Gelbman Brian D, Heguy Adriana, O'Connor Timothy P, Zabner Joseph, Crystal Ronald G
Division of Pulmonary and Critical Care Medicine, Weill Medical College of Cornell University, New York, New York, USA.
Respir Res. 2007 Feb 8;8(1):10. doi: 10.1186/1465-9921-8-10.
Cigarette smoke disrupts the protective barrier established by the airway epithelium through direct damage to the epithelial cells, leading to cell death. Since the morphology of the airway epithelium of smokers does not typically demonstrate necrosis, the most likely mechanism for epithelial cell death in response to cigarette smoke is apoptosis. We hypothesized that cigarette smoke directly up-regulates expression of apoptotic genes, which could play a role in airway epithelial apoptosis.
Microarray analysis of airway epithelium obtained by bronchoscopy on matched cohorts of 13 phenotypically normal smokers and 9 non-smokers was used to identify specific genes modulated by smoking that were associated with apoptosis. Among the up-regulated apoptotic genes was pirin (3.1-fold, p < 0.002), an iron-binding nuclear protein and transcription cofactor. In vitro studies using human bronchial cells exposed to cigarette smoke extract (CSE) and an adenovirus vector encoding the pirin cDNA (AdPirin) were performed to test the direct effect of cigarette smoke on pirin expression and the effect of pirin expression on apoptosis.
Quantitative TaqMan RT-PCR confirmed a 2-fold increase in pirin expression in the airway epithelium of smokers compared to non-smokers (p < 0.02). CSE applied to primary human bronchial epithelial cell cultures demonstrated that pirin mRNA levels increase in a time-and concentration-dependent manner (p < 0.03, all conditions compared to controls). Overexpression of pirin, using the vector AdPirin, in human bronchial epithelial cells was associated with an increase in the number of apoptotic cells assessed by both TUNEL assay (5-fold, p < 0.01) and ELISA for cytoplasmic nucleosomes (19.3-fold, p < 0.01) compared to control adenovirus vector.
These observations suggest that up-regulation of pirin may represent one mechanism by which cigarette smoke induces apoptosis in the airway epithelium, an observation that has implications for the pathogenesis of cigarette smoke-induced diseases.
香烟烟雾通过直接损伤上皮细胞破坏气道上皮建立的保护屏障，导致细胞死亡。由于吸烟者气道上皮的形态通常未显示坏死，因此香烟烟雾导致上皮细胞死亡的最可能机制是凋亡。我们推测香烟烟雾直接上调凋亡基因的表达，这可能在气道上皮凋亡中起作用。
对13名表型正常的吸烟者和9名非吸烟者的匹配队列进行支气管镜检查获取气道上皮，进行微阵列分析，以鉴定受吸烟调节的与凋亡相关的特定基因。上调的凋亡基因中有匹啉（3.1倍，p<0.002），一种铁结合核蛋白和转录辅因子。使用暴露于香烟烟雾提取物（CSE）的人支气管细胞和编码匹啉cDNA的腺病毒载体（AdPirin）进行体外研究，以测试香烟烟雾对匹啉表达的直接影响以及匹啉表达对凋亡的影响。
定量TaqMan RT-PCR证实，与非吸烟者相比，吸烟者气道上皮中匹啉表达增加2倍（p<0.02）。将CSE应用于原代人支气管上皮细胞培养物表明，匹啉mRNA水平以时间和浓度依赖性方式增加（p<0. .03，所有条件与对照相比）。使用载体AdPirin在人支气管上皮细胞中过表达匹啉，与对照腺病毒载体相比，通过TUNEL测定（5倍，p<0.01）和细胞质核小体ELISA（19.3倍，p<0.01）评估的凋亡细胞数量增加。
这些观察结果表明，匹啉上调可能是香烟烟雾诱导气道上皮凋亡的一种机制，这一观察结果对香烟烟雾诱导疾病的发病机制具有重要意义。
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