Loss of FOXF1 expression promotes human lung-resident mesenchymal stromal cell migration via ATX/LPA/LPA1 signaling axis

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.

Prostaglandin-endoperoxide synthase 2 COX2 Cyclooxygenase isoform 2 PGE2 Prostaglandin E2 NFAT1 Nuclear factor of activated T cells 2 HOXA13 Homeobox A13 CDH11 Cadherin 11 or osteoblast-cadherin CXCL10 C-X-C Motif chemokine ligand 10 CXCL11 C-X-C Motif chemokine ligand 11 Mesenchymal cells are an important component of cellular niches in adult organs and are being increasingly recognized to display tissue-specific transcriptome and functions. We have previously demonstrated that human adult lung contains a population of resident, long-lived mesenchymal stromal cells (MSC) with clonal multilineage differentiation potential 1 . MSCs derived from adult lungs exhibit unique mesenchymal transcriptional signature suggesting their lung-specificity and origin from embryonic lung mesenchyme 1,2 . Expansion and mobilization of lung-resident mesenchymal stem cells (LR-MSCs) is noted during conditions of lung injury and fibrosis 2,3 , and the lipid mediator lysophosphatidic acid (LPA) has been identified as a key inducer of LR-MSC migration 4 . LR-MSCs can regulate LPA expression in an autocrine manner by secretion of Autotaxin (ATX), a lysophospholipase D that enzymatically produces LPA from extracellular lysophosphatidylcholine 5 . 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 signaling 5 . While these data shed light on mechanisms of MSC mobilization and activation, transcriptional regulation of MSCs under homeostatic conditions remain to be identified. The Forkhead Box (Fox) family of transcription factors is a group of proteins that share a common DNA binding domain termed a winged-helix or forkhead domain, with FOXF1 being a mesenchyme-specific, putative transcription factor 6,7 , which plays a critical role in lung development. In mice, FOXF1 expression is noted in the lateral mesenchyme at embryonic day 9.5 and its deficiency is associated with defects in branching morphogenesis of the lung 8,9 . 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 LR-MSCs derived from human adult lungs express ~ 35,000-fold higher FOXF1 mRNA than bone-marrow MSCs 5 . However, the significance of FOXF1 expression in adult human LR-MSCs remains to be elucidated.
In this study, we investigated the mechanistic role of FOXF1 in regulating the biologic functions of lungresident mesenchymal stem cells. Our investigations identify FOXF1 as a transcriptional repressor, with loss of FOXF1 promoting an activated mesenchymal phenotype accompanied by intense mitogenic activity, higher rates of cellular migration, proliferation, and the secretion of pro-inflammatory chemokines and cytokines. Importantly, our data demonstrates that FOXF1 regulates the migration of LR-MSCs via its novel role as a transcriptional repressor of ATX.

Materials and methods
Human subjects and ethics statement. Informed consent was obtained directly from all human subjects after a full explanation of the study objectives and procedures. The study was carried out in accordance with relevant guidelines and regulations using a protocol for human studies approved by the University of Michigan Institutional Review Board (approval number HUM00042443) and was in compliance with the Helsinki Declaration.
Isolation and culture conditions of LR-MSCs. LR-MSCs were isolated and characterized as previously described from bronchoalveolar lavage derived from lung transplant recipients with or without chronic lung allograft rejection [1][2][3][4][5]10,11 . LR-MSCs cultured in high-glucose DMEM (11965-118, Gibco) supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, and 0.5% fungizone were utilized at passages 3-6. All methods were carried out in accordance with relevant guidelines and regulations. RNA interference. At 60-70% confluence, LR-MSCs were transfected with 100 nM FOXF1-specific (Stealth RNAi HSS142031, Invitrogen) or scrambled control siRNA (sc-37007, Santa Cruz), using Oligofectamine (12252-011, Invitrogen) and Opti-MEM I reduced serum medium. For double gene silencing, LR-MSCs were transfected with FOXF1-specific siRNA or scrambled control (100 nM each). 24 h later, LR-MSCs were transfected again with 100 nM of ATX-specific siRNA or scrambled control, incubated overnight, and then maintained in serum-free DMEM. Cells were subsequently assayed for migration rates after 48 h. RNA and protein lysates were harvested after 72 h for real-time PCR and immunoblotting analysis. Lentiviral transduction of LPAR1 short hairpin RNA. For lentivirus transduction, LR-MSCs were transfected with FOXF1-specific siRNA or scrambled control (100 nM each) and incubated for 48 h. Subsequently, the cells were infected with lentiviral vectors that contain either a control transduction particles-shRNA (Mission pLKO.1-puro, Sigma) or LPAR1-specific transduction particles-shRNA (Mission: Clone #: NM_057179, Sigma) in serum-free growth medium with 2.5 multiplicities of infection (MOI) using protamine sulphate as linker. After incubating for a period of 48 h, the cells were subsequently assayed for migration rates.
Proliferation assay. LR-MSCs were transfected with FOXF1 siRNA or scrambled control (100 nM each), and 24 h post-transfection, the mesenchymal cells were trypsinized and seeded at 5000 cells/well in 96 well plates. Cells were cultured in medium for 72 h and assayed with a CyQUANT NF Cell Proliferation Assay Kit (C35006, ThermoFisher Scientific) as per manufacturer´s instructions.
Protein measurement in cell supernatant. Mesenchymal cells were transfected with FOXF1-specific or the scrambled siRNA in Opti-MEM I reduced serum medium. After overnight incubation, media was exchanged for serum-free DMEM for 48 h and the conditioned media was measured for CCL5 and CCL7 by ELISA according to the manufacturer's protocols: R&D systems (Minneapolis, MN): Human CCL5/RANTES Quantikine ELISA Kit (DRN00B), Human CCL7/MCP-3 Quantikine ELISA Kit (DCC700). Absorbance was read with a SpectraMax M3 multi-mode microplate reader (Molecular Devices).
Migration assay. Migration rates of LR-MSCs was measured in matrigel-coated transwells as previously described 4 . Briefly, transwells were freshly coated with matrigel overnight at 37 °C. LR-MSCs were transfected Luciferase reporter assay. To examine the transcriptional functionality of 3 potential binding sites of Statistical analysis. All data are presented as Means ± SEM. Statistical significance was assessed with Student's paired two-tailed t test for comparing scrambled and FOXF1-silenced groups, or with one-way ANOVA and a post hoc Bonferroni test for 3 or more groups, unless specified otherwise using GraphPad Prism 8 software (La Jolla, CA). p < 0.05 was considered significant.

Loss of FOXF1 induces migration and the expression and activity of ATX in human LR-MSCs.
In order to ascertain the role of FOXF1 in cellular migration, a functional in vitro assay using a modified Boyden chamber was utilized. Comparision of LR-MSCs transfected with scrambled control or FOXF1-specific siRNA demonstrated an approximate 2.5-fold increase in cell migration following FOXF1-silencing, suggesting that the loss of FOXF1 imparts LR-MSCs with a robust migratory phenotype ( Fig. 2A,B). ATX-LPA axis is key regulator of cellular migration and Autotaxin-encoding gene-ENPP2 was noted to be among the top ten upregulated genes in FOXF1-silenced cells (Fig. 1I). Increased ATX expression in response to FOXF1-silencing was confirmed at mRNA and protein level by real-time PCR and western blotting respectively (Fig. 2C,D). Furthermore, supernatant from FOXF1-silenced LR-MSCs demonstrated significantly higher ATX activity utilizing a fluorimetric substrate, FS-3, compared to scrambled siRNA controls (Fig. 2E). We next overexpressed mFoxf1 in LR-MSCs and assessed ATX protein levels and activity by immunoblotting and fluorimetry, respectively. Efficacy of mFoxf1 overexpression was confirmed by immunoblotting for HA as shown in Fig. 2F. A 40% decrease in ATX protein expression was noted in LR-MSCs overexpressing mFoxf1 (Fig. 2F), with a concordant decrease in ATX activity compared to control vector (Fig. 2G). Together these findings demonstrated that decreased FOXF1 leads to increased LR-MSC migration and ATX secretion and activity.
ATX/LPA/LPA1 signaling axis mediates increased migration rates in FOXF1-silenced LR-MSCs. To further ascertain if increased ATX expression mediates the increase in migration induced by loss of FOXF1, LR-MSCs transfected with FOXF1 siRNA were subjected to subsequent transfection with siRNA specific to ATX or scrambled control (Fig. 3A). Migration was compared between LR-MSCs silenced for FOXF1 alone or in combination with ATX silencing in the modified Boyden chamber migration assay (Fig. 3B). Increased migration noted in response to FOXF1-silencing was abrogated in LR-MSCs subjected to gene silencing for both FOXF1 and ENPP2 (ATX) (Fig. 3C). That ATX is a key factor in mediating the pro-migratory effect of FOXF1 inhibition was further confirmed by using PF8380, a specific pharmacologic inhibitor of ATX. FOXF1-silenced LR-MSCs treated with PF8380 demonstrated significant reduction in migration rates with levels comparable to scrambled control siRNA transfected LR-MSCs (Fig. 3D). ATX regulates cellular migration via its generation of lipid mediator LPA and downstream LPA receptor signaling. We have previously shown that LR-MSCs predominantly express LPA receptor isoform 1 (LPA1) and that migration of LR-MSCs in response to LPA is mediated via LPA1 receptor ligation 4 . To study the pharmacologic blockade of LPA signaling on migration of FOXF1-silenced LR-MSCs, we utilized VPC12249, an LPA1-specific antagonist. FOXF1-silenced LR-MSCs demonstrated a two-fold higher migration, which was significantly diminished by the pharmacologic blockade of LPA1 (Fig. 3E). Furthermore, lentiviral gene silencing of the LPA receptor-LPAR1, using short hairpin RNA (shRNA), was adopted as a complementary approach to determine the effects of LPA signaling on migration. FOXF1-silenced LR-MSCs demonstrated 1.5-fold higher migration, which was significantly mitigated by shRNA-mediated lentiviral repression of LPAR1 gene expression (Fig. 3F). Together, these data demonstrate that loss of FOXF1 promotes LR-MSC migration via ATX/LPA/ LPA1 signaling pathway.
Identification of novel FOXF1 binding sites in the − 1.5 kb upstream region of the ATX promoter (− 1217/− 1127/− 458). Silencing of FOXF1 in LR-MSCs resulted in a robust induction of ATX at mRNA level, so we next sought to investigate whether FOXF1 directly binds to regions of the ATX promoter to influence transcription. Utilizing JASPAR promoter analysis (www.jaspa r.gener eg.net), we observed three putative FOXF1/2 binding sites (RTA AAY A) 21 in the − 1.5 kb upstream region of ATX promoter (Fig. 4A). To study the function of these three potential binding sites, we constructed luc2 luciferase expressing vectors driven by full length or truncated ATX promoters on the pGAL4.23 backbone (Fig. 4B). These reporter constructs were www.nature.com/scientificreports/ co-transfected with control Renilla vector pRL-TK into LR-MSCs and the luciferase activity was measured. As shown in Fig. 4B, deletion of the two most upstream putative FOXF1 binding sites (sites 1 and 2) increased luciferase expression two and fourfold, respectively. Promoter truncation omitting all 3 putative FOXF1 binding sites resulted in a sixfold induction of ATX transcription, suggesting a role for all three binding sites in repressing ATX transcription. Next, we utilized chromatin immunoprecipitation (ChIP) analysis to investigate if FOXF1 can bind these putative sites in the ATX promoter. FOXF1 antibody was used to pull down the FOXF1/ chromosome complexes with goat IgG as the negative control. ChIP data demonstrated an over 20-fold increase in FOXF1 binding at all three sites of the ATX promoter compared to IgG control (Fig. 4C). Collectively, these results suggest that FOXF1 transcriptionally represses expression of ATX by directly binding to regions of the ATX promoter. www.nature.com/scientificreports/

FOXF1-silencing induces proliferation and inflammatory responses in LR-MSCs.
We also further investigated other biological functions which were identified to be significantly altered by FOXF1 silencing by affymetrix analyses (Table 2). Quantitative assessment of cellular proliferation by CyQUANT NF cell proliferation assay demonstrated approximately 75% higher proliferation in FOXF1-silenced LR-MSCs compared to that of the scrambled siRNA control (Fig. 5A). Real-time PCR analyses confirmed upregulation of genes involved in cell cycle progression upon FOXF1 silencing, such as cyclin D1 (CCND1), cyclin B1 (CCNB1), cyclin-dependent kinase 1 (CDK1) and phosphoprotein enriched in astrocytes 15 (PEA15) (Fig. 5B). Additionally, FOXF1-silencing in LR-MSCs demonstrated upregulation of proteins marking proliferation and cell cycle progression such as proliferating cell nuclear antigen (PCNA), phosphorylated histone H3 (Ser 10) and cyclin D1 (Fig. 5C,D). The gene expression pattern demonstrating increased pro-inflammatory cytokines noted in FOXF1-silenced LR-MSCs by Affymetrix analyses was also further confirmed by real-time PCR. An approximately 100-fold increase in the gene expression of CCL5 and tenfold increase in the gene expression of CCL7, and a 150-fold increase in the gene expressions of CXCL10 and CXCL11 was found in FOXF1-silenced LR-MSCs relative to scrambled siRNA control (Fig. 5E). FOXF1-silencing induced increase in cytokine secretion by LR-MSCs was documented by ELISA where higher levels of CCL5 and CCL7 were noted in conditioned media collected from FOXF1-silenced LR-MSCs compared to the respective scrambled controls (Fig. 5F). Real-time PCR also confirmed that loss of FOXF1 induced expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2 or COX2) a key enzyme in prostaglandin biosynthesis (Fig. 5G). Here, we identify a role for transcription factor forkhead protein FOXF1 as a master repressor of key cellular functions in human LR-MSCs. FOXF1 silencing was noted to promote proliferation, migration, and secretory function of LR-MSCs. Furthermore, FOXF1 was identified as a novel transcriptional repressor of ATX, a key enzyme largely responsible for the synthesis of extracellular pro-fibrotic mediator, LPA. Increased ATX secretion followed by subsequent LPA synthesis and autocrine LPA1 signaling, mediated LR-MSC migration in response to decreased FOXF1 expression. Together, these data shed light on novel restraining mechanisms in mesenchymal cells which limit their activation in homeostatic conditions. These findings have significant relevance to understanding both adaptive and mal-adaptive reparative processes in the lung. Our studies provide first evidence for the role of FOXF1 as a transcriptional repressor of key enzyme ATX in human LR-MSCs. ATX, a secreted glycoprotein from the family of ectonucleotide pyrophosphatases/phosphodiesterases, is essential for development and is implicated in a variety of physiologic and pathologic processes 23 . ATX produces majority of the extracellular LPA and the ATX/LPA/LPA1 signaling axis has been shown to play a key role in fibrosis, inflammation, and cancer across various organs 5,[24][25][26][27][28][29][30][31] . ATX-LPA signaling is implicated in fibrotic diseases of the lung 5,30,32 , and we have demonstrated stable increased expression of ATX in mesenchymal cells derived from fibrotic lung allografts 5 . In these studies, ATX mRNA expression was noted to be regulated by nuclear factor of activated T cells 2 (NFAT1). NFAT1 is a known enhancer of ATX transcription with NFAT binding sites described in the ATX promoter region in breast cancer cells 33 . Other transcription factors such as HOXA13, v-JUN, NF-κB and Stat3 have also been identified as transcriptional activators of ATX in various murine and human cellular conditions [33][34][35][36] , however, no ATX repressor has been reported to date. ATX as a  www.nature.com/scientificreports/ target of FOXF1 was identified by global affymetrix analysis where ENPP2 was among the top differentially expressed genes in FOXF1-silenced LR-MSCs. We utilized both FOXF1 silencing and overexpression strategies to confirm regulation of ATX by FOXF1 in LR-MSCs. Silencing of FOXF1 resulted in robust increases in ATX at the transcriptional level as well as increased ATX expression and function-as indicated by increased ATX mRNA, protein, and activity. FOXF1 overexpression was associated with reduced ATX expression at both the RNA and protein level. We identified, previously uncharacterized, three putative FOXF1 binding sites on the ATX promoter. That FOXF1 binds to and is a repressor of the ATX gene ENPP2 was confirmed by its direct binding to the ATX promoter using ChIP analysis. Increases in ATX transcription was noted in luciferase assays with subsequent promoter truncations. Future studies will focus on identifying the exact binding site. Previous studies in NIH3T3 cells have identified FOXF1 as a repressor of the CDH11 gene 37 and other members of the FOX family such as FOXP1 and FOXP2 which are expressed in the lung epithelium have also been characterized as transcriptional repressors 38 .
A key finding of our work is recognition of the role of transcriptional factor FOXF1 as a inhibitory regulator of LR-MSC migration. Downregulation of FOXF1 resulted in a robust migratory phenotype in LR-MSCs which was found to be dependent on ATX secretion and downstream LPA/LPA1 signaling. Mesenchymal cell migration is a key feature of its activated state and its positive regulation by growth factors and biological mediators is well studied in context of tissue repair and fibrosis. However, the fundamental question of what prevents activation of mesenchymal cell migration in a quiescent condition has not been previously explored. Our data demonstrating FOXF1 as a transcriptional repressor of ATX and its loss promoting ATX/LPA/LPA1 signaling axis mediated migration suggests that FOXF1 expression could be critical brake on cellular migration in homeostatic conditions by keeping autocrine ATX expression in check. Loss of FOXF1 has been linked to increased invasiness of hepatocellular cancer cells 39 . FOXF1 has also been identified as a target of p53 in a separate study of human cancer cell lines, with its ectopic expression inhibiting cancer cell invasion and migration and its inactivation of FOXF1 stimulating cell invasion and migration 40 .
MSCs are key components of cellular niches, and regulate biologic processes via their paracrine actions and locally generated ATX has been demonstrated to be important in cellular interactions within tissue microenvironment 41 . Further evidence for the role of FOXF1 in regulating the secretome of the LR-MSCs was provided by affymetrix analysis where a significant change in the cytokine transcriptome was noted with marked upregulation of key chemokines such as CCL5, CCL7, CXCL10 and CXCL11. PTGS2, the enzyme that regulates prostanoid synthesis, was also significantly upregulated in FOXF1-silenced LR-MSCs. This suggests that FOXF1 regulates multiple downstream pathways in human LR-MSCs, the mechanism of which remains to be elucidated. Future studies are needed to identify other transcriptional targets of FOXF1 in LR-MSCs.
Among top upregulated biological processes identified in FOXF1-silenced LR-MSCs by GO analysis were positive regulation of cell proliferation. Mesenchymal cells within the lungs have relatively low turnover 42 , but our previous longitudinal studies of human lung allografts have provided clues regarding conditions associated with LR-MSC proliferation and mobilization 3 . An increase in LR-MSC numbers were noted early post-transplant during an active repair phase and later post-transplant preceding development of allograft fibrosis 3 . Both these conditions are marked by significant epithelial injury and FOXF1 plays a key role in mesenchymal-epithelial interactions during lung development 43 . FOXF1 is a Shh target gene and loss of Shh signaling has been implicated in mesenchymal cell proliferation in murine models 44,45 . Our finding that loss of FOXF1 promotes cellular proliferation suggests that FOXF1 could be a key intermediary for the actions of Shh. That loss of FOXF1 can promote mesenchymal cell activation and contribute to fibrosis is suggested in studies of transgenic mice with myofibroblast-specific deletion of Foxf1, where worse fibrotic remodeling was noted in response to bleomycin 37 . Further investigations are needed to shed more light on the regulation of this novel regulatory mechanism of mesenchymal cell activation in normal reparative and aberrant fibrotic responses within tissue niches in a human lung.
In conclusion, our study elucidates a critical mechanistic role of transcription factor FOXF1 that acts as a master regulator of cellular functions and paracrine actions of resident MSCs in human adult lungs. Furthermore, these studies are novel in their elucidation of the first transcriptional repressor of ATX in any cell type, a finding that has significant implication across various organs and diseases.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.