CUHK electronic theses & dissertations collection

環氧合酶-2(COX-2)是在花生四烯酸(AA)轉化為前列腺素H₂(PGH₂)的過程中最重要的限速酶，PGH2再進一步被合成為各種前列腺素，包括前列腺素E₂(PGE₂), 因此，COX-2在前列腺素的合成中起著舉足輕重的作用。COX-2在受到例如感染和炎症等刺激的情況下被誘導，迅速大量地產生。越來越多的證據證明瞭COX-2在許多細胞反應和病理生理過程中起重要作用, 其中, 對COX-2在炎症中的作用研究最深入。囊性纖維化病(CF)是一種由於編碼囊性纖維化跨膜轉導調節器(CFTR)基因的突變所引起的常染色體隱性遺傳疾病。CFTR是在上皮細胞中廣泛表達的環磷酸腺苷(cAMP)依賴的陰離子通道。愈來愈多的證據顯示, CF的呼吸道上皮處於過量炎症因子和前列腺素的微環境中, 最終導致了在CF肺部病變中觀察到的超炎症反應. 但其中的機制仍未闡明. 本研究觀察到, 相對於野生型人類支氣管上皮細胞系(16HBE14o-), CF的人類支氣管上皮細胞系(CFBE41o-)中NFκB的活化, COX-2的表達和PGE₂的產量增加. 此外, CFTR基因敲除小鼠顯示出升高的NFκB活性和COX-2表達水準, 提示CFTR基因的缺失介導了超炎症反應的信號. 我們還驗證了一條PKA和CREB參與介導的PGE₂產生的正回饋通路. 更重要的是, 在CFBE41o-細胞中過表達CFTR顯著地抑制了COX-2的表達. 用LPS或者PGE₂處理16HBE14o-細胞導致了野生型CFTR表達的顯著升高. 這些實驗結果提示了CFTR可能參與對COX-2/PGE₂的負調節. 因此, CFTR負調節PGE₂介導的炎症反應. 這個調節機制的缺陷可能導致在CF炎症反應的組織中觀察到的過量的NFκB活化和過量PGE₂產生.我們證實了睾丸中也存在這條CFTR負調節COX-2/PGE₂的通路. 由於隱睾處於比陰囊溫度高的腹腔中, 在隱睾中, 我們觀察到了高溫導致的CFTR下調,伴隨著COX-2的上調以及緊密連接蛋白(ZO-1, occludin)的下調. 這種CFTR和COX-2的負相關在小鼠睾丸高熱動物模型以及CFTR基因敲除小鼠模型中也被證實. 為了模擬隱睾的病理狀況, 我們提高原代睾丸支援細胞的培養溫度至37°C. 與在32°C培養條件下的對照細胞相比, 37C培養的支持細胞中CFTR表達顯著下調, 而COX-2表達顯著上調. 用CFTR的抑制劑CFTRinh-172處理支持細胞48小時後, COX-2的表達也上升了. 抑制或者敲除支持細胞中的CFTR都引起了ZO-1和occludin表達水準的下降, 從而損傷了支持細胞間的緊密連接. NFκB或者PGE₂的抑制劑都能逆轉ZO-1和occludin表達水準的下降. PGE₂同樣導致了支援細胞間緊密連接的損傷. 以上結果提示CFTR對緊密連接的調節作用是通過NFκB/COX-2/PGE₂通路實現的. 本研究闡明了在支持細胞中, CFTR通過負調節NFκB/COX-2/PGE₂通路調節緊密連接, 從而參與了隱睾導致的生精障礙的病理過程.總之, 本研究論證了CFTR/COX-2/PGE₂通路在CF呼吸道的超炎症反應以及隱睾導致的生精障礙兩個病理過程中的作用, 說明了CFTR在呼吸系統和男性生殖系統中維持細胞因子穩態的重要作用. CF肺中CFTR的缺失或者隱睾病中CFTR表達水準的下降可能導致了呼吸道中過剩炎症反應和生精障礙.Cyclooxygenase-2 (COX-2) is a pivotal rate-limiting enzyme responsible for the production of prostaglandins by converting arachidonic acid (AA) to prostaglandin H₂ (PGH₂), which is further metabolized to various prostaglandins, including PGE₂. COX-2 is inducible and increases dramatically upon stimulation, such as infection and inflammation. Accumulating evidences have demonstrated the important role of COX-2 in many cellular responses and pathophysiological processes, especially inflammation.Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-dependent anion channel expressed in many epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased COX-2 expression and over-production of prostaglandin E₂ (PGE₂) in human CF bronchial epithelial cell line (CFBE41o-) with elevated NFκB activity compared to a wild-type bronchial epithelial cell line (16HBE14o-). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NFκB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE₂ involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o- cells were challenged with LPS as well as PGE₂, indicating possible involvement of CFTR in the negative regulation of COX-2/PGE₂. These results suggest that CFTR is a negative regulator of PGE₂-mediated inflammatory response, defect of which may result in excessive activation of NFκB, leading to over production of PGE2 as seen in inflammatory CF tissues.This negative regulation of COX-2/PGE₂ pathway by CFTR was also identified in the testis in the present study. Downregulation of CFTR accompanied by upregulation of COX-2/PGE₂ and downregulation of tight junction proteins, including ZO-1 and occludin, were observed in a cryptorchidism mouse model with elevated testis in the abdomen, at which the temperature is several degrees higher than that in the scrotum. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and in CF mice. Culturing primary Sertoli cells at a temperature of 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression compared to the physiological condition of 32°C. Increase of COX-2 expression was also detected 48 hours after administrating CFTRinh-172 to the cells. Inhibition or knockdown of CFTR led to decreased ZO-1 and occludin expression and impaired tight junction in Sertoli cells, which could be mimicked by PGE₂, but reversed by NFκB and COX-2 inhibitors, suggesting that regulation of tight junction by CFTR is mediated by NFκB /COX-2/PGE₂ pathway. This study illustrates that CFTR may be involved in regulating testicular tight junctions through its negative regulation of NFκB/COX-2/PGE₂ pathway in Sertoli cells, defect of which may result in spermatogenesis defect in cryptorchidism.Taken together, the present study has demonstrated the role of CFTR/ NFκB /COX-2/PGE₂ pathway in two pathological processes, exaggerated inflammation in CF airway and defective spermatogenesis in cryptorchidism, indicating that CFTR is critical for maintaining cytokine homeostasis in respiratory system and male reproductive system. Defect of CFTR in CF lung and downregulation of CFTR in cryptorchidism may contribute to the excessive lung inflammation and impaired spermatogenesis respectively.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Chen, Jing.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 109-121).Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.Abstract also in Chinese.ABSTRACT --- p.i摘要 --- p.ivACKNOWLEDGEMENT --- p.viLIST OF PUBLICATIONS --- p.viiABBREVIATIONS --- p.xiiLIST OF FIGURES AND TABLES --- p.xviChapter 1 --- Chpter 1: Overview --- p.1Chapter 1.1 --- CFTR and Cystic Fibrosis --- p.1Chapter 1.1.1 --- Cystic Fibrosis --- p.1Chapter 1.1.2 --- Structure of CFTR --- p.2Chapter 1.1.3 --- Mutations of CFTR --- p.2Chapter 1.1.4 --- Channel and signal transduction function of CFTR --- p.3Chapter 1.1.5 --- Interaction of CFTR with other proteins --- p.4Chapter 1.1.6 --- Regulation of CFTR --- p.5Chapter 1.2 --- COX-2 and PGE₂ --- p.6Chapter 1.2.1 --- Biosynthesis of PGE₂ --- p.6Chapter 1.2.2 --- Pathophysiologic roles of COX-2 and PGE₂ --- p.7Chapter 1.2.3 --- Role of COX-2/PGE₂ in inflammation --- p.7Chapter 1.2.4 --- Regulation of COX-2 --- p.8Chapter 1.2.4.1 --- Regulation of COX-2 by NF-κB --- p.9Chapter 1.2.4.2 --- Regulation of COX-2 by CREB --- p.10Chapter 1.3 --- Link between CFTR and NF-κB --- p.11Chapter 1.4 --- General hypothesis and aims of study --- p.12Chapter 2 --- Chapter 2: CFTR negatively regulates COX-2/PGE₂ positive loop in feedback loop in inflammation --- p.13Chapter 2.1 --- Introduction --- p.13Chapter 2.1.1 --- Airway inflammation in Cystic Fibrosis --- p.13Chapter 2.1.2 --- Current theories on the causes of pulmonary inflammation in CF --- p.13Chapter 2.1.2.1 --- Theory one --- p.14Chapter 2.1.2.2 --- Theory two --- p.16Chapter 2.1.3 --- Role of airway epithelia in CF airway inflammation --- p.16Chapter 2.1.4 --- Link between CFTR and NF-κB in pulmonary inflammation --- p.17Chapter 2.1.5 --- Link between CFTR and COX-2/PGE₂ in pulmonary inflammation --- p.18Chapter 2.1.6 --- Hypothesis and aims of study --- p.18Chapter 2.2 --- Materials and methods --- p.20Chapter 2.2.1 --- Cell culture materials --- p.20Chapter 2.2.2 --- Animals --- p.20Chapter 2.2.3 --- Chemicals, drugs and assay kits --- p.20Chapter 2.2.4 --- Antibodies --- p.22Chapter 2.2.5 --- Cell culture. --- p.22Chapter 2.2.6 --- Animal models and procedures --- p.23Chapter 2.2.7 --- Manipulation of RNA and QRT-PCR --- p.23Chapter 2.2.8 --- Manipulation of protein and Western blot --- p.25Chapter 2.2.9 --- Histological and morphological --- p.27Chapter 2.2.9.1 --- Tissue section. --- p.28Chapter 2.2.9.2 --- Hematoxylin and eosin staining --- p.28Chapter 2.2.9.3 --- Immunohistochemistry --- p.28Chapter 2.2.10 --- PGE₂ EIA --- p.29Chapter 2.2.11 --- Statistical analysis --- p.30Chapter 2.3 --- Results --- p.30Chapter 2.3.1 --- Increased expression of NF-κB and COX-2 in the lung of CF mice --- p.31Chapter 2.3.2 --- Defect of CFTR leads to increased COX-2 expression in CF cell line --- p.31Chapter 2.3.3 --- Increased expression of COX-2 in CF cells is attributed to NF-κB activation --- p.33Chapter 2.3.4 --- A positive feedback loop from PGE₂ to COX-2 is mediated by PGE₂/cAMP/PKA/p-CREB pathway --- p.34Chapter 2.3.5 --- PGE₂ increase the expression of CFTR protein in 16HBE14o- but not in CFBE41o- cells --- p.35Chapter 2.4 --- Discussion --- p.47Chapter 2.5 --- Conclusion --- p.51Chapter 3 --- Chapter 3: Role of CFTR/COX-2/PGE₂ Pathway in the Regulation of Junctional Complex Proteins in Sertoli Cells and its Implication in Spermatogenesis Defect in Cryptorchidism --- p.53Chapter 3.1 --- Introduction --- p.53Chapter 3.1.1 --- Spermatogenesis.p53Chapter 3.1.1.1 --- Structure of the seminiferous tubules --- p.53Chapter 3.1.1.2 --- Role of Sertoli cells in spermatogenesis --- p.55Chapter 3.1.1.3 --- Role of junctional complexes in spermatogenesis --- p.55Chapter 3.1.2 --- Junctional complexes in the testis --- p.59Chapter 3.1.2.1 --- Tight Junction --- p.59Chapter 3.1.2.2 --- Anchoring Junction. --- p.60Chapter 3.1.2.3 --- Cross talk between TJs and AJs --- p.60Chapter 3.1.3 --- Cryptorchidism --- p.61Chapter 3.1.3.1 --- Causes and consequences of Cryptorchidism --- p.61Chapter 3.1.3.2 --- Elevated temperature caused by cryptorchidism greatly contributes to defective spermatogenesis --- p.62Chapter 3.1.3.3 --- Changes of Sertoli cells in cryptorchidim contributing to defective spermatogenesis. --- p.62Chapter 3.1.3.4 --- Disruption of junctional complexes in heat shock and cryptorchidism. --- p.65Chapter 3.1.4 --- CFTR and spermatogenesis --- p.66Chapter 3.1.4.1 --- Expression of CFTR in Sertoli cells in testis --- p.66Chapter 3.1.4.2 --- Temperature sensitive processing of CFTR protein --- p.66Chapter 3.1.4.3 --- CFTR and junctional complex --- p.67Chapter 3.1.4.4 --- CFTR and male reproduction --- p.68Chapter 3.1.4.5 --- Role of CFTR in spermatogenesis --- p.68Chapter 3.1.5 --- Prostaglandins and male fertility --- p.69Chapter 3.1.5.1 --- Expression of COX-2 in testis. --- p.69Chapter 3.1.5.2 --- Role of prostaglandins in spermatogenesis --- p.70Chapter 3.1.5.3 --- Regulation of junctional complexes by PGE₂ --- p.70Chapter 3.1.5.4 --- Prostaglandins in cryptorchidism --- p.72Chapter 3.1.6 --- Hypothesis and aims of study --- p.73Chapter 3.2 --- Materials and Methods --- p.74Chapter 3.2.1 --- Cell culture materials --- p.74Chapter 3.2.2 --- Drugs and Reagents --- p.74Chapter 3.2.3 --- Antibodies --- p.74Chapter 3.2.4 --- Animals --- p.75Chapter 3.2.4.1 --- Mice artificial cryptorchidism model --- p.75Chapter 3.2.4.2 --- Mice testes hyperthermia model --- p.75Chapter 3.2.5 --- Sertoli cell primary culture --- p.76Chapter 3.2.6 --- siRNA against CFTR and transfection --- p.76Chapter 3.2.7 --- Examination of assembly and destruction of assembly of inter-Sertoli TJs --- p.77Chapter 3.2.8 --- Manipulation of RNA and Real-Time Quantitative RT-PCR (QRT-PCR) --- p.77Chapter 3.2.9 --- Manipulation of protein and western blot --- p.77Chapter 3.2.10 --- Histological and morphological studies --- p.78Chapter 3.2.10.1 --- Immunofluorescence of ZO-1 Staining in Sertoli cells --- p.78Chapter 3.2.10.2 --- Immunofluorescent staining of ZO-1, Occludin and β-Catenin in testes --- p.78Chapter 3.2.11 --- PGE₂ EIA --- p.79Chapter 3.2.12 --- Statistical Analysis --- p.79Chapter 3.3 --- Results --- p.79Chapter 3.3.1 --- Downregulation of CFTR is associated with upregulation of COX-2 in mice cryptorchidism model, mice testes hyperthermia model, and CF mice testes --- p.79Chapter 3.3.2 --- Negative regulation of COX-2 by CFTR is mediated by NF-κB --- p.81Chapter 3.3.3 --- Decreased tight junction proteins expression and increased anchoring junction proteins expression in cryptorchid testes. --- p.81Chapter 3.3.4 --- Elevation of culture temperature results in downregulation of CFTR and upregulation of COX-2 in primary cultured rat sertoli cells --- p.82Chapter 3.3.5 --- Defect of functional CFTR leads to increased COX-2 expression. --- p.83Chapter 3.3.6 --- CFTR regulates TJ protein expression and TJ formation through NF-κB/COX-2/PGE₂. --- p.83Chapter 3.4 --- Discussion --- p.100Chapter 3.5 --- Conclusion --- p.104Chapter 4 --- Chapter 4: General Discussion --- p.105Chapter 4.1 --- The immunosuppressive function of PGE₂ in CF lung disease and cryptorchidism-induced infertility. --- p.105Chapter 4.2 --- Importance of CFTR/ NF-κB /COX-2/PGE₂ pathway in inflammation-based diseases. --- p.106Chapter 4.3 --- Possible implications of CFTR/NF-κB /COX-2/PGE₂ pathway in cancer --- p.107Chapter 4.4 --- Concluding remarks --- p.10