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.