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 4

Hypoxia induces Edn1 expression through GCN2 in both mouse lungs and human lung ECs.

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Hypoxia induces Edn1 expression through GCN2 in both mouse lungs and hum...
(A) Representative heatmap of RNA-sequencing analysis of mouse lung tissues (n = 4 mice combined per group). Nx, normoxia; Hx, hypoxia. (B) Western blotting demonstrating Edn1 protein levels were upregulated in lung tissues of hypoxic WT mice, which were significantly reduced in hypoxic KO mice. (C) Quantitative RT-PCR analysis verifying Edn1 mRNA upregulation in WT hypoxia mouse lungs but not in KO hypoxia mouse lungs. N = 5–8/group. (D) Quantitative RT-PCR analysis demonstrating GCN2 siRNA–mediated (siGCN2-mediated) knockdown of GCN2 in HLMVECs. N = 5/group. CTL, control siRNA. (E) Quantitative RT-PCR analysis demonstrating hypoxia-induced EDN1 mRNA expression was mediated by GCN2 in HLMVECs. N = 6/group. (F) ELISA of EDN1 secreted in culture medium demonstrating hypoxia-induced EDN1 protein expression was reduced by GCN2 silencing in HLMVECs. N = 6/group. (G) Western blotting confirmation of reduced HIF-2α expression in GCN2-deficient HLMVECs compared with control cells under hypoxia exposure. (H) Quantitative RT-PCR analysis demonstrating inhibited HIF2A mRNA expression in GCN2-deficient HLMVECs. Control n = 6, siGCN2 n = 8. (I) Quantitative RT-PCR analysis demonstrating GCN2 mediated hypoxia-induced EDN1 expression through HIF-2α rather than HIF-1α. HLMVECs were transfected with either siGCN2 or control siRNA (CtlsiRNA), and HIF1A, HIF2A, or vector plasmid, and then challenged with hypoxia or maintained in normoxia. At 48 hours after hypoxia challenge or normoxia, the cells were lysed for RNA isolation for quantitative RT-PCR analysis. N = 6–10/group. Data are shown as means + SD. *, P < 0.05; ****, P < 0.0001. Unpaired 2-tailed t test (D and H); 2-way ANOVA with Tukey’s multiple comparisons test (C, E, and F); 1-way ANOVA with Tukey’s multiple comparisons test (I).