Regulated gene expression in cultured type II cells of adult human lung

Abstract

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.

Fig. 1.

Immunostaining of freshly isolated adult type II cell preparation. Most cells are positive for punctate intracellular staining for SP-B (left) and negative for vimentin as a marker for fibroblasts (right). Nuclei are shown by DAPI staining (blue). Images are representative of 5 experiments.

Fig. 2.

Morphology and staining of induced proteins in day 5 cells. Adult type II cells were cultured for 5 days without (control) or with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) for the last 3 days on collagen-coated chamber slides or culture dishes. Representative areas are shown for punctate immunostaining for SP-B (A, control; B, DCI) and DC-LAMP (C, control; D, DCI) as lamellar body markers, diffuse immunostaining for membrane protein Duox1 (E, control; F, DCI), phase microscopy (G, control; H, DCI), and toluidine blue staining at 2 magnifications (I and K, control; J and L, DCI). Treated cells show more refractile inclusions (G vs. H), prominent lamellar bodies (K vs. L), and increased intensity and proportion of cells staining for SP-B, DC-LAMP, and Duox1 compared with control cells. Counting of cells in the immunostaining experiment (B) indicated that 92% were positive for SP-B.

Fig. 3.

Transmission electron microscopy of day 5 cells. Adult type II cells were cultured for 5 days without (A, control) or with DCI for the last 3 days (B). Membranous vesicles with loosely packed lamellar or amorphous material were observed in both control and DCI-treated cells, and lamellar bodies (LB) were more common in treated cells. Prominent lysomes (Ly) were observed in some cells.

Fig. 4.

Transcript abundance in adult compared with fetal type II cells. Mean data from 5 experiments are shown for 11,176 probes that had a significant signal in adult cells. Signals for fetal cells that were called absent were assigned a value of 20 fluorimetric units (f.u.). Colored symbols indicate adult:fetal ratio >10-fold different as indicated. Slope = 0.69 and r = 0.88; the line of identity is shown.

Fig. 5.

Surfactant-related genes induced by DCI: transcript abundance on day 5 compared with freshly isolated day 0 cells. The gene list includes 4 surfactant proteins, PCG (pro-SP-B processing), 9 genes involved in de novo lipogenesis, and 7 genes related to lipid substrate or uptake. For the 21 genes, the induced transcript level on day 5 is greater than for day 0 for 7 genes, similar for 12 genes and less than day 0 for 2. Data are means + SE (n = 5); *P ≤ 0.01.

Fig. 6.

Venn diagram of regulated genes in fetal and adult type II cells. Approximately 300 genes were induced by hormones in both fetal and adult cells, with 127 genes in common; 25 of these genes were also downregulated during control culture of adult cells and represent candidate genes for hormonal regulation in vivo.

Fig. 7.

Validation of selected induced genes. Adult type II cells and fetal lung epithelial cells were cultured with or without DCI for qPCR (A) and Western (B and C) analysis. A: mRNA content for control and DCI-treated cells normalized to 18S rRNA. All transcripts were significantly induced by DCI except for TTF-1 in adult cells. mRNA content of adult DCI-treated cells was similar to that in fetal type II cells except for CEPBD (decreased) and Duox1 (increased). Data are means + SE, n = 3; *P < 0.05 adult DCI vs. fetal DCI. B: immunoreactive protein is shown for a representative Western of adult (left) and fetal (right) cells with GAPDH used as loading control. A stronger signal is observed for DCI-treated cells vs. control (C) for each protein; day 0 = day 0 cells. Nonspecific bands are seen above PGC and below Duox1. C: fold induction by densitometry in 3 experiments for proteins of panel B. Data are means + SE; *P < 0.05 for DCI-treated vs. control.