。
研究了 TRAPS 患者（n=16）和健康对照者（HC）（n=22）的外周血单核细胞，以及表达野生型-TNFR1 或 TRAPS 相关突变的 HEK293T 细胞。通过流式细胞术测量未折叠蛋白反应（UPR）相关蛋白（蛋白激酶样内质网激酶，PERK）、磷酸化-PERK（p-PERK）、磷酸化肌醇需求酶 1α（p-IRE1α）和剪接 X 盒结合蛋白 1（sXBP1））。通过逆转录 PCR/实时定量 PCR 评估 XBP1 剪接和 UPR 相关转录物的表达。通过流式细胞术使用 CM-H（2）DCFDA 和 MitoSOX Red 测量患者单核细胞或 HEK293T 细胞中的 ROS 水平。
表达突变型 TNFR1 的 HEK293T 细胞表现出与细胞内聚集相关的 TNFR1 表达增加。与 HC 相比，TRAPS 患者的 sXBP1 转录本增加（p<0.01）。发现升高的 p-PERK 蛋白，表明 UPR，但其他 UPR 相关转录本正常。与 HC 相比，TRAPS 单核细胞中的 ROS 水平升高（p<0.02）；在受到 IL-6 刺激后，这些水平进一步升高（p<0.01）。TRAPS 患者而非 HC 的外周血单核细胞经脂多糖刺激后，sXBP1 水平升高（p<0.01），抗氧化剂治疗后降低（p<0.05）。
TRAPS 患者表现出 sXBP1 和 PERK 表达增加的证据，但没有其他经典 UPR 的迹象，并且 ROS 生成增加，这可能导致与 TRAPS 相关的促炎状态。作者提出了一种非传统的 XBP1 途径，增强的 sXBP1 作为 TRAPS 的一种新的疾病致病机制。
