Novel Roles Of Aquaporin 5 In Lung Cancer And Pulmonary Epithelial Defense Function Against P. Aeruginosa

Objective:To explore roles of AQP5 in lung cancer oncogenesis and development, the effects of AQP5 on lung cancer cell proliferation and migration and metastasis were investigated on the AQP5 stably expressed SPC-A1 and PC-9 cell line in vitro.Methods:Lung cancer cells with different AQP5 expression were used to study cell proliferation and migration potential. Immunofluorescent assay was used to detect AQP5 expression levels in SPC-A1 cells. DNA vector with insert targeting on the sequence of AQP5 were successfully constructed and transfected into SPC-A1 and PC-9 cells via Lipofectamine 2000. Cells were subcultured till day 7 after transfection and AQP5-neg-transfected group (as control group), and stably transfected cell lines were selected. Low level expression of AQP5 by gene silencing (siRNA), AQP5 levels were quantified by real-time quantitative PCR and western blot analysis. Osmotic water permeability measurement, immunohistochemistry, immunofluorescence, and further in vitro study including tumor cell migration and invasion, wound healing assays etc were performed.Results:AQP5 stably transfected cells were selected, and tested. We found enhanced invasion and migration potential in cancer cells with high AQP5 expression, while reduced metastasis potential in cancer cells with low AQP5 expression. Tumor cell migration and invasion test, wound healing assay showed significantly accelerated migration, invasion and wound healing in AQP5 over-expression cells (AQP5 cells) vs AQP5-neg-transfected group (control tumor cells). AQP5 transfected cells also showed significant increased MUC5AC mucin expression, which might contribute to the enhanced metastasis potential of lung cancer. AQP5 over-expression resulted in enhanced activation of epidermal growth factor receptor (EGFR), extracellular receptor kinase (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) pathway in cancer cells.Conclusions:AQP5-facilitated lung cancer cell proliferation and migration, possibly through activation of EGFR/ERK/p38 MAPK signaling pathway in part.Objective:According to the increased invasion and migration potential of AQP5 over-expressing lung cancer cells, to explore roles of AQP5 in lung cancer oncogenesis and development in vivo, the effects of AQP5 on lung cancer cell proliferation and metastasis was investigated.Methods:1×106 SPC-A1 cells including AQP5 over-expression cells (AQP5 cells) and AQP5-neg-transfected group (control tumor cells) were injected i.v. through tail vein into nude mice, respectively. Mice were then monitored for potential lung tumor colonization and metastases by in vivo Fluorescence imaging. Metastatic lesions were identified by fluorescence signals, and analyzed by WinLight32 Software. Mice were sacrificed after 2w and 4w, and lungs were harvested for hemotoxylin/eosin staining and AQP5 immunofluorescence staining. The metastatic lesions were identified by histology and fluorescence analyses:Paraffin-embedded sections were stained with hematoxylin and eosin (HE staining). Lung frozen sections were observed directly or for immunofluorescence detection by standard procedures. The number of tumor colonies in lungs was counted. In another experiment,106 SPC-A1 cells including AQP5 cells and control tumor cells were injected s.c. between the shoulders, respectively. Tumor length (L) and width (W) were measured with a caliper for estimation of tumor volume as 0.52xLxW2 every 3 days for 30 days. AQP5 levels were quantified by real-time quantitative PCR and western blot analysis, immunohistochemistry and immunofluorescence detection were performed.Results:Lung cancer cells with high and low expressions of AQP5 were used to study cell proliferation and migration potential, which are two important parameters for tumor cell biology. Enhanced proliferation and migration potential in cancer cells with high AQP5 expression, while reduced proliferation and metastasis potential in cancer cells with low AQP5 expression were found. Oncogene analysis showed significantly increased PCNA and c-myc expression in AQP5 transfected cells. AQP5 transfected cells also showed significant increased MUC5AC mucin expression, which might contribute to the enhanced metastasis potential of lung cancer. AQP5 over-expression resulted in enhanced activation of epidermal growth factor receptor (EGFR), extracellular receptor kinase (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) pathway in cancer cells. Moreover, deletion of AQP5 demonstrated declined activation of EGFR/ERK/p38 MAPK pathway in AQP5 knockout mice lungs, while deletion of AQP1 or AQP3 did not exhibit significant changes on the activation of EGFR/ERK/p38 MAPK pathway in lung tissue.Conclusions:The results provide evidence for AQP5-facilitated lung cancer cell proliferation and migration, possibly through activation of EGFR/ERK/p38 MAPK signaling pathway in part, but why AQP5 but not other aquaporin expression could affect the activation of EGFR/ERK/p38 MAPK pathway still needs further exploration.Objective:The present study aimed at investigating potential effects of AQP5 in P. aeruginosa (PA) infection induced lung injury in mice. To explore AQP5 might play protective roles in acute lung injury induced by P. aeruginosa, deletion of AQP5 might impair bacteria clearance ability in lung.Methods:Wild mice (AQP5+/+) were used as time-matched controls. Lung injury model was induced by intra-tracheal instillation of P. aeruginosa (1×10(?)6cfu) in wild type (AQP5+/+) and AQP5 knockout mice (AQP5-/-), respectively. Two and 6 hours later, Blood and lung lysate were cultured to detect blood dissemination. Bronchoalveolar lavage (BAL) fluid, plasma samples, and lung tissue were collected for monocyte chemoattractant protein-1 (MCP-1), Monocyte chemotactic protein (MCP)-2 and tumor necrosis factor-α(TNF-α)) measurement, inflammatory cells differentiation count and histology analysis, Protein leakage and Evans blue dye extravasation were evaluated for pulmonary barrier function.Results:Results showed AQP5 deficiency reduced neutrophil recruitment in distal airways and alveoli, increased bacterial blood dissemination and reduced mucin (MUC5AC, MUC5B) production. In vivo, PA infection induced lung injury was characterized by an increased microvascular permeability and myeloperoxidase (MPO) activity, etc. Nuclear factor-κB (NF-κB) in infected lungs was activated, accompanied with increased levels of MCP-1 and MIP-2 secretion. MMP9, but not MMP2, mainly correlates with the alteration of alveolar-capillary permeability in the early stage of PA-induced lung injury. AQP5 deletion.Improved activity of Matrix metalloproteinases 9 (MMP9) may be correlated alveolar-capillary permeability alteration. AQP5 deficiency showed declined activation of Mitogen-activated protein kinase (MAPK) and Nuclear factor-kappa B (NF-κB) pathway in lungs before and after upon P. aeruginosa infection. The increases in MAPK/NF-κB pathway, inflammatory cytokines were significantly suppressed by especially in the early stage of PA infection in AQP5-/- mice.Conclusions:The data demonstrated for the first time that AQP5 may play a protective role in the maintenance of pulmonary barrier function against P. aeruginosa infection. Attenuated MAPK/NF-κB pathway mediated expression of inflammatory cytokines may contribute to the decreased ability of bacterial clearance partly.