Background Although the expression of the glycophosphatidylinositol-linked protein Thy-1 has been suggested to be important in alveolar fibroblast proliferation and myofibroblast (MYF) differentiation, its role in chronic lung disease of the premature infant is not known. We have previously demonstrated that hyperoxia augments spontaneous transdifferentiation of alveolar lipofibroblasts (LIFs) to MYFs when passaged in culture. We hypothesized that Thy-1 is differentially expressed by LIFs and MYFs and that exposure to hyperoxia would specifically affect Thy-1 expression by alveolar interstitial fibroblasts.
Objectives To characterize the expression of Thy-1 and other well-established markers of LIFs and MYFs phenotypes in spontaneously passaged alveolar interstitial fibroblasts and to determine the effect of hyperoxia on their expression.
Design/Methods Fetal rat lung fibroblasts (FRLF) from embryonic (e) day 19 (term = e22) Sprague-Dawley rat fetuses were isolated, maintained, and passaged in Minimum Essential Medium + 10% fetal bovine serum in 21% O2/5% CO2 at 37°C. At passage (P) 1, 5, and 10, cells were also exposed to 95% O2/5% CO2 for 24 h, following which the expression of the well-characterized intermediates of the parathyroid hormone-related protein (PTHrP receptor and peroxisome proliferator-activated receptorγ) and Wnt (αsmooth muscle actin, calponin, and SMAD3) signaling pathways were determined by Western hybridization. Thy-1 expression was established by Western hybridization, flow cytometry, and semiquantitative RT-PCR. These data were also complemented by genome-wide microarray analysis of the RNA extracted from normoxia- and hyperoxia-exposed FRLFs.
Results Normoxia-exposed P1 FRLFs were Thy-1 (-), whereas P5 and P10 cells expressed Thy-1 robustly. Exposure to hyperoxia increased the expression of Thy-1 at all passages. Exposure to hyperoxia, in general, decreased the expression of the PTHrP signaling pathway intermediates while concomitantly increasing the expression of the Wnt signaling intermediates. Cluster analysis of the microarray data suggests that hyperoxia decreases the expression of the cholesterol and fatty acid synthesis genes while increasing the expression of the fatty acid degradation enzymes- and Wnt-related genes.
Conclusions These data suggest an association of the Thy-1 (+) phenotype with the canonical Wnt signaling pathway and a possible role for Thy-1 expression in fibroblast to MYF transdifferentiation.
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