Roles Of Rho GTPases And Effector IQGAP1 In The Injury And Repair Of Airway Epithelial Cell

Part I Effects of phosphorylation of IQGAP1 induced by mechanical scraching on wound and repair in airway epithelial cellsBackground:Airway (trachea and bronchi) epithelium is frequently injured because of its exposure to the external environment. After injury, the airway epithelium initiates a wound repair process to keep normal lung function, which requires spreading, migration, and eventually proliferation of airway epithelial cells into the injured area. Damage to airway epithelium is critical to the pathogenesis of airway disorders such as chronic bronchitis and COPD, so the ability of the airway epithelium to repair itself is an important step in the resolution of airway disease. However, the molecular mechanism involved in the injury and repair of airway epithelium has not been well understood.IQGAP1 (IQ domain GTPase-activating protein) is a key actor in a series of cell processes, but has not been identified in lung epithelial cells. Phosphorylation is a major post-translational method for regulating protein function, including that of the cytoskeleton. The serine residues of IQGAP1 are phosphorylated, which promote neurite outgrowth. PKCεmay be one of the candidates responsible for phosphorylation of IQGAP1. We suppose that phosphorylation of IQGAP1 may play roles in the process of injury and repair.Objective:In this study, we utilized a widely used model in vitro by scratching bronchial epithelial cells (BECs) and investigated the effects of phosphorylation of IQGAP1 in the process of wound closure. Method:We established an in vitro injury and repair model by scratching a monolayer of BECs. Then the following tests were undertaken: wound assays, immunohistochemistry, western blot, confocal laser sanning, immunoprecipitation, transient transfection and so on. We aimed to study phosphorylation of IQGAP1, the association between IQGAP1 and PKCεand the effects of PKCεfor wound closure after activation or inhibition its activity.Results:Cell morphological observation demonstrated cultured BECs showed a classic cobblestone epithelial morphology that was three-dimensional, slightly raised and closely adherent. After scratching, bronchial epithelial cells moved unidirectionally as sheets or groups, perpendicular to the direction of the wound. A polarized morphology developed 6 h following scratching and became pronounced after 12 h. The wound gap was approximately closed in 24 h after scratching.Our results showed that IQGAP1 was abundant in BECs of several species. It was colocalized with tubulin cytoskeleton, but was destroyed by microtubule disassembled reagent of nocodazole. Soon after scratching, phosphorylation of IQGAP1 increased and reached a maximum at 1 h, lasting for at least 9 h. Overexpression of PKCεor treatment with PMA (the PKC activator) increased the amounts of phosphorylation of IQGAP1. On the contrary, phosphorylation of IQGAP1 induced by scratching was blocked by GF109203X (the PKC inhibitor). We next found that scratching induced close association between IQGAP1 and PKCεby immunofluorescence and immunoprecipitation methods. Our data from wound assays suggested that the wound gap closed within 24 h in control, while overexpression of PKCεand treatment with PMA promoted wound closure. On the other hand, the group treated with GF109203X was shown to impair the wound closure and still had 32% of original width. These results suggested that phosphorylation of IQGAP1 mediated the process of wound closure in BECs. Conclusion:Our results showed that IQGAP1 was generously expressed in BECs and suggested that PKCεmediated phosphorylation of IQGAP1 was involved in the early stage of wound closure. Part II IQGAP1 is involved in the proliferation process of injury and repair in BECs independent of Rac1 and Cdc42Background:Airway epithelium is repeatedly subjected to injury by cigarette smoke, pathogens, toxicants and infection. Immediately after injury, the airway epithelium initiates a multiple wound healing process to keep normal lung function, which involves so much signaling pathways and factors including cell extension, migration and proliferation. Rho GTPases are main branches of the Ras small GTPase superfamily, in which RhoA, Rac1 and Cdc42 are widely studied. Most GTPases cycle between the GTP-bound active and GDP-bound inactive forms. Through the effectors such as IQGAP1, PAK (p21 activated kinase) and WASP (Wiskott-Aldrich syndrome protein), Rho GTPases play critical roles in many processes, such as organization, chemotaxis and injury and healing. As an effector of Rac1 and Cdc42, IQGAP1 is an integral protein of cytoskeletal organization and interacts with cytoskeleton, cell adhesion complex and microtubule associated proteins (MAPs), thus IQGAP1 is considered to play roles in cell adhesion, polarization and migration. The mechanisms of Rho GTPases and their effector IQGAP1 are poor known in the process of airway epithelium. We supposed that Rho GTPases-IQGAP1 signal regulated the process of injury and repair in BECs and discussed the possible mechanism.Objective:We aim to study whether and how Rac1, Cdc42 and IQGAP1 work in injury and repair of BECs in vitro. The dynamic expressions of these factors during different stages of injury and repair were detected and their roles involved in the process were discussed. This study may bring theoretical and experimental evidences for further exploring the mechanism of injury and repair.Methods:In the model of injury and repair by scratching a monolayer of BECs in vitro, we detected the activities of Rac1 and Cdc42 by pull down assay. Next, we observed and examined the localizations and expressions of IQGAP1 and related factors in the model of injury and repair by immunofluorescence, RT-PCR, fluorescence quantitative PCR, western blot, isolation of cytoplasmic and nuclear proteins, transient transfection and RNA interference methods.Results:We found that scratching decreased GTP-bound Rac1 and Cdc42, but increased the amounts of IQGAP1 in both mRNA and protein at different time points. Next, we confirmed that IQGAP1 interacted with constitutively active Rac1 (Rac1V12) and Cdc42 (Cdc42V12) rather than dominant negative Rac1 (Rac1N17) and Cdc42 (Cdc42N17). Overexpressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was mutated at 1050-1052 amino acids instead for alanine and was defective to interact with Rho GTPase, induced translocation ofβ-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and induced expressions of its target genes of c-myc and cyclin D1. As similar, scratching increased the amounts of c-myc and cyclin D1. On the other hand, scratching altered the associations of IQGAP1 withβ-catenin, adenmatous polyposis coli (APC) and cytoplasmic linker protein-170 (CLIP-170). Depletion of IQGAP1 by small interference RNA (siRNA) blocked the wound closure. We conclude that IQGAP1 signal is involved in injury and repair and promotes cell proliferation in a manner independent of Rac1 and Cdc42 in the process of BECs.Conclusion:Our results indicated that: (1) Increasing amounts of IQGAP1 should play role in the process of wound closure. (2) IQGAP1 promotedβ-catenin translocation, activated Tcf and induced expressions of its target genes of c-myc and cyclin-D1 in a manner independent of Rac1 and Cdc42. (3) Scratching induced weak associations betweenα- andβ-catenin, but strong associations between IQGAP1 andβ-catenin. (4) IQGAP1 was shown to have strong associations with APC and CLIP-170 after scratching. (5) Depletion of IQGAP1 blocked the scratched wound closure.Our data suggested that: The process of wound closure in airway epithelial cells involves several coordinated events that are dependent on up-regulating of IQGAP1. The participation of Rac1 or Cdc42 is not necessary for this effect, but phosphylation of IQGAP1 is important at the early stage of injury and repair. These results may help to elucidate the mechanisms of the diseases in respiratory system and arrest their progression.