Mariko Umemura

Regulation of the human CHOP gene promoter by the stress response transcription factor ATF5 via the AARE1 site in human hepatoma HepG2 cells.

AIMS Activating transcription factor (ATF) 5 is a member of the cAMP response element-binding protein (CREB)/ATF family of transcription factors. We have shown that ATF5 is a stress response transcription factor that responds to amino acid limitation, arsenite exposure, or cadmium exposure. In this study we investigated whether ATF5 is involved in the regulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) gene expression. MAIN METHODS We used a transient transfection system to express ATF5 and analyzed the regulation of CHOP gene promoter in human hepatoma, HepG2 cells. We also studied the effect of ATF5 knockdown on arsenite-induced CHOP protein expression and arsenite-induced cell death of HepG2 cells. KEY FINDINGS We showed that ATF5 activates the CHOP gene promoter in HepG2 cells. Both deletion analysis and point mutations of the promoter revealed that amino acid response element (AARE) 1 is responsible for ATF5-dependent promoter activation. Furthermore, the existence of either AARE1 or activating protein-1 (AP-1) site is sufficient for transcriptional activation of the CHOP gene promoter by arsenite exposure, although complete induction requires the existence of both elements. We also demonstrated that knockdown of ATF5 reduced arsenite-induced CHOP protein expression and arsenite-induced cell death of HepG2 cells. SIGNIFICANCE These results suggested that the CHOP gene is a potential target for ATF5, and that ATF5 raises the arsenite-induced CHOP gene expression level via the AARE1 site in HepG2 cells.

The 5′‐untranslated region regulates ATF5 mRNA stability via nonsense‐mediated mRNA decay in response to environmental stress

We previously reported that activating transcription factor 5 (ATF5) mRNA increases in response to amino acid limitation, and that this increase is dependent on mRNA stabilization. The ATF5 gene allows transcription of mRNAs with two alternative 5′‐UTRs, 5′‐UTRα and 5′‐UTRβ, derived from exon 1α and exon 1β. 5′‐UTRα contains the upstream open reading frames uORF1 and uORF2. Phosphorylation of eukaryotic initiation factor 2α during the integrated stress response had been previously shown to lead to bypassing of uORF2 translation and production of ATF5 protein. Translation of uORF2 is expected to result in translational termination at a position 125 nucleotides upstream of the exon junction, and this fits the criterion of a nonsense‐mediated decay target mRNA. We investigated the potential role of 5′‐UTRα in the control of mRNA stabilization, and found that 5′‐UTRα reduced the stability of ATF5 mRNA. 5′‐UTRα‐regulated destabilization of mRNA was suppressed by knockdown of the nonsense‐mediated decay factors Upf1 and Upf2. Mutation of the downstream AUG (uAUG2) rendered mRNA refractory to Upf1 and Upf2 knockdown. Moreover, 5′‐UTRα‐regulated down‐regulation was hindered by amino acid limitation and tunicamycin treatment, and stress‐induced phosphorylation of eukaryotic initiation factor 2α was involved in stabilization of ATF5 mRNA. These studies show that ATF5 mRNA is a naturally occurring normal mRNA target of nonsense‐mediated decay, and provide evidence for linkage between stress‐regulated translational regulation and the mRNA decay pathway. This linkage constitutes a mechanism that regulates expression of stress response genes.

Phenolic Diterpenes from Rosemary Suppress cAMP Responsiveness of Gluconeogenic Gene Promoters

The cAMP/protein kinase A/cAMP response element (CRE)‐binding protein pathway is important for various physiological aspects including regulation of gluconeogenic gene expression. Rosemary, a well‐known herb, has been reported to decrease blood glucose levels. We found that methanol extracts of rosemary suppressed forskolin (FSK)‐stimulated luciferase expression under the control of CRE, as well as the promoters for cytosolic phosphoenolpyruvate carboxykinase (PEPCK‐C) and glucose‐6‐phosphatase (G6Pase) catalytic subunit genes in human hepatoma HepG2 cells. Three abietane‐type diterpenes and two flavonoids were isolated from the rosemary extracts. Among these, 7‐O‐methylrosmanol (1) and royleanonic acid (3) effectively suppressed FSK‐induced luciferase expression under the control of the CRE, PEPCK‐C and G6Pase gene promoters. PEPCK‐C and G6Pase, which play a key role in the homeostatic regulation of blood glucose levels, are important for managing type II diabetes mellitus. Therefore, the ability of rosemary and its components to suppress cAMP responsiveness of the PEPCK‐C or G6Pase gene may contribute to its antihyperglycemic activity. Copyright

Activating transcription factor 5 (ATF5) is essential for the maturation and survival of mouse basal vomeronasal sensory neurons

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of transcription factors, which is highly expressed in olfactory chemosensory tissues, the main olfactory epithelium and vomeronasal epithelium (VNE) in mice. The vomeronasal sensory neurons in the VNE detect pheromones in order to regulate social behaviors such as mating and aggression; however, the physiological role of ATF5 in the vomeronasal sensory system remains unknown. In this study, we found that the differentiation of mature vomeronasal sensory neurons, assessed by olfactory marker protein expression, was inhibited in ATF5-deficient VNE. In addition, many apoptotic vomeronasal sensory neurons were evident in ATF5-deficient VNE. The vomeronasal sensory neurons consist of two major types of neuron expressing either vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo. We demonstrated that the differentiation, survival and axonal projection of V2r/Gαo-type rather than V1r/Gαi2-type vomeronasal sensory neurons were severely inhibited in ATF5-deficient VNE. These results suggest that ATF5 is one of the transcription factors crucial for the vomeronasal sensory formation.

Activating transcription factor 5 is required for mouse olfactory bulb development via interneuron

Activating transcription factor 5 (ATF5) is a stress response transcription factor of the cAMP-responsive element-binding/ATF family. Earlier, we reported that ATF5 expression is up-regulated in response to stress, such as amino acid limitation or arsenite exposure. Although ATF5 is widely expressed in the brain and the olfactory epithelium, the role of ATF5 is not fully understood. Here, the olfactory bulbs (OBs) of ATF5-deficient mice are smaller than those of wild-type mice. Histological analysis reveals the disturbed laminar structure of the OB, showing the thinner olfactory nerve layer, and a reduced number of interneurons. This is mainly due to the reduced number of bromodeoxyuridine-positive proliferating cells in the subventricular zone, where the interneuron progenitors are formed and migrate to the OBs. Moreover, the olfaction-related aggressive behavior of ATF5-deficient mice is reduced compared to wild-type mice. Our data suggest that ATF5 plays a crucial role in mouse OB development via interneuron. Graphical abstract ATF5-deficient mice (ATF5-/-) have smaller olfactory bulbs (OBs) showing the thinner olfactory nerve layer and a reduced number of interneurons.

Comprehensive Behavioral Analysis of Activating Transcription Factor 5-Deficient Mice

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/-) mice demonstrated abnormal olfactory bulb development due to impaired interneuron supply. Furthermore, ATF5-/- mice were less aggressive than ATF5+/+ mice. Although ATF5 is widely expressed in the brain, and involved in the regulation of proliferation and development of neurons, the physiological role of ATF5 in the higher brain remains unknown. Our objective was to investigate the physiological role of ATF5 in the higher brain. We performed a comprehensive behavioral analysis using ATF5-/- mice and wild type littermates. ATF5-/- mice exhibited abnormal locomotor activity in the open field test. They also exhibited abnormal anxiety-like behavior in the light/dark transition test and open field test. Furthermore, ATF5-/- mice displayed reduced social interaction in the Crawley’s social interaction test and increased pain sensitivity in the hot plate test compared with wild type. Finally, behavioral flexibility was reduced in the T-maze test in ATF5-/- mice compared with wild type. In addition, we demonstrated that ATF5-/- mice display disturbances of monoamine neurotransmitter levels in several brain regions. These results indicate that ATF5 deficiency elicits abnormal behaviors and the disturbance of monoamine neurotransmitter levels in the brain. The behavioral abnormalities of ATF5-/- mice may be due to the disturbance of monoamine levels. Taken together, these findings suggest that ATF5-/- mice may be a unique animal model of some psychiatric disorders.

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB.

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK‐C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element‐binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea MAXIM. var. kiushiana Makino was found to suppress cAMP‐enhanced PEPCK‐C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin‐stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK‐C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright

N-terminal hydrophobic amino acids of activating transcription factor 5 (ATF5) protein confer interleukin 1β (IL-1β)-induced stabilization.

Background: The N-terminal region of ATF5 is responsible for its CdCl2- and NaAsO2-induced expression. Results: IL-1β stabilizes ATF5 protein and elevates the translation efficiency of ATF5 mRNA. Conclusion: The N-terminal hydrophobic amino acids of ATF5 are important for protein stabilization and responsiveness to IL-1β. Significance: This study provides new insights about the roles of ATF5 in immune response. Activating transcription factor 5 (ATF5) is a stress-response transcription factor that responds to amino acid limitation and exposure to cadmium chloride (CdCl2) and sodium arsenite (NaAsO2). The N-terminal amino acids contribute to the destabilization of the ATF5 protein in steady-state conditions and serve as a stabilization domain in the stress response after CdCl2 or NaAsO2 exposure. In this study, we show that interleukin 1β (IL-1β), a proinflammatory cytokine, increases the expression of ATF5 protein in HepG2 hepatoma cells in part by stabilizing the ATF5 protein. The N-terminal domain rich in hydrophobic amino acids that is predicted to form a hydrophobic network was responsible for destabilization in steady-state conditions and served as an IL-1β response domain. Furthermore, IL-1β increased the translational efficiency of ATF5 mRNA via the 5′ UTRα and phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). ATF5 knockdown in HepG2 cells up-regulated the IL-1β-induced expression of the serum amyloid A 1 (SAA1) and SAA2 genes. Our results show that the N-terminal hydrophobic amino acids play an important role in the regulation of ATF5 protein expression in IL-1β-mediated immune response and that ATF5 is a negative regulator for IL-1β-induced expression of SAA1 and SAA2 in HepG2 cells.