C/EBPβ Regulation of the Type 1 IFN Response; Sensitizing Keratinocytes to Direct Activators of Cytosolic PRRs and DNA Damage-Induced Cell Death

细胞应激、DNA损伤、病原体损伤和免疫机制可以激活分子途径， 导致受调控的细胞死亡。增加我们对调节细胞死亡决定的途径的理解 在细胞应激反应中是至关重要的，因为失调的细胞死亡与癌症、神经变性和 自身免疫性疾病表皮是皮肤微生物、病毒和细菌的第一道防线， 环境损伤和角质形成细胞在宿主先天性免疫应答中起关键作用， I型干扰素（IFN-I）。除了抗微生物和抗病毒反应外，这种相同的IFN-I反应还 介导不同的细胞和生物反应，如增殖，凋亡，衰老和DNA 损伤响应本实验室最近的数据表明，CCAAT/增强子结合蛋白-β （C/EBPβ）转录因子是一种新的调节角质形成细胞型IFN-I反应的因子。观察了 小鼠表皮中C/EBPβ的条件性缺失（CKOβ）导致IFNb表达增加， 包括胞质模式识别受体（cPRR）的许多ISG。cPRRs检测病毒/病原体 RNA和DNA以及受损的宿主胞质DNA和RNA以触发IFN-1应答。CKOβ 用胞质PRR的直接活化剂处理的角质形成细胞显示出大大增加的IFN-Ⅰ应答， 令人惊讶地通过激活半胱天冬酶-8和半胱天冬酶-3导致细胞凋亡增加。作为breifly 如前所述，DNA损伤可以激活先天免疫反应。DNA损伤剂可导致 RNA和DNA中的修饰和结构变化可能被胞质PRR感知。我们 观察到CKOβ角质形成细胞受到DNA损伤后，caspase 8介导的 细胞凋亡依赖于干扰素a/B受体（INPAR）。IFN-I型应答的激活可 导致具有凋亡功能的ISGs表达增加，其激活caspase-8介导的凋亡 通过死亡受体信号传导。本提案的目的是了解C/EBPβ 负性调节IFN-1应答和caspase-8介导的细胞凋亡， 在小鼠表皮和原代角质形成细胞中的细胞溶质PRR和DNA损伤。我们假设 C/EBPβ负性调节角质形成细胞对IFN-Ⅰ的反应， 通过病原体RNA/DNA或受损的宿主RNA/DNA直接激活细胞溶质PRR， 诱导促凋亡ISG和半胱天冬酶8介导凋亡。这些实验将增加我们的 了解C/EBPβ调节cPRR激活和IFN-I应答的新途径 控制细胞死亡的决定，以应对细胞应激，包括DNA损伤。这种增加的 了解可以确定新的治疗靶点，以恢复细胞死亡的适当调节， 癌症、皮肤感染性疾病和皮肤自身免疫性疾病。

Cellular stress, DNA damage, pathogen insult, and immune mechanisms can activate molecular pathways that result in regulated cell death. Increasing our understanding of the pathways that regulate cell death decisions in response to cellular stress is critical as misregulated cell death is linked to cancer, neurodegeneration and autoimmune diseases. The epidermis is the first line of defense to cutaneous microbes, viruses and environmental insults, and keratinocytes play a critical role in the host innate immune response mediated by type I interferons (IFN-I). In addition to anti-microbial and anti-viral responses this same IFN-I response also mediates diverse cellular and biological responses such as proliferation, apoptosis, senescence and the DNA damage response. Recent data from out laboratory suggest that the CCAAT/enhancer-binding protein-β (C/EBPβ) transcription factor is a novel regulator the keratinocyte type IFN-I response. We observed the conditional deletion of C/EBPβ in mouse epidermis (CKOβ) resulted in increased expression of IFNb and numerous ISGs which included cytosolic pattern recognition receptors (cPRRs). cPRRs sense viral/pathogen RNA and DNA as well as damaged host cytosolic DNA and RNA to trigger a IFN-I response. CKOβ keratinocytes treated with direct activators of cytosolic PRRs displayed a greatly increased IFN-I response that surprisingly resulted in increased apoptosis via the activation of caspase-8 and caspase-3. As breifly mentioned, DNA damage can activate the innate immune response. DNA damaging agents can result in modifications and structural changes in RNA and DNA that could potentially be sensed by cytosolic PRRs. We observed that CKOβ keratinocytes challenged with DNA damage displayed increase caspase 8-mediated apoptosis that was dependent on the interferon a/b receptor (INFAR). Activation of a type IFN-I response can result in the increased expression of ISGs with apoptotic functions that activate caspase-8 mediated apoptosis via death receptor signaling. The objective of this proposal is to understand molecular basis for how C/EBPβ negatively regulates the IFN-I response and caspase-8 mediated apoptosis in response to direct activators of cytosolic PRRs and DNA damage in vivo in mouse epidermis and in primary keratinocytes. We hypothesize that C/EBPβ negatively regulates the IFN-I response in keratinocytes and the loss of C/EBPβ sensitizes keratinocytes to the direct activation of cytosolic PRRs by pathogen RNA/DNA or damaged host RNA/DNA to induce pro-apoptotic ISGs and caspase 8-mediated apoptosis. These experiments will increase our understanding of a novel pathway by which C/EBPβ regulates activation of cPRRs and the IFN-I response to control cell death decisions in response to cellular stress including DNA damage. This increased understanding could identify new therapeutic targets to restore proper regulation of cell death to treat skin cancer, skin infectious diseases, and skin autoimmune diseases.