Abstract
Forkhead box F1 (FOXF1) is a lung embryonic mesenchyme-associated transcription factor that demonstrates persistent expression into adulthood in mesenchymal stromal cells. However, its biologic function in human adult lung-resident mesenchymal stromal cells (LR-MSCs) remain to be elucidated. Here, we demonstrate that FOXF1 expression acts as a restraint on the migratory function of LR-MSCs via its role as a novel transcriptional repressor of autocrine motility-stimulating factor Autotaxin (ATX). Fibrotic human LR-MSCs demonstrated lower expression of FOXF1 mRNA and protein, compared to non-fibrotic controls. RNAi-mediated FOXF1 silencing in LR-MSCs was associated with upregulation of key genes regulating proliferation, migration, and inflammatory responses and significantly higher migration were confirmed in FOXF1-silenced LR-MSCs by Boyden chamber. ATX is a secreted lysophospholipase D largely responsible for extracellular lysophosphatidic acid (LPA) production, and was among the top ten upregulated genes upon Affymetrix analysis. FOXF1-silenced LR-MSCs demonstrated increased ATX activity, while mFoxf1 overexpression diminished ATX expression and activity. The FOXF1 silencing-induced increase in LR-MSC migration was abrogated by genetic and pharmacologic targeting of ATX and LPA1 receptor. Chromatin immunoprecipitation analyses identified three putative FOXF1 binding sites in the 1.5 kb ATX promoter which demonstrated transcriptional repression of ATX expression. Together these findings identify FOXF1 as a novel transcriptional repressor of ATX and demonstrate that loss of FOXF1 promotes LR-MSC migration via the ATX/LPA/LPA1 signaling axis.
Highlights
Abbreviations Forkhead box F1 (FOXF1) Forkhead box isoform 1 ATX Autotaxin ENPP2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 lysophosphatidic acid (LPA) Lysophosphatidic acid LPA1 LPA receptor isoform 1 lung-resident mesenchymal stem cells (LR-mesenchymal stromal cells (MSC)) Lung-resident mesenchymal cells mFoxf[1] Murine FOXF1 overexpressing vector multiplicities of infection (MOI) Multiplicities of infection DMEM Dulbecco’s minimum essential medium FBS Fetal bovine serum proliferating cell nuclear antigen (PCNA) Proliferating cell nuclear antigen HRP Horseradish peroxidase ChIP Chromatin immunoprecipitation CCL5/RANTES Chemokine ligand 5 CCL7/MCP-3 Chemokine ligand 7 STRING Search tool for the retrieval of interacting genes/proteins GO Gene ontology
We have recently demonstrated that ATX expression is upregulated in lung-resident mesenchymal stem cells (LR-MSCs) derived from diseased lungs and can drive β-catenin activation via downstream LPA/LPA1 s ignaling[5]
FOXF1 is unique among the embryonic lung mesenchyme-associated transcription factors in that it demonstrates persistent expression in the mesenchymal cells of an adult lung, and we have reported that LRMSCs derived from human adult lungs express ~ 35,000-fold higher FOXF1 mRNA than bone-marrow M SCs5
Summary
Abbreviations FOXF1 Forkhead box isoform 1 ATX Autotaxin ENPP2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 (encodes for ATX) LPA Lysophosphatidic acid LPA1 LPA receptor isoform 1 LR-MSCs Lung-resident mesenchymal cells mFoxf[1] Murine FOXF1 overexpressing vector MOI Multiplicities of infection DMEM Dulbecco’s minimum essential medium FBS Fetal bovine serum PCNA Proliferating cell nuclear antigen HRP Horseradish peroxidase ChIP Chromatin immunoprecipitation CCL5/RANTES Chemokine ligand 5 CCL7/MCP-3 Chemokine ligand 7 STRING Search tool for the retrieval of interacting genes/proteins GO Gene ontology. We have recently demonstrated that ATX expression is upregulated in LR-MSCs derived from diseased lungs and can drive β-catenin activation via downstream LPA/LPA1 s ignaling[5]. While these data shed light on mechanisms of MSC mobilization and activation, transcriptional regulation of MSCs under homeostatic conditions remain to be identified. FOXF1 is unique among the embryonic lung mesenchyme-associated transcription factors in that it demonstrates persistent expression in the mesenchymal cells of an adult lung, and we have reported that LRMSCs derived from human adult lungs express ~ 35,000-fold higher FOXF1 mRNA than bone-marrow M SCs5. The significance of FOXF1 expression in adult human LR-MSCs remains to be elucidated
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