Opposing regulation of human alveolar type II cell differentiation by nitric oxide and hyperoxia

Abstract

Clinical trials demonstrated decreasing rates of bronchopulmonary dysplasia in preterm infants with hypoxic respiratory failure treated with inhaled nitric oxide (iNO). However, the molecular and biochemical effects of iNO on developing human fetal lungs remain vastly unknown. By using a well-characterized model of human fetal alveolar type II cells, we assessed the effects of iNO and hyperoxia, independently and concurrently, on NO-cGMP signaling pathway and differentiation. Exposure to iNO increased cGMP levels by 40-fold after 3 d and by 8-fold after 5 d despite constant expression of phosphodiesterase-5 (PDE5). The levels of cGMP declined significantly on exposure to iNO and hyperoxia at 3 and 5 d, although expression of soluble guanylyl cyclase (sGC) was sustained. Surfactant proteins B and C (SP-B, SP-C) and thyroid transcription factor (TTF)-1 mRNA levels increased in cells exposed to iNO in normoxia but not on exposure to iNO plus hyperoxia. Collectively, these data indicate an increase in type II cell markers when undifferentiated lung epithelial cells are exposed to iNO in room air. However, hyperoxia overrides these potentially beneficial effects of iNO despite sustained expression of sGC.

Figure 1

Panel A, cGMP in cell lysates from day 3 and 5 DCI treated cells. Panel B and C, stable NO-metabolites in cell media (B), and cell lysates (C). Exposures include: normoxia (□), hyperoxia (■), normoxia plus NO (), or hyperoxia plus NO (); *p < 0.05 compared to normoxia on day 3 and ^†p < 0.05 compared to corresponding treatment group on day 3, n=4 for all treatment groups.

Figure 2

Near-infrared quantification of sGC levels in cells exposed to various gases in DCI media at day 3 (Panel A) and at day 5 (Panel B). Exposure conditions: normoxia (□), hyperoxia (■), normoxia plus NO (), or hyperoxia plus NO ().

Figure 3

The levels of PDE5 were quantified by western blotting and near-infrared detection at day 3 (Panel A) and day 5 (Panel B). Exposure groups: normoxia (□), hyperoxia (■), normoxia NO (), or hyperoxia plus NO ().

Figure 4

mRNAs for Type II cell SP-B (A), SP-C (B), and TTF-1 (C) were measured after 5 days of culture in DCI media and either normoxia (□), hyperoxia (■), normoxia plus NO (), or hyperoxia plus NO (); *p < 0.05, n=3 for all treatment groups.

Figure 5

Panel A, Representative SP-B blots. Lanes: (1–2) normoxia controls with DCI media at days 3 and 5 respectively; (3–4) cells exposed to respective gases as indicated without DCI media at days 3 and 5; (5–6) cells exposed to respective gases as indicated with DCI media at days 3 and 5 respectively. Panel B, SP-B protein levels normalized to GAPDH loading controls after 5 days of culture in DCI media and either normoxia (□), hyperoxia (■), normoxia plus NO (), or hyperoxia plus NO (); data is expressed as percent of the corresponding normoxia control *p < 0.05, n=5 for all treatment groups.