TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4

Abstract

Tribbles homolog 3 (TRB3) is a pseudokinase the level of which is increased in response to various stresses. We and other researchers have previously shown that TRB3 interacts with activating transcription factor 4 (ATF4) and may function as a negative feedback regulator of ATF4. In the present study, we investigate the effect of ATF4 and TRB3 on cell growth and viability, using both the enforced expression and silencing of the genes. HEK293 cells overexpressing ATF4 show retarded growth in the complete medium and decreased viability in the glucose-free medium. The enforced expression of ATF4 increases the level of reactive oxygen species (ROS) and the supplementation of the medium with ROS scavenging and reducing compounds supports the growth and survival of cells overexpressing ATF4. The deleterious effects of elevated ATF4 are suppressed by the coexpression of TRB3, which downregulates ATF4 transcriptional activity and results in the decrease of intracellular ROS. Also, the coexpression of TRB3 rescues postmitotic neuronally differentiated PC12 cells from the apoptosis evoked by ATF4 overexpression. The silencing of ATF4 and TRB3 genes by RNA interference reveals that endogenous ATF4 promotes and TRB3 suppresses the death of glucose-deprived SaOS2 cells. Together, the results indicate that TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4.

Introduction

TRB3 (also known as NIPK, SKIP3, TRIB3 and SINK) was first described as a protein encoded by a gene which is activated during the neuronal cell death induced by nerve growth factor deprivation and the exposure to calcium ionophore A23187 [1]. Further studies have revealed that TRB3 expression is upregulated in many cell types in response to various stresses, including nutrient deficiency, endoplasmic reticulum stress, hypoxia and oxidative stress [2], [3], [4], [5], [6], [7], [8], [9], and that TRB3 is overexpressed in multiple primary tumors and cancer cell lines [5], [10]. TRB3, as well as Drosophila protein Tribbles and its two other mammalian homologs, TRB1 and TRB2, belong to a group of proteins named pseudokinases or kinase-like proteins, because they contain a region with a considerable similarity to subdomains VI-XI of the common catalytic core structure of the protein kinase family [11], but due to strong deviations from the kinase active site consensus sequence, it is likely that Tribbles and its homologs have no kinase activity [12], [13], [14]. TRB3 interacts with a number of proteins and has been implicated in several processes. TRB3 associates with the E3 ubiquitin ligase COP1 and recruits it to acetyl-coenzyme A carboxylase (ACC), triggering the degradation of ACC and stimulating lipolysis [15]. It has been reported that TRB3 is involved in the regulation of insulin signal transduction [16], [17] and muscle differentiation [18] by binding to serine–threonine kinase Akt/PKB and blocking its activation. However, recent articles question the role of TRB3 in the insulin signaling pathway [19], [20]. Kiss-Toth et al. [21], [22] have reported that TRB3 controls mitogen-activated protein kinase (MAPK) cascades by binding to MAPK kinases, and Selim et al. [23] have suggested a role for TRB3 in the cell cycle arrest and the cellular depletion of lymphocytes. We and other researchers have shown that TRB3 interacts with ATF4 and inhibits its transcriptional activation activity, and that ATF4 participates in the transcriptional upregulation of TRB3 [3], [4], [5], [6]. Therefore, TRB3 may serve as a negative feedback regulator of ATF4 [4], [6]. Recently, Jousse et al. [24], studying the regulation of gene expression by amino acid limitation, have reached the same conclusion.

ATF4, a basic leucine-zipper transcription activator, carries out a variety of functions. It plays a major role in the control of gene expression during the integrated stress response (ISR) [25], [26] and is crucial for the long-term memory formation [27], the proliferation of fetal hematopoietic progenitors [28], the differentiation of osteoblasts [29] and the survival of lens cells at the embryonic stage [30], [31]. The ISR is activated by divergent cellular stresses (protein malfunction in endoplasmic reticulum, amino acid starvation, viral infection, heme deficiency) through the signaling pathways that converge on a single event—phosphorylation of the translation initiation factor eIF2α, resulting in general translational pause with selective increase in ATF4 mRNA translation and the subsequent stimulation of expression of ATF4 target genes [26]. The ISR facilitates the cells to adapt to the stresses, as revealed by the increased sensitivity of cells with inactivated ISR signaling to glucose starvation [32] and hypoxia [33], and ATF4-deficient cells to amino acid depletion and oxidative stress [25]. However, the target genes of ATF4 that are upregulated in the conditions of stress also include in addition to cytoprotective genes (such as heme oxygenase-1 [34]) genes known to be proapoptotic (such as CHOP [35]), suggesting that ATF4 may contribute to the initiation of cell death program.

In the present paper, we characterize the growth and viability properties of ATF4 and its negative regulator TRB3 in proliferating and postmitotic cells. The results indicate that the uncontrolled or excessive expression of ATF4 is growth inhibitory or cytotoxic, and that the coexpression of TRB3 suppresses the deleterious effect of ATF4.