Mechanistic Study Of Aquaporin3in Photoaging Of Normal Human Skin Fibroblasts

Abstract:AQP3is a kind of membrane integral protein which can fast transport water, urea and glycerol. AQP3is significant to epidermal barrier and water retention. Researchers found AQP3participate not only in cell proliferation,cell migrations and the process of cell photoaging, but also in regulating transmembrane absorbtion of H2O2,which effects on intracellular signalingways.Autophagy is a fundamental salvage pathway, is involved in aging and various pathological processes. In old organs, the function of autophagy is weakened, causes the ability of cells in adapting to the external environment reduced, cell damages and a large amount of oxygen free radicals and other reactive oxygen compounds communicated and can not be effectively removed, make cellular homeostasis disturbed. In the process of cell photoaging, ROS produced by UVA radiation can induce autophagy and cell senescence, and autophagy decreased in aged cells, which causing ROS continuous accumulation, further aggravating the cell senescence. Thus, we hypothesized that, AQP3and autophagy in NHSFs may have some relations in the process of photoaging.In the first part of this study, primary normal human skin fibroblast(NHSFs) cultures were prepared from the circumcision foreskin of6to8year-old boys. Firstly, the photoaging model of NHSFs was established using UVA irradiation of320-400nm. Secondly, to change the AQP3expression in NHSFs, construction of AQP3shRNA and AQP3cDNA lentivirus stably transfected with NHSFs were performed. Lastly, the levels of P16and AQP3, the autophagic marker microtubule-associated protein1A/1B-light chain3(LC3Ⅱ/Ⅰ) were examined using Western blotting and RT-PCR.Aging level was also detected by β-galactosidase staining. Transmission electron microscope detected autophagosome, Immune fluorescent staining (IF) detected the changes of cell autophagy spots. In this part, we found (1) Compared with the normal control group,(3-galactosidase staining rate and the expression of P16INK4a in NHSFs have no significant difference after5J/cm2UVA radiation, while in5J/cm2/d radiation repeated for5days,(3-galactosidase staining rate and the expression of P16INK4a were significantly increased.(2) Compared with the normal control group,the level of AQP3is increased in the5J/cm2UVA radiation group while decreased in the5J/cm2/d repeated5days group.(3) Compared with the blank control group,the expression of P16INK4a and (3-galactosidase staining rate in NHSFs-AQP3shRNA cells were increased,in5J/cm2UVA radiation of NHSFs-AQP3shRNA, the (3-galactosidase staining rate and P16INK4a expression increased significantly. Compared with the blank control group,the expression of P16INK4a and (3-galactosidase staining rate expression in NHSFs-AQP3cDNA cells have no apparent decrease; but after radiation of5J/cm2/d repeated5days, compared with the blank group,(3-galactosidase staining rate and increased P16INK4a expression decreased obviously.(4) The results of IF showed that5J/cm2UVA radiation increased autophagy spots in NHSFs, while spots significantly reduced after the treatment of5J/cm2/d repeated5days. The turnover of LC3Ⅰ/Ⅱ detected by WB has the same trends.(5)Transmission electron microscope showed that compared with blank control group, autophagosome in NHSFs-AQP3shRNA cells was reduced, and further reduced after5J/cm2UVA radiation; autophagosome in NHSFs-AQP3cDNA cells was increased, after radiation of5J/cm2/d repeated5days autophagosome was decreased,but less than in the blank control group. IF and WB results showed that changes of autophagy level has the same trends.To further clarify the mechanism that AQP3influences autophagy is involved in the process of photoaging in NHSFs, in the second part of our study, the above cell models were used.10nmol/lU1060(ERK inhibitor), lOnmol/lJNK inhibitors,20nmol/lP38MAPK inhibitors, or mTOR inhibitors, and H2O2inhibitor catalase (catalase) and DPI (Nox inhibitor), H2O2kits for the detection of intracellular H2O2concentration were used. Phosphorylation/total expression of ERK1/2, JNK/SAPK, p38MAPK and mTOR were detected by WB. We found:(1) AQP3takes part in regulation of the intracellular H2O2concentration in NHSFs;(2) JNK/SAPK and P38MAPK pathway are involved in the MAPK/mTOR/LC3/P16INK4a, AQP3through which regulating autophagic effect on the photoaging process, while the ERK1/2MAPK pathway unrelated with this effect; and the level of cellular H2O2mediated by AQP3influenced this two MAPKs pathways.According to the above research results, we draw the following conclusions:AQP3plays a protective role in the process of photoaging in NHSFs, which adjusted by autophagy level, and AQP3regulates autophagy via ROS-MAPKs-mTOR pathways.