Department of Surgery, University of California, San Francisco, California, USA.
Gastroenterology. 2011 Nov;141(5):1864-74.e1-3. doi: 10.1053/j.gastro.2011.07.035. Epub 2011 Jul 28.
BACKGROUND & AIMS: Although proteases control inflammation and pain, the identity, cellular origin, mechanism of action, and causative role of proteases that are activated during disease are not defined. We investigated the activation and function of cysteine cathepsins (Cat) in colitis.
Because protease activity, rather than expression, is regulated, we treated mice with fluorescent activity-based probes that covalently modify activated cathepsins. Activated proteases were localized by tomographic imaging of intact mice and confocal imaging of tissues, and were identified by electrophoresis and immunoprecipitation. We examined the effects of activated cathepsins on excitability of colonic nociceptors and on colonic pain, and determined their role in colonic inflammatory pain by gene deletion.
Tomography and magnetic resonance imaging localized activated cathepsins to the inflamed colon of piroxicam-treated il10(-/-) mice. Confocal imaging detected activated cathepsins in colonic macrophages and spinal neurons and microglial cells of mice with colitis. Gel electrophoresis and immunoprecipitation identified activated Cat-B, Cat-L, and Cat-S in colon and spinal cord, and Cat-S was preferentially secreted into the colonic lumen. Intraluminal Cat-S amplified visceromotor responses to colorectal distension and induced hyperexcitability of colonic nociceptors, which required expression of protease-activated receptor-2. Cat-S deletion attenuated colonic inflammatory pain induced with trinitrobenzene sulfonic acid.
Activity-based probes enable noninvasive detection, cellular localization, and proteomic identification of proteases activated during colitis and are potential diagnostic tools for detection of predictive disease biomarkers. Macrophage cathepsins are activated during colitis, and Cat-S activates nociceptors to induce visceral pain via protease-activated receptor-2. Cat-S mediates colitis pain and is a potential therapeutic target.
蛋白酶可控制炎症和疼痛，但在疾病发生时被激活的蛋白酶的种类、细胞起源、作用机制和因果关系尚不清楚。本研究旨在探讨结肠炎中半胱氨酸蛋白酶（Cat）的激活和功能。
由于蛋白酶的活性而非表达受到调控，我们用荧光活性探针处理小鼠，该探针可与被激活的组织蛋白酶发生共价结合。通过对完整小鼠进行断层扫描成像和组织的共聚焦成像，来定位被激活的蛋白酶，并通过电泳和免疫沉淀来对其进行鉴定。我们研究了被激活的组织蛋白酶对结肠感觉神经元兴奋性和结肠疼痛的影响，并通过基因敲除来确定其在结肠炎症性疼痛中的作用。
断层扫描和磁共振成像将被激活的组织蛋白酶定位到吡罗昔康处理的 il10(-/-) 小鼠的发炎结肠中。共聚焦成像检测到结肠炎小鼠的结肠巨噬细胞和脊髓神经元及小胶质细胞中存在被激活的组织蛋白酶。凝胶电泳和免疫沉淀鉴定到了在结肠和脊髓中被激活的 Cat-B、Cat-L 和 Cat-S，且 Cat-S 优先分泌到结肠腔中。腔内的 Cat-S 增强了对结直肠扩张的内脏运动反应，并诱导了结肠感觉神经元的超兴奋性，该过程需要蛋白酶激活受体-2 的表达。Cat-S 缺失可减轻三硝基苯磺酸诱导的结肠炎症性疼痛。
活性探针可实现非侵入性检测、细胞定位和对结肠炎中被激活的蛋白酶的蛋白质组学鉴定，是探测预测性疾病生物标志物的潜在诊断工具。巨噬细胞组织蛋白酶在结肠炎中被激活，Cat-S 通过蛋白酶激活受体-2 激活感觉神经元，引发内脏疼痛。Cat-S 介导结肠炎疼痛，是一个潜在的治疗靶点。
