白细胞介素-13通过15-脂氧合酶/G蛋白偶联受体激酶2机制使人气道上皮细胞中的β2-肾上腺素能受体脱敏。

IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism.

作者信息

Albano Giusy D, Zhao Jinming, Etling Emily B, Park Seo Young, Hu Haizhen, Trudeau John B, Profita Mirella, Wenzel Sally E

机构信息

University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa; Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Palermo, Italy.

University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa.

出版信息

J Allergy Clin Immunol. 2015 May;135(5):1144-53.e1-9. doi: 10.1016/j.jaci.2015.02.006. Epub 2015 Mar 24.

DOI:10.1016/j.jaci.2015.02.006

PMID:25819984

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4426258/

Abstract

BACKGROUND

β2-Adrenergic receptor (β2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of β2-agonists is well studied, type 2 inflammation could also affect β2AR function.

OBJECTIVE

We sought to evaluate the effect of the type 2 cytokine IL-13 on β2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2).

METHODS

HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and β2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2.

RESULTS

Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in β2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients.

CONCLUSION

IL-13 treatment of HAECs leads to β2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) β2ARs, suggesting that the beneficial effects of β2-agonists could be blunted in patients with type 2 associated asthma.

摘要

背景

β2肾上腺素能受体（β2AR）激动剂是哮喘的关键治疗药物。然而，受体脱敏可导致治疗效果丧失。尽管对重复使用β2激动剂的脱敏作用已得到充分研究，但2型炎症也可能影响β2AR功能。

目的

我们试图评估2型细胞因子白细胞介素-13（IL-13）对人气道上皮细胞（HAECs）中β2AR脱敏的影响，并确定15-脂氧合酶-1（15LO1）与磷脂酰乙醇胺结合蛋白1（PEBP1）的结合是否通过释放G蛋白受体激酶2（GRK2）促成脱敏。

方法

对气液界面培养的HAECs进行预处理，分别给予或不给予IL-13（48小时）或盐酸异丙肾上腺素（ISO；30分钟），然后用ISO刺激10分钟。使用酶联免疫吸附测定法测量环磷酸腺苷（cAMP）水平，使用蛋白质免疫印迹法测量β2AR和GRK2的磷酸化水平。使用小干扰RNA敲低15LO1。通过免疫沉淀/蛋白质免疫印迹法和免疫荧光检测GRK2、PEBP1和15LO1的相互作用。对来自对照受试者和哮喘患者的HAECs及气道组织进行15LO1、PEBP1和GRK2评估。

结果

与单独给予10分钟ISO相比，用ISO或IL-13预处理可降低ISO诱导的cAMP生成，同时β2AR和GRK2磷酸化增加。ISO处理10分钟后，GRK2与PEBP1结合，同时GRK2低磷酸化（pGRK2）水平出现。相反，在存在IL-13加ISO（10分钟）的情况下，GRK2与PEBP1的结合减少，而15LO1结合和pGRK2水平增加。敲低15LO1可恢复ISO诱导的cAMP生成。这些发现也在哮喘患者细胞和组织中新鲜刷取的HAECs中得到证实。