Hypoxia-inducible factor regulates expression of surfactant protein in alveolar type II cells in vitro

Abstract

Alveolar type II (ATII) cells cultured at an air-liquid (A/L) interface maintain differentiation, but they lose these properties when they are submerged. Others showed that an oxygen tension gradient develops in the culture medium as ATII cells consume oxygen. Therefore, we wondered whether hypoxia inducible factor (HIF) signaling could explain differences in the phenotypes of ATII cells cultured under A/L interface or submerged conditions. ATII cells were isolated from male Sprague-Dawley rats and cultured on inserts coated with a mixture of rat-tail collagen and Matrigel, in medium including 5% rat serum and 10 ng/ml keratinocyte growth factor, with their apical surfaces either exposed to air or submerged. The A/L interface condition maintained the expression of surfactant proteins, whereas that expression was down-regulated under the submerged condition, and the effect was rapid and reversible. Under submerged conditions, there was an increase in HIF1α and HIF2α in nuclear extracts, mRNA levels of HIF inducible genes, vascular endothelial growth factor, glucose transporter-1 (GLUT1), and the protein level of pyruvate dehydrogenase kinase isozyme-1. The expression of surfactant proteins was suppressed and GLUT1 mRNA levels were induced when cells were cultured with 1 mM dimethyloxalyl glycine. The expression of surfactant proteins was restored under submerged conditions with supplemented 60% oxygen. HIF signaling and oxygen tension at the surface of cells appears to be important in regulating the phenotype of rat ATII cells.

Figure 1.

The amount of apical fluid alters surfactant protein (SP) levels. Rat ATII cells were adhered for 1 day and cultured for 6 days in 5% rat serum (RS) with 10 ng/ml keratinocyte growth factor (KGF) and different amounts of apical fluid, with or without rocking. Cells were harvested on Day 7, and protein levels of SPs were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. m, mature.

Figure 2.

The submerged condition decreases, and the A/L interface condition maintains, the expression of surfactant proteins. (A) Rat ATII cells were adhered for 1 day and cultured for 2, 4, or 6 days in 5% RS and 10 ng/ml KGF. Cells were harvested on Day 3, 5, or 7, and levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of five independent experiments. (B) Levels of surfactant proteins from the immunoblotting data were normalized to β-actin and analyzed according to NIH Image (Bethesda, MD) (n = 5). (C) The mRNA concentrations of surfactant proteins were measured by real-time PCR from five independent experiments. These levels were normalized to the constitutive probe cyclophilin B (CyB). Values represent means ± SEMs of five independent experiments. A/L, air–liquid interface condition; Sub, submerged condition. *P < 0.05.

Figure 2.

The submerged condition decreases, and the A/L interface condition maintains, the expression of surfactant proteins. (A) Rat ATII cells were adhered for 1 day and cultured for 2, 4, or 6 days in 5% RS and 10 ng/ml KGF. Cells were harvested on Day 3, 5, or 7, and levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of five independent experiments. (B) Levels of surfactant proteins from the immunoblotting data were normalized to β-actin and analyzed according to NIH Image (Bethesda, MD) (n = 5). (C) The mRNA concentrations of surfactant proteins were measured by real-time PCR from five independent experiments. These levels were normalized to the constitutive probe cyclophilin B (CyB). Values represent means ± SEMs of five independent experiments. A/L, air–liquid interface condition; Sub, submerged condition. *P < 0.05.

Figure 3.

The expression of surfactant proteins is reduced by the addition of apical fluid. (A) Rat ATII cells were adhered for 1 day and cultured for 6 days in 5% RS and 10 ng/ml KGF under the A/L interface condition. They were then cultured for additional 8, 24, or 48 hours, under either the A/L interface or submerged condition, and levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of five independent experiments. (B) Levels of surfactant proteins from immunoblotting data were normalized to β-actin and analyzed according to NIH Image (n = 5). Values represent means ± SEMs of five independent experiments. *P < 0.05.

Figure 3.

The expression of surfactant proteins is reduced by the addition of apical fluid. (A) Rat ATII cells were adhered for 1 day and cultured for 6 days in 5% RS and 10 ng/ml KGF under the A/L interface condition. They were then cultured for additional 8, 24, or 48 hours, under either the A/L interface or submerged condition, and levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of five independent experiments. (B) Levels of surfactant proteins from immunoblotting data were normalized to β-actin and analyzed according to NIH Image (n = 5). Values represent means ± SEMs of five independent experiments. *P < 0.05.

Figure 4.

The A/L interface condition restores surfactant protein expression from the submerged condition. (A) Rat ATII cells were adhered for 1 day and cultured for 6 days in 5% RS and 10 ng/ml KGF under the submerged condition. They were then cultured for an additional 8, 24, or 48 hours in either the submerged condition or the A/L interface condition, and levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of five independent experiments. (B) Levels of surfactant proteins from immunoblotting data were normalized to β-actin and analyzed according to NIH Image (n = 5). Values represent means ± SEMs of five independent experiments. *P < 0.05.

Figure 4.

The A/L interface condition restores surfactant protein expression from the submerged condition. (A) Rat ATII cells were adhered for 1 day and cultured for 6 days in 5% RS and 10 ng/ml KGF under the submerged condition. They were then cultured for an additional 8, 24, or 48 hours in either the submerged condition or the A/L interface condition, and levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of five independent experiments. (B) Levels of surfactant proteins from immunoblotting data were normalized to β-actin and analyzed according to NIH Image (n = 5). Values represent means ± SEMs of five independent experiments. *P < 0.05.

Figure 5.

Increased expression of hypoxia inducible factor (HIF) and HIF-inducible genes under the submerged condition. (A) Rat ATII cells were adhered for 1 day and cultured for 2, 4, or 6 days in 5% RS and 10 ng/ml KGF. Cells were harvested on Day 3, 5, or 7, and mRNA concentrations of VEGF and GLUT1 were measured by real-time PCR from five independent experiments. These levels were normalized to the constitutive probe cyclophilin B. Values represent means ± SEMs of five independent experiments. (B) Rat ATII cells were harvested on Day 7, and protein levels of pyruvate dehydrogenase kinase isozyme–1 (PDK1) were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. Left, Representative blot of four independent experiments. Right, Protein levels of PDK1 from immunoblotting data analyzed according to NIH Image (n = 4). Protein levels were normalized to β-actin. Values represent means ± SEMs of four independent experiments. (C) Rat ATII cells were adhered for 1 day and cultured for 18 hours in 5% RS and 10 ng/ml KGF, and protein levels of HIF1α and HIF2α from nuclear extract were analyzed by immunoblotting. This blot is representative of three independent experiments. *P < 0.05.

Figure 6.

Dimethyloxalyl glycine (DMOG), an HIF stabilizer, decreases the expression of surfactant proteins. (A) Rat ATII cells were adhered for 1 day and cultured for 6 days in 5% RS, 10 ng/ml KGF, 0.58% ethanol, and PBS for 6 days under A/L interface, submerged, or A/L interface conditions with 1 mM DMOG dissolved in 0.58% ethanol and PBS. This blot is representative of four independent experiments. Each condition is represented by two lanes from duplicate wells. Protein levels were normalized by β-actin. (B) Levels of surfactant proteins from immunoblotting data were normalized to β-actin and analyzed according to NIH Image (n = 4). Values represent means ± SEMs of four independent experiments. *P < 0.05.

Figure 7.

Supplemental oxygen restores the expression of surfactant proteins under the submerged condition and prevents the decrease because of submersion. (A) Rat ATII cells were adhered for 1 day and cultured in 5% RS and 10 ng/ml KGF under submerged conditions with 21% oxygen for 6 days. They were then cultured for an additional 48 hours in the submerged condition with 21% oxygen, in the A/L interface condition with 21% oxygen, or in the submerged condition with 60% oxygen. Levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of three independent experiments. (B) Rat ATII cells were adhered for 1 day and cultured in 5% RS and 10 ng/ml KGF under A/L interface conditions with 21% oxygen for 6 days. They were then cultured for additional 24 hours under the A/L interface condition with 21% oxygen, under the submerged condition with 21% oxygen, or under the submerged condition with 60% oxygen. Levels of surfactant proteins were analyzed by immunoblotting. Each condition is represented by two lanes from duplicate wells. This blot is representative of three independent experiments.