GSK3在吸烟致气道上皮和肺泡Ⅱ型上皮细胞损伤修复中的作用研究

发布时间：2018-04-21 10:00

本文选题：糖原合成酶激酶3 + AP-1　；参考：《华中科技大学》2006年博士论文

【摘要】： 第一部分糖原合成酶激酶3在吸烟诱导的猪气道上皮细胞鳞状分化中的作用研究气道(气管和支气管)上皮的鳞状细胞化生常见于慢性支气管炎症、致癌剂刺激(如吸烟)等,一般被认为是对慢性损伤的一种适应性反应,也是肺鳞癌的癌前病变,其分子机制尚未完全阐明。糖原合成酶激酶3(glycogen synthase kinase 3,GSK3)是一种多功能的蛋白激酶,有结构和功能相似的两种亚型GSK3α和GSK3β,在静息细胞内呈组成性激活,其N末端丝氨酸残基(Ser21-GSK3α,Ser9-GSK3β)的磷酸化可导致其活性抑制。多年来大量的研究表明,GSK3具有广泛的底物,包括代谢酶、翻译起始因子、转录因子、细胞周期相关蛋白、癌基因产物和细胞骨架蛋白等,可参与糖代谢、蛋白质合成以及细胞的增殖、分化、凋亡和运动等多种生命活动。近来研究显示GSK3及其转录因子底物AP-1(activator protein-1)可能参与气道上皮鳞状分化的发生。因此,本研究对GSK3的表达、GSK3和AP-1信号在吸烟诱导的气道上皮细胞鳞状分化中的作用进行了初步探讨。本实验首先用免疫组化和免疫细胞荧光法检测GSK3在人和多种实验动物(大鼠、小鼠和猪)的肺组织及培养的猪气道上皮细胞中的表达,结果显示GSK3α和GSK3β广泛表达于人、鼠和猪的肺组织中,定位于胞浆;它们在几种动物肺组织中的表达分布大致相似,主要见于各级支气管上皮细胞、肺泡上皮细胞、粘膜平滑肌细胞和粘膜下腺体;但GSK3α在软骨细胞中表达明显强于GSK3p;几种哺乳动物肺组织中均未检测到GSK3α/β的磷酸化。在培养的猪支气管上皮细胞中有丰富的GSK3α、β的表达,磷酸化的GSK3α/β信号弱。其次,细胞毒性实验和形态学观察显示香烟烟雾提取物和尼古丁处理可抑制猪气道上皮细胞的增殖并导致细胞出现较为伸展和扁平、融合减缓、细胞间隙增宽等形态学改变。Western blot和RT-PCR检测发现,香烟烟雾提取物和尼古丁处理后鳞状分化标记物外皮蛋白(involucrin)和小脯氨酸丰富蛋白(small proline-richprotein,SPRP)的表达增强,证实了吸烟能够诱导猪气道上皮细胞的鳞状分化。进一步的研究显示,香烟烟雾提取物和尼古丁处理后,抑制性磷酸化GSK3α/β(Ser-21-GSK3α/Ser-9-GSK3β)的水平升高、GSK3p表达降低,表明香烟成分可抑制GSK3的表达和活性;而且,用GSK3的抑制剂SB216763处理细胞后检测外皮蛋白的表达变化,结果显示外皮蛋白的表达升高并呈现浓度和时间依赖性。这提示GSK3可能在香烟成分诱导猪气道上皮的鳞状分化中起重要作用。最后,转录因子活性检测显示香烟烟雾提取物和尼古丁处理可显著增强转录因子AP-1与外皮蛋白基因上游调节区域的结合活性;而用GSK3的抑制剂LiCl和SB216763模拟香烟成分的作用也得到了类似的结果,提示GSK3可能通过负向调节AP-1的活性从而介导了香烟成分诱导的猪气道上皮鳞状分化。小结上述结果表明:(1)香烟成分可抑制猪气道上皮细胞的增殖并诱导其发生鳞状分化。(2)GSK3在气道上皮细胞中有丰富的表达,可能通过负向调节AP-1的活性从而介导了香烟成分诱导的猪气道上皮鳞状分化。第二部分吸烟致肺泡Ⅱ型上皮细胞损伤中GSK3p的活性变化对 β-catenin/TCF信号途径的影响大量研究证明,吸烟能够从多个方面损伤肺泡上皮细胞,阻碍其迁移、增生和分化以修复受损区域,因而有助于吸烟相关疾病(如慢性阻塞性肺疾病和支气管源性肺癌等)的发生与发展。然而,吸烟致肺泡上皮细胞损伤的分子机制还远未阐明。 β-连环素(p-catenin)是一种多功能蛋白,既可作为细胞骨架蛋白与E-钙粘附素结合,参与组成中间连接,维持上皮细胞的极性和组织结构的完整性;又是Wnt信号途径的重要成员,可与TCF/LEF(T cell factor/lymphoid enhancer factor)结合形成转录因子复合体,调节多种基因的表达,参与细胞和组织的发育分化、损伤修复以及肿瘤的发生发展等生物学过程。最近研究显示,尼古丁和香烟致癌物NNK可促进糖原合成酶激酶3(glycogen synthase kinase 3,GSK3)的抑制性磷酸化,而GSK3β可磷酸化调节p-catenin,在经典的Wnt信号途径中起关键性抑制作用。我们课题组的早期工作发现,在吸烟导致的气道上皮损伤修复过程中β-catenin的表达和定位发生了变化。但未知GSK3β和p-catenin/TCF信号是否介导了吸烟诱导的肺泡上皮损伤。因此,本实验利用人肺泡Ⅱ上皮细胞株(A549)对吸烟致肺泡上皮损伤中GSK3p的活性变化及其对p-catenin/TCF信号的影响进行了初步探讨。本实验首先采用免疫细胞荧光检测发现GSK3β在肺泡Ⅱ型上皮细胞(A549细胞)中高表达。然后用不同浓度的香烟烟雾提取物(CSE)处理细胞24h后,进行Western blot分析。结果显示,GSK3β的表达降低、抑制性磷酸化GSK3β的水平升高,并呈现浓度依赖性,表明CSE可抑制肺泡Ⅱ型上皮细胞中GSK3β的表达和活性。其次,为分析CSE对p-catenin的影响,用不同浓度的CSE处理细胞24h后提取细胞总蛋白,进行Western blot检测发现CSE促进了β-catenin的表达并呈现浓度依赖性。为进一步阐明表达增强的p-catenin是否向核内转位,用4%CSE处理细胞24h后分别提取细胞浆和细胞核蛋白进行检测,结果显示细胞浆和细胞核中β-catenin的表达均增加,表明CSE促进了β-catenin向核内转位。接着,为分析CSE处理后β-catenin/TCF信号的状态,用含有TCF/LEF结合序列的荧光素酶报告基因质粒(pGL3-OT,突变型质粒pGL3-OT作为对照)转染细胞,再行CSE处理后,检测荧光素酶活性。结果显示,4%CSE处理组的荧光素酶活性明显高于对照组,差异有显著意义(P0.05),提示CSE可激活肺泡Ⅱ型上皮细胞中β-catenin/TCF信号。最后,为进一步分析GSK3p是否在其中发挥了作用,用持续激活突变型GSK3β(GSK3βS9A,不受GSK3的上游激酶下调而持续激活)转染细胞后检测GSK3β和β-catenin的水平,结果显示GSK3p的表达明显增强,而β-catenin的表达则显著降低,证实了肺泡Ⅱ型上皮细胞中GSK3β的表达和活性增强可促进β-catenin的降解;与此同时,也证明所用质粒GSK3βS9A是可靠的。然后,将GSK3βS9A与pGL3-OT共转染后,再加入CSE处理,进行荧光报告基因分析。分析发现,与仅用CSE处理组相比,共转染GSK3βS9A组的荧光素酶活性显著降低,差异有极显著意义(P0.01),说明细胞转染GSK3βS9A后表达的突变型GSK3p不受CSE的抑制而持续激活,促进了β-catenin的降解,因而导致β-catenin/TCF信号的抑制。上述结果表明,在肺泡Ⅱ型上皮细胞中,CSE可通过抑制GSK3β而增强β-catenin/TCF的转录活性。小结本实验表明:(1)CSE可抑制肺泡Ⅱ型上皮细胞中GSK3β的表达和活性。(2)CSE可促进肺泡Ⅱ型上皮细胞中β-catenin的表达和转位从而激活β-catenin/TCF信号。(3)在肺泡Ⅱ型上皮细胞中,CSE可通过抑制GSK3β而增强β-catenin/TCF的转录活性。本实验提示:GSK3β可通过对β-catenin/TCF信号途径的调节,参与吸烟致肺泡上皮细胞的损伤,进而可能介导吸烟相关疾病的发生与发展。
[Abstract]:Part one
Role of glycogen synthase kinase 3 in smoking induced squamous cell differentiation of porcine airway epithelial cells
Squamous cell metaplasia of the airway (trachea and bronchus) is common in chronic bronchitis, carcinogenic stimulant (such as smoking), which is generally considered an adaptive response to chronic injury, and is also a precancerous lesion of squamous cell carcinoma of the lung. Its molecular mechanism has not been fully elucidated. Glycogen synthase kinase 3 (glycogen synthase kinase)
3, GSK3) is a multifunctional protein kinase with two subtypes of structure and function similar to GSK3 alpha and GSK3 beta, which are active in resting cells. The phosphorylation of the N terminal serine residue (Ser21-GSK3 alpha, Ser9-GSK3 beta) can lead to the inhibition of its activity. Many studies have shown that GSK3 has a wide range of substrates, including metabolic enzymes, and turns over many years. Translation initiation factors, transcription factors, cell cycle related proteins, oncogene products and cytoskeleton proteins are involved in glycometabolism, protein synthesis, cell proliferation, differentiation, apoptosis and exercise. Recent studies have shown that the GSK3 and its transcription factor substrate AP-1 (activator protein-1) may be involved in the squamous cell differentiation of the airway epithelium. Therefore, the role of GSK3 expression, GSK3 and AP-1 signaling in smoking induced squamous cell differentiation of airway epithelial cells is discussed in this study.
In this experiment, the expression of GSK3 in lung tissues and cultured porcine airway epithelial cells in human and a variety of experimental animals (rats, mice and pigs) was detected by immunohistochemistry and immunofluorescence. The results showed that GSK3 alpha and GSK3 beta were widely expressed in the lung tissues of human, rat and pig, and located in the cytoplasm; their expression in several animal lung tissues was expressed. The distribution is roughly similar, mainly in bronchial epithelial cells at all levels, alveolar epithelial cells, mucous smooth muscle cells and submucosal glands, but the expression of GSK3 alpha in chondrocytes is obviously stronger than that of GSK3p; there are no phosphorylation of GSK3 alpha / beta in several mammalian lung tissues. There are abundant GSK3 alpha, beta in the cultured porcine bronchial epithelial cells. The expression of phosphorylated GSK3 alpha / beta signal is weak.
Secondly, cytotoxicity test and morphological observation showed that cigarette smoke extract and nicotine treatment could inhibit the proliferation of porcine airway epithelial cells and lead to more extensional and flat cells, slow fusion and widening of intercellular space, such as.Western blot and RT-PCR detection, cigarette smoke extract and nicotine treated scales. The expression of involucrin and small proline rich protein (small proline-richprotein, SPRP) was enhanced, which confirmed that smoking can induce the squamous differentiation of porcine airway epithelial cells. Further studies showed that the inhibitory phosphorylation of GSK3 alpha / beta (Ser-21-GSK3 alpha /Ser-9-GS) after cigarette smoke extract and nicotine treatment. The level of K3 beta and the expression of GSK3p decreased, indicating that the cigarette composition could inhibit the expression and activity of GSK3. Moreover, the expression of the outer skin protein was detected with the GSK3 inhibitor SB216763 treated cells. The results showed that the expression of the outer skin protein increased and showed a concentration and time dependence. This suggests that GSK3 may induce the pig airway in the cigarette composition. The scaly differentiation of the skin plays an important role.
Finally, the activity detection of transcription factor showed that cigarette smoke extract and nicotine treatment could significantly enhance the binding activity of the transcription factor AP-1 and the upstream regulation region of the outer skin protein gene, while the effect of GSK3 inhibitor LiCl and SB216763 on the simulation of cigarette composition was similar, suggesting that GSK3 may regulate the activity of AP-1 by negative direction. Sex thus mediated the differentiation of porcine airway epithelium induced by cigarette components.
Summary
The above results show that: (1) cigarette composition can inhibit the proliferation and induce squamous differentiation of porcine airway epithelial cells. (2) GSK3 has a rich expression in the airway epithelial cells, which may mediate the squamous differentiation of porcine airway epithelium induced by cigarette composition by negatively regulating the activity of AP-1.
The second part is about the change of GSK3p activity in alveolar type II epithelial cells induced by smoking.
The effect of beta -catenin/TCF signal pathway
A large number of studies have shown that smoking can damage alveolar epithelial cells from many aspects, obstruct their migration, proliferation and differentiation to repair the damaged areas, thus contributing to the occurrence and development of smoking related diseases such as chronic obstructive pulmonary disease and bronchogenic lung cancer. However, the molecular mechanism of smoking induced alveolar epithelial cell damage is far from being explained. Ming.
Beta catenin (P-catenin) is a multifunctional protein that can be used as a cytoskeleton and E- calcium adhesion element to form intermediate connections to maintain the polar and tissue integrity of epithelial cells. It is also an important member of the Wnt signaling pathway and can be combined with TCF/LEF (T cell factor/lymphoid enhancer factor) to form a transcription factor complex. The recent study shows that nicotine and cigarette carcinogen NNK can promote the inhibitory phosphorylation of glycogen synthase kinase 3 (glycogen synthase kinase 3, GSK3), while GSK3 beta phosphorylation regulates p-caten. In, which plays a key inhibitory role in the classic Wnt signaling pathway, has been found in our early work group that changes in the expression and localization of beta -catenin during the repair of airway epithelial injury caused by smoking. But the unknown GSK3 beta and p-catenin/TCF signals mediate the alveolar epithelial injury induced by smoking. Therefore, this experiment is used in this experiment. The effect of human alveolar epithelial cell line (A549) on GSK3p activity and p-catenin/TCF signaling in cigarette smoke induced alveolar epithelial injury is discussed.
This experiment first used immunofluorescence to detect the high expression of GSK3 beta in the alveolar type II epithelial cells (A549 cells). Then the Western blot analysis was carried out after the treatment of cell 24h with different concentrations of cigarette smoke extract (CSE). The results showed that the expression of GSK3 beta was reduced, the level of GSK3 beta phosphorylated, and the concentration dependence of the inhibitory phosphorylation of GSK3 beta. It indicates that CSE can inhibit the expression and activity of GSK3 beta in alveolar type II epithelial cells.
Secondly, in order to analyze the effect of CSE on P-catenin, the total protein of cell was extracted with 24h of different concentrations of CSE, and Western blot detection found that CSE promoted the expression of beta -catenin and showed a concentration dependence. The results showed that the expression of beta -catenin in cytoplasm and nucleus increased, indicating that CSE promoted the translocation of beta -catenin into the nucleus.
Then, in order to analyze the state of the beta -catenin/TCF signal after CSE treatment, the luciferase activity was detected by using the luciferase reporter gene plasmid containing the TCF/LEF binding sequence (pGL3-OT, the mutant plasmid pGL3-OT as the control). The luciferase activity was detected after CSE treatment. The results showed that the luciferase activity of the 4%CSE treatment group was significantly higher than that of the control group, and the difference was found. Significant (P0.05) indicates that CSE can activate the beta -catenin/TCF signal in alveolar type II epithelial cells.
Finally, in order to further analyze whether GSK3p played a role in it, the level of GSK3 beta and beta -catenin was detected by continuous activation of mutant GSK3 beta (GSK3 beta S9A, not down regulated by the upstream kinase of GSK3). The results showed that the expression of GSK3p was obviously enhanced and the expression of beta -catenin decreased significantly, which confirmed the type II of alveolar type. The expression and activity enhancement of GSK3 beta in epithelial cells can promote the degradation of beta -catenin; at the same time, it is also proved that the plasmid GSK3 beta S9A is reliable. Then, after CO transfection of GSK3 beta S9A and pGL3-OT, then CSE treatment is added to the fluorescence report gene analysis. The analysis found that the luciferase of GSK3 beta S9A group was co transfected to the GSK3 beta S9A group compared with the CSE treatment group. The activity significantly decreased, and the difference was significant (P0.01), indicating that the mutant GSK3p expressed after the transfection of GSK3 beta S9A was not inhibited by CSE, which promoted the degradation of beta -catenin and resulted in the inhibition of beta -catenin/TCF signal. The results showed that CSE could enhance the beta -cate in the alveolar type II epithelial cells by inhibiting the GSK3 beta. The transcriptional activity of nin/TCF.
Summary
This experiment shows: (1) CSE can inhibit the expression and activity of GSK3 beta in the alveolar type II epithelial cells. (2) CSE can promote the expression and transposition of beta -catenin in the alveolar type II epithelial cells and activate the beta -catenin/TCF signal. (3) in the alveolar type II epithelial cells, CSE can enhance the transcriptional activity of beta -catenin/TCF by inhibiting the GSK3 beta.
This experiment suggests that GSK3 beta may be involved in the injury of alveolar epithelial cells induced by smoking by regulating the signaling pathway of beta -catenin/TCF, and may also mediate the occurrence and development of smoking related diseases.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

【参考文献】