Abstract
The amino acid response (AAR) and unfolded protein response (UPR) pathways converge on eIF2α phosphorylation, which is catalyzed by Gcn2 and Perk, respectively, under different stresses. This close interconnection makes it difficult to specify different functions of AAR and UPR. Here, we generated a zebrafish model in which loss of threonyl-tRNA synthetase (Tars) induces angiogenesis dependent on Tars aminoacylation activity. Comparative transcriptome analysis of the tars-mutant and wild-type embryos with/without Gcn2- or Perk-inhibition reveals that only Gcn2-mediated AAR is activated in the tars-mutants, whereas Perk functions predominantly in normal development. Mechanistic analysis shows that, while a considerable amount of eIF2α is normally phosphorylated by Perk, the loss of Tars causes an accumulation of uncharged tRNAThr, which in turn activates Gcn2, leading to phosphorylation of an extra amount of eIF2α. The partial switchover of kinases for eIF2α largely overwhelms the functions of Perk in normal development. Interestingly, although inhibition of Gcn2 and Perk in this stress condition both can reduce the eIF2α phosphorylation levels, their functional consequences in the regulation of target genes and in the rescue of the angiogenic phenotypes are dramatically different. Indeed, genetic and pharmacological manipulations of these pathways validate that the Gcn2-mediated AAR, but not the Perk-mediated UPR, is required for tars-deficiency induced angiogenesis. Thus, the interconnected AAR and UPR pathways differentially regulate angiogenesis through selective functions and mutual competitions, reflecting the specificity and efficiency of multiple stress response pathways that evolve integrally to enable an organism to sense/respond precisely to various types of stresses.
Highlights
Stress-induced angiogenesis contributes enormously to both normal development and pathogenesis of various diseases including cancer, metabolic, and cardiovascular disorders[1]
Among many stress response pathways implicated in the regulation of angiogenesis, the amino acid response (AAR)[2] and unfolded protein response (UPR) pathways[3,4] are closely interconnected, as they converge on the common target, eukaryotic initiation factor 2 subunit α, the key regulator of protein translation[5,6]
Our GSEA results showed that: (i) the KEGG_RNA_DEGRADATION gene set was significantly enriched in the upregulated genes by Perk knockdown in normal embryos (Supplementary Fig. S3a, left), whereas Gcn[2] knockdown (Supplementary Fig. S3a, middle) or tars knockout (Supplementary Fig. S3a, right) showed no significant enrichment; and (ii) when the Perk knockdown was performed in tars−/−, the enrichment of this gene set was completely abolished (Supplementary Fig. S3b). These results suggest that Perk mainly functions in homeostatic states but, in the stress condition its function might be largely overwhelmed by activation of the general control nonderepressible 2 (Gcn2)-mediated AAR
Summary
Stress-induced angiogenesis contributes enormously to both normal development and pathogenesis of various diseases including cancer, metabolic, and cardiovascular disorders[1]. To compare the role of AAR and UPR in tars-deficiency-induced angiogenesis, we designed an approach combining comparative gene expression profiling and quantitative phenotype analysis of zebrafish embryos with different genotypes and under specific treatments.
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