Berger John P, Simet Samantha M, DeVasure Jane M, Boten Jessica A, Sweeter Jenea M, Kharbanda Kusum K, Sisson Joseph H, Wyatt Todd A
Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, 985910 Nebraska Medical Center, Omaha, NE 68198-5910, USA.
Research Service, VA Nebraska-Western Iowa Health Care System, 4101 Woolworth Avenue, Omaha, NE 68105, USA; Gastroenterology and Hepatology Division, Department of Internal Medicine, 988090 Nebraska Medical Center, Omaha, NE 68198-8090, USA.
Alcohol. 2014 Aug;48(5):493-500. doi: 10.1016/j.alcohol.2014.02.005. Epub 2014 May 11.
Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3-7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3 to 7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.
同时接触香烟烟雾和乙醇会产生丙二醛和乙醛，随后可能导致醛加合物蛋白的形成。我们之前已经表明，将支气管上皮细胞暴露于丙二醛 - 乙醛（MAA）加合物蛋白会增加蛋白激酶C（PKC）活性和促炎细胞因子的释放。一种清道夫受体A（SRA）的特异性配体，岩藻依聚糖，可阻断这种效应。我们推测MAA加合物蛋白通过SRA与支气管上皮细胞结合。将人支气管上皮细胞（BEAS - 2B）暴露于MAA加合物蛋白（牛血清白蛋白[BSA - MAA]或表面活性蛋白D[SPD - MAA]），并使用共聚焦显微镜、荧光激活细胞分选（FACS）和免疫沉淀法检测SRA。通过气液界面培养的分化小鼠气管上皮细胞（MTEC）用于检测MAA刺激的PKC活性和角质形成细胞衍生趋化因子（KC）的释放。在暴露于MAA 3 - 7分钟后，特异性细胞表面膜染料与上调的SRA共定位，并在20分钟时消退。同样，MAA加合物蛋白在3至7分钟内与SRA共定位，随后在10分钟时MAA内化。这些结果通过FACS分析得到证实，并显示3分钟后SRA的平均荧光降低。此外，在用MAA处理3分钟后，通过蛋白质印迹法在免疫沉淀的SRA样品中可以检测到MAA加合物蛋白的量增加。MAA刺激野生型小鼠而非SRA基因敲除小鼠中PKCε介导的KC释放。这些数据表明，肺部的醛加合物蛋白在气道上皮中MAA加合物蛋白刺激PKC依赖性炎症细胞因子释放之前，迅速与SRA结合并使该受体内化。
