Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.

Cold-induced thermogenesis in endotherms demands adaptive thermogenesis fueled by mitochondrial respiration and Ucp1-mediated uncoupling in multilocular brown adipocytes (BAs). However, dietary regulation of thermogenesis in BAs isn't fully understood. Here, we describe that the deficiency of Leucine-rich pentatricopeptide repeat containing-protein (Lrpprc) in BAs reduces mtDNA-encoded ETC gene expression, causes ETC proteome imbalance, and abolishes the mitochondria-fueled thermogenesis. BA-specific Lrpprc knockout mice are cold resistant in a 4°C cold-tolerance test in the presence of food, which is accompanied by the activation of transcription factor 4 (ATF4) and proteome turnover in BAs. ATF4 activation genetically by BA-specific ATF4 overexpression or physiologically by a low-protein diet feeding can improve cold tolerance in wild-type and Ucp1 knockout mice. Furthermore, ATF4 activation in BAs improves systemic metabolism in obesogenic environment regardless of Ucp1's action. Therefore, our study reveals a diet-dependent but Ucp1-independent thermogenic mechanism in BAs that is relevant to systemic thermoregulation and energy homeostasis.

ATF4-mediated transcriptional regulation protects against ß-cell loss during endoplasmic reticulum stress in a mouse model.

OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.

Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.

OBJECTIVE: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. METHODS: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor, rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. CONCLUSIONS: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). OBJECTIVE: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. METHODS: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. RESULTS: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. CONCLUSIONS: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Identification of novel cerebellar developmental transcriptional regulators with motif activity analysis.

BACKGROUND: The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development. RESULTS: We used the HeliScopeCAGE library sequencing on cerebellar samples over 8 embryonic and 4 early postnatal times. This study showcases temporal expression pattern changes during cerebellar development. Through a bioinformatics analysis that focused on transcription factors, their promoters and binding sites, we identified genes that appear as strong candidates for involvement in cerebellar development. We selected several candidate transcriptional regulators for validation experiments including qRT-PCR and shRNA transcript knockdown. We observed marked and reproducible developmental defects in Atf4, Rfx3, and Scrt2 knockdown embryos, which support the role of these genes in cerebellar development. CONCLUSIONS: The successful identification of these novel gene regulators in cerebellar development demonstrates that the FANTOM5 cerebellum time series is a high-quality transcriptome database for functional investigation of gene regulatory networks in cerebellar development.

ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides.

BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

ATF4 Regulates CD4+ T Cell Immune Responses through Metabolic Reprogramming.

T cells are strongly regulated by oxidizing environments and amino acid restriction. How T cells reprogram metabolism to adapt to these extracellular stress situations is not well understood. Here, we show that oxidizing environments and amino acid starvation induce ATF4 in CD4+ T cells. We also demonstrate that Atf4-deficient CD4+ T cells have defects in redox homeostasis, proliferation, differentiation, and cytokine production. We further reveal that ATF4 regulates a coordinated gene network that drives amino acid intake, mTORC1 activation, protein translation, and an anabolic program for de novo synthesis of amino acids and glutathione. ATF4 also promotes catabolic glycolysis and glutaminolysis and oxidative phosphorylation and thereby provides precursors and energy for anabolic pathways. ATF4-deficient mice mount reduced Th1 but elevated Th17 immune responses and develop more severe experimental allergic encephalomyelitis (EAE). Our study demonstrates that ATF4 is critical for CD4+ T cell-mediated immune responses through driving metabolic adaptation.

MIF-2/D-DT enhances proximal tubular cell regeneration through SLPI- and ATF4-dependent mechanisms.

Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions that is produced by several organs and cell types. Depending on the target cell and the inflammatory context, MIF can engage its two component receptor complex CD74 and CD44 and the chemokine receptors CXCR2/4. MIF is constitutively expressed in renal proximal tubular cells, stored in intracellular preformed pools, and released at a low rate. Recently, a second MIF-like protein (i.e., MIF-2/D-DT) has been characterized in mammals. Our study was aimed at examining the role of MIF-2/D-DT, which mediates tissue protection in the heart, in tubular cell regeneration from ischemia-reperfusion injury. We found that Mif-/-, Mif-2-/-, and Cd74-/- mice had significantly worse tubular injury compared with wild-type (WT) control mice and that treatment with MIF-2/D-DT significantly improved recovery of injured epithelial cells. RNAseq analysis of kidney tissue from the ischemia-reperfusion injury model revealed that MIF-2/D-DT treatment stimulates secretory leukocyte proteinase inhibitor (SLPI) and cyclin D1 expression. MIF-2/D-DT additionally activates of eukaryotic initiation factor (eIF) 2α and activating transcription factor (ATF) 4, two transcription factors involved in the integrated stress response (ISR), which is a cellular stress response activated by hypoxia, nutrient deprivation, and oxygen radicals. MIF-2/D-DT also inhibited apoptosis and induced autophagy in hypoxia-treated mouse proximal tubular (MPT) cells. These results indicate that MIF-2/D-DT is an important factor in tubular cell regeneration and may be of therapeutic utility as a regenerative agent in the clinical setting of ischemic acute kidney injury.

Double minute amplification of mutant PDGF receptor α in a mouse glioma model.

In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf(-/-) background, mutant animals frequently developed brain tumors resembling anaplastic human gliomas (WHO grade III). Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.

The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness.

Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4-/- littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4-/- mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4-/- mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness.

ATF4 deficiency protects hepatocytes from oxidative stress via inhibiting CYP2E1 expression.

Activating transcription factor (ATF) 4 is involved in the regulation of oxidative stress in fibroblasts and neurons. The role of ATF4 in hepatocytes, however, is unknown. The aim of this study was to investigate the role of ATF4 in hepatocytes in oxidative stress under a high-fat diet (HFD). Here, we showed that palmitate-stimulated reactive oxygen species (ROS) production and triglyceride (TG) accumulation is blocked by ATF4 deficiency in primary hepatocytes. Consistently, HFD-induced oxidative stress, TG accumulation and expression of cytochrome P450, family 2, subfamily, polypeptide 1 (CYP2E1) are also blocked by knocking down ATF4 expression in the mouse liver. This suggests that ATF4 might regulate oxidative stress via CYP2E1 under an HFD. In addition, we observed that expression of CYP2E1 is indirectly regulated by ATF4 in a cAMP-responsive element binding protein (CREB)-dependent manner, which can directly activate the CYP2E1 promoter activity. Notably, ATF4-stimulated ROS production is inhibited in vivo by treatment with diallyl sulphide, a selective CYP2E1 inhibitor. Finally, we showed that ATF4 expression in the liver is responsible for the protective effects against HFD-induced CYP2E1 expression, oxidative stress, and TG accumulation. Taken together, these observations suggest that ATF4 is a novel regulator of oxidative stress as well as accumulation of TG in response to HFD.

Functional contribution of the transcription factor ATF4 to the pathogenesis of amyotrophic lateral sclerosis.

Endoplasmic reticulum (ER) stress represents an early pathological event in amyotrophic lateral sclerosis (ALS). ATF4 is a key ER stress transcription factor that plays a role in both adaptation to stress and the activation of apoptosis. Here we investigated the contribution of ATF4 to ALS. ATF4 deficiency reduced the rate of birth of SOD1(G86R) transgenic mice. The fraction of ATF4(-/-)-SOD1(G85R) transgenic mice that were born are more resistant to develop ALS, leading to delayed disease onset and prolonged life span. ATF4 deficiency completely attenuated the induction of pro-apoptotic genes, including BIM and CHOP, and also led to quantitative changes in the ER protein homeostasis network. Unexpectedly, ATF4 deficiency enhanced mutant SOD1 aggregation at the end stage of the disease. Studies in the motoneuron cell line NSC34 demonstrated that knocking down ATF4 enhances mutant SOD1 aggregation possibly due to alteration in the redox status of the cell. Our results support a functional role of ATF4 in ALS, offering a novel target for disease intervention.

Lipopolysaccharide preconditioning protects hepatocytes from ischemia/reperfusion injury (IRI) through inhibiting ATF4-CHOP pathway in mice.

BACKGROUND: Low-dose lipopolysaccharide (LPS) preconditioning-induced liver protection has been demonstrated during ischemia-reperfusion injury (IRI) in several organs but has not been sufficiently elucidated underlying causal mechanism. This study investigated the role of low-dose LPS preconditioning on ATF4-CHOP pathway as well as the effects of the pathway on tissue injury and inflammation in a mouse model of liver partial-warm IRI. METHODS: LPS (100 µg/kg/d) was injected intraperitoneally two days before ischemia. Hepatic injury was evaluated based on serum alanine aminotransferase levels, histopathology, and caspase-3 activity. The ATF4-CHOP pathway and its related apoptotic molecules were investigated after reperfusion. The role of LPS preconditioning on apoptosis and ATF4-CHOP pathway was examined in vitro. Moreover, the effects of the ATF4-CHOP pathway on apoptosis, Caspase-12, and Caspase-3 were determined with ATF4 small interfering RNA (siRNA). Inflammatory cytokine expression was also checked after reperfusion. Inflammatory cytokines and related signaling pathways were analyzed in vitro in macrophages treated by LPS preconditioning or ATF4 siRNA. RESULTS: LPS preconditioning significantly attenuated liver injury after IRI. As demonstrated by in vitro experiments, LPS preconditioning significantly reduced the upregulation of the ATF4-CHOP pathway and inhibited Caspase-12 and Caspase-3 activation after IRI. Later experiments showed that ATF4 knockdown significantly suppressed CHOP, cleaved caspase-12 and caspase-3 expression, as well as inhibited hepatocellular apoptosis. In addition, in mice pretreated with LPS, TNF-α and IL-6 were inhibited after reperfusion, whereas IL-10 was upregulated. Similarly, low-dose LPS significantly inhibited TNF-α, IL-6, ATF4-CHOP pathway, NF-κB pathway, and ERK1/2 in high-dose LPS-stimulated macrophages, whereas IL-10 and cytokine signaling (SOCS)-3 suppressor were induced. Importantly, ATF4 siRNA is consistent with results of LPS preconditioning in macrophages. CONCLUSIONS: This work is the first time to provide evidence for LPS preconditioning protects hepatocytes from IRI through inhibiting ATF4-CHOP pathway, which may be critical to reducing related apoptosis molecules and modulating innate inflammation.

ATF4 promotes bone angiogenesis by increasing VEGF expression and release in the bone environment.

Activating transcription factor 4 (ATF4) is a critical transcription factor for bone remodeling; however, its role in bone angiogenesis has not been established. Here we show that ablation of the Atf4 gene expression in mice severely impaired skeletal vasculature and reduced microvascular density of the bone associated with dramatically decreased expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in osteoblasts located on bone surfaces. Results from in vivo studies revealed that hypoxia/reoxygenation induction of HIF-1α and VEGF expression leading to bone angiogenesis, a key adaptive response to hypoxic conditions, was severely compromised in mice lacking the Atf4 gene. Loss of ATF4 completely prevented endothelial sprouting from embryonic metatarsals, which was restored by addition of recombinant human VEGF protein. In vitro studies revealed that ATF4 promotion of HIF-1α and VEGF expression in osteoblasts was highly dependent upon the presence of hypoxia. ATF4 interacted with HIF-1α in hypoxic osteoblasts, and loss of ATF4 increased HIF-1α ubiquitination and reduced its protein stability without affecting HIF-1α mRNA stability and protein translation. Loss of ATF4 increased the binding of HIF-1α to prolyl hydroxylases, the enzymes that hydroxylate HIF-1a protein and promote its proteasomal degradation via the pVHL pathway. Furthermore, parathyroid hormone-related protein (PTHrP) and receptor activator of NF-κB ligand (RANKL), both well-known activators of osteoclasts, increased release of VEGF from the bone matrix and promoted angiogenesis through the protein kinase C- and ATF4-dependent activation of osteoclast differentiation and bone resorption. Thus, ATF4 is a new key regulator of the HIF/VEGF axis in osteoblasts in response to hypoxia and of VEGF release from bone matrix, two critical steps for bone angiogenesis.

Activating transcription factor 4 regulates stearate-induced vascular calcification.

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate.

Platelet-derived growth factor-BB mediates cell migration through induction of activating transcription factor 4 and tenascin-C.

The acute response to vascular cell injury, which underpins vasculo-occlusive pathologies such as atherogenesis and restenosis after percutaneous coronary intervention, involves a complex series of molecular events that alter patterns of gene expression and favor a synthetic phenotype. One transcription factor that has been implicated in this process is the evolutionarily conserved mammalian stress response pathway regulator activating transcription factor 4 (ATF-4). Here, we show for the first time that both mRNA and protein levels of ATF-4 are induced in smooth muscle cells (SMCs) by the potent migratory factor PDGF-BB through PDGFR-ß. PDGF-BB also stimulates the expression of tenascin-C (TN-C), an extracellular matrix glycoprotein that regulates the activity of focal adhesion complexes, facilitating the SMC migration that underlies negative vascular remodeling in response to injury. Overexpression of ATF-4 increased transcript levels of the four TN-C isoforms in rat vascular SMCs, and ATF-4 knockdown inhibited PDGF-BB-inducible TN-C expression in vitro and injury-inducible TN-C protein expression in the balloon-injured rat artery wall. Furthermore, we show that ATF-4 is required for PDGF-BB-inducible SMC migration in response to injury. PDGF-BB-induced migration was also compromised in ATF-4 null mEFs, and this effect was rescued by the addition of TN-C. Our findings thus demonstrate the role of ATF-4 in both injury- and PDGF-BB-inducible TN-C expression and cell migration.

Activation of the unfolded protein response enhances motor recovery after spinal cord injury.

Spinal cord injury (SCI) is a major cause of paralysis, and involves multiple cellular and tissular responses including demyelination, inflammation, cell death and axonal degeneration. Recent evidence suggests that perturbation on the homeostasis of the endoplasmic reticulum (ER) is observed in different SCI models; however, the functional contribution of this pathway to this pathology is not known. Here we demonstrate that SCI triggers a fast ER stress reaction (1-3 h) involving the upregulation of key components of the unfolded protein response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR transcription factors, leads to a reduced locomotor recovery after experimental SCI. The effects of UPR inactivation were associated with a significant increase in the number of damaged axons and reduced amount of oligodendrocytes surrounding the injury zone. In addition, altered microglial activation and pro-inflammatory cytokine expression were observed in ATF4 deficient mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition.

ATF4 deficiency protects mice from high-carbohydrate-diet-induced liver steatosis.

Chronic feeding of HCD (high-carbohydrate diet) is one of the major contributors to the prevailing of metabolic diseases. ATF4 (activating transcription factor 4) has been shown to play an important role in the regulation of glucose metabolism and obesity development; however, it is unclear how ATF4(-/-) mice respond to HCD. In the present study, we show that 8 weeks of HCD results in significant higher accumulation of TAGs (triacylglycerols) in livers and impairment in glucose tolerance in ATF4(+/+) mice, but not in ATF4(-/-) mice, compared with those on a normal diet. Meanwhile, energy expenditure is further enhanced by HCD in ATF4(-/-) mice. Moreover, we show that ATF4 deficiency suppresses HCD-induced SCD1 (stearoyl-CoA desaturase 1) expression, furthermore, oral supplementation of the main product of SCD1 oleate (18:1) increases TAG accumulation in livers of ATF4(-/-) mice. Taken together, these results suggest that ATF4 deficiency is protective for HCD-induced hepatic steatosis and impairment of glucose tolerance and insulin sensitivity. Furthermore, the resistance to hepatic steatosis is at least in part due to suppression of SCD1 expression under HCD.

Negative regulation of IRF7 activation by activating transcription factor 4 suggests a cross-regulation between the IFN responses and the cellular integrated stress responses.

Cells react to viral infection by exhibiting IFN-based innate immune responses and integrated stress responses, but little is known about the interrelationships between the two. In this study, we report a linkage between these two host-protective cellular mechanisms. We found that IFN regulatory factor (IRF)7, the master regulator of type I IFN gene expression, interacts with activating transcription factor (ATF)4, a key component of the integrated stress responses whose translation is induced by viral infection and various stresses. We have demonstrated that IRF7 upregulates ATF4 activity and expression, whereas ATF4 in return inhibits IRF7 activation, suggesting a cross-regulation between the IFN response and the cellular integrated stress response that controls host innate immune defense against viral infection.

The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts.

The recent demonstration that osteoblasts have a role in controlling energy metabolism suggests that they express cell-specific regulatory genes involved in this process. Activating transcription factor 4 (ATF4) is a transcription factor that accumulates predominantly in osteoblasts, where it regulates virtually all functions linked to the maintenance of bone mass. Since Atf4-/- mice have smaller fat pads than littermate controls, we investigated whether ATF4 also influences energy metabolism. Here, we have shown, through analysis of Atf4-/- mice, that ATF4 inhibits insulin secretion and decreases insulin sensitivity in liver, fat, and muscle. Several lines of evidence indicated that this function of ATF4 occurred through its osteoblastic expression. First, insulin sensitivity is enhanced in the liver of Atf4-/- mice, but not in cultured hepatocytes from these mice. Second, mice overexpressing ATF4 in osteoblasts only [termed here alpha1(I)Collagen-Atf4 mice] displayed a decrease in insulin secretion and were insulin insensitive. Third, the alpha1(I)Collagen-Atf4 transgene corrected the energy metabolism phenotype of Atf4-/- mice. Fourth, and more definitely, mice lacking ATF4 only in osteoblasts presented the same metabolic abnormalities as Atf4-/- mice. Molecularly, ATF4 favored expression in osteoblasts of Esp, which encodes a product that decreases the bioactivity of osteocalcin, an osteoblast-specific secreted molecule that enhances secretion of and sensitivity to insulin. These results provide a transcriptional basis to the observation that osteoblasts fulfill endocrine functions and identify ATF4 as a regulator of most functions of osteoblasts.