Publication Date
2010
Document Type
Thesis
Committee Members
Steven Berberich (Committee Member), Madhavi Kadakia (Advisor), Michael Leffak (Committee Member)
Degree Name
Master of Science (MS)
Abstract
Skin cancers, such as squamous cell carcinoma (SCC), develop from accumulated mutations as a result of excessive exposure to Ultraviolet B (UVB) radiation. Intriguingly, UVB also catalyzes the synthesis of 1alpha, 25-dihydroxy Vitamin D3 (VD3), the hormonally active form of Vitamin D. Downstream VD3 signaling has been associated with promoting the inhibition of cell cycle progression, regulating calcium homeostasis, and inducing differentiation and apoptosis. VD3 mediates these processes via genomic mechanisms through interaction with its cognate receptor, the Vitamin D Receptor, (VDR). In addition, it was recently discovered that VD3 reduces UVB-mediated phosphorylation of the SAPK/c-Jun N-terminal kinase (JNK), which correlated with a reduction in apoptosis and an increase in cell survival. Furthermore, VD3 treatment of keratinocytes also promoted up-regulation of the pro-survival p63 isoform, deltaNp63alpha. Expression of deltaNp63alpha in the basal progenitor layer of the epidermis is required for maintaining epidermal integrity by promoting continual proliferation and early commitment to stratification. VDR has also been shown to be essential for maintaining the integrity of the epidermis as exhibited by VDR null mice, which display skin defects. Although the downstream effects of VDR and deltaNp63alpha are well documented, their upstream regulation has been underexplored. In this study, we hypothesized that deltaNp63alpha and VDR are regulated by VD3 and UV signaling. To address this, mouse embryonic fibroblasts (MEFs) and transformed keratinocyte cell lines were treated with VD3 in the presence or absence of UV radiation. We confirmed that UV treatment resulted in the phosphorylation and activation of the JNK pathway, and that this effect is reduced in keratinocytes pre-treated with VD3. Moreover, we observed that UV exposure resulted in a reduction in VDR protein levels, and led to an electrophoretic mobility shift in deltaNp63alpha. Although, a mobility shift in deltaNp63alpha upon UV treatment was previously attributed to phosphorylation by p38, it did not rule out JNK as an alternate kinase and upstream regulator. While VD3 treatment inhibited UV induced p-JNK and VDR degradation, it did not reverse the mobility shift in deltaNp63alpha. Furthermore, VD3 treatment did not result in a significant elevation of p63 or VDR RNA, but increased the protein levels of VDR and p63. We attributed the increased levels of deltaNp63alpha and VDR protein to increased stability since VD3 significantly increased the half-life of both proteins. Finally, we observed that siRNA knockdown of VDR did not affect the ability of VD3 to inhibit the formation of p-JNK, but instead resulted in reduced total JNK levels independent of VD3 treatment. Our data shows that UV and VD3 signaling merge to regulate both deltaNp63alpha and VDR, and the balance between the two signaling pathways could determine whether cells survive or undergo apoptosis following UV-mediated DNA damage.
Page Count
91
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
2010
Copyright
Copyright 2010, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
