Abstract
ABSTRACTThe alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker), exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC), whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.
Highlights
Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), represent a significant health burden worldwide owing to their progressive nature and the current lack of curative treatments
Using a proteomic approach, the authors reported for the first time carbonyl reductase 2 (CBR2), enolase 1 (ENO1) and protein disulfide isomerase associated 3 (PDIA3) as functional alveolar epithelial cell proteins
Expression of ENO1, protein disulfide-isomerase associated 3 (PDIA3) and T1α decreased upon inhibition of Wnt/β-catenin signaling during Alveolar epithelial type II (ATII)-to-Alveolar epithelial type I (ATI) trans-differentiation, whereas CBR2 levels were stabilized
Summary
Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), represent a significant health burden worldwide owing to their progressive nature and the current lack of curative treatments. The alveolar epithelium consists largely of two morphologically distinct epithelial cell types, which are crucial to maintain lung homeostasis. ATI cells are not the most abundant cell type, they cover the largest surface area of the distal lung (Stone et al, 1992; Weibel, 2015). Alveolar epithelial type II (ATII) cells, which exhibit a cuboidal cell morphology, account for a much larger number of cells in the distal lung while covering a significant lower surface area (Stone et al, 1992). Recent studies utilizing linage tracing technology have established that ATII cells are capable of long-term self-renewal, indicating that these cells represent a major stem-cell population in the adult alveolar epithelium (Barkauskas et al, 2013; Desai et al, 2014)
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