Abstract:Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII) cell line, A549. After cloning A549 subpopulations, eac… Show more
“…An inverse relationship to SFTPC expression was observed for the ATI marker AQP5 , which was significantly upregulated 1.5 fold from 72 to 120 h (P ≤ 0.01), increasing further at 168 h to a 4.3 fold change in expression relative to that of 72 h (P ≤ 0.01). The inverse and dynamic relationship between SFTPC and AQP5 points to differentiation of ATII to ATI, a key feature of the role of ATII cells as progenitors of ATI 22 , 23 . Figure 5 Comparative analysis of gene expression, comparing SFTPC (ATII marker) with that of AQP5 (ATI marker).…”
Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including roles in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during regeneration of the alveolar epithelium. Their secretion of surfactant protein helps to maintain homeostasis in the distal lung and exert protective, antimicrobial properties. Despite the cell’s crucial roles, they remain difficult to study, in part due to inefficient and expensive isolation methods, a propensity to differentiate into alveolar type I cells in culture and susceptibility to fibroblast overgrowth from primary isolations. Published methods of isolation often require specialist technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis (BTB), a serious re-emerging disease in both animals and humans worldwide. We present here a simple and cost-effective method that may be utilised in the generation of bovine primary ATII cells. These exhibit an ATII phenotype in 2D and 3D culture in our studies and are conducive to further study of the role of ATII cells in bovine respiratory diseases.
“…An inverse relationship to SFTPC expression was observed for the ATI marker AQP5 , which was significantly upregulated 1.5 fold from 72 to 120 h (P ≤ 0.01), increasing further at 168 h to a 4.3 fold change in expression relative to that of 72 h (P ≤ 0.01). The inverse and dynamic relationship between SFTPC and AQP5 points to differentiation of ATII to ATI, a key feature of the role of ATII cells as progenitors of ATI 22 , 23 . Figure 5 Comparative analysis of gene expression, comparing SFTPC (ATII marker) with that of AQP5 (ATI marker).…”
Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including roles in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during regeneration of the alveolar epithelium. Their secretion of surfactant protein helps to maintain homeostasis in the distal lung and exert protective, antimicrobial properties. Despite the cell’s crucial roles, they remain difficult to study, in part due to inefficient and expensive isolation methods, a propensity to differentiate into alveolar type I cells in culture and susceptibility to fibroblast overgrowth from primary isolations. Published methods of isolation often require specialist technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis (BTB), a serious re-emerging disease in both animals and humans worldwide. We present here a simple and cost-effective method that may be utilised in the generation of bovine primary ATII cells. These exhibit an ATII phenotype in 2D and 3D culture in our studies and are conducive to further study of the role of ATII cells in bovine respiratory diseases.
“…Differentiation of ATII cells into ATI cells may be studied by following the expression of the ATI marker aquaporin 5 (Aqp5). The increase of AQP5 mRNA expression is concomitant with a corresponding decrease in SFTPC in submerged 2D cultures over time, a key indicator of the dynamic and inverse relationship between these two cell markers (Kondo, Miyoshi, Sakiyama, Tangoku, & Noma, 2015;Lee et al, 2018).…”
“…A549 cells are derived from alveolar type II epithelium, which synthesizes pulmonary surfactant lipid–protein complex, stores it in LBs, and secretes it at the plasma membrane (Kondo, Miyoshi, Sakiyama, Tangoku, & Noma, ; Robin, Sarah, Peter, Teresa, & Molly, ; Wang, Russo, Muligeta, & Beers, ). LBs express lysosomal markers and are now recognized as secretory lysosomes or lysosome‐related organelles.…”
Phosphatidylinositol 3-phosphate (PI(3)P) is the predominant phosphoinositide species in early endosomes and autophagosomes, in which PI(3)P dictates traffic of these organelles. Phosphoinositide levels are tightly regulated by lipid-kinases and -phosphatases; however, a phosphatase that converts PI(3)P back to phosphatidylinositol in the endosomal and autophagosomal compartments is not fully understood. We investigated the subcellular distribution and functions of myotubularin-related protein-4 (MTMR4), which is distinct among other MTMRs in that it possesses a PI(3)P-binding FYVE domain, in lung alveolar epithelium-derived A549 cells. MTMR4 was localized mainly in late endosomes and autophagosomes. MTMR4 knockdown markedly suppressed the motility, fusion, and fission of PI(3)P-enriched structures, resulting in decreases in late endosomes, autophagosomes, and lysosomes, and enlargement of PI(3)P-enriched early and late endosomes. In amino acid- and serum-starved cells, MTMR4 knockdown decreased both autophagosomes and autolysosomes and markedly increased PI(3)P-containing autophagosomes and late endosomes, suggesting that the fusion with lysosomes of autophagosomes and late endosomes might be impaired. Notably, MTMR4 knockdown inhibited the nuclear translocation of starvation stress responsive transcription factor-EB (TFEB) with reduced expression of lysosome-related genes in starved cells. These findings indicate that MTMR4 is essential for the integrity of endocytic and autophagic pathways.
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