Amino Acid Availability Controls TRB3 Transcription in Liver through the GCN2/eIF2α/ATF4 Pathway

In mammals, plasma amino acid concentrations are markedly affected by dietary or pathological conditions. It has been well established that amino acids are involved in the control of gene expression. Up to now, all the information concerning the molecular mechanisms involved in the regulation of gene transcription by amino acid availability has been obtained in cultured cell lines. The present study aims to investigate the mechanisms involved in transcriptional activation of the TRB3 gene following amino acid limitation in mice liver. The results show that TRB3 is up-regulated in the liver of mice fed a leucine-deficient diet and that this induction is quickly reversible. Using transient transfection and chromatin immunoprecipitation approaches in hepatoma cells, we report the characterization of a functional Amino Acid Response Element (AARE) in the TRB3 promoter and the binding of ATF4, ATF2 and C/EBPβ to this AARE sequence. We also provide evidence that only the binding of ATF4 to the AARE plays a crucial role in the amino acid-regulated transcription of TRB3. In mouse liver, we demonstrate that the GCN2/eIF2α/ATF4 pathway is essential for the induction of the TRB3 gene transcription in response to a leucine-deficient diet. Therefore, this work establishes for the first time that the molecular mechanisms involved in the regulation of gene transcription by amino acid availability are functional in mouse liver

Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib

The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α–ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α–ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP–ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP–ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α–ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib

The transcription factor CHOP, an effector of the integrated stress response, is required for host sensitivity to the fungal intracellular pathogen <i>Histoplasma capsulatum</i>

<div><p>The ability of intracellular pathogens to manipulate host-cell viability is critical to successful infection. Some pathogens promote host-cell survival to protect their replicative niche, whereas others trigger host-cell death to facilitate release and dissemination of the pathogen after intracellular replication has occurred. We previously showed that the intracellular fungal pathogen <i>Histoplasma capsulatum</i> (<i>Hc</i>) uses the secreted protein Cbp1 to actively induce apoptosis in macrophages; interestingly, <i>cbp1</i> mutant strains are unable to kill macrophages and display severely reduced virulence in the mouse model of <i>Hc</i> infection. To elucidate the mechanism of Cbp1-induced host-cell death, we performed a comprehensive alanine scanning mutagenesis and identified all amino acid residues that are required for Cbp1 to trigger macrophage lysis. Here we demonstrate that <i>Hc</i> strains expressing lytic <i>CBP1</i> alleles activate the integrated stress response (ISR) in infected macrophages, as indicated by an increase in eIF2α phosphorylation as well as induction of the transcription factor <i>CHOP</i> and the pseudokinase <i>Tribbles 3</i> (<i>TRIB3</i>). In contrast, strains bearing a non-lytic allele of <i>CBP1</i> fail to activate the ISR, whereas a partially lytic <i>CBP1</i> allele triggers intermediate levels of activation. We further show that macrophages deficient for <i>CHOP</i> or <i>TRIB3</i> are partially resistant to lysis during <i>Hc</i> infection, indicating that the ISR is critical for susceptibility to <i>Hc</i>-mediated cell death. Moreover, we show that CHOP-dependent macrophage lysis is critical for efficient spread of <i>Hc</i> infection to other macrophages. Notably, <i>CHOP</i> knockout mice display reduced macrophage apoptosis and diminished fungal burden and are markedly resistant to <i>Hc</i> infection. Together, these data indicate that Cbp1 is required for <i>Hc</i> to induce the ISR and mediate a CHOP-dependent virulence pathway in the host.</p></div

Lytic <i>CBP1</i> alleles cause a decrease in phospho-Akt levels in infected macrophages.

<p>BMDMs were infected with the indicated <i>Hc</i> strains, and phospho-Akt (Thr308) and total Akt were assessed by Western blot at <b>(A)</b> 12 hpi and <b>(B)</b> 48 hpi. Representative blots are shown. The average signal intensity of phospho-Akt relative to total Akt from three replicates is shown in the bar graphs.*p<0.05, **p<0.01, compared to wildtype, two-tailed t-test.</p

Model of Cbp1-mediated host-cell death during <i>Hc</i> infection.

<p>During infection, <i>Hc</i> yeast produce a large amount of the secreted protein Cbp1 (red triangle). Cbp1 induces phosphorylation of the mammalian protein eIF2α through an unknown mechanism. The increase in phospho-eIF2 leads to the preferential translation of the transcription factor ATF4, which leads to the expression of <i>CHOP</i> and <i>TRIB3</i>. The pseudokinase Tribbles 3 inhibits Akt phosphorylation, thereby promoting Bax/Bak oligomerization, ultimately resulting in caspase-3/7 activation, which is enhanced by caspase-8 activation. The activation of the executioner caspases ultimately results in macrophage death, allowing for the release of live fungal cells.</p

CHOP is required for optimal spread of <i>Hc</i> during macrophage infection.

<p><b>(A)</b> Uninfected wildtype BMDMs were seeded in 6-well plates. Above them, transwells with wildtype or <i>CHOP</i>^<i>-/-</i> BMDMs, either uninfected or infected with wildtype <i>Hc</i>, were placed with the macrophages on the underside of the transwell. After the onset of lysis of the infected macrophages, the transwells were removed. The fungal burdens immediately after transwell removal <b>(B)</b> and lysis kinetics <b>(C)</b> of the bottom macrophages were assessed by CFU enumeration and LDH release assay, respectively. For CFUs, ***p<0.005 by two-tailed t-test; for macrophage lysis, *p<0.05 by two-tailed Mann-Whitney.</p

Lytic <i>CBP1</i> alleles activate the integrated stress response in infected macrophages.

<p>BMDMs were treated with 2.5 μg/mL tunicamycin (Tm), infected with indicated <i>Hc</i> strains at an MOI of 5, or mock infected (uninf). Phosphorylated eIF2α (Ser51) <b>(A)</b> and ATF4 <b>(B)</b> were assessed by Western blot at 12 hpi. Representative blots are shown. The average signal intensity of phospho-eIF2α or ATF4 relative to the corresponding α-tubulin loading control from three replicates is shown in the bar graphs.*p<0.05, **p<0.01, compared to wildtype, two-tailed t-test. <b>(C)</b> Relative abundances of <i>CHOP</i> and <i>TRIB3</i> transcripts were assessed by RT-qPCR at the indicated time points and normalized to uninfected BMDMs at 3 hpi.</p

The host genes <i>CHOP</i> and <i>TRIB3</i> are necessary for robust caspase-3/7 activity and macrophage death during <i>Hc</i> infection.

<p><b>(A)</b> Wildtype, <i>CHOP</i>^-/-, and <i>TRIB3</i>^-/- macrophages were mock infected or infected with wildtype <i>Hc</i> at an MOI of 5 or 10. Caspase-3/7 activity was measured 24 hpi and normalized to the activity in uninfected macrophages for each genotype. Each value is an average of triplicate wells ± standard deviation. *p<0.05, compared to wildtype, two-tailed t-test. <b>(B)</b> Wildtype, <i>CHOP</i>^-/-, and <i>TRIB3</i>^-/- BMDMs (MΦ) were infected with wildtype <i>Hc</i> at an MOI of 1 or mock infected (uninf), and macrophage lysis was measured by LDH release. BMDM lysis is presented as the percentage of total LDH in the supernatant and lysate of uninfected macrophages at 3 hpi. The lysis at each time point is an average of duplicate measurements of wells infected in triplicate, resulting in six total measurements, ± standard deviation.</p

<i>Hc</i> does not cause ER stress in infected BMDMs.

<p>BMDMs were infected with the indicated <i>Hc</i> strains at an MOI of 5, mock infected (uninf), or treated with 2.5 μg/mL tunicamycin (Tm). <b>(A)</b> Unspliced (<i>Xbp1u</i>) and spliced (<i>Xbp1s</i>) isoforms of <i>Xbp1</i> were detected by non-quantitative RT-PCR. <b>(B-D)</b> Relative abundances of <b>(B)</b> <i>ERdj4</i>, <b>(C)</b> <i>SEL1L</i>, and <b>(D)</b> <i>BiP</i> were measured by RT-qPCR. <b>(E)</b> Phosphorylated PERK (Thr 980) was detected by Western blot at 12 hpi. <b>(F)</b> BMDMs were treated with the PERK-specific inhibitor GSK2606414 (3 μM) or vehicle (DMSO) for 12 hpi, and caspase-3/7 activity was assessed and normalized to vehicle-treated uninfected cells. Each value is an average of triplicate wells ± standard deviation. **p<0.01, two-tailed t test.</p

<i>CHOP</i>^<i>-/-</i> mice are resistant to <i>Hc</i> infection.

<p><b>(A)</b> C57BL/6 and <i>CHOP</i>^<i>-/-</i> mice were mock infected (uninf) or infected with 1x10⁶ <i>Hc</i> yeast and the percentage of apoptotic (viability dye⁺ and caspase-3/7⁺) alveolar macrophages (CD45⁺ SiglecF⁺ CD11b^mid CD11c⁺ F4/80⁺) was measured at 3 dpi. *p<0.05, as determined by ANOVA. (B) CFUs from lungs and spleens of C57BL/6 and CHOP-/- mice infected with 3 x 10⁵ <i>Hc</i> yeast. *p<0.05, **p<0.005, as determined by independent 1-way ANOVA analyses of log transformed CFUs at each time point. (C) Kaplan-Meier survival curves of C57BL/6 and <i>CHOP</i>^-/- mice mock infected (uninf; n = 2) or infected with 1 x 10⁶ <i>Hc</i> yeast (n = 11). *p<0.05, logrank test.</p