FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy

FOXF1, a member of the forkhead box family of transcription factors, has been previously shown to be critical for lung development, homeostasis, and injury responses. However, the role of FOXF1 in lung regeneration is unknown. Herein, we performed partial pneumonectomy, a model of lung regeneration, in mice lacking one Foxf1 allele in endothelial cells (PDGFb-iCre/Foxf1fl/+ mice). Endothelial cell proliferation was significantly reduced in regenerating lungs from mice deficient for endothelial Foxf1. Decreased endothelial proliferation was associated with delayed lung regeneration as shown by reduced respiratory volume in Foxf1-deficient lungs. FACS-sorted endothelial cells isolated from regenerating PDGFb-iCre/Foxf1fl/+ and control lungs were used for RNAseq analysis to identify FOXF1 target genes. Foxf1 deficiency altered expression of numerous genes including those regulating extracellular matrix remodeling (Timp3, Adamts9) and cell cycle progression (Cdkn1a, Cdkn2b, Cenpj, Tubb4a), which are critical for lung regeneration. Deletion of Foxf1 increased Timp3 mRNA and protein, decreasing MMP14 activity in regenerating lungs. ChIPseq analysis for FOXF1 and histone methylation marks identified DNA regulatory regions with the Cd44, Cdkn1a, and Cdkn2b genes, indicating they are direct FOXF1 targets. Thus FOXF1 stimulates lung regeneration following partial pneumonectomy via direct transcriptional regulation of genes critical for extracellular matrix remodeling and cell cycle progression. ChIPseq on quiescent MFLM-91U cells; RNAseq on FACS-sorted endothelial cells (CD45-CD31+CD326-) from Foxf1fl/+ and PDGFb-iCre Foxf1fl/+ lungs 4 days after partial pneumonectomy surgery.

FOXF1作为叉头框（forkhead box）转录因子家族成员，既往研究已证实其在肺发育、稳态维持及损伤应答中发挥关键作用。然而，FOXF1在肺再生过程中的功能尚不明确。本研究于内皮细胞单等位基因Foxf1缺失的小鼠（PDGFb-iCre/Foxf1fl/+小鼠）中构建部分肺切除术模型，该模型是肺再生研究的经典模型。实验结果显示，在内皮细胞Foxf1缺陷小鼠的再生肺组织中，内皮细胞增殖水平显著降低。Foxf1缺陷小鼠肺组织的呼吸容量下降，提示内皮增殖受损与肺再生延迟密切相关。研究人员从再生的PDGFb-iCre/Foxf1fl/+小鼠及对照小鼠肺组织中分离经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）的内皮细胞，通过RNA测序（RNAseq）分析鉴定FOXF1的靶基因。Foxf1缺陷可改变大量基因的表达谱，其中包括调控细胞外基质（extracellular matrix）重塑（Timp3、Adamts9）与细胞周期进程（Cdkn1a、Cdkn2b、Cenpj、Tubb4a）的关键基因，而这些过程对肺再生至关重要。Foxf1缺失会上调Timp3的mRNA及蛋白表达水平，进而降低再生肺组织中基质金属蛋白酶14（MMP14）的活性。针对FOXF1及组蛋白甲基化修饰的染色质免疫共沉淀测序（ChIPseq）分析，在Cd44、Cdkn1a及Cdkn2b基因位点鉴定到DNA调控区域，证实上述基因为FOXF1的直接靶标。综上，FOXF1可通过直接转录调控细胞外基质重塑与细胞周期进程相关的关键基因，促进部分肺切除术后的肺再生过程。本研究的测序数据集包括：对静止态MFLM-91U细胞的ChIPseq，以及对部分肺切除术术后4天的Foxf1fl/+与PDGFb-iCre Foxf1fl/+小鼠肺组织中经FACS分选的内皮细胞（CD45⁻CD31⁺CD326⁻）的RNAseq。