Ultraviolet B (UVB) irradiation is an environmental carcinogen and the most common risk factor for the development of skin cancers, which account for the highest percentage of all cancers diagnosed in the United States each year. Narrow band UVB has also been approved for the treatment of several skin diseases such as psoriasis, vitiligo, and eczema. Therefore, a better understanding of how human skin cells respond to UVB at the molecular level is required for improvement of skin disease therapies. One way that cells protect themselves from environmental stress is by rapid modulation of protein synthesis through phosphorylation of eukaryotic initiation factor 2 (eIF2~P). There are four eIF2 kinases that are each activated by different environmental stresses. Because multiple stresses elicit translational control this pathway is referred to as the Integrated Stress Response (ISR). eIF2~P causes a global repression of translation initiation, coincident with preferential translation of select gene transcripts, such as the transcription factors ATF4 and downstream CHOP. We previously showed that repression of protein synthesis through eIF2~P provides keratinocytes resistance to UVB-induced cell death. However ATF4 is repressed following UVB, and forced ATF4 expression with the small molecule salubrinal results in increased UVB-induced apoptosis. Therefore, the protective aspects of eIF2~P and translational control are independent of ATF4. We hypothesize that eIF2~P elicits positive effects on cell viability following UVB through translational control of mRNAs involved in key protective pathways, such as DNA repair and cell cycle control. In order to investigate this idea, we will subject ISR-deficient keratinocytes to UVB irradiation, followed by immunoblot analysis of thymine dimers as well as cell cycle analysis via synthetic nucleotide labeling followed by flow cytometry. We predict that ISR-deficient keratinocytes will exhibit a reduced ability to repair DNA damage and arrest in the G1 phase of the cell cycle. Secondly, we also propose a genome-wide study of UVB irradiated keratinocytes with ribosome profiling, which will determine changes in ribosome occupancies of individual mRNAs and provide a snapshot of translational changes in the cell following UVB. We predict that ribosome profiling will identify translationally regulated mRNAs that have not previously been investigated in the context of UVB irradiation and skin disease. Together the proposed aims will determine mechanisms by which eIF2~P and translational provides for cytoprotection to UVB irradiation, with the goal of identifying targets for improved skin disease therapies.
UVB irradiation is both an environmental carcinogen as well as a tool for phototherapy to treat psoriasis; therefore, identification of UVB-induced molecular changes are crucial for improvement of skin disease therapies. Regulation of protein synthesis levels is a key means by which cells protect themselves from environmental stress, which marks it as a promising target for therapies that utilize UVB. An analysis of how translational control regulates UVB-induced cellular processes as well as a genome-wide study of UVB-induced changes in translation will provide novel information to increase our understanding of how UVB affects human skin.
|Collier, Ann E; Wek, Ronald C; Spandau, Dan F (2017) Human Keratinocyte Differentiation Requires Translational Control by the eIF2? Kinase GCN2. J Invest Dermatol 137:1924-1934|
|Loesch, Mathew M; Collier, Ann E; Southern, David H et al. (2016) Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo. Mol Oncol 10:1245-54|