Gene expression and integrated stress response in HepG2/C3A cells cultured in amino acid deficient medium

Abstract

The integrated stress response (ISR), a defense mechanism cells employ when under stress (e.g., amino acid deprivation), causes suppression of global protein synthesis along with the paradoxical increased expression of a host of proteins that are useful in combating various stresses. Genes that were similarly differentially expressed under conditions of either leucine- or cysteine-depletion were identified. Many of the genes known to contain an amino acid response element and to be induced in response to eIF2α phosphorylation and ATF4 heterodimer binding (ATF3, C/EBPβ, SLC7A1, SLC7A11, and TRIB3), as well as others shown to be induced downstream of eIF2α phosphorylation (C/EBPγ, CARS, SARS, CLCN3, CBX4, and PPP1R15A) were among the upregulated genes. Evidence for the induction of the ISR in these cells also included the increased phosphorylation of eIF2α and increased protein abundance of ATF4, ATF3, and ASNS in cysteine- and leucine-depleted cells. Based on genes highly differentially expressed in both leucine- and cysteine-deficient cells, a list of 67 downregulated and 53 upregulated genes is suggested as likely targets of essential amino acid deprivation in mammalian cells.

Autophagy modulates amino acid signaling network in myotubes: differential effects on mTORC1 pathway and the integrated stress response

“…The amino acid store can also be mobilized by the autophagy-lysosome and ubiquitin-proteasome proteolytic systems (10,11,26), and amino acid starvation potently induces the integrated stress response in various cell models to activate the amino acid transcriptional program (15)(16)(17). However, it remains to be demonstrated whether autophagy differentially regulates the mTORC1 pathway and the integrated stress response.…”

“…Although numerous studies have shown that ER stress activates autophagy to mitigate cellular stress (19,20), our results revealed that autophagy is required to prevent the integrated stress response. Mammalian cells activate the integrated stress response to cope with various metabolic challenges including amino acid starvation and ER stress, and eIF2a plays a central role in mediating the translational and transcriptional adaptations of this stress response (15)(16)(17). Phosphorylation of eIF2a diminishes general protein translation but paradoxically increases translation of selected mRNA species, including the transcription factor ATF4, which in turn induces the expression of multiple genes involved in amino acid transport and synthesis (18).…”

“…Numerous translational and transcriptional adaptations of the integrated stress response are regulated by eIF2a, including the expression of genes involved in amino acid transport and synthesis (15)(16)(17). Given that autophagy inhibition consistently activated eIF2a, regardless of amino acid availability (Figs.…”

“…For example, general control nonderepressible 2 is activated by amino acid starvation, whereas protein kinase-like endoplasmic reticulum (ER) kinase is activated by ER stress (17). The stress kinases in turn phosphorylate eukaryotic initiation factor 2a (eIF2a), and the collective effects of eIF2a activation are termed the integrated stress response (16). Phosphorylation of eIF2a leads to inhibition of general protein synthesis but paradoxically increases the translation of selective mRNA such as ATF4 (18).…”

“…Another system that integrates amino acid balance with cell physiology is the integrated stress response network (15)(16)(17). In this system, specific kinases are activated in response to different cellular stress.…”