The forkhead box transcription factor-2 ( Foxa 2 ) and lung disease

Foxa2 [The fork head box transcription factor-2; also known as HNF3B (hepatocyte nuclear factors-3), or TCF3B (transcription factor-3b)], is a member of the Fork head family of nuclear transcription factors that is highly expressed in respiratory epithelial cells lining conducting airways and in alveolar type II cells in the development and mature lung. Foxa2 plays critical roles in lung development, which is required for surfactant protein and lipid synthesis, normal lung maturation, epithelial differentiation, and cross talking between epithelium and innate immune. Foxa2 can determine gene expression and cell fate in the lung, and is required for the suppression of Th2 immunity, mucus production, and goblet cell metaplasia in the developing lung in a process determined in part by its regulation of the Cysteinyl leukotriene (CysLT) pathway. Recent studies have implicated that Foxa2 is a suppressor of lung cancer. This review will summarize the role of Foxa2 in lung development, pulmonary diseases, and also the possible signaling pathways regulated by Foxa2.


FOXA2
Foxa2 is a member of the forkhead nuclear transcription factors family that contains a modified helix-turn-helix, known as the winged helix DNA binding domain [1] and a unique AKT2/PKB phosphorylation site at the N terminus of the forkhead domain [2,3] .Foxa2 from human, located on chromosome 20p11.21,was first identified as a liver-enriched transcription factor regulating liver-specific gene expression [4,5] .Expression of Foxa2 is restricted mainly to endoderm derived tissues including lung, liver, stomach, small intestine and is essential participants in liver and lung specific gene transcription [6,[8][9][10] .Interestingly, Foxa2 mRNA was also found in notochord cells and ventral neural epithelium, indicating additional functions for Foxa2 in mesoderm and neural axis formation [11] .In the mice, Foxa2 gene is small and contains only three exons with conserved intro-exon boundaries [12] .The full length of Foxa2 cDNA from mouse was shown to be 2.3kb in size and comprises 459 amino acids with a molecular mass of 48.5 kDa [12][13][14] .The amino acid sequence of mouse and rat Foxa2 is highly conserved.The proximal promoter of the mouse Foxa2 gene is remarkably well similarity in comparison with that of rat, with 116 of the 120 first nucleotides being identical [12] .
The Foxa proteins including Foxa1, Foxa2, Foxa3 (previously termed HNF-3α, -3β, and -3γ respectively), were first identified by their ability to bind to important promoter REVIEW elements in the α1-antitrypsin and transthyretin genes [5] .The Foxa proteins share strong homology ( 95% identical ) in their DNA binding domains and possess conserved residues at the carboxyl terminus (regions II and III) and the amino terminus (region IV) [15,16] .Three proteins bind to DNA in the form of a monomer through the winged helix motif, which is also utilized by a number of developmental regulators, such as the Drosophila homeotic forkhead (fkh) protein.Fkh shares striking similarities within the Foxa2 DNA binding domain, with 100 of 110 amino acids are identical [17,18] .Foxa2 protein binds to distinct DNA sites and its specificity in protein recognition is determined by a 20-amino-acid region, located adjacent to the DNA recognition helix, which alteration in the site contributes to DNA-binding specificity [1] .Foxa2 carboxyl terminus, which includes conserved regions II and III, will function as a position-independent transcriptional activation domain [19,20] .Foxa2 amino terminus, region IV, a second HNF-3β activation domain [21] , is composed of 52 amino acids which enriches serine and tyrosine residues [22] and contains two putative casein kinase I (CKI) phosphorylation sites [23] , which was shown to be important for transcriptional activation (Fig. 1).The activity of Foxa2 amino terminus requires the participation of carboxyl terminus regions II and III domain to activate transcription, thus suggesting an interaction between these activation domains [21] .

FOXA2 AND LUNG DEVELOPMENT
The transcription factors, including thyroid transcription factor (TTF)-1, GATA binding protein (GATA)-6, Sam Pointed Domain Ets-like Factor 9 (SPDEF ) and FOXA2, play important roles in directing respiratory epithelial cell fate during lung development and in diseases.They are expressed in a regional and cell-selective manner in the mature lung where they interact in a complex transcriptional network that regulates airway epithelial cell differentiation and function in the mature lung [24] .

Foxa2 expression in development
Foxa2 is the first gene of the family to be activated during embryogenesis.In the mouse, expression of Foxa2 is detected in the anterior primitive streak and the node at embryonic day 6.5 (E6.5) [11,12,15,25] , shortly after found in the onset of gastrulation [26] .Thereafter, expression of Foxa2 in the notochord, gut endoderm and ventral midline of the central nervous system (CNS) is observed at E7.5 [27] , strongly on Days 9.5 and 10.5 [3,7,28] , weakly detected during the next 2 days, and returns to an intermediate level of expression on Day 15.5 [11] .Expression of Foxa2 was analyzed in conducting and alveolar epithelial cells at embryonic days 12.5 [29] .Later in the endoderm during embryonic development, expression of Foxa2 gives rise to the lung buds, where they continue to be expressed in the conducting and pulmonary epithelium to adulthood [6,30,31] .The expression of the transcription factors SOX (SRY-related HMG-box)-17 and FOXA2 has been used to as a marker of formation of "definitive endoderm" [24] .The temporal-spatial distribution of Foxa2 restricted to respiratory epithelial cells is regulated precisely during lung development [32] .
Foxa2 was confined to bronchiolo-alveolar portals and Type II cells in human nonfetal lung, which also has critical role in human lung diseases.Assessed the temporal-spatial expression of Foxa2 in human infants with BPD found that Foxa2 was abundantly increased in regenerating epithelial cells at the periphery of lung lobules [32] .Foxa2 plays a critical role in regulating epithelial cell differentiation in the process of repair of the postnatal lung.

Foxa2 is required during lung development
Foxa2 is required during early mouse development.The role of Foxa2 in lung development was evaluated by genetic means in mice.In the early stage, using Foxa2 +/-mice model found that adiposity on a high-fat diet was increased and adipocyte glucose uptake and glycolysis were correspondingly decreased, indicating that Foxa2 plays an important role in lipid and glucose metabolism [33] .Thereafter, Mouse embryos with null mutation of Foxa2 die by E10-E11 and exhibit severe defects in structures related to all three germ layers before the onset of lung morphogenesis, including absence of the notochord, abnormalities of the neural tube and somites, and failure to form the gut tube, although endoderm cells are present [34][35][36] .In the early pseudo glandular stage, abnormal expression of Foxa2 in distal respiratory epithelial cells caused disruption of  [15] .Foxa2 possess three transactivations: regions II (372-385) and III (445-456) at the carboxyl terminus and the region IV (1-52) at amino terminus respectively.Region IV contains two putative casein kinase I (CKI) phosphorylation sites, which was critical for transcriptional activation.Foxa2 contains a DNA binding domain, which is located in region I (157-257), its specificity of protein recognition is dependent on a 20-amino-acid region, a conserved residues located adjacent to region I.
branching morphogenesis and blocks differentiation of distal peripheral epithelial cells differentiation [31,37] , supporting an essential role of precise regulation of Foxa2 expression in the lung development.Differentiation of respiratory epithelial cells is required for normal expression of Foxa2 in the respiratory epithelium.In recent study, using a conditional Cre/loxP recombination system to specifically delete Foxa2 in respiratory epithelial cells during lung development demonstrated that peripheral lung saccules and alveolar decreased at PN3 and focal neutrophilic infiltrations, goblet-cell hyperplasia and extensive airspace enlargement were observed at PN16 and later, although lung morphology was not perturbed at E16.5-18.5 in Foxa2 ∆/∆ pups [29] , supporting that Foxa2 is an indispensable transcription factor in the mouse embryonic lung development and is necessary for normal alveolarization and postnatal lung homeostasis, but is not required for branching morphogenesis.In addition, specific loss of Foxa2 in the epithelial cells of the developing mouse lung suppressed the differentiation of respiratory epithelial cells and caused neonatal respiratory failure with the feature of Acute Respiratory Distress Syndrome (ARDS) in preterm infants [38] , suggesting that cellular diversity of developing respiratory epithelium and lung morphogenesis are influenced by Foxa2.To sum up, the precise temporal-spatial pattern of Foxa2 expression is essential to respiratory epithelial cell differentiation and lung development.

Foxa2 interacted with other genes in regulating lung development
Functional analyses of several lung-specific genes verified the most important function of Foxa2 in lung development through regulating the transcription of genes that influence lung morphogenesis and homeostasis, normal lung maturation, surfactant protein, lipid synthesis, epithelial differentiation, and a cross talking between epithelium and innate immune.
Foxa2 regulates perinatal lung maturation through interaction with other transcription factor in a network.It has been supposed to that TTF-1, Foxa1, Foxa2, and GATA6 interact with each other and have essential roles in the morphogenesis of lung epithelial cells and the expression of significant molecules of surfactant proteins [28,39] .Foxa2, TTF-1, and C/EBPα (CCAAT enhancer binding protein α) reciprocally interact to regulate transcriptional targets of surfactant synthesis and peripheral lung maturation [40] .TTF-1 and Foxa2 are expressed in conducting and alveolar epithelial cells, where they are required for normal cell differentiation [38,39,41] .Foxa2 binding sites in promoter regions of TTF-1, SP (surfactant protein)-B, and CCSP (Clara cell secretory protein) have been identified [8,10,42,43] .Therefore, Foxa2 influences the expression of TTF-1, SP-B, and CCSP may depend on regulation of the promoter activity of these genes [37,44] .Although Foxa2 binding sites were indentified within 5' region of SP-B and CCSP promoter, the possibility of Foxa2 cooperating with other transcription factors or indirectly regulating expression of target genes was not be ruled out.C/EBPα, a member of the family of basic leucine zipper (bZIP) transcription factors [45] , is essential for cell differentiation and proliferation, lipid metabolism and normal tissue development [46] [47-49] .C/EBPα was expressed in respiratory epithelial cells prior to birth.Thereafter, the abundant expression of C/EBPα was observed in alveolar type II cells in the peripheral lung [50] .Specific deletion of C/EBPα in respiratory epithelial cells in fetal mice caused respiratory failure at birth, decreased the expression of SP-A, SP-B, SP-C, and SP-D mRNAs, delayed structural and biochemical maturation of the lung, inhibited lung epithelial cell proliferation and differentiation by increasing transforming growth factor (TGF) -β2 expression [51] , supporting that C/EBPα plays a crucial role in the maturation of the respiratory epithelium in late gestation, and which is not only necessary for the production of surfactant lipids and proteins, but also required for lung maturation.In certain conditions, Foxa2 and TTF-1 both separately regulate expression of surfactant protein, suggesting that regulation of lung differentiation by them may through sharing same transcriptional targets or together participating in regulatory programs.
Assessing C/EBPα expression by immunohistochemistry in the lungs from Foxa2 Δ/Δ or TTF-1 -/-at E18.5 demonstrated that Foxa2 or TTF-1 were required for expression of C/EBPα in respiratory epithelial cells [38,52] .Deletion of Foxa2 or TTF-1 given rise to a markedly decreased or absent of C/EBPα staining in the respiratory epithelium, providing evidence that presence of Foxa2 or TTF-1 is the key for maintaining the normal expression of C/EBPα [51] .Consistently, Foxa2 expression was decreased in the lungs from Cebpa Δ/Δ mice [51] .Thereafter, assessed the potential role of C/EBPα in Foxa2 promoter activity found that C/EBPα enhanced the activity of Foxa2 promoter in both HeLa and H441 cells.All of these indicate the concept that Foxa2, TTF-1, Cebpa participate in a network regulating perinatal lung maturation.C/EBPα, Foxa2, and TTF-1 share transcriptional targets crucial for surfactant synthesis, cell differentiation and host defense [51] .
Foxa1 and Foxa2 in a complementary manner regulate branching morphogenesis during lung development.Expression of Foxa1 in the respiratory epithelial cells of conducting and peripheral airways is similar to the temporal-spatial pattern of Foxa2 [6] .Alterations in branching morphogenesis were not observed in single knock-out Foxa1 -/-or Foxa2 Δ/Δ mice [29,31] .However, both deletion of Foxa1 and Foxa2 perturbed the formation of small peripheral acinar tubules and terminal saccules in the Foxa1 -/-/Foxa2 Δ/Δ mice [31] .Recent study demonstrated that loss of Foxa1 and Foxa2 influence mesenchymal cell differentiation and lung branching morphogenesis partly depending on inhibiting Shh (sonic hedgehog) expression [53,54] .Expression of epithelial cell markers, including SP-A, SP-B, and CCSP, were decreased in mice with specific deletion of Foxa2, but their expression was not entirely dependent on Foxa2 [29] .A study reported that immunostaining of SP-B, SP-C, CCSP, and Foxj1 was absent in Foxa1 -/-/Foxa2 Δ/Δ mice confirmed that expression of SP-A, SP-B, and CCSP is required for Foxa1 and Foxa2 [31] .Single deletion of Foxa1 resulted in increased level of Foxa2 mRNA, Similarly, Foxa1 expression was induced after loss of Foxa2 in pulmonary epithelial cells [31] .In short, Foxa1 and Foxa2 play commentary roles in the regulation of branching morphogenesis.

FOXA2 AND TH2 INFLAMMATION
Foxa2 is involved in the process of asthma.By quantifying expression of Foxa2 in airway biopsies which obtained from subjects with mild or moderate asthma observed that Foxa2 expression was significantly reduced [55]   .Th2-mediate cytokines is also associated with Foxa2.Interleukin (IL)-4 and IL-13 over-expression in mouse airways and IL-13 stimulation human bronchial epithelial cells both down-regulated Foxa2 expression [29,56] .Mucous cell metaplasia, a prominent feature of asthma, is associated with decreased expression of the transcription factor Foxa2. Challenging transgenic mice that specific expression Foxa2 gene in airway epithelial cells and exposure to ovalbuminin found that Foxa2 transgenic mice had little or no mucin visible in airway epithelium [55] .Foxa2 expression was negatively correlated with the production of MUC5AC in asthma.A luciferase assay reports that Foxa2 can inhibit the transcriptional activity of MUC5AC in NCI-H292 human lung mucoepidermoid cells, supporting the ability of Foxa2 inhibiting mucous metaplasia, at least in part, mediated by reducing MUC5AC transcriptional activity and decreasing production of MUC5AC [55] .This result indicated Foxa2 regulates the mucus gene transcription directly.All the results suggest a potential therapeutic approach that normal expression and function of Foxa2 could be beneficial for reducing mucous metaplasia in the asthma.
Foxa2 is a key regulator in the progress of Th2-mediated inflammation.Selective deletion of Foxa2 allele in the respiratory epithelium during embryonic stage (E6.5-E12.5)was found to cause asthma-like phenotype in the mice 2 weeks after birth: eosinophilic inflammation and goblet cell metaplasia.Deletion of Foxa2 increased the expression of mRNAs encoding components of the lipoxygenase signaling pathway and expression of goblet cell transcription factor Spdef, and increased the production of Th2 cytokines and chemokines, including IL-4, IL-13, IL-5 and thymus and activation-regulated chemokine (TARC), and enhanced myeloid dendritic-cell (DC) recruitment and activation, and promoted the leukotriene (LT) signaling pathway activation [29, 57, 58] .However, the mechanisms underlying this inflammation remain unknown.LTs, the most important inflammatory mediators, are considered to be involved in the pathogenesis of asthma.Studies reported that there is an interaction between LTs and Th2 cytokines: LTs can promote the production of Th2 cytokines, and Th2 cytokines activate the LTs pathway too [59] .In our study, loss of Foxa2 increases the production of LTs and Th2 cytokines and causes goblet-cell metaplasia in the mice lungs during the first 2 weeks after birth.Increased production of LTs induced by loss of Foxa2 occurred as early as PN5, at a time when the expression of Th2 cytokines was similar in control and Foxa2 Δ/Δ mice, supports the concept that activation of LT components was independent of Th2 cytokines in Foxa2 Δ/Δ mice.Therefore, the suppression of LTs by Foxa2 is a critical determinant of pulmonary Th2 polarization in this model.However, mechanisms by which Foxa2 controls airway epithelial differentiation and Th2 immunity are incompletely known.Eosinophilic inflammation in Foxa2 Δ/Δ mice was significantly inhibited by the anti-IL-4R antibody, indicating that the IL-4R pathway plays a key role in this Th2-mediated pulmonary inflammation [57] .However, the finding that activation of Th2 cytokines depends on the up-regulation of the LT pathway in the Foxa2 Δ/Δ mice indicating that LTs signaling pathway may play more important role in this animal mode.The inhibition of the Cysteinyl leukotriene 1 (CysLT1) receptor (CysLT1 R) with montelukast inhibited Th2 inflammation induced by deletion of Foxa2 in pulmonary epithelial cells.Montelukast inhibited the expression of genes regulating Th2 inflammation and mucus metaplasia.In addition, LTs can regulate DC activation, migration, and function [57] , the activation of DCs in Foxa2 Δ/Δ mice may result from spontaneous activation of the LT pathway in the absence of Foxa2.Together, we conclude that the LT pathway plays an important role in Th2-mediated pulmonary inflammation during lung development, and Foxa2 is required for the suppression of Th2 immunity and mucus metaplasia in the developing lung in a process determined in part by its regulation of the CysLT pathway.

FOXA2 AND GOBLET CELL HYPERPLASIA
FOXA2 is required for the maintenance of airway epithelial cell differentiation in the postnatal lung.Selective deletion of Foxa2 allele in the respiratory epithelium was found to cause obvious asthma-like phenotype and goblet cell metaplasia in the neonatal mice [29] .Foxa2 regulates goblet cell hyperplasia main dependenting on that Foxa2 can regulate the expression of genes associated with goblet cell phenotype such as Spdef.Spdef, a transcription factor critical for mucous cell differentiation and mucous production in the lung, normally expressed at low level in tracheal and bronchial epithelium, is significantly increased in goblet cells caused by Th2-mediated inflammation signaling during allergen exposure and by IL-13 [41] .Mechanism of Spdef regulate goblet cell differentiation and pathogenesis of asthma is that Spdef can promote the expression of Muc16, Agr2, Clca1, Ptger3, Muc5ac, and a group of genes mediating packaging, synthesis of mucin, and also its glycosylation.Spdef induces goblet cell metaplasia by inhibiting both TTF-1 and Foxa2 [60] .In mouse, loss of Spdef led to the absence of goblet cells in tracheal-laryngeal submucosal glands and in the surface epithelium after exposure pulmonary to allergen.Selectional expression Spdef in clara cells driven by CCSP-rtTA recombinantion system led to the presence of goblet cell hyperplasia and the absent of Foxa2 at sites of goblet cell hyperplasia [60] .Consistent with the counter-regulatory roles of Spdef and TTF-1/Foxa2, loss of Foxa2 can induce Spdef expression [29] , and conditional expression of Foxa2 can inhibit expression of Spdef and goblet cell differentiation after allergen exposure indicating that Foxa2 and Spdef are interacting in a genetic network that was related to mucus hyperproduction and goblet cell differentiation, a process that is highly relevant to the pathogenesis of asthma, cystic fibrosis, and other inflammatory lung diseases.Expression of Spdef is in a mutually exclusive manner with Foxa2.
However, the mechanism of controlling goblet cell metaplasia induced by loss of Foxa2 in airway epithelial cells during postnatal lung maturation remained largely unknown.Recent studies show that loss of Foxa2 induced goblet cell metaplasia is mediated by recruiting and activating myeloid dendritic cells (mDCs) and Th2 cells in the lung, and increasing production of Th2 cytokines and chemokines, resulting in increasing expression of goblet cell transcription factor Spdef [61] .LTs, which is thought to play a role in goblet-cell metaplasia, can regulate DC activation, migration, function, and up-regulate production of Th2 cytokines suggesting a potential role of LTs in a possess of goblet-cell metaplasia [57] .As a result, loss of Foxa2 induces the goblet cell metaplasia may result from spontaneous activation of the LT pathway in the absence of Foxa2.Markers associated with the production of airway mucus, Spdef, Muc5ac, Arg2, and Foxa3 mRNAs were significantly decreased in Foxa2 Δ/Δ mice with montelukast treatment [58] supporting that the goblet-cell metaplasia induced by Foxa2 deletion during neonatal lung development is, at least in part, regulated by the LT pathway.
Then which gene can regulate Foxa2 to induce goblet cell metaplasia?According to the studies, epithelial cell dysplasia and goblet cell differentiation induced by β-catenin was associated with inhibition of Foxa2 expression in the conducting airways [62] .β-catenin was expressed in respiratory epithelial cells of the developing lung, which also plays a critical role in epithelial cell differentiation during the normal lung morphogenesis and formation [63,64] .β-catenin is required for the formation of alveoli.Study reported that deletion of β-catenin prevented peripheral alveolar epithelial cell differentiation during lung morphogenesis [64] .Restricted to respiratory epithelial cells of the conducting and peripheral airways, activation of β-catenin blocked epithelial cell differentiation, caused goblet cell metaplasia and air space enlargement [62] .Foxa2 suppress mucin expression and maintain airway epithelial cell differentiation in the normal postnatal lung.Absence of Foxa2 staining was observed at sites of goblet cell differentiation in Catnb Δ(ex3) -expressing mice.And consistent with that the transcriptional activity of Foxa2 was negatively regulated by both activated and wild type β-catenin in vitro [62] .Thus provides a potential mechanism that goblet cell differentiation and epithelial cell dysplasia caused by Catnb Δ(ex3) was dependent, at least in part, on its inhibitory effects on Foxa2 gene expression in airway epithelial cells [62] .

FOXA2 AND LUNG CANCER
It has been determined that Foxa2, an airway transcription factor, plays a critical role in the pathogenesis of lung cancer.Several studies have confirmed that low or absent levels of Foxa2 is a frequent event in lung cancer cell lines and non-small cell lung carcinomas (NSCLCs) [65] .Expression of Foxa2 in the NCI-H358 NSCLC cell line led to growth reduction, proliferation arrest and increased apoptosis [65] .This result indicates Foxa2 has a potential role as tumor inhibitor in some NSCLCs.However, the mechanisms of down-regulation of Foxa2 have never been thoroughly studied in the NSCLC.The main mechanism of down-regulation or loss of Foxa2 in NSCLCs may be a result of epigenetic silencing through promoter hypermethylation [66]   .Considering the important roles that Foxa2 plays, low or absent expression of Foxa2 probably contributes to the lung cancer.Therefore, Foxa2 expression may be a good prognostic factor in NSCLC.However, the mechanism of Foxa2 as a suppressor of lung cancer largely remains unknown at present.The recent study showed that Foxa2 functions as a suppressor of tumor metastasis by inhibition of epithelial-to-mesenchymal transition (EMT) in human lung cancers [67] .Foxa2 protein level was decreased in A549 cells with TGF-β1 treatment.Decreased expression of Foxa2 accelerated EMT and invasion of lung cancer cells, whereas increased expression of Foxa2 disrupted TGF-β1-induced EMT and reduced the invasion, suggesting that Foxa2 is a key regulator of TGF-β1 in controlling EMT [67] .The reasons for the close association between Foxa2 expression and EMT in lung cancer cell lines are not well understood.Recent study have reported that over-expression of Foxa2 in A549, H446, H129 suppressed the TGF-β1-induced EMT through inhibition of the down-regulation of E-cadherin, the increase of fibronectin, and the upregulation of zonula occludens-1 (ZO-1), and inhibition of the ability of cell migration/invasion, but not influenced cell proliferation rate.Slug, a negative regulator of E-cadherin expression, plays a key role in EMT induction in human lung cancer cells [68] .The transcriptional activity of the slug promoter can be negatively regulated by Foxa2 through binding its one or two conserved putative site [67] .TGF-β1 treatment can abolish the interaction between Foxa2 and slug by decreasing Foxa2 protein level [67] .Take together, Foxa2 acts as a tumor metastasis suppressor characteristic by suppression of EMT and inhibition of the cell migration/invasion ability.
Although dysregulation of Foxa2 has been reported to promote the progression of certain cancers, the precise regulation f Foxa2 in lung tumor progression not clear.A study reported that IκB kinase (IKK)-α, an important downstream kinase of tumor necrosis factor (TNF)-α, inhibits Foxa2 transactivation activity through interacting with and phosphorylates Foxa2 at S107/S111, and then giving rise to decreased Numb expression, and further caused activation of the NOTCH pathway and promoted cell proliferation and tumorigenesis [69] .Comparably, another study reported that the levels of pFoxa2 (S107/S111), IKKα and activated NOTCH were significantly higher in hepatocellular carcinoma tumors [70] , providing us a new insight that Foxa2 as a lung tumor suppressor may dependent on Notch signaling.

LTs signaling pathway
LTs, the metabolic products of arachidonic acid, are important lipid mediators of airway inflammation and bronchospasm in asthma, allergic inflammation and innate immunity.LTs are synthesized through multiple enzymatic steps from membrane phospholipids via phospholipase A2 [71]   .
Arachidonate 15-lipoxygenase (LO)-1 [72]   , 5-lipoxygenase (5-LO) [73] and 5-lipoxygenase activating protein (FLAP) [74] are the encoding key lipoxygenases in LTs pathway, which expressed in many of the cells implicated in allergic inflammation and asthma pathology.Recent evidence indicates that LT pathway is required for the Th2-mediated pulmonary inflammation caused by deletion of Foxa2 in airway epithelial cells [58] .Loss of Foxa2 in pulmonary epithelium caused activation of LTs pathway, which partly contributed to pulmonary inflammation.The amount of key lipoxygenases in LTs pathway are significantly increased in the brochchial tissue of patients with asthma [75] .In our study we observed that as early as 5 days after birth, the loss of Foxa2 increases the production of LTs including leukotriene C4 (LTC4), leukotriene D4 (LTD4), leukotriene E4 (LTE4) in bronchoalveolar lavage (BAL) and the activity of LTs-related enzyme in the lungs tissues of Foxa2 gene-targeted mice.Then increased the production of LTs resulted in increased production of Th2 cytokines and chemokines to develop asthma-like eosinophilic inflammation and goblet cell metaplasia in the neonatal Foxa2 Δ/Δ mice.Thus indicate a potential mechanism that Foxa2 is required for the suppression of Th2 immunity and mucus metaplasia in the developing lung in a process determined in part by its regulation of the CysLT pathway.Expression of Foxa2 directly inhibited Alox15 gene transcription in vitro.A selective antagonist of the CysLT1 receptor, montelukast, inhibited the Th2-mediated inflammation and goblet cell differentiation caused by the deletion of Foxa2 in the developing mouse lung supporting that LT pathway plays an important role in Th2-mediated pulmonary inflammation during lung development.We conclude that Foxa2 can regulate the LT pathway to suppress Th2-mediated inflammation in the lung.

Hipp signaling pathway
Hippo pathway regulates lung maturation dependent on Foxa2.Mammalian sterile 20-like kinase 1 and 2 (Mst1/2), a upstream kinase in the Hippo signaling pathway, involves in organ development and cancer [76] .Using Mst1 and Mst2 generated conditional double-knockout mice (dKO) in the developing lung epithelium revealed that loss of Mst1/2 in lung epithelial cells impaired surfactant homeostasis and delayed type I and II pneumocyte differentiation, resulting in abnormal lung maturation, supporting that Mst1/2 plays an essential role in the regulation of surfactant homeostasis and peripheral lung maturation [77] .Expression of Foxa2 remarkably reduced in the Mst1/2-dKO lung tissue and increased expression of Mst1 in A549 cells significantly enhanced the stability of Foxa2 protein [77] suggesting Mst1/2 may act through modulation of Foxa2 to regulate lung development.To confirm the role of Foxa2 in Mst1/2-mediated synthesis of surfactant protein, over-expression Foxa2 in the absent of Mst1/2 cells found that decrease of SP-B and SP-C is partially rescued, indicating that Mst1/2 regulate surfactant protein, at least in part, relying on Foxa2.However, Mst1/2 did not phosphorylate the forkhead domain of Foxa2 demonstrating that Mst1/2 regulates Foxa2 independently of canonical Hippo pathway [77] .In summary, these data indicate that regulation of Mst1/2 in lung maturation may through modulation of Foxa2 in the noncanonical Hippo pathway which is independent of YAP.

STAT6 signaling pathway
Signal transducer and activator of transcription (STAT6), a member of signal transducers and activator of transcription factor family, has a link between cytokines receptors and cytokines induced gene transcription [78] .STAT6-deficient mice failed to develop airway hyper responsiveness (AHR) induced by allergen provocation, and consistent with lacking Th2 cytokine response [79] , suggesting STAT6 signaling is essential in the production of Th2 cytokines in response to antigen challenge.Expression of Foxa2 in the respiratory epithelium, is inhibited during goblet cell metaplasia and Th2 inflammation induced by IL-13 and IL-4 intratracheal administration [57,80] .Effects of allergens or Th2 cytokines on goblet cell hyperplasia, Foxa2 and pulmonary inflammation are dependent on STAT6 in a process associated with the induction of Spdef, Foxa2, and Foxa3 [29] .Decreased or absent of Foxa2 staining was observed in goblet cells after exposing to Th2 cytokines by intratracheal administration.Although IL-4 caused goblet cell hyperplasia in wild-type, neither decreased Foxa2 staining nor goblet cell hyperplasia were observed in STAT6 -/-mice supporting the concept that goblet cell hyperplasia induced by deletion of Foxa2 in pulmonary epithelial cells increasing the production of Th2 cytokines is depended on STAT6 signaling pathway [79]   .In addition, repression of STAT6 can inhibit SPDEF and MUC5AC expression induced by IL-13 [81] .However, the precise mechanism of STAT6 on regulation of inflammation and goblet cell metaplasia remains unknown at present.

Conclusions
Foxa2 plays a key role in the control of specific gene expression in the pulmonary epithelial cells and thus contributing to regulate the lung development and diseases.Recent studies have suggested that Foxa2 may act as a tumor suppressor gene in lung cancer.Considering the important roles that Foxa2 plays in the lung development, abnormal expression of Foxa2 probably contributes to the pathogenesis of lung disease.A better understanding of agents and pathways and molecular mechanisms that control Foxa2 expression in the lung will be useful for the development of novel treatment strategies for lung diseases such as Th2-mediated pulmonary inflammation, asthma, lung cancer and others.

Figure 1 .
Figure 1.Foxa2 protein organization[15] .Foxa2 possess three transactivations: regions II (372-385) and III (445-456) at the carboxyl terminus and the region IV (1-52) at amino terminus respectively.Region IV contains two putative casein kinase I (CKI) phosphorylation sites, which was critical for transcriptional activation.Foxa2 contains a DNA binding domain, which is located in region I (157-257), its specificity of protein recognition is dependent on a 20-amino-acid region, a conserved residues located adjacent to region I.