Abstract
PART Ι. Proteomic analysis of UVB-induced protein expression and thiol-reactivity alterations in skin fibroblasts using lysine- and cysteine-labeling two-dimensional differential gel electrophoresis UVB is the most energetic and DNA-damaging of the Ultraviolet (UV) irradiation that reaches the earth surface. Many previous reports suggested that exposure to the UVB irradiation causes skin pathologies like photoaging and skin cancer due to the direct damage on DNA molecules and the generation of reactive oxygen species (ROS) which interfere downstream signaling cascades via post-translational modifications of cysteine-residues in target proteins resulting in losing their biological functions. However, the detailed molecular and biochemical mechanisms that lead to skin cancer by UVB are yet to be clarified. In this study, we treated normal skin fibroblast cells (CCD-966SK) with various doses of UVB and monitored the protein expression and thiol-reactivity changes with lysine- and cysteine-labeling 2-D DIGE and MALDI-TOF MS. Our approaches revealed that 89 and 37 identified proteins showed significant changes in protein expression and thiol-reactivity of cysteine residues, respectively. Many identified proteins involving in protein folding (NPM), redox-regulation (Prxns), nucleotide biosynthesis, growth regulation (PHB) and cell migration (cofilin-1) are up-regulated under UVB irradiation. In contrast, proteins responsible for biosynthesis and protein degradation (PIAS 1) are down-regulated. In addition, proteins involving cytoskeleton, metabolism and signal transduction were shown to be altered in their thiol-reactivity. Furthermore, the UVB-induced protein expression and thiol-reactivity changes were validated with western blotting, ELISA and immunoprecipitation as well as discussed their roles on the early event of UVB-induced skin cancer formation. To sum up, we have identified numerous cellular proteins and redox-regulated proteins which are modulated by UVB irradiation. These identified proteins might play important roles on the early stages of skin cancer formation induced by UVB irradiation and might be potential targets for the rational design of drug to prevent UVB-induced tumorigenesis and potoaging. PART Π. Hyaluronic acid-dependent protection on alkali-damaged corneal cells Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan served as an essential extracellular matrix component and involved in many biological functions including cell proliferation, migration and wound healing. Cornea locates at the outer surface of eye, which suffers more alkali-injuries from household detergents or eye drop augmentations. Many reports revealed that hualuronic acid promote the wound healing of alkali-damaged corneal cells in animal models but the in vitro experiments and detail mechanisms were not to be clarified. The aim of this study is to in vitro investigate the protective process of hualuronic acid in alkali-damaged corneal cells. Accordingly, human corneal epithelial cell lines were treated with NaOH and two different molecular weights hyaluronic acid (100 kDa and 1000 kDa) in various concentrations. Many functional assays and proteomic analysis were performed to investigate the cell responses to hyaluronic acid treatment. Our study suggested that hyaluronic acid indeed stimulates corneal cell viability and wound healing ability. After alkali-damage, high molecular weight hyaluronic acid significantly reduced alkali-induced cell damage and enhances cell migration and wound healing which were modulated by cytoskeleton in the immune fluorescent data. Moreover, 2-D DIGE coupled with MALDI-TOF-MS and MALDI-TOF/TOF-MS analysis showed that high molecular weight hyaluronic acid protects corneal cell against alkali-injuries and stimulates cell wound healing might be due to the modulation of multiple cellular pathways including protein biosynthesis, degradation, trafficking and cell migration. To sum up, we demonstrate that high molecular weight hyaluronic acid facilitates corneal epithelial wound healing after alkali-injuries and suggest the possible mechanisms of hyaluronic acid-induced repair in corneal cells.