Role of ASK1 and PKR in Fancc hematopoiesis

描述(申请人提供)：我们的长期目标是确定范可尼贫血(FA)蛋白在保护造血干/祖细胞(HS/Ps)免受凋亡方面的机制(S)，以便设计有针对性的预防/治疗FA骨髓衰竭的疗法。80%的FA患者死亡是进行性骨髓衰竭的直接结果。因此，了解FA HS/Ps的凋亡易感性机制具有重要意义和临床意义。目前，关于单个FA蛋白在维持HS/PS存活中的作用(S)知之甚少。使用C型FA小鼠模型，我们以前发现Fancc-/-干细胞的再繁殖能力显著降低，Fame-/-前体细胞表现出增强的抑制性细胞因子诱导的凋亡。初步数据还表明，Fancc/-前体细胞对氧化剂高度敏感。我们的中心假设是，通过改变抑制性细胞因子和氧化剂凋亡信号导致的IIS/Ps丢失在FA-C患者的骨髓衰竭的发展中起着至关重要的作用。先前在细胞系中的结构-功能研究显示了两个FANCC功能，可由FANCC突变体分离。一种功能是保护细胞免受基因毒素的伤害，另一种功能是通过抑制双链RNA依赖的激酶(PKR)介导的细胞凋亡来提高干扰素-γ/肿瘤坏死因子-α治疗后的存活率。虽然这些研究开始阐明细胞系中不同的FANCC功能，但一个关键但尚未回答的问题是，这两个功能是否在提高原发HS/P的存活率方面发挥同样重要的作用。此外，我们的初步数据表明，FANCC可能通过依赖于凋亡信号调节激酶1(ASK1)的途径来保护细胞免受肿瘤坏死因子-α和氧化剂诱导的细胞凋亡。我们假设Fancc-/-细胞同时表现出ASK1和PKR凋亡信号的改变，这与氧化剂或抑制性细胞因子处理后Fancc-/HS/Ps的促凋亡表型有关。本研究的目的是：1)确定PKR依赖和非依赖的FANCC功能是否增强Fancc-/-干细胞在体内的再填充能力并保护其免受抑制性细胞因子和基因毒素的治疗；2)确定ASK1是否参与Fancc-/-HS/Ps对凋亡刺激和再填充能力的超敏；以及3)研究原代Fancc-/-细胞的抑制性细胞因子超敏是否涉及ASK1和PKR凋亡信号的改变。

DESCRIPTION (provided by applicant): Our long-term goal is to identify the mechanism(s) that Fanconi anemia (FA) proteins have in protecting hematopoietic stem/progenitor cells (HS/Ps) from apoptosis in order to design targeted therapies for prevention/treatment of bone marrow failure in FA. 80% of FA patient deaths are a direct result of a progressive marrow failure. Thus, understanding mechanisms involved in the apoptotic predisposition of FA HS/Ps is of critical importance and clinically relevant. Currently, little is known regarding the function(s) that individual FA proteins have in maintaining HS/Ps survival. Using a murine model of FA type C, we previously showed that Fancc -/- stem cells have a marked reduction in repopulating ability and Fame -/- progenitors exhibit enhanced inhibitory cytokine-induced apoptosis. Preliminary data also show that Fancc /- progenitors are hypersensitive to oxidants. Our central hypothesis is that loss of IIS/Ps via altered inhibitory cytokine and oxidant apoptotic signaling has a crucial role in the development of marrow failure in FA-C patients. Previous structure-function studies in cell lines demonstrated 2 FANCC functions, separable by FANCC" mutants. One (1) function was to protect from genotoxins, and the other was to enhance survival after IFN-y/TNF-a treatment by inhibiting double-stranded RNA-dependent kinase (PKR)-mediated apoptosis. While these studies begin to clarify distinct FANCC functions in cell lines, a critical yet unanswered question is whether these 2 functions exhibit equally important roles in enhancing the survival of primary HS/Ps. Furthermore, our preliminary data suggest that FANCC may protect cells from TNF-a and oxidant induced apoptosis through an apoptosis signal-regulating kinase 1 (ASK1) dependent pathway. We hypothesize that Fancc -/- cells exhibit both altered ASK1 and PKR apoptotic signaling, which contribute to the pro-apoptotic phenotype of Fancc -/ HS/Ps after oxidant or inhibitory cytokine treatment. The goals of this application are, 1) to determine whether PKR-dependent and -independent FANCC functions enhance Fancc -/- stem cell repopulating ability and protect from inhibitory cytokine and genotoxin treatment in vivo, 2) to determine whether ASK1 participates in Fancc -/- HS/Ps hypersensitivity to apoptotic stimuli and repopulating ability, and 3) to investigate whether inhibitory cytokine hypersensitivity in primary Fancc -/- cells involves alterations in both ASK1 and PKR apoptotic signaling.