GRP78 regulates ER homeostasis and distal epithelial cell survival during lung development

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated mice (cGrp78f/f) with lung epithelial cell-specific knockout (KO) of Grp78, a gene encoding the ER chaperone 78-kDa glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78f/f pups died immediately after birth, likely due to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-β (TGF-β)/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress and TGF-β/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone taursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78f/f lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress and suggest the UPR as a potential therapeutic target in BPD. Whole-genome expression profiling was performed using MouseRef-8 v2.0 Expression BeadChips (Illumina) on RNA isolated from lungs of four Grp78f/f and three cGrp78f/f mice at E18.支气管肺发育不良（Bronchopulmonary dysplasia, BPD）作为一种早产儿慢性肺部疾病，已被证实与内质网（endoplasmic reticulum, ER）应激密切相关。为探究内质网应激在BPD发病机制中的因果作用，我们构建了肺上皮细胞特异性敲除Grp78基因的小鼠模型（cGrp78f/f）：Grp78基因编码内质网分子伴侣78 kDa葡萄糖调节蛋白（GRP78），后者是内质网稳态与未折叠蛋白反应（unfolded protein response, UPR）的核心调控因子。肺上皮特异性Grp78敲除会破坏肺脏形态发生过程，引发发育停滞，导致围产期肺组织中肺泡Ⅱ型上皮细胞凋亡增加，同时使表面活性蛋白与Ⅰ型肺泡上皮细胞标志物的表达水平下调。cGrp78f/f幼鼠出生后即刻死亡，推测死因为呼吸窘迫。值得注意的是，Grp78敲除可触发未折叠蛋白反应激活，促凋亡转录因子C/EBP同源蛋白（C/EBP homologous protein, CHOP）的表达显著上调。氧化应激与细胞死亡相关基因的表达水平升高，而抗氧化酶编码基因的表达则出现降低，这提示氧化应激参与了肺泡上皮细胞（alveolar epithelial cell, AEC）的凋亡过程。Smad3磷酸化水平升高，以及转化生长因子-β（transforming growth factor-β, TGF-β）/Smad3下游靶基因Cdkn1a（编码p21）和Gadd45a的表达上调，表明未折叠蛋白反应的凋亡通路、氧化应激与TGF-β/Smad信号通路之间的相互作用，共同介导了Grp78敲除诱导的肺泡上皮细胞凋亡与发育停滞。化学分子伴侣牛磺去氧胆酸（taursodeoxycholic acid）可降低体外培养的cGrp78f/f肺组织中的未折叠蛋白反应激活与细胞凋亡水平，证实内质网应激在上述肺泡上皮细胞异常表型中发挥了关键作用。本研究结果表明，GRP78通过调控内质网应激，在肺发育过程中对肺泡上皮细胞存活与基因表达起到关键调控作用，并提示未折叠蛋白反应可作为BPD潜在的治疗靶点。本研究采用MouseRef-8 v2.0 Expression BeadChips（Illumina），对胚胎第18天（E18）时4只Grp78f/f小鼠与3只cGrp78f/f小鼠的肺组织分离所得RNA进行了全基因组表达谱分析。