Wnt signaling regulates trans-differentiation of stem cell like type 2 alveolar epithelial cells to type 1 epithelial cells
Type 2 alveolar epithelial cells (AT2s) behave as stem cells and show clonal proliferation upon alveolar injury followed by trans-differentiation (TD) into Type 1 alveolar epithelial cells (AT1s). In the present study we identified signaling pathways involved in the physiological AT2-to-AT1 TD process.
AT2 cells can be isolated from human lungs and cultured in vitro where they undergo TD into AT1s. In the present study we identified signaling pathways involved in the physiological AT2-to-AT1 TD process using Affymetrix microarray, qRT-PCR, fluorescence microscopy, and an in vitro lung aggregate culture.
Affymetrix microarray revealed Wnt signaling to play a crucial role in the TD process. Wnt7a was identified as a ligand regulating the AT1 marker, Aquaporin 5 (AQP5). Artificial Neural Network (ANN) analysis of the Affymetrix data exposed ITGAV: Integrin alpha V (ITGAV), thrombospondin 1 (THBS1) and epithelial membrane protein 2 (EMP2) as Wnt signaling targets.
Wnt signaling targets that can serve as potential alveolar epithelial repair targets in future therapies of the gas exchange surface after injury. As ITGAV is significantly increases during TD and is regulated by Wnt signaling, ITGAV might be a potential target to speed up the alveolar healing process.
KeywordsWnt signaling Alveolar epithelial cell Transdifferentiation
Ankyrin repeat domain 1
Artificial neural network
Alveolar Type 1 Cell
Alveolar Type 2 Cell
BMP and activin membrane-bound inhibitor
Bone morphogenic protein
Cytochrome P450 family 4, subfamily B, polypeptide 1
Epithelial membrane protein 2
Epithelial mesenchymal transition
Epithelial Cell Adhesion Molecule
Fluorescent activated cell sorting
Fibroblast growth factor
Integrin alpha V
Membrane bound O-acyltransferase
Normal Human Lung Fibroblasts
Protein ANalysis THrough Evolutionary Relationships program
Receptor for Advanced Glycation End-Products
Receptor Tyrosine Kinase Like Orphan Receptor 2
Small airway epithelial cells
Soluble frizzled receptor peptide
Surfactant protein A
Surfactant protein C
VANGL planar cell polarity protein 1
WNT1 Inducible Signaling Pathway Protein
Understanding the molecular regulation of alveolar regeneration is of high clinical importance. Mechanical injury of the alveoli induced by ventilation  or loss of gas exchange surface due to accumulation of environmental damage during aging  could both be treated if the process is understood and the appropriate molecular targets are identified for drug development .
The enormous alveolar surface of the lung has a significant and physiological regeneration capacity [4, 5]. Type 2 alveolar epithelial cells (AT2s) have been suspected to act as progenitor cells in the alveoli and recent genetic fate-tracking experiments in transgenic mice provided evidence that AT2s are indeed function as stem cells and show clonal proliferation in response to injury . About 95% of the alveolar surface area is covered by flat and thin Type 1 alveolar epithelial cells (AT1) that die by apoptosis upon injury leaving a denuded alveolar basement membrane behind. The cuboid AT2s are more resistant to injury, they proliferate, migrate and spread over the basement membrane then transdifferentiate into AT1 cells . The above process can happen in vitro also that was established mainly in animal studies [8, 9]. Recent organ regeneration studies suggest that reactivation of developmental mechanisms occur during injury repair  involving BMP, FGF, Notch and Wnt  signaling pathways.
Wnt/beta-catenin signaling is an evolutionarily conserved, versatile and highly complex pathway. Activation of this pathway leads to the accumulation of beta-catenin in the cytosol and translocation to the nucleus where it promotes transcription of various genes. Regulation of beta-catenin protein stability is dependent on its phosphorylation at various phosphorylation sites that either promotes protein degradation (Ser33, Ser37 and Thr41) or its stabilization and nuclear localization (Ser675) . The Wnt family of secreted glycoproteins are known regulators of cell proliferation, differentiation, polarity, adhesion and migration during lung development . While the Wnt/beta-catenin signaling is necessary for alveolar morphogenesis it is not essential for the development of proximal airways . Several additional Wnt ligands, such as Wnt5a , Wnt7b  and most Frizzled (Fzd) receptors  are also central to the regulation of lung development. During aging deregulated Wnt ligand composition can alter alveolar epithelial differentiation  and can give rise to modified molecular microenvironments that promote emphysema and other diseases . Although it is recognized that Wnt signaling must have a critical role in pulmonary regeneration, its precise involvement in the trans-differentiation (TD) processes remain obscure. Particularly so, as most of the studies were performed in cell line, mouse and rat models using immunostaining [20, 21] which did now allow a preconception free approach to understand the role of Wnt signaling of AT2-to-AT1 TD in the human lung. To investigate the process, cellular transformation of primary human AT2 cells was studied in vitro and data was compared to gene expression of AT1 and AT2 cells freshly isolated from primary human lung tissues. The effects of the identified Wnt ligands were tested in three-dimensional (3D) human lung aggregate cultures  to confirm their roles in the TD process and in the regulation of their downstream targets recognized by artificial neural network (ANN) analysis.
Materials and methods
Cell isolation from lung tissue
Human primary lung epithelial cells were isolated from tissue samples from lobectomy patients (n = 26) with normal lung function. All patients gave written informed consent for the use of their tissues and clinical data for research purposes (ethics 07/MRE08/42). Patient characteristics are summarized in Additional file 1: Table S1. Cells were isolated and cultured as described previously . Briefly, lung tissue was rinsed with saline, then digested using warm trypsin, followed by mincing in the presence of DNase (Sigma-Aldrich, St. Louis, Missouri, USA) and foetal calf serum (FCS) (Lonza, Basel, Switzerland). The cell suspension was passed through a cell strainer (mesh size 40 μm) and the freshly isolated cells were either processed for Fluorescent activated cell sorting (FACS) or cultured for longer term .
In vitro TD of primary pulmonary epithelial cells
Pulmonary epithelial cells were seeded into collagen coated plates and cultured in DCCM-1 medium (Biological Industries Ltd. Kibbutz Beit-Haemek, Israel) containing 10% FCS for 3–6 days. On day 3 and day 6, cells were lysed and total RNA was isolated and processed for microarray analysis or real-time qPCR.
Sorting of freshly isolated lung epithelial cells
Freshly isolated lung cells (n = 12) were washed with PBS containing 0.1% BSA (Sigma-Aldrich, St. Louis, Missouri, USA) and 0.1% Na-Azide (Sigma-Aldrich, St. Louis, Missouri, USA), then antibodies were added for 30 min. EpCAM-FITC, CD208-PE and Podoplanin-APC conjugated antibodies were used to differentiate between AT1-like (EpCAM+ Podoplanin+ CD208- population)  and AT2-like (EpCAM+ Podoplanin- CD208+ population)  epithelial cells. Cells were sorted with a Beckman-Coulter MoFlo XDP high-speed cell sorter (Additional file 1: Figure S1). Average yield of AT1- and AT2-like cells were 4.43 × 104 and 1.39 × 105, respectively. Cells were then lysed, RNA was isolated using RNeasy kit (Qiagen, Hilden, Germany) and cDNA was generated from 200 ng total RNA using a WT Expression Kit (Ambion, Thermo Fisher Scientific, Waltham, USA).
cDNA of in vitro cultured cells of n = 3 patients (days 3 and 6 of culturing) was fragmented and fluorescently labeled using the GeneChip WT terminal Labeling Kit (Affymetrix, Santa Clara, USA). cDNA was hybridized to Human Gene 1.0 ST arrays (Affymetrix, Santa Clara, USA). Probe cell intensity data (CEL) from the microarrays were analysed using the Expression Console software with the default settings of the RMA-sketch workflow. Differentially expressed probe sets were identified using the limma package in Bioconductor project.
Protein analysis through evolutionary relationships (PANTHER)
The PANTHER Classification System (supported by research grants from the National Human Genome Research Institute and the National Science Foundation, and maintained by the Thomas lab at the University of Southern California) was designed to classify proteins (and their genes) in order to facilitate high-throughput analysis. Details of the methods can be found in [27, 28]. PANTHER is part of the Gene Ontology Phylogenetic Annotation Project.
Real time qRT-PCR
Total RNA was isolated from cultured lung epithelial cells isolated from patients (n = 11) and from sorted freshly isolated AT2- and AT1-like cells (n = 12) using the NucleoSpin RNA isolation kit with on-column DNase digestion (Macherey-Nagel, Düren, Germany). cDNA synthesis was performed using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, USA) following manufacturer’s protocols. For real-time qPCR experiments, master mixes with or without SYBR Green were used (Roche, Basel, Switzerland). Primer sequences are listed in Additional file 1: Table S2. PCR experiments were performed on a Light Cycler 480 Instrument (Roche, Basel, Switzerland). In the plots reverse dCt values versus GAPDH expression are presented; the following formula was used for calculation: dCt = Ct target gene-CtGAPDH. Data was presented as relative quantity (RQ).
3D tissues and treatment with recombinant human Wnt proteins
Normal primary human small airway epithelial cells (SAEC) and normal human lung fibroblast (NHLF) were purchased from Lonza (Basel, Switzerland), isolated from anonymous donors of different ages and sexes. All cells were cultured at 37 °C and 5% CO2 in primary cell culture media. Both cell types were sub-cultured and mixed at 1:1 ratio then dispensed 3*105 cells/well onto a low-attachment 96-well U-bottom plates (Corning, New York, USA) (Additional file 1: Figures S3 and S4). 3D aggregates were formed as described previously (Kovacs et al., 2014). Aggregates were treated with 0.1 μg/ml of recombinant human protein Wnt4, Wnt5a or Wnt7a, respectively for 48 h, then collected for total RNA isolation for TaqMan based PCR application (n = 3 biological repeats).
Fluorescence staining of 3D aggregates
3D lung aggregates were embedded into TissueTek embedding media, frozen and 8 μm thick cryostat sections were cut and fixed in 4% para-formaldehyde (PFA) (Sigma-Aldrich, St. Louis, Missouri, USA). AQP5 was detected using an anti-AQ5 rabbit polyclonal IgG (sc-28,628) (Santa Cruz Biotechnology, Dallas, USA) (dilution 1∶100). ITGAV was detected using an anti-CD51 polyclonal goat antibody (PA5–47096), Thermo Fisher Scientific (Waltham, USA) (dilution 1:100). The secondary antibody was a goat anti-rabbit IgG antibody (Alexa Fluor® 568) (ab175471) (1:2000) (Abcam Plc, Cambridge, United Kingdom) and the anti-mouse antibody was an Alexa Fluor® 488 conjugated IgG (Thermo Fisher Scientific, Waltham, USA) (dilution 1:200). Nuclei were counterstained with Dapiprazole hydrochloride (DAPI)(ab142859) (1:1000) (Abcam Plc, Cambridge, United Kingdom). Images were acquired using Nikon Eclipse Ti-U microscope (Nikon GmbH CEE, Vienna, Austria) equipped with CCD camera (AndorZyla 5.5) then densitometry was performed using ImageJ.
Artificial neural network (ANN) analysis
Evaluation of Wnt signaling pathway on AT2-to-AT1 TD was carried out using a feed forward artificial neural network (ANN) (Neurosolutions 6, NeuroDimension Inc.) software. Gene expression data were obtained with Affymetrix array using pooled cDNA samples of AT2 as controls and AT1 cell samples. Mean sensitivity was determined and set as to 1.0, all other sensitivity values are also shown accordingly in heat map format.
Statistical analysis was performed with SPSS version 20 software. Data are presented as mean ± standard deviation (STDEV), and statistical analysis was performed using Mann-Whitney non-parametric tests. p < 0.05 was considered as significant.
Wnt signaling pathways are the most active during AT2-to-AT1 TD in vitro
Three dimensional (3D) aggregate cultures confirm a role of Wnt ligands in TD
ANN analysis of microarray data reveals Wnt pathway targets during AT2-to-AT1 TD
In the present study, three Wnt ligands were identified to play important roles in the AT2-to-AT1 TD process, Wnt4, Wnt5a and Wnt7a. All three ligands were identified as down-regulators of SPC and Wnt7a as an inducer of the AT1 type differentiation marker AQP5. Previous studies support our discoveries. During the pulmonary aging process Wnt4 and Wnt5a were identified as inhibitors of lipid uptake and therefore surfactant production , while Wnt7a triggered differentiation and reduced proliferation of lung adenocarcinoma cell lines , respectively. The three Wnt ligands during AT2-to-AT1 TD are involved in a complex regulatory link with other genes identified by ANN. However, the only gene that was directly affected by an individual Wnt ligand, was ITGAV. ITGAV is strongly affected by PORCN (Porcupine) that is a membrane bound O-acyltransferase (MBOAT) involved in the acylation and secretion of Wnt proteins . ITGAV in general plays an important role in the regulation of cancer growth, metastasis and tissue remodeling , but upregulation of ITGAV not just increases cellular adhesion but plays an inhibitory role in lipid transport that is essential for surfactant production . As ITGAV is significantly increases during the TD process but decreases upon Wnt5a ligand treatment, it was assumed that elevated levels of ITGAV aids AT1 differentiation via blocking surfactant production. Additional analysis of ITGAV protein levels have, however, demonstrated that Wnt5a can cell type specifically modify ITGAV expression. While in the mesenchymal fibroblasts ITGAV levels increased, in the epithelial cell layers ITGAV levels significantly decreased corresponding to decreased mRNA levels in the aggregate cultures following rhWnt5a treatment. Such results support previous findings that Wnt5a triggers ITGAV expression in the mesenchyme  and also that SPC production is associated with fibroblast differentiation . Consequently, we can hypothesize that ITGAV and not directly Wnt ligands are responsible for regulation of SPC levels.
The other genes identified by ANN analysis are more difficult to explain as in follow-up experiments neither Wnt4, Wnt5a or Wnt7a affected individually the expression of THBS1, TGM2 or EMP2. The most strongly affected by the modified Wnt microenvironment is the up-regulated THBS1, that is a secreted glycoprotein involved in wound healing, angiogenesis and inflammatory response  as well as in inhibition of tumor growth . Upregulation of THBS1 during a physiological regeneration process could be a built-in molecular protection mechanism against carcinogenesis. The significantly downregulated TGM2 gene encodes an ubiquitously expressed enzyme capable of catalyzing protein cross-links and regulate extracellular matrix integrity . Down-regulation of TGM2, however, fits into the envisaged TD process, as loosening the extracellular matrix is probably needed to facilitate AT2 spreading over the basal membrane. Increased expression of EMP2 has been linked to cancer progression by controlling cell membrane composition  and blood vessel growth . So, elevated EMP2 expression during the physiological AT2-to-AT1 TD potentially facilitates capillary blood vessel formation. Additionally, ANN revealed that EMP2 is most sensitive to inhibitors of the canonical and the PCP Wnt pathways like VANGL1, DKK2, SFRP4, receptors like ROR2 and the Wnt2 associated receptor, Fzd3. Simultaneously, EMP2 is unaffected by a number of genes with similar inhibitory characteristics (CER1, SFRP2, Fzd9 and BAMBI) indicating the existence of a so far unidentified regulatory network of alveolar regeneration. Finally, upregulation of CYP4B1 is also a characteristic marker of cellular –mainly bronchial- differentiation of the lung . Upregulation of CYP4B1 during AT2-to-AT1 TD is affected by a specific ligand Wnt3a, that plays an important role in in lung cancer .
Investigation of gene expression during AT2-to-AT1 TD not only identified Wnt ligands that can accelerate AT1 type differentiation. We have also identified Wnt pathway associated genes that are affected by the cumulative changes in the Wnt microenvironment. The balance of the microenvironment, however, is crucial as most of the target genes are important regulators of carcinogenesis or cancer progression. In the light of our research data it is not surprising that in recent years Wnt signaling has become a target of investigation for both cancer  and regenerative therapies .
Concept and design: DB, JEP, DRT, Cell preparation, laboratory work and data analysis: EMMA, JR, DF, VC, DB, JEP, Patient recruitment: DRT, DB, Preparation of manuscript & figures: EMMA, JR, DB, SP, DRT, JEP. All authors have read and approved the manuscript.
DRT was funded by the Medical Research Council UK and The Wellcome Trust. DB was funded by an FP7-Marie Curie Intra-European Fellowship (FP7-PEOPLE-IEF 300371 and an ERS long term training fellowship (ERS-LTRF 2011–131). JEP was funded by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TAMOP4.2.4.A/ 2–11/1–2012-0001 ‘National Excellence Program’ as well as by the European Union co-financed by the European Regional Development Fund GINOP 2.3.2-15-2016-00022.
Ethics approval and consent to participate
All procedures in this study were performed in accordance with approval from the local research ethics committee at the University of Birmingham. All patients included in this study gave written informed consent for the use of their tissue and clinical data for research purposes. Ethics committee approval number is 07/mre08/42 amendment 2 dated 2/5/2009.
Consent for publication
The authors declare that they have no competing interest.
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