Department of Physiology & Pharmacology, HRIC 4AC60, University of Calgary Faculty of Medicine, 3280 Hospital Drive N,W,, Calgary, AB T2N 4Z6, Canada.
Respir Res. 2013 Aug 30;14(1):88. doi: 10.1186/1465-9921-14-88.
Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Cigarette smoking is the leading risk factor for the development of COPD and 25% of asthmatics smoke. Smoking asthmatics have worse symptoms and more frequent hospitalizations compared to non-smoking asthmatics. The degree of neutrophil recruitment to the airways correlates with disease severity in COPD and during viral exacerbations of asthma. We have previously shown that HRV and cigarette smoke, in the form of cigarette smoke extract (CSE), each induce expression of the neutrophil chemoattractant and activator, CXCL8, in human airway epithelial cells. Additionally, we demonstrated that the combination of HRV and CSE induces expression of levels of CXCL8 that are at least additive relative to induction by each stimulus alone, and that enhancement of CXCL8 expression by HRV+CSE is regulated, at least in part, via mRNA stabilization. Here we further investigate the mechanisms by which HRV+CSE enhances CXCL8 expression.
Primary human bronchial epithelial cells were cultured and treated with CSE alone, HRV alone or the combination of the two stimuli. Stabilizing/destabilizing proteins adenine/uridine-rich factor-1 (AUF-1), KH-type splicing regulatory protein (KHSRP) and human antigen R (HuR) were measured in cell lysates to determine expression levels following treatment. siRNA knockdown of each protein was used to assess their contribution to the induction of CXCL8 expression following treatment of cells with HRV and CSE.
We show that total expression of stabilizing/de-stabilizing proteins linked to CXCL8 regulation, including AUF-1, KHSRP and HuR, are not altered by CSE, HRV or the combination of the two stimuli. Importantly, however, siRNA-mediated knock-down of HuR, but not AUF-1 or KHSRP, abolishes the enhancement of CXCL8 by HRV+CSE. Data were analyzed using one-way ANOVA with student Newman-Keuls post hoc analysis and values of p≤ 0.05 were considered significant.
Induction of CXCL8 by the combination of HRV and CSE is regulated by mRNA stabilization involving HuR. Thus, targeting the HuR pathway may be an effective method of dampening CXCL8 production during HRV-induced exacerbations of lower airway disease, particularly in COPD patients and asthmatic patients who smoke.
人类鼻病毒（HRV）可引发哮喘和慢性阻塞性肺疾病（COPD）加重。吸烟是 COPD 发展的主要危险因素，25%的哮喘患者吸烟。与非吸烟哮喘患者相比，吸烟哮喘患者的症状更严重，住院次数更多。气道中中性粒细胞的募集程度与 COPD 疾病的严重程度以及哮喘病毒加重期间的中性粒细胞募集程度相关。我们之前已经表明，HRV 和香烟烟雾（以香烟烟雾提取物（CSE）的形式）都会诱导人呼吸道上皮细胞中中性粒细胞趋化因子和激活剂 CXCL8 的表达。此外，我们还证明 HRV 和 CSE 的组合诱导的 CXCL8 表达水平至少与每种单独刺激诱导的表达水平相加，并且 HRV+CSE 增强 CXCL8 表达的作用至少部分是通过 mRNA 稳定化调节的。在这里，我们进一步研究了 HRV+CSE 增强 CXCL8 表达的机制。
原代人支气管上皮细胞培养并单独用 CSE、HRV 或两种刺激物处理。用细胞裂解物中腺嘌呤/尿嘧啶丰富因子-1（AUF-1）、KH 剪接调节蛋白（KHSRP）和人抗原 R（HuR）来测量稳定/不稳定蛋白，以确定处理后表达水平。用 siRNA 敲低每种蛋白，以评估它们对 HRV 和 CSE 处理后 CXCL8 表达的诱导作用。
我们表明，与 CXCL8 调节相关的稳定/不稳定蛋白的总表达，包括 AUF-1、KHSRP 和 HuR，不受 CSE、HRV 或两种刺激物的影响。然而，重要的是，siRNA 介导的 HuR 敲低，但不是 AUF-1 或 KHSRP，消除了 HRV+CSE 对 CXCL8 的增强作用。数据采用单向方差分析和学生纽曼-凯斯事后分析，p 值≤0.05 被认为具有统计学意义。
HRV 和 CSE 的组合诱导的 CXCL8 表达受涉及 HuR 的 mRNA 稳定化调节。因此，靶向 HuR 途径可能是一种有效的方法，可以在 HRV 诱导的下呼吸道疾病加重期间，特别是在 COPD 患者和吸烟哮喘患者中，抑制 CXCL8 的产生。
