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Idiopathic pulmonary fibrosis (IPF) is a chronic disease with high mortality and is refractory to treatment. Pulmonary macrophages can both promote and repress fibrosis, however molecular mechanisms regulating macrophage functions during fibrosis remain poorly understood. FOXM1 is a transcription factor and is not expressed in quiescent lungs. Herein, we show that FOXM1 is highly expressed in pulmonary macrophages within fibrotic lungs of IPF patients and mouse fibrotic lungs. Macrophage-specific deletion of Foxm1 in mice (myFoxm1-/-) exacerbated pulmonary fibrosis. Inactivation of FOXM1 in vivo and in vitro increased p38 MAPK signaling in macrophages and decreased DUSP1, a negative regulator of p38 MAPK pathway. FOXM1 directly activated Dusp1 promoter. Overexpression of DUSP1 in FOXM1-deficient macrophages prevented activation of p38 MAPK pathway. Adoptive transfer of wild-type monocytes to myFoxm1-/- mice alleviated bleomycin-induced fibrosis. Altogether, contrary to known pro-fibrotic activities in lung epithelium and fibroblasts, FOXM1 has anti-fibrotic function in macrophages by regulating p38 MAPK.

Pulmonary endothelial progenitor cells (EPCs) are critical for neonatal lung angiogenesis and represent a subset of general capillary cells (gCAPs). Molecular mechanisms through which EPCs stimulate lung angiogenesis are unknown. Herein, we used single-cell RNA sequencing to identify the BMP9/ACVRL1/SMAD1 pathway signature in pulmonary EPCs. BMP9 receptor, ACVRL1, and its downstream target genes were inhibited in EPCs from Foxf1 WT/S52F mutant mice, a model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Expression of ACVRL1 and its targets were reduced in lungs of ACDMPV subjects. Inhibition of FOXF1 transcription factor reduced BMP9/ACVRL1 signaling and decreased angiogenesis in vitro. FOXF1 synergized with ETS transcription factor FLI1 to activate ACVRL1 promoter. Nanoparticle-mediated silencing of ACVRL1 in newborn mice decreased neonatal lung angiogenesis and alveolarization. Treatment with BMP9 restored lung angiogenesis and alveolarization in ACVRL1-deficient and Foxf1 WT/S52F mice. Altogether, EPCs promote neonatal lung angiogenesis and alveolarization through FOXF1-mediated activation of BMP9/ACVRL1 signaling. The molecular mechanisms through which pulmonary endothelial progenitor cells stimulate lung angiogenesis are not clear. Here, authors show that these cells stimulate the growth of alveolar capillaries and alveoli of newborn mice through FOXF1 and FLI1 nuclear protein-activation of the BMP9/ACVRL1/SMAD1 signaling pathway.

The liver possesses a high regenerative capacity. Liver regeneration is a compensatory response overcoming disturbances of whole-body homeostasis provoked by organ defects. Here we show that a vagus-macrophage-hepatocyte link regulates acute liver regeneration after liver injury and that this system is critical for promoting survival. Hepatic Foxm1 is rapidly upregulated after partial hepatectomy (PHx). Hepatic branch vagotomy (HV) suppresses this upregulation and hepatocyte proliferation, thereby increasing mortality. In addition, hepatic FoxM1 supplementation in vagotomized mice reverses the suppression of liver regeneration and blocks the increase in post-PHx mortality. Hepatic macrophage depletion suppresses both post-PHx Foxm1 upregulation and remnant liver regeneration, and increases mortality. Hepatic Il-6 rises rapidly after PHx and this is suppressed by HV, muscarinic blockade or resident macrophage depletion. Furthermore, IL-6 neutralization suppresses post-PHx Foxm1 upregulation and remnant liver regeneration. Collectively, vagal signal-mediated IL-6 production in hepatic macrophages upregulates hepatocyte FoxM1, leading to liver regeneration and assures survival. The mechanisms underlying the regenerative capacity of the liver are not fully understood. Here, the authors show that the acute regenerative response to liver injury in mice is regulated by the communication involving the vagus nerve, macrophages, and hepatocytes, leading to hepatic FoxM1 activation and promotion of overall survival.

Cleft palate is among the most common birth defects in humans. Previous studies have shown that Shh signaling plays critical roles in palate development and regulates expression of several members of the forkhead-box (Fox) family transcription factors, including Foxf1 and Foxf2, in the facial primordia. Although cleft palate has been reported in mice deficient in Foxf2, whether Foxf2 plays an intrinsic role in and how Foxf2 regulates palate development remain to be elucidated. Using Cre/loxP-mediated tissue-specific gene inactivation in mice, we show that Foxf2 is required in the neural crest-derived palatal mesenchyme for normal palatogenesis. We found that Foxf2 mutant embryos exhibit altered patterns of expression of Shh, Ptch1, and Shox2 in the developing palatal shelves. Through RNA-seq analysis, we identified over 150 genes whose expression was significantly up- or down-regulated in the palatal mesenchyme in Foxf2-/- mutant embryos in comparison with control littermates. Whole mount in situ hybridization analysis revealed that the Foxf2 mutant embryos exhibit strikingly corresponding patterns of ectopic Fgf18 expression in the palatal mesenchyme and concomitant loss of Shh expression in the palatal epithelium in specific subdomains of the palatal shelves that correlate with where Foxf2, but not Foxf1, is expressed during normal palatogenesis. Furthermore, tissue specific inactivation of both Foxf1 and Foxf2 in the early neural crest cells resulted in ectopic activation of Fgf18 expression throughout the palatal mesenchyme and dramatic loss of Shh expression throughout the palatal epithelium. Addition of exogenous Fgf18 protein to cultured palatal explants inhibited Shh expression in the palatal epithelium. Together, these data reveal a novel Shh-Foxf-Fgf18-Shh circuit in the palate development molecular network, in which Foxf1 and Foxf2 regulate palatal shelf growth downstream of Shh signaling, at least in part, by repressing Fgf18 expression in the palatal mesenchyme to ensure maintenance of Shh expression in the palatal epithelium.

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF. Pulmonary fibrosis results from dysregulated lung repair, but the role of endothelial cells (EC) in fibrosis is unclear. Here, the authors show that FOXF1/R-Ras signalling in EC inhibits profibrotic mediators and that ECspecific nanoparticle FOXF1 gene therapy decreases lung fibrosis in mice.

SAM-pointed domain-containing ETS transcription factor (SPDEF) is expressed in normal prostate epithelium. While its expression changes during prostate carcinogenesis (PCa), the role of SPDEF in prostate cancer remains controversial due to the lack of genetic mouse models. In present study, we generated transgenic mice with the loss- or gain-of-function of SPDEF in prostate epithelium to demonstrate that SPDEF functions as tumor suppressor in prostate cancer. Loss of SPDEF increased cancer progression and tumor cell proliferation, whereas over-expression of SPDEF in prostate epithelium inhibited carcinogenesis and reduced tumor cell proliferation in vivo and in vitro. Transgenic over-expression of SPDEF inhibited mRNA and protein levels of Foxm1, a transcription factor critical for tumor cell proliferation, and reduced expression of Foxm1 target genes, including Cdc25b, Cyclin B1, Cyclin A2, Plk-1, AuroraB, CKS1 and Topo2alpha. Deletion of SPDEF in transgenic mice and cultures prostate tumor cells increased expression of Foxm1 and its target genes. Furthermore, an inverse correlation between SPDEF and Foxm1 levels was found in human prostate cancers. The two-gene signature of low SPDEF and high FoxM1 predicted poor survival in prostate cancer patients. Mechanistically, SPDEF bound to, and inhibited transcriptional activity of Foxm1 promoter by interfering with the ability of Foxm1 to activate its own promoter through auto-regulatory site located in the -745/-660 bp Foxm1 promoter region. Re-expression of Foxm1 restored cellular proliferation in the SPDEF-positive cancer cells and rescued progression of SPDEF-positive tumors in mouse prostates. Altogether, SPDEF inhibits prostate carcinogenesis by preventing Foxm1-regulated proliferation of prostate tumor cells. The present study identified novel crosstalk between SPDEF tumor suppressor and Foxm1 oncogene and demonstrated that this crosstalk is required for tumor cell proliferation during progression of prostate cancer in vivo.

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro‐inflammatory mediators and undergoing epithelial‐to‐mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation‐induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation‐induced pneumonitis and pulmonary fibrosis, and increased the expression of IL‐1 β, Ccl2 , Cxcl5 , Snail1 , Zeb1 , Zeb2 and Foxf1 . Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation‐induced pulmonary fibrosis and prevented the increase in EMT‐associated gene expression. siRNA‐mediated inhibition of Foxm1 prevented TGF‐β‐induced EMT in vitro . Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF‐β‐induced loss of E‐cadherin in Foxm1‐deficient cells in vitro . Lineage‐tracing studies demonstrated that Foxm1 increased EMT during radiation‐induced pulmonary fibrosis in vivo . Foxm1 is required for radiation‐induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT. This study establishes the in vivo relevance of FoxM1 in the context of radiation‐induced fibrosis. FoxM1 ablation in the respiratory epithelium supports a regulatory role during EMT and pulmonary inflammation that could become of therapeutic relevance.

Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease. Mutations in FOXF1 , a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins. Here, the authors discovered four genomic regions that control cell type-specific activity of Foxf1 during lung development and show that disrupting these regions via genetic deletions leads to alveolar capillary dysplasia.

Lung cancer remains one of the most prominent public health challenges, accounting for the highest incidence and mortality among all human cancers. While pulmonary invasive mucinous adenocarcinoma (PIMA) is one of the most aggressive types of non-small cell lung cancer, transcriptional drivers of PIMA remain poorly understood. In the present study, we found that Forkhead box M1 transcription factor (FOXM1) is highly expressed in human PIMAs and associated with increased extracellular mucin deposition and the loss of NKX2.1. To examine consequences of FOXM1 expression in tumor cells in vivo, we employed an inducible, transgenic mouse model to express an activated FOXM1 transcript in urethane-induced benign lung adenomas. FOXM1 accelerated tumor growth, induced progression from benign adenomas to invasive, metastatic adenocarcinomas, and induced SOX2, a marker of poorly differentiated tumor cells. Adenocarcinomas in FOXM1 transgenic mice expressed increased MUC5B and MUC5AC, and reduced NKX2.1, which are characteristics of mucinous adenocarcinomas. Expression of FOXM1 in KrasG12D transgenic mice increased the mucinous phenotype in KrasG12D-driven lung tumors. Anterior Gradient 2 (AGR2), an oncogene critical for intracellular processing and packaging of mucins, was increased in mouse and human PIMAs and was associated with FOXM1. FOXM1 directly bound to and transcriptionally activated human AGR2 gene promoter via the -257/-247 bp region. Finally, using orthotopic xenografts we demonstrated that inhibition of either FOXM1 or AGR2 in human PIMAs inhibited mucinous characteristics, and reduced tumor growth and invasion. Altogether, FOXM1 is necessary and sufficient to induce mucinous phenotypes in lung tumor cells in vivo.

Under insulin-resistant conditions such as obesity, pancreatic β-cells proliferate to prevent blood glucose elevations. A liver–brain–pancreas neuronal relay plays an important role in this process. Here, we show the molecular mechanism underlying this compensatory β-cell proliferation. We identify FoxM1 activation in islets from neuronal relay-stimulated mice. Blockade of this relay, including vagotomy, inhibits obesity-induced activation of the β-cell FoxM1 pathway and suppresses β-cell expansion. Inducible β-cell-specific FoxM1 deficiency also blocks compensatory β-cell proliferation. In isolated islets, carbachol and PACAP/VIP synergistically promote β-cell proliferation through a FoxM1-dependent mechanism. These findings indicate that vagal nerves that release several neurotransmitters may allow simultaneous activation of multiple pathways in β-cells selectively, thereby efficiently promoting β-cell proliferation and maintaining glucose homeostasis during obesity development. This neuronal signal-mediated mechanism holds potential for developing novel approaches to regenerating pancreatic β-cells. Neuronal signals, in particular those transmitted via the vagal nerve, regulate both β-cell function and proliferation. Here, Yamamoto et al. show that the forkhead box M1 pathway is required for vagal signal-mediated induction of β-cell proliferation during obesity.