Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle

Abstract

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

Keywords: 2-methoxyestradiol; CYP1a1; airway smooth muscle; bronchopulmonary dysplasia; estradiol; hyperoxia; neonate.

Fig. 1.

Comparison of protein expression in human fetal airway smooth muscle (fASM) cells. A: protein expression of metabolizing enzymes as well as estrogen receptors was unchanged with hyperoxia exposure. CYP1b1, cytochrome P450 1B1; COMT, catechol-O-methyl transferase; ERα estrogen receptor-α; ERβ, estrogen receptor-β. B: CYP1a1 expression by Western analysis (normalized for GAPDH) was significantly increased following 24 h of 50% hyperoxia exposure. C: CYP1a1 enzyme activity, as detected by luminescence (RLU, relative light units), is increased in hyperoxia. 3-Methylcholanthrene (3-MC), a known inducer of CYP1a1 expression, is included as a positive control. Values are means ± SE. *Significant effect of hyperoxia (P < 0.05).

Fig. 2.

Proliferation of human fASM cells. Cellular proliferation of human fASM cells in the presence of estradiol (E2) (A), 2-Methoxyestradiol (2-ME) (C), or CYP inhibitor, 1- aminobenzotriazole (1-AB) (D), was evaluated using a fluorescent CyQuant assay (see materials and methods). A: under normoxic conditions, E2 significantly increased fASM proliferation even at low concentrations of 100 pM-1 nM to extents comparable to that induced by the mitogen PDGF. However, exposure to 50% hyperoxia for 24 h prevented E2-induced proliferation but did not affect PDGF influences. B: separately, estradiol exposure increased proliferating cell nuclear antigen (PCNA) expression consistent with increased cell proliferation. C: in contrast to E2, the metabolite 2-ME did not affect fASM cell proliferation and, if anything, blunted proliferation at high concentrations, regardless of the oxygen concentration. D: inhibition of CYP1a1 with 1-AB restored the effect of E2 on proliferation in the presence of hyperoxia. Data were performed in triplicate and expressed as means ± SE. *Significant effect for hyperoxia, #significant effect for E2 or 2-ME effect (P < 0.05).

Fig. 3.

Markers of apoptosis and reactive oxygen species (ROS) generation in fASM exposed to hyperoxia, E2, and 2-ME. A: changes in protein expression of active caspase 3 and procaspase 9 with estradiol treatment under hyperoxic conditions reflect an increase in apoptosis. These results were reversed with the addition of the CYP1a1 inhibitor 1-AB, suggesting that the metabolite 2-ME contributes to the increase in apoptosis. ROS generation can contribute to increased apoptosis. The addition of 2-ME to fASM does not show any increase in ROS; rather, 2-ME showed a dose-dependent decrease in ROS generated (B and C). fASM under hyperoxic conditions exposed to E2 or with the CYP1a1 inhibitor 1-AB also supports the observation that 2-ME does not increase ROS (C). Data were performed in triplicate and expressed as means ± SE. #Significant effect for hyperoxia, *significant effect for normoxia (P < 0.05).