Heme oxygenase-1 (HO-1) is well known for being involved in antioxidant defence. BTB and CNC homology1 protein (Bach1) is involved in the transcriptional regulation of the HO-1 gene as a negative regulator. In human skin cells, HO-1 induction protects against UVA-induced cellular damage. Previous studies from this laboratory have found that during this process, suppressing the Bach1 level regulates HO-1 expression level. However little is known about whether repression of Bach1 would enhance HO-1 protection against UVA damage. In this study, we silenced Bach1 by specific siBach1 RNA and showed that Bach1 repression does not protect against UVA induced damage in cultured human skin fibroblasts. However, silencing of Bach1 in skin fibroblasts was shown to be involved in the process of UVA-induced ROS generation. We observed that UVA-induced ROS generation is independent of HO-1 induction. This led us to investigate further the mechanism by which UVA-induces ROS in cultured human skin cells; we targeted NADPH oxidase (NOX) as the major source of UVA-induced ROS generation in human skin cells, and showed that following UVA irradiation, NOX is activated to different extents in two cultured human skin cell lines (i.e. FEK4 cells and HaCaT cells). The possible involvement of different NOX proteins was then studied and the results showed that NOX1 and NOX4 both contribute to the NOX activation process but to different extents. NOX1 and NOX4 protein expression levels were also induced to different extents following UVA irradiation of FEK4 cells and HaCaT cells. The NOX general inhibitor, diphenylene iodonium (DPI), was used in this study to repress NOX, and we showed that this general inhibitor of the enzyme reduced ROS generation following UVA irradiation in human fibroblasts. This data is consistent with a previous study showing DPI inhibition of UVA-induced ROS generation in keratinocytes. However, using specific siRNA knock-down reagents, NOX1 and NOX4 were found to have only a limited contribution to the UVA-induced ROS generation process in human skin fibroblasts, a result consistent with the involvement of additional NOX proteins in this pathway.
|Date of Award||19 Nov 2014|
|Supervisor||Rex Tyrrell (Supervisor)|