Activating Transcription Factor 4 Knockout Mice Research Articles

Intestinal activating transcription factor 4 regulates stress-related behavioral alterations via paraventricular thalamus in male mice

Chronic stress induces depression- and anxiety-related behaviors, which are common mental disorders accompanied not only by dysfunction of the brain but also of the intestine. Activating transcription factor 4 (ATF4) is a stress-induced gene, and we previously show that it is important for gut functions; however, the contribution of the intestinal ATF4 to stress-related behaviors is not known. Here, we show that chronic stress inhibits the expression of ATF4 in gut epithelial cells. ATF4 overexpression in the colon relieves stress-related behavioral alterations in male mice, as measured by open-field test, elevated plus-maze test, and tail suspension test, whereas intestine-specific ATF4 knockout induces stress-related behavioral alterations in male mice. Furthermore, glutamatergic neurons are inhibited in the paraventricular thalamus (PVT) of two strains of intestinal ATF4-deficient mice, and selective activation of these neurons alleviates stress-related behavioral alterations in intestinal ATF4-deficient mice. The highly expressed gut-secreted peptide trefoil factor 3 (TFF3) is chosen from RNA-Seq data from ATF4 deletion mice and demonstrated decreased in gut epithelial cells, which is directly regulated by ATF4. Injection of TFF3 reverses stress-related behaviors in ATF4 knockout mice, and the beneficial effects of TFF3 are blocked by inhibiting PVT glutamatergic neurons using DREADDs. In summary, this study demonstrates the function of ATF4 in the gut-brain regulation of stress-related behavioral alterations, via TFF3 modulating PVT neural activity. This research provides evidence of gut signals regulating stress-related behavioral alterations and identifies possible drug targets for the treatment of stress-related behavioral disorders.

ATF4 activation promotes hepatic mitochondrial dysfunction by repressing NRF1–TFAM signalling in alcoholic steatohepatitis

ObjectiveMitochondrial dysfunction plays a dominant role in the pathogenesis of alcoholic liver disease (ALD); however, the underlying mechanisms remain to be fully understood. We previously found that hepatic activating transcription...

Liver-specific Gene Inactivation of the Transcription Factor ATF4 Alleviates Alcoholic Liver Steatosis in Mice

Although numerous biological functions of the activating transcription factor 4 (ATF4) have been identified, a direct effect of ATF4 on alcoholic liver steatosis has not been described previously. The aim of our current study is to investigate the role of ATF4 in alcoholic liver steatosis and elucidate the underlying mechanisms. Here, we showed that the expression of ATF4 is induced by ethanol in hepatocytes in vitro and in vivo, and liver-specific ATF4 knock-out mice are resistant to ethanol-induced liver steatosis, associated with stimulated hepatic AMP-activated protein kinase (AMPK) activity. Furthermore, adenovirus-mediated AMPK knockdown significantly reversed the suppressive effects of ATF4 deficiency on ethanol-induced liver steatosis in mice. In addition, ethanol-fed ATF4 knock-out mice exhibit AMPK-dependent inhibition of fatty acid synthase and stimulation of carnitine palmitoyltransferase 1 (CPT1) in the liver. Moreover, hepatic Tribbles homolog 3 (TRB3) expression was stimulated by ethanol in an ATF4-dependent manner, and adenovirus-mediated TRB3 knockdown blocked ATF4-dependent ethanol-induced AMPK inhibition and triglyceride accumulation in AML-12 cells. Finally, TRB3 directly interacted with AMPK to suppress its phosphorylation. Taken together, these results identify the ATF4-TRB3-AMPK axis as a novel pathway responsible for ethanol-induced liver steatosis.

ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells.

BackgroundMonocyte chemoattractant protein-1 (MCP-1) is a major chemokine that recruits monocyte/macrophage to the site of tissue injury and plays a critical role in microvascular complications of diabetes. However, the mechanisms underlying the regulation of MCP-1 are not fully understood. The present study aims to explore the role of activating transcription factor 4 (ATF4), an ER stress-inducible transcription factor, in regulation of MCP-1 expression and production in brain and retinal microvascular endothelial cells.MethodsFor in vitro study, primary brain microvascular endothelial cells isolated from ATF4 knockout mice or mouse retinal endothelial cells were treated with lipopolysaccharide (LPS) to induce MCP-1 expression. ATF4 expression/function was manipulated by adenoviruses expressing wild type ATF4 (Ad-ATF4) or a dominant negative mutant of the protein (Ad-ATF4DN). For in vivo study, MCP-1 expression was induced by intravitreal injection of LPS or Ad-ATF4 in heterozygous ATF4 knockout or wild type mice.ResultsLPS treatment induced a dose- and time-dependent increase in ATF4 expression, ER stress and MCP-1 production in brain and retinal microvascular endothelial cells. Overexpression of ATF4 in endothelial cells significantly increased the secretion of MCP-1 and promoted THP-1 monocyte-endothelial cell adhesion. Conditioned medium from ATF4-overexpressiing endothelial cells significantly enhanced THP-1 cell migration. Consistently, intravitreal injection of Ad-ATF4 remarkably enhanced retinal levels of MCP-1 and promoted inflammatory cell infiltration into the vitreous and retina. In contrast, LPS-induced MCP-1 upregulation was markedly attenuated in ATF4-deficient endothelial cells and in retinas of ATF4 knockout mice, suggesting that ATF4 is essential for LPS-induced MCP-1 production in endothelial cells and in the retina. Mechanistically, overexpression of ATF4 enhanced, while inhibition of ATF4, attenuated the basal and LPS-stimulated phosphorylation of NF-κB, P38, and JNK. Furthermore, pharmacological inhibition of NF-κB, P38, or JNK significantly reduced ATF4-stimulated MCP-1 secretion from endothelial cells.ConclusionsTaken together, our results suggest a critical role of ATF4 in the regulation of MCP-1 production in retinal and brain microvascular endothelial cells, which may contribute to inflammation-related endothelial injury in diseases such as diabetic retinopathy.

Specific downregulation of hippocampal ATF4 reveals a necessary role in synaptic plasticity and memory.

Prior studies suggested that the transcription factor ATF4 negatively regulates synaptic plastic and memory. By contrast, we provide evidence from direct in vitro and in vivo knockdown of ATF4 in rodent hippocampal neurons and from ATF4-null mice that implicate ATF4 as essential for normal synaptic plasticity and memory. In particular, hippocampal ATF4 downregulation produces deficits in long-term spatial memory and behavioral flexibility without affecting associative memory or anxiety-like behavior. ATF4 knockdown or loss also causes profound impairment of both long-term potentiation (LTP) and long-term depression (LTD) as well as decreased glutamatergic function. We conclude that ATF4 is a key regulator of the physiological state necessary for neuronal plasticity and memory.

MicroRNA-214 Suppresses Gluconeogenesis by Targeting Activating Transcriptional Factor 4

Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis.

General Transcription Factor IIA-γ Increases Osteoblast-specific Osteocalcin Gene Expression via Activating Transcription Factor 4 and Runt-related Transcription Factor 2

ATF4 (activating transcription factor 4) is an osteoblast-enriched transcription factor that regulates terminal osteoblast differentiation and bone formation. ATF4 knock-out mice have reduced bone mass (severe osteoporosis) throughout life. Runx2 (runt-related transcription factor 2) is a runt domain-containing transcription factor that is essential for bone formation during embryogenesis and postnatal life. In this study, we identified general transcription factor IIA gamma (TFIIA gamma) as a Runx2-interacting factor in a yeast two-hybrid screen. Immunoprecipitation assays confirmed that TFIIA gamma interacts with Runx2 in osteoblasts and when coexpressed in COS-7 cells or using purified glutathione S-transferase fusion proteins. Chromatin immunoprecipitation assay of MC3T3-E1 (clone MC-4) preosteoblast cells showed that in intact cells TFIIA gamma is recruited to the region of the osteocalcin promoter previously shown to bind Runx2 and ATF4. A small region of Runx2 (amino acids 258-286) was found to be required for TFIIA gamma binding. Although TFIIA gamma interacts with Runx2, it does not activate Runx2. Instead, TFIIA gamma binds to and activates ATF4. Furthermore, TFIIA gamma together with ATF4 and Runx2 stimulates osteocalcin promoter activity and endogenous mRNA expression. Small interfering RNA silencing of TFIIA gamma markedly reduces levels of endogenous ATF4 protein and Ocn mRNA in osteoblastic cells. Overexpression of TFIIA gamma increases levels of ATF4 protein. Finally, TFIIA gamma significantly prevents ATF4 degradation. This study shows that a general transcription factor, TFIIA gamma, facilitates osteoblast-specific gene expression through interactions with two important bone transcription factors ATF4 and Runx2.