Primary Alveolar Epithelial Type II Cells Research Articles

MICALL2 participates in the regulation of epithelial-mesenchymal transition in alveolar epithelial cells – Potential roles in pulmonary fibrosis

Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is a vital process in idiopathic pulmonary fibrosis (IPF), which results in the accumulation of fibroblasts and myofibroblasts and excessive extracellular matrix deposition. Based on RNA sequencing analysis and GEO dataset reanalysis, we screened out MICALL2, a gene upregulated in the lungs of IPF mice and alveolar epithelial type II (ATII) cells from IPF patients, and aimed to explore its role in IPF. We validated the expression of MICALL2 in bleomycin (BLM)-induced IPF mice and TGF-β1-stimulated ATII cells (primary murine ATII cells and A549 cells), and explored the role of MICALL2 in IPF by knockdown of MICALL2 in BLM-induced mice and TGF-β1-stimulated ATII cells. We found that MICALL2 was upregulated in the lungs of BLM-induced mice and TGF-β1-stimulated ATII cells. MICALL2-deficient mice had reduced fibrogenesis and restrained EMT upon BLM challenge. Knockdown of MICALL2 restrained the EMT process, in vitro, through impeding β‐catenin nuclear translocation. Mechanistically, we demonstrated that NPAS2 is directly bound to the promoter of MICALL2. Altogether, our data revealed transactivation of MICALL2 induced by NPAS2, contributing to activation of the Wnt/β-catenin pathway in ATII cells, thus leading to the EMT process and subsequent pulmonary fibrosis. Interfering with MICALL2 may represent an innovative therapeutic target to mitigate the extent of IPF.

PAI-1 Regulation of p53 Expression and Senescence in Type II Alveolar Epithelial Cells.

Cellular senescence contributes importantly to aging and aging-related diseases, including idiopathic pulmonary fibrosis (IPF). Alveolar epithelial type II (ATII) cells are progenitors of alveolar epithelium, and ATII cell senescence is evident in IPF. Previous studies from this lab have shown that increased expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, promotes ATII cell senescence through inducing p53, a master cell cycle repressor, and activating p53-p21-pRb cell cycle repression pathway. In this study, we further show that PAI-1 binds to proteasome components and inhibits proteasome activity and p53 degradation in human lung epithelial A549 cells and primary mouse ATII cells. This is associated with a senescence phenotype of these cells, manifested as increased p53 and p21 expression, decreased phosphorylated retinoblastoma protein (pRb), and increased senescence-associated beta-galactose (SA-β-gal) activity. Moreover, we find that, although overexpression of wild-type PAI-1 (wtPAI-1) or a secretion-deficient, mature form of PAI-1 (sdPAI-1) alone induces ATII cell senescence (increases SA-β-gal activity), only wtPAI-1 induces p53, suggesting that the premature form of PAI-1 is required for the interaction with the proteasome. In summary, our data indicate that PAI-1 can bind to proteasome components and thus inhibit proteasome activity and p53 degradation in ATII cells. As p53 is a master cell cycle repressor and PAI-1 expression is increased in many senescent cells, the results from this study will have a significant impact not only on ATII cell senescence/lung fibrosis but also on the senescence of other types of cells in different diseases.

THE WNK4/SPAK PATHWAY STIMULATES ALVEOLAR FLUID CLEARANCE BY UPREGULATION OF EPITHELIAL SODIUM CHANNEL IN MICE WITH LIPOPOLYSACCHARIDE-INDUCED ACUTE RESPIRATORY DISTRESS SYNDROME.

ABSTRACTWith-No lysine Kinases (WNKs) have been newly implicated in alveolar fluid clearance (AFC). Epithelial sodium channels (ENaCs) serve a vital role in AFC. The potential protective effect of WNK4 in acute respiratory distress syndrome (ARDS), mediated by ENaC-associated AFC was investigated in the study. A model of lipopolysaccharide (LPS)-induced ARDS was established in C57BL/6 mice. WNK4, Sterile 20-related proline-alanine-rich kinase (SPAK), small interfering RNA (siRNA)-WNK4 or siRNA-SPAK were transfected into mouse lung or primary alveolar epithelial type II (ATII) cells. AFC, bronchoalveolar lavage fluid and lung histomorphology were determined. The expression of ENaC was determined to investigate the regulation of AFC by WNK4-SPAK signaling pathway. Activation of WNK4-SPAK signaling improved lung injury and survival rate, with enhanced AFC and reduced pulmonary edema via the upregulation of ENaC in ARDS. In primary rat ATII cells, gene-silencing by siRNA transfection reduced ENaC expression and the level of WNK4-associated SPAK phosphorylation. Immunoprecipitation revealed that the level of neural precursor cell-expressed developmentally downregulated gene 4 (Nedd4-2) binding to ENaC was decreased as a result of WNK4-SPAK signaling. The present study demonstrated that the WNK4/SPAK pathway improved AFC during LPS-induced ARDS, which is mainly dependent on the upregulation of ENaC with Nedd4-2-mediated ubiquitination.

The Oncogene ECT2 Contributes to a Hyperplastic, Proliferative Lung Epithelial Cell Phenotype in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) and lung cancer are progressive lung diseases with a poor prognosis. IPF is a risk factor for the development of lung cancer, and the incidence of lung cancer is increased in patients with IPF. The disease pathogenesis of IPF and lung cancer involves common genetic alterations, dysregulated pathways, and the emergence of hyperplastic and metaplastic epithelial cells. Here, we aimed to identify novel, common mediators that might contribute to epithelial cell reprogramming in IPF. Gene set enrichment analysis of publicly available non-small cell lung cancer and IPF datasets revealed a common pattern of misregulated genes linked to cell proliferation and transformation. The oncogene ECT2 (epithelial cell transforming sequence 2), a guanine nucleotide exchange factor for Rho GTPases, was highly enriched in both IPF and non-small cell lung cancer compared with nondiseased controls. Increased expression of ECT2 was verified by qPCR and Western blotting in bleomycin-induced lung fibrosis and human IPF tissue. Immunohistochemistry demonstrated strong expression of ECT2 staining in hyperplastic alveolar epithelial type II (ATII) cells in IPF, as well as its colocalization with proliferating cell nuclear antigen, a well-known proliferation marker. Increased ECT2 expression coincided with enhanced proliferation of primary mouse ATII cells as analyzed by flow cytometry. ECT2 knockdown in ATII cells resulted in decreased proliferation and collagen I expression in vitro. These data suggest that the oncogene ECT2 contributes to epithelial cell reprogramming in IPF, and further emphasize the hyperplastic, proliferative ATII cell as a potential target in patients with IPF and lung cancer.

Reduced Frizzled Receptor 4 Expression Prevents WNT/β-Catenin-driven Alveolar Lung Repair in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/β-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored. To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD. FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/β-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components. FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/β-catenin activity. Inhibition of FZD4 decreased WNT/β-catenin-driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/β-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo. Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells.

Because of the many energy-demanding functions they perform and their physical location in the lung, alveolar epithelial type II (ATII) cells have a rapid cellular metabolism and the potential to influence substrate availability and bioenergetics both locally in the lung and throughout the body. A thorough understanding of ATII cell metabolic function in the healthy lung is necessary for determining how metabolic changes may contribute to pulmonary disease pathogenesis; however, lung metabolism is poorly understood at the cellular level. Here, we examine lactate utilization by primary ATII cells and the ATII model cell line, MLE-15, and link lactate consumption directly to mitochondrial ATP generation. ATII cells cultured in lactate undergo mitochondrial respiration at near-maximal levels, two times the rates of those grown in glucose, and oxygen consumption under these conditions is directly linked to mitochondrial ATP generation. When both lactate and glucose are available as metabolic substrate, the presence of lactate alters glucose metabolism in ATII to favor reduced glycolytic function in a dose-dependent manner, suggesting that lactate is used in addition to glucose when both substrates are available. Lactate use by ATII mitochondria is dependent on monocarboxylate transporter (MCT)-mediated import, and ATII cells express MCT1, the isoform that mediates lactate import by cells in other lactate-consuming tissues. The balance of lactate production and consumption may play an important role in the maintenance of healthy lung homeostasis, whereas disruption of lactate consumption by factors that impair mitochondrial metabolism, such as hypoxia, may contribute to lactic acid build-up in disease.

WNT/β-Catenin Signaling Induces IL-1β Expression by Alveolar Epithelial Cells in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. It is characterized by alterations of the alveolar epithelium, myofibroblast activation, and increased extracellular matrix deposition. Recently, reactivation of the developmental WNT/β-catenin pathway has been linked with pulmonary fibrosis. The cell-specific mechanisms and mediators of WNT/β-catenin signaling in the lung, however, remain elusive. Here, we applied an unbiased gene expression screen to identify epithelial cell-specific mediators of WNT/β-catenin signaling. We found the proinflammatory cytokine IL-1β to be one of the most up-regulated genes in primary murine alveolar epithelial Type II (ATII) cells after WNT3a treatment. Increased transcript and protein expression of IL-1β upon WNT3a treatment was further detected in primary ATII cells by quantitative RT-PCR (log fold change, 2.0 ± 0.5) and ELISA (1.8-fold increase). We observed significant up-regulation of IL-1β and IL-6 in bronchoalveolar lavage fluid (BALF) in bleomycin-induced lung fibrosis in vivo. Importantly, primary fibrotic ATII cells isolated from lungs subjected to bleomycin secreted enhanced IL-1β and IL-6 in vitro. Furthermore, the orotracheal application of recombinant WNT protein in the Tcf optimal promoter (TOP)-β-galactosidase reporter animals led to WNT/β-catenin activation in epithelial cells, along with significant increases in IL-1β and IL-6 in vivo (2.7-fold and 6.0-fold increases, respectively). Finally, we found increased WNT3a protein in fibrotic alveolar epithelia, accompanied by enhanced IL-1β and IL-6 concentrations in BALF from patients with IPF. Taken together, our findings reveal that the alveolar epithelium is a relevant source of proinflammatory cytokines induced by active WNT/β-catenin in pulmonary fibrosis. Thus, WNT/interleukin signaling represents a novel link between developmental pathway reactivation and inflammation in the development of pulmonary fibrosis.

Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles

The present study aimed at developing a drug delivery system targeting the densest site of tuberculosis infection, the alveolar macrophages (AMs). Rifampicin (RFP)-loaded solid lipid nanopar- ticles (RFP-SLNs) with an average size of 829.6 ± 16.1 nm were prepared by a modified lipid film hydration method. The cytotoxicity of RFP-SLNs to AMs and alveolar epithelial type II cells (AECs) was examined using MTT assays. The viability of AMs and AECs was above 80 % after treatment with RFP-SLNs, which showed low toxicity to both AMs and AECs. Confocal Laser Scanning Microscopy was employed to observe the interaction between RFP- SLNs and both AMs and AECs. After incubating the cells with RFP-SLNs for 2 h, the fluorescent intensity in AMs was more and remained longer (from 0.5 to 12 h) when compared with that in AECs (from 0.5 to 8 h). In vitro uptake characteristics of RFP-SLNs in AMs and AECs were also investigated by detection of intracellular RFP by High performance liquid chro- matography. Results showed that RFP-SLNs delivered markedly higher RFP into AMs (691.7 ng/mg in cultured AMs, 662.6 ng/mg in primary AMs) than that into AECs (319.2 ng/mg in cultured AECs, 287.2 ng/mg in primary AECs). Subsequently, in vivo delivery efficiency and the selectivity of RFP-SLNs were further verified in Sprague-Dawley rats. Under pulmonary administration of RFP-SLNs, the amount of RFP in AMs was significantly higher than that in AECs at each time point. Our results demonstrated that solid lipid nanoparticles are a promising strategy for the delivery of rifampicin to alveolar macrophages selectively.

Keratinocyte Growth Factor and Dexamethasone Plus Elevated cAMP Levels Synergistically Support Pluripotent Stem Cell Differentiation into Alveolar Epithelial Type II Cells

Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application.

WNT1-inducible signaling protein–1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/beta-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein-1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.

Transgelin is a direct target of TGF‐β/Smad3‐dependent epithelial cell migration in lung fibrosis

Enhanced transforming growth factor (TGF) -beta signaling contributes to idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease characterized by alveolar epithelial type II (ATII) cell hyperplasia, (myo)fibroblast accumulation, and excessive extracellular matrix deposition. TGF-beta is a potent inducer of lung fibrosis, and it regulates the ATII cell phenotype; however, direct TGF-beta target genes controlling the ATII cell phenotype remain elusive. Here, we identified the transgelin (tagln) gene as a novel immediate target of TGF-beta/Smad3-dependent gene expression in ATII cells using a Smad3 chromatin immunoprecipitation (ChIP) screen. Direct ChIP confirmed the rapid and specific binding of Smad3 to the tagln promoter. Luciferase assays demonstrated transactivation of the tagln promoter by activin-like kinase (Alk) 5-mediated TGF-beta signaling. TGF-beta treatment resulted in rapid up-regulation of tagln, but not tagln2, mRNA and protein expression, assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. In vivo, tagln expression was significantly increased in ATII cells of mice during bleomycin-induced lung fibrosis, as well as in lung specimen obtained from IPF patients, as assessed by RT-PCR and immunohistochemistry. Knockdown of tagln using siRNA inhibited TGF-beta-induced migration of lung epithelial A549 cells, as well as primary ATII cells. We thus identified tagln as a novel target of TGF-beta/Smad3-dependent gene expression in ATII cells. Increased ATII cell expression of tagln in experimental and idiopathic pulmonary fibrosis may contribute to TGF-beta-dependent ATII cell injury, repair, and migration in lung fibrosis.

Induction of CXCL5 During Inflammation in the Rodent Lung Involves Activation of Alveolar Epithelium

The lung is continuously exposed to bacteria and their products, and has developed a complex defense mechanism, including neutrophil recruitment. In mice, keratinocyte cell-derived chemokine and macrophage inflammatory protein-2 are the major chemokines for neutrophil recruitment into the lung. We have previously described a role for C-X-C chemokine (CXCL5) in neutrophil trafficking during lipopolysaccharide (LPS)-induced lung inflammation in mice. The aims of the present study were to identify the cellular origin of CXCL5 and to determine the signaling cascades that regulate its expression in the lung during LPS-induced inflammation and in isolated LPS-stimulated CXCL5-expressing cells. Our immunohistochemical analysis indicates that alveolar epithelial type II (AEII) cells are the primary source of CXCL5 in the rodent lung. These in vivo observations were confirmed with primary AEII cells. In addition, our data indicate that the Toll-like receptor 4 (TLR4) signaling cascade involving TLR4, myeloid differentiation factor 88, and Toll-IL-1R domain-containing adapter protein is required to induce CXCL5 expression in the lung. Furthermore, p38 and c-Jun N-terminal kinases are involved in lung CXCL5 expression. Similarly, TLR4, and p38 and c-Jun N-terminal kinases, are associated with LPS-induced CXCL5 expression in AEII cells. These novel observations demonstrate that activation of AEII cells via TLR4-dependent signaling is important for the production of CXCL5 in the lung exposed to LPS.

Caveolin-1 Expression and Caveolae Biogenesis during Cell Transdifferentiation in Lung Alveolar Epithelial Primary Cultures

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-“like” phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.