GCN2 kinase activation mediates pulmonary vascular remodeling and pulmonary arterial hypertension
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
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Research Article Vascular biology

GCN2 kinase activation mediates pulmonary vascular remodeling and pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart failure. Recessive mutations of EIF2AK4 gene (encoding general control nonderepressible 2 kinase, GCN2) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients but rarely in patients with PAH. The role of GCN2 kinase activation in the pathogenesis of PAH remains unclear. Here, we show that GCN2 was hyperphosphorylated and activated in pulmonary vascular endothelial cells (ECs) of hypoxic mice, monocrotaline-treated rats, and patients with idiopathic PAH. Unexpectedly, loss of GCN2 kinase activity in Eif2ak4–/– mice with genetic disruption of the kinase domain induced neither PVOD nor pulmonary hypertension (PH) but inhibited hypoxia-induced PH. RNA-sequencing analysis suggested endothelin-1 (Edn1) as a downstream target of GCN2. GCN2 mediated hypoxia-induced Edn1 expression in human lung ECs via HIF-2α. Restored Edn1 expression in ECs of Eif2ak4–/– mice partially reversed the reduced phenotype of hypoxia-induced PH. Furthermore, GCN2 kinase inhibitor A-92 treatment attenuated PAH in monocrotaline-treated rats. These studies demonstrate that GCN2 kinase activation mediates pulmonary vascular remodeling and PAH at least partially through Edn1. Thus, targeting GCN2 kinase activation is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss-of-function mutations.

Authors

Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao

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Figure 3

Hypoxia induces GCN2 phosphorylation and activation.

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Hypoxia induces GCN2 phosphorylation and activation. (A) Representative ...
(A) Representative micrographs of anti–phospho-GCN2 immunostaining showing GCN2 hyperphosphorylation by hypoxia challenge in primary cultures of HLMVECs. Fixed cells at indicated times after hypoxia (1% O2) challenge (Hx) or normoxia (Nx) were immunostained with anti-Thr899 phospho-GCN2 (red). Nuclei were counterstained with DAPI (blue). (B) Quantification of cytoplasmic over nuclear GCN2-Pi at 2-hour and 4-hour hypoxia exposure. N = 5/group. (C–G) Western blotting demonstrating hypoxia-induced GCN2 phosphorylation and activation. HLMVECs were lysed for Western blotting with anti-Thr899 phospho-GCN2 (GCN2-Pi) antibody and anti-Ser51 phospho-EIF2α (EIF2α-Pi) antibody. Total GCN2 and EIF2α levels were assessed by anti-GCN2 antibody and anti-EIF2α antibody, respectively, while anti–β-actin was used as a loading control (C). The band intensities of phospho-GCN2 (GCN2-Pi) and phospho-EIF2α (EIF2α-Pi) were quantified (D–G). (H–J) Western blotting demonstrating hypoxia-induced GCN2 phosphorylation was inhibited by PDK1 inhibitor (GSK, GSK2334470) treatment in a dose-dependent manner. HLMVECs in complete growth medium were treated with GSK2334470 at 10 or 1 μM or control vehicle under 2-hour hypoxia challenge (1% O2). Then cells were lysed for Western blotting with anti-Thr899 phospho-GCN2 (GCN2-Pi) antibody. Total GCN2 levels were assessed by anti-GCN2 antibody while anti-tubulin was used as a loading control (H). The band intensities of phospho-GCN2 were quantified (I and J). N = 3 repeat studies. Data are shown as means + SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Unpaired 2-tailed t test (B); 1-way ANOVA with Dunnett’s multiple comparisons test (D–G), with Holm-Šídák multiple comparisons test (I), or with Tukey’s multiple comparisons test (J).