Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity

Disruption of alveolar epithelial cell (AEC) differentiation is implicated in peripheral lung diseases strongly impacting morbidity and mortality worldwide, such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. However, to date no study has comprehensively characterized the dynamic epigenomic alterations that facilitate this critical process in humans. We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for dozens of transcription factors, including NKX2-1 and FOXA family members, as well as transcription factors with previously uncharacterized roles in lung differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations between transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching may be involved in facilitating differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that human mammary epithelial cells (HMEC) have a similar epigenomic structure to alveolar epithelium, with NKX2-1 serving as a distinguishing feature of distal lung differentiation. Taken together, our results suggest that enhancer regions with dynamic transcription factor interactions are hotspots of epigenomic alteration that help to facilitate AEC differentiation.

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Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity

BMC Genomics ◽

10.1186/s12864-021-08152-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

B. Zhou ◽

T. R. Stueve ◽

E. A. Mihalakakos ◽

L. Miao ◽

D. Mullen ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Epithelial Cell ◽

Lung Diseases ◽

Regulatory Networks ◽

Alveolar Epithelial Cell ◽

Chronic Obstructive ◽

Differentiation Process ◽

Alveolar Epithelial ◽

Distal Lung

Abstract Background Disruption of alveolar epithelial cell (AEC) differentiation is implicated in distal lung diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma that impact morbidity and mortality worldwide. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. Previous studies have focused on changes of individual transcription factors, and to date no study has comprehensively characterized the dynamic, global epigenomic alterations that facilitate this critical differentiation process in humans. Results We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression data. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for many transcription factors, including NKX2–1 and FOXA family members, as well as transcription factors with less well characterized roles in AEC differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations among transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching in driving differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that AECs have similar epigenomic structures to other profiled epithelial cell types, including human mammary epithelial cells (HMECs), with NKX2–1 serving as a distinguishing feature of distal lung differentiation. Conclusions Enhancer regions are hotspots of epigenomic alteration that regulate AEC differentiation. Furthermore, the differentiation process is regulated by dynamic networks of transcription factors acting in concert, rather than individually. These findings provide a roadmap for understanding the relationship between disruption of the epigenetic state during AEC differentiation and development of lung diseases that may be therapeutically amenable.

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Molecular biomarkers in idiopathic pulmonary fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00014.2014 ◽

2014 ◽

Vol 307 (9) ◽

pp. L681-L691 ◽

Cited By ~ 101

Author(s):

Brett Ley ◽

Kevin K. Brown ◽

Harold R. Collard

Keyword(s):

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Alveolar Epithelial Cell ◽

Underlying Disease ◽

Current Approach ◽

Molecular Biomarkers ◽

Alveolar Epithelial ◽

Cell Dysfunction ◽

Collaborative Efforts ◽

Intended Use

Molecular biomarkers are highly desired in idiopathic pulmonary fibrosis (IPF), where they hold the potential to elucidate underlying disease mechanisms, accelerated drug development, and advance clinical management. Currently, there are no molecular biomarkers in widespread clinical use for IPF, and the search for potential markers remains in its infancy. Proposed core mechanisms in the pathogenesis of IPF for which candidate markers have been offered include alveolar epithelial cell dysfunction, immune dysregulation, and fibrogenesis. Useful markers reflect important pathological pathways, are practically and accurately measured, have undergone extensive validation, and are an improvement upon the current approach for their intended use. The successful development of useful molecular biomarkers is a central challenge for the future of translational research in IPF and will require collaborative efforts among those parties invested in advancing the care of patients with IPF.

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Silica directly increases permeability of alveolar epithelial cells

Journal of Applied Physiology ◽

10.1152/jappl.1990.68.4.1354 ◽

1990 ◽

Vol 68 (4) ◽

pp. 1354-1359 ◽

Cited By ~ 11

Author(s):

R. K. Merchant ◽

M. W. Peterson ◽

G. W. Hunninghake

Keyword(s):

Epithelial Cells ◽

Cell Injury ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Dependent Manner ◽

Alveolar Epithelial ◽

Lactate Dehydrogenase Release ◽

Capillary Membrane

Alveolar epithelial cell injury and increased alveolar-capillary membrane permeability are important features of acute silicosis. To determine whether silica particles contribute directly to this increased permeability, we measured paracellular permeability of rat alveolar epithelium after exposure to silica, in vitro, using markers of the extracellular space. Silica (Minusil) markedly increased permeability in a dose- and time-dependent manner. This was not the result of cytolytic injury, because lactate dehydrogenase release from monolayers exposed to silica was not increased. Pretreatment of the silica with serum, charged dextrans, or aluminum sulfate blocked the increase in permeability. Scanning electron microscopy demonstrated adherence of the silica to the surface of the alveolar epithelial cells. Thus silica can directly increase permeability of alveolar epithelium.

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Cell cycle kinetics in the alveolar epithelium

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.6.l1031 ◽

1997 ◽

Vol 272 (6) ◽

pp. L1031-L1045 ◽

Cited By ~ 62

Author(s):

B. D. Uhal

Keyword(s):

Epithelial Cell ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelium ◽

Cell Loss ◽

Type Ii ◽

Alveolar Epithelial ◽

Pathological Conditions ◽

Type Ii Cell ◽

The Relationship

The type II alveolar epithelial cell has important metabolic and biosynthetic functions but also serves as the stem cell of the alveolar epithelium. Much of the evidence underlying this premise was obtained before 1980 and provided the basis for a working model that has not been reconsidered for more than fifteen years. With the exceptions to be discussed below, little evidence has accumulated in the interim to suggest that the model requires significant alteration. Important questions remain unanswered, however, and some components of the model need to be supplemented, particularly in light of recent investigations that have provided insights not possible in earlier work. In particular, in vitro studies have suggested that the relationship between the parent type II cell and its progeny may not be as straightforward as originally thought. In addition, the rate of epithelial cell loss was recognized long ago to be an important factor in the regulation of this system, but its kinetics and mechanisms have received little attention. These and other unresolved issues are critical to our understanding of the homeostasis of the alveolar epithelium under normal and pathological conditions.

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Voltage-gated proton channels help regulate pHi in rat alveolar epithelium

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00299.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. L398-L408 ◽

Cited By ~ 20

Author(s):

Ricardo Murphy ◽

Vladimir V. Cherny ◽

Deri Morgan ◽

Thomas E. DeCoursey

Keyword(s):

Epithelial Cells ◽

Ph Regulation ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Proton Channel ◽

Resting Cells ◽

Proton Channels ◽

Voltage Gated ◽

Alveolar Epithelial

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pHi) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO3−-free solutions [at external pH (pHo) 7.4]. Cells were acid-loaded by the NH4+ prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 μM ZnCl2, a proton channel inhibitor, slowed recovery about twofold. In addition, the pHi minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl2 was observed at pHo 7.4 and pHo 8.0 and in normal and high-K+ Ringer solutions. The observed rate of Zn2+-sensitive pHi recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pHi recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl2 had no effect on pHi in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pHi recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

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Activation of L-type Ca2+ channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2)

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.269.6.l873 ◽

1995 ◽

Vol 269 (6) ◽

pp. L873-L883 ◽

Cited By ~ 2

Author(s):

P. Dietl ◽

T. Haller ◽

B. Wirleitner ◽

H. Volkl ◽

F. Friedrich ◽

...

Keyword(s):

Epithelial Cell ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelium ◽

Bay K 8644 ◽

Epithelial Cell Line ◽

Apparent Dissociation Constant ◽

Whole Cell ◽

Fura 2 ◽

Alveolar Epithelial ◽

Ca2 Channels

In the alveolar epithelium, ATP increases the intracellular Ca2+ concentration ([Ca2+]i) and stimulates the secretion of surfactant. We investigated the effects of extracellular ATP on the membrane potential (Vm), the whole cell current, and [Ca2+]i in a cloned rat alveolar epithelial cell line (L2). In microelectrode experiments, ATP caused a sustained depolarization of Vm, resulting from the activation of cation and Cl- conductances, as revealed by ion replacements. The depolarizing phase of the Vm shift was superimposed by Ca(2+)-dependent depolarizing spikes. Spikes were also induced by depolarizing Vm with charybdotoxin or maitotoxin. Replacement of bath Ca2+ with Ba2+ or Sr2+ also evoked repetitive spikes. Ca2+ (Ba2+, Sr2+)-induced spikes were unaffected by pretreatment with ionomycin or thapsigargin. They were, however, completely abolished by (+)-isradipine (100 nM) and stimulated by BAY K 8644 (100 nM). Whole cell L-type Ca2+ (Ba2+, Sr2+) currents were similarly abolished by (+)-isradipine and enhanced by BAY K 8644. L-type Ca2+ channels were further confirmed by demonstrating high-affinity dihydropyridine receptors stereoselectively labeled by (+)-[3H]-isradipine, apparent dissociation constant < 1 nM. In fura 2 experiments, ATP evoked a transient elevation of [Ca2+]i in the absence of Ca2+ and a biphasic sustained elevation in the presence of Ca2+, indicating intracellular Ca2+ release and Ca2+ entry. The ATP-induced fura 2 signals were unaffected by (+)-isradipine. We conclude that in L2 cells, L-type Ca2+ channels are activated after purinoceptor stimulation by ATP. The overall [Ca2+]i response is, however, mediated by Ca2+ entry through and (+)-isradipine-insensitive mechanism and by intracellular Ca2+ release.

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Pneumocystis carinii Activates the NF-κB Signaling Pathway in Alveolar Epithelial Cells

Infection and Immunity ◽

10.1128/iai.73.5.2766-2777.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 2766-2777 ◽

Cited By ~ 39

Author(s):

Jing Wang ◽

Francis Gigliotti ◽

Sanjay Maggirwar ◽

Carl Johnston ◽

Jacob N. Finkelstein ◽

...

Keyword(s):

Cell Line ◽

Signaling Pathway ◽

Nuclear Translocation ◽

Inflammatory Responses ◽

Alveolar Epithelial Cell ◽

Pneumocystis Carinii ◽

Specific Inhibitor ◽

Alveolar Epithelium ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial

ABSTRACT Pneumocystis carinii pneumonia (PcP) is a clinically important infection of immunocompromised patients. Although the interaction of Pneumocystis with the alveolar epithelium has been well documented, very little information regarding the epithelial response to Pneumocystis is currently available. In order to study Pneumocystis-epithelium interactions, a murine cell line derived specifically from an alveolar epithelial cell (AEC) was utilized. The coculture of murine AECs with mouse Pneumocystis induced a dose- and time-dependent release of the CXC chemokine MIP-2. Importantly, the specific removal of Pneumocystis from the preparation, or the pretreatment of AECs with sulfasalazine, a potent and specific inhibitor of NF-κB, nearly completely abrogated the chemokine response to Pneumocystis. Since the murine MIP-2 promoter contains consensus κB binding sequences, the ability of Pneumocystis to stimulate NF-κB signaling in AECs was examined. Pneumocystis stimulation of an AEC line stably transfected with a κB-dependent reporter construct triggered the NF-κB signaling pathway and reporter production. These data were confirmed in gel shift assays, providing direct evidence that Pneumocystis induced the nuclear translocation of the p50/p65 heterodimeric form of NF-κB. Maximal NF-κB activation was dependent upon direct contact with viable Pneumocystis organisms. These data demonstrate that Pneumocystis activates NF-κB signaling in AECs and establish a reporter cell line for studying NF-κB activation in AECs. Given the global regulatory functions of the NF-κB family, these findings suggest that Pneumocystis directly alters AEC gene expression in a manner that promotes pulmonary immune and inflammatory responses.

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Suppression of Rcn3 in the alveolar epithelial cells may have beneficial effect against COPD

10.21203/rs.3.rs-20953/v1 ◽

2020 ◽

Author(s):

Qianyu Zhang ◽

Tong Wang ◽

Jiawei Jin ◽

Xiaoqian Shi ◽

Zhenru Ma ◽

...

Keyword(s):

Beneficial Effect ◽

Secretory Pathway ◽

Alveolar Epithelial Cell ◽

Critical Role ◽

Airflow Obstruction ◽

Alveolar Epithelial Cells ◽

Conditional Knockout ◽

Chronic Obstructive ◽

Alveolar Epithelial ◽

Copd Patients

Abstract Background: Chronic obstructive pulmonary disease (COPD) is a highly prevalent lung disease worldwide and imposes increasing disease burdens globally. COPD is characterized by irreversible airflow obstruction. Emphysema is one of the primary pathological features causing the irreversible decline of pulmonary function, while the precise mechanisms behind emphysema remain unclear. Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein localized in the secretory pathway of living cells. We have reported that Rcn3 in type II alveolar epithelial cell (AECIIs) plays a critical role in perinatal lung development and bleomycin-induced lung injury-repair. Since is associated with alveolar epithelial disruption, Rcn3 might be involved in the development of emphysema during COPD. Materials and Methods : We examined Rcn3 expression in lung specimens from patients (44 COPD patients and 26 non-COPD control patients) undergoing lung lobectomy or pneumonectomy. Mouse models of COPD and emphysema were established by cigarette smoke (CS) exposure and intratracheal installation of pancreatic porcine elastase (PPE), respectively. Rcn3 expression was detected in the lung tissues from these mice. Furthermore, conditional knockout (CKO) mice with Rcn3 deletion specific to AECIIs, were used to explore the role of Rcn3 in PPE-induced emphysema progression. Rcn3 protein expression in lung tissues were evaluated by Western blot and immunohistochemistry. Rcn3 mRNA expression in lung tissues was detected by qPCR.Results : Compared with non-COPD patients, Rcn3 expression was significantly increased in the lung specimens from COPD patients. Rcn3 expression was also significantly up-regulated in the lung tissues from COPD mice and emphysematous mice. Moreover, selective ablation of Rcn3 in AEC IIs significantly alleviated severity of the mouse emphysema in response to intratracheal installation of PPE.Conclusions: Our data, for the first time, indicated that Rcn3 in AECIIs might play a role in modulating the progression of emphysema, and thus be involved in the development of COPD. Suppression of Rcn3 expression in AECIIs may have a beneficial effect on COPD.This work was supported by National Natural Science Foundation of China (No. 81641004).

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Transcription factor enrichment analysis (TFEA): Quantifying the activity of hundreds of transcription factors from a single experiment

10.1101/2020.01.25.919738 ◽

2020 ◽

Author(s):

Jonathan D. Rubin ◽

Jacob T. Stanley ◽

Rutendo F. Sigauke ◽

Cecilia B. Levandowski ◽

Zachary L. Maas ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Regulatory Networks ◽

Time Series Data ◽

Effective Means ◽

Enrichment Analysis ◽

Cost Effective ◽

Computational Method ◽

Series Data ◽

Single Experiment

1AbstractDetecting differential activation of transcription factors (TFs) in response to perturbation provides insight into cellular processes. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects differential activity of hundreds of TFs given any set of perturbation data. TFEA draws inspiration from GSEA and detects positional motif enrichment within a list of ranked regions of interest (ROIs). As ROIs are typically inferred from the data, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent (eg. PRO-Seq), CAGE, ChIP-Seq, and accessibility (e.g. ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.

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