Translational control in human keratinocytes following UVB irradiation

UVB 照射后人类角质形成细胞的翻译控制

• 批准号: 9199053
• 负责人: Annie Collier
• 金额: $ 1.63万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9199053
• 关键词: Address; Affect; Alpha Cell; Apoptosis; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Cytoprotection; DNA Damage; DNA Repair; Data; Development; Disease; Eczema; Environmental Carcinogens; Epidermis; Eukaryotic Cell; Eukaryotic Initiation Factor-2; Exhibits; Feedback; Flow Cytometry; G1 Phase; Gene Expression; Gene Targeting; Genes; Genetic Translation; Goals; Healthcare Systems; Human; Immunoblot Analysis; Incidence; Individual; Label; Messenger RNA; Metabolism; Molecular; Pathway interactions; Phosphorylation; Phosphotransferases; Photosensitization; Phototherapy; Play; Process; Protein Biosynthesis; Protein Dephosphorylation; Proteins; Psoriasis; Regulation; Repression; Resistance; Ribosomes; Risk Factors; Role; Skin; Skin Cancer; Skin Carcinoma; Stress; Techniques; The Sun; Thymine Dimers; Transcript; Transcription Coactivator; Translation Initiation; Translational Regulation; Translations; UV protection; UVB induced; United States; Vitiligo; Work; biological adaptation to stress; cancer diagnosis; cell type; experience; genome-wide; genome-wide analysis; improved; innovation; insight; irradiation; keratinocyte; novel; nutrient deprivation; overexpression; protein folding; public health relevance; remediation; repaired; response; ribosome profiling; skin disorder; skin organogenesis; small molecule; small molecule inhibitor; synthetic nucleotide; targeted treatment; tool; transcription factor; transcriptome sequencing; ultraviolet

 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.

 描述（由申请人提供）：紫外线B（UVB）辐射是一种环境致癌物，也是皮肤癌发生的最常见风险因素，在美国每年诊断的所有癌症中，皮肤癌占比最高。窄波段UVB也已被批准用于治疗几种皮肤病，如牛皮癣、白癜风和湿疹。因此，需要更好地了解人类皮肤细胞如何在分子水平上对UVB做出反应，以改善皮肤病治疗。细胞保护自身免受环境应激的一种方式是通过真核起始因子2（eIF 2 ~P）的磷酸化来快速调节蛋白质合成。有四种eIF 2激酶，每种都被不同的环境应激激活。由于多重应激引起翻译控制，因此该途径被称为整合应激反应 （ISR）。eIF 2 ~P导致翻译起始的全面抑制，与选择基因转录物的优先翻译一致，如转录因子ATF 4和下游CHOP。我们以前的研究表明，通过eIF 2 ~P抑制蛋白质的合成提供了角质形成细胞抵抗UVB诱导的细胞死亡。然而，UVB后ATF 4被抑制，并且用小分子salubrinal强制ATF 4表达导致UVB诱导的细胞凋亡增加。因此，eIF 2 ~P的保护作用和翻译调控不依赖于ATF 4。我们推测eIF 2 ~P通过转录调控DNA修复和细胞周期调控等关键保护途径中的mRNAs，对UVB照射后的细胞活力产生积极影响。为了研究这一想法，我们将ISR缺陷的角质形成细胞进行UVB照射，然后通过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的细胞周期分析，然后进行流式细胞术。我们预测，ISR缺陷的角质形成细胞将表现出降低的能力，修复DNA损伤和停滞在G1期的细胞周期。其次，我们还提出了一个全基因组的研究UVB照射角质形成细胞与核糖体分析，这将确定在核糖体occupancy的变化的单个mRNA，并提供了一个快照的翻译变化的细胞后UVB。我们预测，核糖体分析将确定以前没有在UVB照射和皮肤病的背景下进行过研究的转录调控的mRNA。共同提出的目标将确定eIF 2 ~P和翻译提供UVB照射的细胞保护的机制，目的是确定改善皮肤病治疗的靶点。