Huang Hong, McIntosh Jane L, Fang Lanyan, Szabo Csaba, Hoyt Dale G
Division of Pharmacology, Ohio State University College of Pharmacy, 500 West Twelfth Avenue, Columbus, OH 43210, USA.
Int J Mol Med. 2003 Oct;12(4):533-40.
Endotoxin (LPS) is a cause of adult respiratory distress syndrome (ARDS), a disease which is preceded by acute lung injury involving the pulmonary endothelium. Experimentally, LPS causes acute DNA strand breakage in mouse lung endothelial cells (MLEC). Engagement of integrin cell adhesion receptors inhibits acute DNA breakage, which could be of use in reducing lung injury. Because integrins presumably inhibit DNA damage or activate repair, we hypothesized that the DNA-damage response protein, poly(ADP-ribose) polymerase-1 (PARP-1), regulates the protective action of integrins, as well as sensitivity to LPS. Therefore, the effect of LPS on MLEC cultured from wild-type and PARP-1 knockout mice was determined. Fluorescence microscopic measures were used to assess plasma membrane integrity, PARP activity, DNA strand breakage and DNA repair in attached cells. LPS caused a concentration-dependent increase in the permeability of wild-type MLEC. Engagement of beta1 integrins with an antibody protected wild-type MLEC from this LPS-induced injury. Wild-type cells treated with the PARP-inhibitor, 3-aminobenzamide, and PARP-1 knockout MLEC were also resistant. LPS caused acute DNA breakage in both wild-type and knockout MLEC, but PARP was activated only in wild-type cells. LPS-induced DNA breakage was inhibited by 3-aminobenzamide, but not by knockout. Anti-beta1 integrin antibody also inhibited the DNA breakage and PARP activation caused by LPS in wild-type MLEC. However, integrin engagement did not prevent DNA breakage in PARP-1 knockout cells, despite a similar level of beta1 integrin in wild-type and knockout cells. Thus, integrin engagement, 3-aminobenzamide, and PARP-1 deletion protected MLEC from increases in membrane permeability caused by LPS. PARP-1 deletion also impaired the ability of integrin engagement to inhibit LPS-induced DNA breakage, suggesting that knockout may affect nuclear factors necessary for integrin-mediated suppression of LPS-induced DNA breakage.
内毒素（脂多糖）是成人呼吸窘迫综合征（ARDS）的病因之一，ARDS之前会出现涉及肺内皮的急性肺损伤。在实验中，脂多糖会导致小鼠肺内皮细胞（MLEC）出现急性DNA链断裂。整合素细胞粘附受体的结合可抑制急性DNA断裂，这可能有助于减轻肺损伤。由于整合素可能抑制DNA损伤或激活修复，我们推测DNA损伤反应蛋白聚（ADP - 核糖）聚合酶-1（PARP - 1）调节整合素的保护作用以及对脂多糖的敏感性。因此，我们测定了脂多糖对野生型和PARP - 1基因敲除小鼠培养的MLEC的影响。采用荧光显微镜测量法评估贴壁细胞的质膜完整性、PARP活性、DNA链断裂和DNA修复情况。脂多糖导致野生型MLEC的通透性呈浓度依赖性增加。用抗体结合β1整合素可保护野生型MLEC免受这种脂多糖诱导的损伤。用PARP抑制剂3 - 氨基苯甲酰胺处理的野生型细胞和PARP - 1基因敲除的MLEC也具有抗性。脂多糖在野生型和基因敲除的MLEC中均导致急性DNA断裂，但PARP仅在野生型细胞中被激活。脂多糖诱导的DNA断裂被3 - 氨基苯甲酰胺抑制，但基因敲除细胞不受影响。抗β1整合素抗体也抑制了脂多糖在野生型MLEC中引起的DNA断裂和PARP激活。然而，尽管野生型和基因敲除细胞中β1整合素水平相似，但整合素结合并不能阻止PARP - 1基因敲除细胞中的DNA断裂。因此，整合素结合、3 - 氨基苯甲酰胺和PARP - 1缺失可保护MLEC免受脂多糖引起的膜通透性增加的影响。PARP - 1缺失也损害了整合素结合抑制脂多糖诱导的DNA断裂的能力，表明基因敲除可能影响整合素介导的抑制脂多糖诱导的DNA断裂所需的核因子。
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