Stress and Growth Related Keratinocyte Pathways

Cellular stress and damage response mechanisms play a crucial role in physiological defense against hyperproliferative diseases, genotoxic injury, mutations and malignancy. An important first step in the cellular stress response is upregulation of immediate early genes that initiate and coordinate subsequent cellular events. IEX-1 is a novel immediate early gene that has been shown to be induced by gamma irradiation, phorbol ester treatment and ultraviolet irradiation. Goal of this thesis was to more specifically characterize the role of IEX-1 in keratinocytes. Special emphasis was directed to elucidating the cellular response to stress and mitogenic stimulation and the role of IEX-1 in mediating apoptosis as well as localizing the protein within the cell during these events. By northern blot analysis, it could be shown that gamma irradiation of primary human keratinocytes results in a time dependent, rapid induction of IEX-1 expression followed by prompt downregulation, similar to previous findings in a squamous cell carcinoma cell line. In addition, UV-irradiation or treatment with the reactive oxygen species hydrogen peroxide also induced IEX-1 expression rapidly and transiently. Further incubation with the mitogenic factor for keratinocyte Epidermal Growth Factor (EGF) resulted in increased steady state levels of IEX-1 mRNA. Compared to normal keratinocytes, similar observations were made in the non-tumorigenic keratinocyte cell line HaCaT. Regulation of IEX-1 gene expression by the EGF-Receptor (EGFR) was investigated using an IEX-1-promoter-luciferase-reporter-gene assay and blockage of EGFR by the highly specific EGFR-tyrosine kinase inhibitor PD153035. Blockage of the receptor was followed by a marked decrease in IEX-1 promoter activity. Effects of IEX-1 overexpression in HaCaT cells were investigated. IEX-1 overexpression enhanced proliferation, as confirmed by [3H]-thymidine incorporation assay. To examine the effects of IEX-1 on apoptosis induced by various agents, IEX-1 over-expressing HaCaT were challenged with ultraviolet radiation, the DNA damaging agent camptothecin or serum-deprival. Cell survival, caspase 3-activity and nucleosome formation were measured to assess apoptosis. There was no difference observed in baseline apoptotic activity of cells cultured under non-stressed conditions, when comparing the IEX-1 overexpressing and the control cell line. However, upon stress-induction, the IEX-1 overexpressing cells showed markedly higher levels of apoptosis. Data characterizing the intracellular localization of IEX-1 were obtained by immunohistochemical staining as well as by molecular biological methods. IEX-1 was predominantly located within the cell nucleus, forming several intranuclear patches. Treatment with various stress-inducing agents did not significantly alter the localization or cause translocation of the IEX-1 protein. Distribution of IEX-1 within the skin and epidermis was assessed by immunocytochemistry of human skin specimens and revealed predominant localization of IEX-1 within the nucleus and cytoplasm of the basal epidermal and suprabasal layers. These findings implicate IEX-1 in the control of apoptosis upon cell stress as well as promotion of cell replication during favorable growth conditions. The function of IEX-1 seems to be closely linked to the differentiation status of the keratinocyte. As an immediate early gene product, IEX-1 is a novel regulator of keratinocyte growth and survival, similar to other critical cell cycle control proteins, such as p53, p21Waf1, c-myc and related proteins. This suggests that IEX-1 is another crucial element in the pattern of regulatory pathways of cell growth and defense against malignant transformation.