Translational control in human keratinocytes following UVB irradiation

 描述(申请人提供)：紫外线B(UVB)辐射是一种环境致癌物质，也是皮肤癌发展的最常见风险因素，皮肤癌在美国每年诊断的所有癌症中所占比例最高。窄波中波紫外线也被批准用于治疗几种皮肤病，如牛皮癣、白癜风和湿疹。因此，在分子水平上更好地了解人类皮肤细胞对UVB的反应，对于皮肤病治疗的改进是必要的。细胞保护自身免受环境胁迫的一种方式是通过真核细胞起始因子2(eIF2~P)的磷酸化来快速调节蛋白质的合成。有四种eIF2激酶，每一种都被不同的环境压力激活。由于多重应激引起翻译控制，这一途径被称为整合应激反应 (ISR)。EIF2~P导致翻译起始的全局性抑制，与转录因子ATF4和下游CHOP等选择性基因转录本的优先翻译一致。我们先前的研究表明，通过eIF2~P抑制蛋白质合成可以使角质形成细胞抵抗UVB诱导的细胞死亡。然而，UVB后ATF4被抑制，并在小分子药物作用下强迫ATF4表达，导致UVB诱导的细胞凋亡增加。因此，eIF2~P的保护作用和翻译调控是独立于ATF4的。我们假设eIF2~P通过翻译控制参与关键保护通路的mRNAs，如DNA修复和细胞周期控制，对UVB后的细胞活力产生积极的影响。为了研究这一想法，我们将对ISR缺陷的角质形成细胞进行UVB辐射，然后进行胸腺嘧啶二聚体的免疫印迹分析，以及通过合成核苷酸标记和流式细胞术进行细胞周期分析。我们预测，ISR缺陷的角质形成细胞修复DNA损伤的能力将降低，并停滞在细胞周期的G1期。其次，我们还建议利用核糖体图谱对UVB辐射后的角质形成细胞进行全基因组研究，这将确定单个mRNAs核糖体占有率的变化，并提供UVB后细胞翻译变化的快照。我们预测，核糖体图谱将识别以前没有在UVB辐射和皮肤病背景下研究过的翻译调控的mRNAs。建议的目标将共同确定eIF2~P和翻译提供UVB辐射的细胞保护的机制，目标是确定改进皮肤病治疗的靶点。

 DESCRIPTION (provided by applicant): 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.