Effect Of AQP5 On Osmotic Water Permeability And MUC5AC Mucin Synthesis And Secretion In Human Airway Submucosal Gland Cells

The 1^st part:Down regulation of aquaporin 5, induced by vector-based shorthairpin RNA in human airway submucosal gland cells[Abstract] Objective In this study, we investigated whether vector-based shRNA could efficiently inhibit the expression of AQP5 in human airway submucosal gland cells (SPC-A1). Methods Immunofluorescent assay was used to detect whether AQP5 and MUC5AC located in SPC-A1 cells. DNA vector with insert targeting on the sequence of AQP5 were successfully constructed and transfected into SPC-A1 cells via Lipofectamine 2000. Cells were subcultured till day 7 after transfection and compared with control group and non-specific transfected group. mRNA of AQP5 were reverse transcribed and quantified by real-time PCR. Protein expression were assessed by FACS (Fluorescent Activated Cell Sorts) and Western blot. Results AQP5 and MUC5AC were both expressed in SPC-A1 cells. After transfection of pShAQP5, mRNA of AQP5 decreased remarkably on day 1 and day 2, while the suppression of protein expression didn't appear until day 5. Conclusion Our results indicate vector-based shRNA is a potential tool to inhibit expression of AQP5 and its effect on AQP5 protein is markedly latter than AQP5 mRNA. The vector-based RNAi technique, if widely used in other AQPs, will further the scope of our knowledge of the physiological function of aquaporins. The 2^nd partEffect of inhibition of AQP5 on osmotic water permeabilityin SPC-A1 cells[Abstract] Objective In this study, we determined whether inhibition of AQP5 had effect onosmotic water permeability in SPC-A1 cells. Methods Cells were divided into 3 groups: Con group, shNeg group and shAQP5 group. Loaded with volume-sensitive fluorescent indicator calcein-AM, intracellular fluorescent concentration in response to varied osmotic solution were monitored by confocal microscopy and osmotic water permeability coefficient (Pf) could be deduced according to a series of equations. Hypotonic shock induced regulated volume decrease phenomena were investigated among 3 groups. Results Calcein-AM quenching didn't exist in SPC-A1 cells after several perfusion experiments with 5 series of loading concentration. Hypertonic perfusion producing cell shrinkage resulted in enhancement of fluorescent concentration, while cell swelling in response to hypotonic shock resulted in decrease of fluorescent concentration. Osmotic water permeability coefficient (Pf) in shAQP5 group were 0.8×l0^-2 cm/s and remarkably decreased compared with Con and shNeg groups. The rebounding time of RVD in shAQP5 group was longer than that of Con group and shNeg group. Cell volume decrease capability in shAQP5 group was significantly decreased as well. Conclusions Our studies indicate confocal microscopy is a potential utility in making quantitative Pf measurement in living cells through real time monitoring changes of cell volume and fluorescent concentration. Osmotic water permeability and regulatory volume decrease capability were reduced after downregulation of AQP5 expression in SPC-A1 cells.The 3^rd partRegulation of MUC5AC synthesis and secretion by depletion of AQP5 and their regulatory mechanism in SPC-A1 cells[Abstract] Objective The present study aimed to examine whether AQP5 was involved in modulation of mucin expression and secretion in airway submucosal gland cells (SPC-A1) and further study the regulatory mechanism between AQP5 and MUC5AC. Methods A recombinant plasmid (pShAQP5) containing shRNA (small hairpin RNA) expression cassette targeting AQP5 sequence was constructed. G418 were used to select stably transfected clones.MUC5AC mRNA were determined by Real-time PCR and the protein level of MUC5AC were measured by Western blots. ELISA was established to detect MUC5AC synthesis and secretion in supernatant and cell lysate. Cells loaded with fura-2 was used to monitor changes of [Ca²⁺]i in non-stimulated and stimulated status. Expression level of PKC and p-PKC in cytoplasm were detected by Western blot. Results After the treatment of G418 for one and a half month, 5 stably transfected clones were selected. In pShAQP5 transiently tranfected cells, MUC5AC mRNA were markedly increased by 120% and 65.7% on 3～4 day after pShAQP5 transfection and the results of ELISA showed MUC5AC synthesis and secretion were increased by 57.9% and 85.3% respectively on day 5 after pShAQP5 transfection. While in 5 stably transfected clones (shAQP5-Gl,G2,G3,A2,A5), the upregulated levels of MUC5AC mRNA were 118%, 165%, 65%, 123% and 38% respectively. The elevated levels of MUC5AC synthesis and secretion varied from 59-156% and 33-166% respectively. In non-stimulated status, [Ca²⁺]i in shAQP5 group were similar with Con and shNeg groups, while pilocarpine induced enhancement of [Ca²⁺]i in shAQP5 group were remarkable, compared with the other 2 groups. shAQP5 cells had almost the same level of intracellular PKC as that of Con group. p-PKC in shAQP5 cells were steadily increased in time-dependent way compared with Con cells. Conclusion This is the first reliable investigation of regulation of MUC5AC mucin secretion by silencing AQP5 and the upregulation of MUC5AC might be mediated via intracellular Ca²⁺-PKC pathway in response to the changes of AQP5 expression in human airway submucosal gland cells.