Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury

Abstract

Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11-induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O(2) caused TUNEL(+) cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O(2). In A1-null mice, IL-11-induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11- and VEGF-induced cytoprotection.

Figure 1

Regulation of A1 by IL-11 and 100% O₂. (A) RPA was used to assess the levels of mRNA encoding A1 and related proteins in whole-lung mRNA from 2-month-old Tg(–) and CC10-IL-11 Tg(+) mice. (B) RPA was used to assess the levels of mRNA encoding A1 and related proteins in lungs from WT mice exposed to 100% O₂ for the noted intervals (hours). The noted experiments are representative of a minimum of 4 similar evaluations. P, multiprobe template.

Figure 2

Role of A1 in hyperoxia. (A) Two-month-old WT mice (black squares) and IL-11 Tg(+) mice with (+/+) (black circles), (+/–) (gray circles), and (–/–) (white circles) A1 loci were exposed to 100% O₂, and survival was assessed. The noted values represent the survival of a minimum of 8 animals at each time point. *P < 0.05; **P < 0.01. (B) Two-month-old Tg(–) mice with (+/+) (black squares), (+/–) (gray squares), and (–/–) (white squares) A1 loci were exposed to 100% O₂, and survival was assessed. The noted values represent the survival of a minimum of 8 animals at each time point. *P < 0.05; **P < 0.01.

Figure 3

Role of A1 in hyperoxia-induced TUNEL⁺ cell death and DNA injury. Tg(–) and Tg(+) mice with (+/+) and (–/–) A1 loci were exposed for 72 hours to room air (white bars) or 100% O₂ (black bars), and TUNEL evaluations were undertaken. The TUNEL evaluations in A–D are representative of a minimum of 9 similar evaluations. The arrows highlight representative TUNEL⁺ cells. The values in E are the mean ± SEM of evaluations in a minimum of 9 mice. *P < 0.05; **P < 0.01.

Figure 4

Effects of A1 on pulmonary caspases. Tg(–) and Tg(+) mice with (+/+) and (–/–) A1 loci were incubated in room air or 100% O₂ for 72 hours. The levels of caspase mRNA (real-time RT-PCR) (A), caspase bioactivity (B), and caspase protein (Western blot) (C) were assessed. The values in A and B represent the mean ± SEM of evaluations in a minimum of 5 mice. C is representative of a minimum of 4 similar evaluations. *P < 0.05; **P < 0.01.

Figure 5

Effects of A1 on cell death regulators. Tg(–) and Tg(+) mice with (+/+) and (–/–) A1 loci were incubated in room air or 100% O₂ for 72 hours. The noted moieties were evaluated by Western blot. Each panel is representative of a minimum of 4 similar evaluations.

Figure 6

Effects of hyperoxia and rIL-11 on epithelial cell A1 in vitro. MLE cells were incubated under normoxic or hyperoxic (95% O₂) conditions (A and B) or in the presence or absence of rIL-11 (25 ng/ml) (C and D) for the noted periods of time, and the levels of mRNA encoding A1 (A and C) and the levels of A1 protein (B and D) were assessed by Northern and Western blot, respectively. The panels are representative of a minimum of 4 similar evaluations.

Figure 7

A1 regulation of hyperoxia-induced apoptosis and necrosis in vitro. MLE cells were stably transfected with an expression construct alone (pcDNA3.1) (vector) or the same construct containing cDNA encoding A1a (A1a-pcDNA3.1) or A1d (A1d-pcDNA3.1). The cells were incubated under normal conditions (5% CO₂ and air) or under hyperoxic conditions (95% O₂ and 5% CO₂). (A) Annexin V and propidium iodide (PI) staining are analyzed with FACS to compare the apoptosis and necrosis induced by 72 hours in 95% O₂. (B) We compare the caspase activities in these cells before (0 hours) and at intervals after the start of 95% O₂ exposure. The FACS diagrams are representative of a minimum of 4 similar evaluations. The values in B are the mean ± SEM of evaluations in a minimum of 5 mice. *P < 0.05; **P < 0.01.

Figure 8

Induction of A1 and its role in VEGF cytoprotection. Lungs were obtained from Tg(–) and VEGF Tg(+) mice on doxycycline water for 1 month. (A) A1 mRNA was quantitated by RPA. (B) A1 protein was evaluated by Western blot analysis. (C) The survival of Tg(–) mice (open squares) and VEGF Tg(+) mice with (+/+) (filled diamonds) and (–/–) (open diamonds) A1 loci in 100% O₂. A and B are representative of a minimum of 4 similar evaluations. The values in C illustrate the survival of a minimum of 6 mice at each time point. P, multiprobe template. **P < 0.05.