Zhang Hui, Sweezey Neil B, Kaplan Feige
Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada;
Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; Departments of Pediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada.
Am J Physiol Lung Cell Mol Physiol. 2015 Feb 15;308(4):L391-402. doi: 10.1152/ajplung.00119.2014. Epub 2014 Dec 5.
Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.
新气体交换单位的快速生长和形成（肺泡形成）是围产期肺的特征。支气管肺发育不良（BPD）在极早产儿中很常见，其特征是肺泡形成过早停滞。间充质细胞（成纤维细胞）通过间充质-上皮相互作用调节肺分支和肺泡形成。妊娠晚期肺1/富含半胱氨酸的分泌蛋白LD2（LGL1/CRISPLD2）在肺成纤维细胞中表达并分泌，其时间或空间缺陷会损害肺分支和肺泡形成（LGL1表示妊娠晚期肺1蛋白；LGL1表示人类基因；Lgl1表示小鼠/大鼠基因）。Lgl1缺失在胚胎期致死。在BPD的氧中毒大鼠模型中，Lgl1水平显著降低，杂合Lgl1(+/-)小鼠表现出类似人类BPD的特征。为了探索LGL1在发育中的肺的间充质-上皮相互作用中的作用，我们在人胎儿肺成纤维细胞（MRC5(LGL1KD)）中建立了一种强力霉素（DOX）诱导的RNA介导的LGL1敲低细胞模型。我们评估了LGL1对细胞增殖、细胞迁移、细胞凋亡和伤口愈合的影响。DOX诱导的MRC5(LGL1KD)抑制细胞生长，并增加膜联蛋白V(+)染色细胞的凋亡和半胱天冬酶3/7活性。LGL1条件培养基增加了胎鼠原代肺上皮细胞和人气道上皮细胞的迁移。添加LGL1条件培养基可减轻伤口模型中MRC5(LGL1KD)细胞愈合受损的情况。LGL1的抑制与细胞外基质基因（MMP1、ColXVα1和弹性蛋白下调）和促凋亡基因（BAD、BAK、CASP2和TNFRSF1B上调）的失调以及4-4/42MAPK磷酸化的抑制有关。我们的研究结果确定了LGL1在成纤维细胞扩增和迁移、上皮细胞迁移以及间充质-上皮信号传导中的作用，这些都是胎儿肺发育中的关键过程。
