【作者】 田丹；

【导师】 吴人亮；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2006， 博士

【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GSK3β;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-rich protein,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、GSK3β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GSK3β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(β-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β-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β-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型上皮细胞株(A549)对吸烟致肺泡上皮损伤中GSK3β的活性变化及其对β-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β-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β-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(P<0.05),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GSK3β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GSK3β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(P<0.01),说明细胞转染GSK3βS9A后表达的突变型GSK3β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。更多还原

【Abstract】 Part I Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine airway epithelial cellSquamous differentiation of airway (tracheobronchial) epithelium induced by diverse stimuli, such as cigarette smoke, has been thought to be an adaptive response to chronic injury as well as a precancerous lesion of lung squamous carcinoma. The molecular mechanisms of squamous differentiation have not been fully elucidated. Glycogen synthase kinase 3 (GSK3) is a multifunctional protein kinase that plays important roles in metabolism, cell proliferation, differentiation, apoptosis and cell motility. There are two mammalian GSK3 isoforms, designated GSK3αand GSK3β. GSK3 is constitutively active in resting cell, and can be inactivated by phosphorylation of Ser-21 in GSK3αor Ser-9 in GSK3β. GSK3 acts as a key and negative regulator of numerous signal pathways, including Wnt/β-catenin and activator protein-1 (AP-1) signaling pathway. Recent researches indicated that GSK3 and AP-1 signaling might be implicated in squamous differentiation of airway epithelium induced by cigarette smoke. In the present study, we examined the expression of GSK3 in lung tissue of several experimental animals and cultured porcine airway epithelial cells (PAECs), and further investigated the role of GSK3 and AP-1 signaling in squamous differentiation of airway epithelium induced by cigarette smoke in vitro.Immunostaining showed that both GSK3αand GSK3βwere prominently expressed in plasm of airway epithelial cells, submucosal gland cells, smooth muscle cells and alveolar epithelial cells of human, rat, mouse or pig. However, there were unexpected high levels of GSK3αin cartilage lacuna cells when compared with those of GSK3β. No signals of phosphorylated GSK3α/βwere observed in lung tissues. Immunofluorescence demonstrated that the expression of GSK3αand GSK3βwere high but phosphorylated GSK3α/βwas low in PAECs.Cytotoxicity assay and cell morphological observation demonstrated cigarette smoke components inhibited the growth of PAECs and resulted in morphological changes, which showed delayed confluence, more widely spread and flattened appearance with widened cell-cell interspaces, compared with the classic cobblestone epithelial morphology. As expected from previous studies, it was confirmed by Western blot and RT-PCR in PAECs that cigarette smoke components enhance the expression of involucrin protein and small proline-rich protein mRNA, two markers of squamous differentiation.Moreover, it was found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) by increasing inactive phosphorylated GSK3α/βand reducing GSK3βexpression. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine airway epithelial cells.In addition, Transcription factor activity assay showed that cigarette smoke components significantly promoted AP-1 binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components.Taken together, these data suggest: (1) cigarette smoke components can inhibit the growth and induce squamous differentiation of PAECs. (2) GSK3 is highly expressed in airway epithelium, and involved in involucrin expression induced by cigarette smoke in PAEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke. Part II Role of glycogen synthase kinase 3 andβ-catenin/TCF signaling in alveolar type II epithelial cell injury caused by cigarette smokeCigarette smoke is known to have various injurious effects on alveolar epithelial cells. It suppresses migration, proliferation and differentiation of the cells so that they can not cover the defects resulted from the injury, and inhibits surfactant secretion and collagen production. Thus injury of alveolar epithelial cells may contribute to the development of lung diseases induced by cigarette smoke. However, the molecular mechanisms of alveolar epithelial cell injury caused by cigarette smoke remain unclear.β-cantenin is a multifunctional protein that plays an important role in cellular development, cell adhesion, repair and injury, cell cycle regulation and tumor formation. It is not only associated with E-cadherin to maintain strong cell-cell adhesion and tissue integrity in epithelium, but also a key component in Wnt signaling to regulate the expression of a variety of genes by its translocation to the nucleus and interaction with transcription factor TCF/LEF (T cell factor/lymphoid enhancer factor). Recent researches demonstrated that nicotine and NNK, two components of cigarette smoke, increased phosphorylation of glycogen synthase kinase 3 (GSK3) in vitro. GSK3βacts as a key and negative regulator of the classical Wnt/β-catenin signaling pathway, and a primary kinase responsible for phosporylation and down-regulation ofβ-cantenin levels. Our former studies showed that the expression and location ofβ-cantenin altered in the injury and repair process of airway epithelium induced by cigarette smoke. But it is unclear whether GSK3βandβ-cantenin/TCF signaling are involved in alveolar epithelial cell injury caused by cigarette smoke. In the present study, we examined the expression of GSK3βin cultured alveolar epithelial cell line (A549), and further investigated the role of GSK3βandβ-cantenin/TCF signaling in alveolar epithelial cell injury caused by cigarette smoke in vitro. Immunofluorescence demonstrated that the expression of GSK3βwas high in A549 cells. Western blot analysis showed that cigarette smoke extract (CSE) notably inhibited GSK3βby reducing GSK3βexpression and increasing inactive phosphorylated GSK3βin a dose-dependent manner.Moreover, it was found by Western blot that CSE increased the expression ofβ-catenin protein in a dose-dependent manner and induced nuclear translocation ofβ-catenin. It was also demonstrated that CSE activatedβ-cantenin/TCF signaling by transient transfection of TCF luciferase reporter plasmids(pGL3-OT)followed by CSE treatment, using the mutant of TCF luciferase reporter plasmids (pGL3-OF) as controls.Finally, we further studied the role of GSK3βin the activation ofβ-cantenin/TCF signaling induced by CSE. We detected the expression of GSK3βandβ-catenin after transient transfection of a stable mutant of GSK3β(GSK3βS9A), which is continuously active and unable to be inhibited by the upstream kinase of GSK3β. It was observed that the GSK3βprotein level was increased dramatically and the expression ofβ-catenin was reduced significantly. These suggest that the high expression of active GSK3βpromote the degradation ofβ-catenin. Then, we examined theβ-catenin/TCF transcriptional activity after cotransfected GSK3βS9A with the TCF luciferase reporter plasmids followed by CSE treatment. We demonstrated that active GSK3βinhibitedβ-catenin/TCF transcriptional activity induced by CSE (P<0.01, compared with the group of CSE treatment). This result suggests that CSE activatesβ-cantenin/TCF signaling via inhibiting GSK3βactivity.Taken together, these data suggest: (1) CSE can inhibit GSK3βby reducing GSK3βexpression and increasing inactive phosphorylated GSK3βin A549 cells. (2) CSE can increase the expression ofβ-catenin and induce nuclear translocation ofβ-catenin in A549 cells. (3) CSE activatesβ-cantenin/TCF signaling via inhibiting GSK3βactivity, implying a possible mechanism responsible for alveolar epithelial cell injury caused by cigarette smoke.更多还原