Role of Fgfr2 signaling in bladder injury and regeneration

项目摘要/摘要： 环磷酰胺(CPP)对膀胱尿路上皮的损伤可导致危及生命的健康状况 包括出血性膀胱炎和膀胱癌。该应用程序的广泛长期目标是确定 CPP膀胱损伤的驱动机制及治疗靶点。CPP诱导细胞凋亡引起的尿路上皮丢失 48小时内坏死，14天后完成增殖修复。而成纤维细胞的生长 因子受体2(FGFR2)在CPP诱导的尿路上皮损伤中的作用 不清楚。初步数据显示，FGFR2：1)抑制尿路上皮细胞丢失，2)促进尿路上皮修复 CPP损伤后。关于细胞丢失，注射角质形成细胞生长因子(KGF)，这是上皮Fgfr2的配体， 野生型小鼠CPP前24小时可阻断尿路上皮细胞的凋亡丢失。AKT，成纤维细胞生长因子信号转导和 已知的细胞凋亡抑制因子在KGF后在尿路上皮细胞中上调。关于再生，小鼠有 FGFR2在所有膀胱尿路上皮层中的条件性缺失具有损伤后尿路上皮修复的缺陷。在3天内 CPP后，对照组出现尿路上皮增生，尿路蛋白(屏障蛋白)染色显著恢复， 炎症和出血的主要消退，而突变膀胱的增生较少，变弱 尿路蛋白染色，持续出血和炎症。而突变体和对照都有扩展 在CPP后3天跨基底层的角蛋白14(Krt14)阳性祖细胞中，突变的Krt14+细胞 细胞核肥大，提示细胞周期缺陷。细胞周期分析和DNA检测 内容提示突变的Krt14+细胞有异常的内复制(DNA复制没有完成 有丝分裂，导致多倍体)。进行明显内复制的FGFR2突变细胞也有证据表明 损伤后3天DNA损伤/复制应激增加。鉴于FGFR2刺激ERK可以抑制 细胞周期进入和内复制，ERK及其读数被评估，并且在突变体3中两者都减少 CPP后数天。再生缺陷，包括大的(可能是多倍体)Krt14+细胞，在CPP后10天持续存在。 总之，假设FGFR2信号通过抑制细胞凋亡来改善CPP所致的尿路上皮损伤 通过Akt，并通过ERK抑制尿路上皮的内复制，促进CPP后的再生。为了测试这一点 假设，提出以下目标：目的1：研究刺激FGFR2信号如何阻断CPP- 诱导膀胱尿路上皮细胞丢失。全动物和基于细胞的检测将阐明FGFR2/Akt在阻断FGFR2/Akt中的作用 CPP诱导的细胞凋亡损伤。AKT抑制剂将在KGF之前使用，以显示Akt在FGFR2下游的作用。 目的2：探讨成纤维细胞生长因子2(FGFR2)在慢性前列腺炎(CPP)后促进膀胱尿路上皮再生的作用。整个动物和 基于细胞的分析将阐明FGFR2/Erk抑制内复制和驱动再生的作用，包括 在突变体中用具有结构性活性的ERK进行遗传拯救，在对照中用化学拯救来加速修复。目标 3：明确CPP损伤后膀胱尿路上皮中FGFR2信号的细胞特异性作用。FGFR2将被删除 在特定的尿路上皮层中，以确定CPP损伤后的细胞特异性行为。

Project Summary/Abstract: Cyclophosphamide (CPP)-induced injury to bladder urothelium can lead to life-threatening health conditions including hemorrhagic cystitis and bladder cancer. The application’s broad long-term objectives are to identify mechanisms driving CPP bladder injury and thus therapeutic targets. CPP induces urothelial loss from apoptosis and necrosis within 48 hours, followed by proliferation and repair finishing by 14 days. While fibroblast growth factor receptor 2 (Fgfr2) activity blunts injury in other tissues, roles of Fgfr2 in CPP-induced urothelial injury are unclear. Preliminary data shows that Fgfr2: 1) dampens urothelial cell loss and 2) enhances urothelial repair after CPP injury. Regarding cell loss, injection of keratinocyte growth factor (KGF), a ligand for epithelial Fgfr2, 24 hours prior to CPP in wildtype mice blocks apoptotic urothelial cell loss. Akt, a transducer of Fgf signaling and known repressor of apoptosis, is upregulated in urothelium after KGF. Regarding regeneration, mice with conditional deletion of Fgfr2 in all bladder urothelial layers have defective urothelial repair after injury. At 3 days post-CPP, controls had urothelial hyperplasia, significant restoration of uroplakin (barrier protein) staining and major resolution of inflammation and hemorrhage, while mutant bladders had less hyperplasia, attenuated uroplakin staining, and ongoing hemorrhage and inflammation. While both mutants and controls had expansion of Keratin 14 (Krt14)-positive putative progenitor cells across basal layers 3 days after CPP, mutant Krt14+ cells were hypertrophic with enlarged nuclei suggesting a cell cycle defect. Cell cycle profiling and assays for DNA content suggest that mutant Krt14+ cells have aberrant endoreplication (DNA replication without completion of mitosis, leading to polyploidy). Fgfr2-mutant cells undergoing apparent endoreplication also had evidence of increased DNA damage/replication stress 3 days after injury. Given that Fgfr2 stimulates Erk that can suppress cell cycle entry and endoreplication, Erk and its readouts were assessed and both were reduced in mutants 3 days after CPP. Regeneration defects, including large (likely polyploid) Krt14+ cells, persisted 10 days after CPP. Together, the hypothesis is that Fgfr2 signaling ameliorates urothelial injury from CPP by suppressing apoptosis via Akt and promotes regeneration after CPP by repressing endoreplication of urothelium via Erk. To test this hypothesis, the following Aims are proposed: Aim 1: Investigate how stimulation of Fgfr2 signaling blocks CPP- induced bladder urothelial cell loss. Whole animal and cell-based assays will elucidate roles of Fgfr2/Akt to block CPP-induced apoptotic injury. Akt inhibitors will be used prior to KGF to show roles of Akt downstream of Fgfr2. Aim 2: Determine how Fgfr2 promotes regeneration of bladder urothelium after CPP-injury. Whole animal and cell-based assays will elucidate roles of Fgfr2/Erk to suppress endoreplication and drive regeneration, including genetic rescue with constitutively active Erk in mutants and chemical rescue to accelerate repair in controls. Aim 3: Identify the cell-specific roles of Fgfr2 signaling in the bladder urothelium after CPP injury. Fgfr2 will be deleted in specific urothelial layers to identify cell-specific actions after CPP-injury.