Activating Transcription Factor Research Articles

Navigating the landscape of the unfolded protein response in CD8+ T cells.

Endoplasmic reticulum stress occurs due to large amounts of misfolded proteins, hypoxia, nutrient deprivation, and more. The unfolded protein is a complex intracellular signaling network designed to operate under this stress. Composed of three individual arms, inositol-requiring enzyme 1, protein kinase RNA-like ER kinase, and activating transcription factor-6, the unfolded protein response looks to resolve stress and return to proteostasis. The CD8+ T cell is a critical cell type for the adaptive immune system. The unfolded protein response has been shown to have a wide-ranging spectrum of effects on CD8+ T cells. CD8+ T cells undergo cellular stress during activation and due to environmental insults. However, the magnitude of the effects this response has on CD8+ T cells is still understudied. Thus, studying these pathways is important to unraveling the inner machinations of these powerful cells. In this review, we will highlight the recent literature in this field, summarize the three pathways of the unfolded protein response, and discuss their roles in CD8+ T cell biology and functionality.

Role and mechanism of ginsenoside Rg1 in ameliorating sepsis-induced acute lung injury based on PERK/eIF2α/ATF4/CHOP-induced alveolar epithelial cell apoptosis

The study investigates the therapeutic effects and mechanisms of ginsenoside Rg_1(GRg_1) on sepsis-induced acute lung injury(SALI). A murine model of SALI was created using cecal ligation and puncture(CLP) surgery, and mice were randomly assigned to groups for GRg_1 intervention. Survival and body weight changes were recorded, lung function was assessed with a non-invasive lung function test system, and lung tissue damage was evaluated through HE staining. The content and expression of inflammatory factors were measured by ELISA and qRT-PCR. Apoptosis was examined using flow cytometry and TUNEL staining. The activation and expression of apoptosis-related molecules cysteinyl aspartate specific proteinase 3(caspase-3), B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax), and endoplasmic reticulum stress-related molecules protein kinase R-like endoplasmic reticulum kinase(PERK), eukaryotic initiation factor 2α(eIF2α), activating transcription factor 4(ATF4), and C/EBP homologous protein(CHOP) were studied using Western blot and qRT-PCR. In addition, an in vitro model of lipopolysaccharide(LPS)-induced lung alveolar epithelial cell injury was used, with the application of the endoplasmic reticulum stress inducer tunicamycin to validate the action mechanism of GRg_1. RESULTS:: indicated that, when compared to the model group, GRg_1 intervention significantly enhanced the survival time of CLP mice, mitigated body weight loss, and improved impaired lung function indices. The GRg_1-treated mice also displayed reduced lung tissue pathological scores, a reduced lung tissue wet-to-dry weight ratio, and lower protein content in the bronchoalveolar lavage fluid. Serum levels of interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α), as well as the mRNA expressions of these cytokines in lung tissues, were decreased. There was a notable decrease in the proportion of apopto-tic alveolar epithelial cells, and down-regulated expressions of caspase-3, Bax, PERK, eIF2α, ATF4, and CHOP and up-regulated expression of Bcl-2 were observed. In vitro findings showed that the apoptosis-lowering and apoptosis-related protein down-regulating effects of GRg_1 were significantly inhibited with the co-application of tunicamycin. Altogether, GRg_1 reduces apoptosis of alveolar epithelial cells, inhibits inflammation in the lungs, alleviates lung injury, and enhances lung function, possibly through the PERK/eIF2α/ATF4/CHOP pathway.

C-phycocyanin reinforces autophagy to block pulmonary fibrogenesis by inhibiting lncIAPF biogenesis

Pulmonary fibrosis is a chronic and irreversible progressive lung disease caused by various factors, such as age and environmental pollution. With countries stepping into an aging society and the seriousness of environmental pollution caused by global industrialization, the incidence of pulmonary fibrosis is annually increasing. However, no effective drug is available for pulmonary fibrosis treatment. C-phycocyanin (C-PC), extracted from blue-green algae, has good water solubility and antioxidation. This study elucidated that C-PC reinforces autophagy to block pulmonary fibrogenesis by inhibiting long noncoding RNA (lncRNA) biogenesis in vivo and in vitro. Cleavage under targets and release using nuclease (CUT & RUN)-PCR, co-immunoprecipitation (Co-IP), and nuclear–cytoplasmic separation experiments clarified that C-PC blocked the nuclear translocation of activating transcription factor 3 (ATF3) to prevent the binding between ATF3 and transcription factor Smad3, thereby hindering lncIAPF transcription. Human antigen R (HuR) truncation experiment and RNA binding protein immunoprecipitation (RIP) were then performed to identify the binding domain with lncIAPF in the 244–322 aa of HuR. lncIAPF exerted its profibrogenic function through the binding protein HuR, a negative regulator of autophagy. In summary, C-PC promoted autophagy via down-regulating the lncIAPF–HuR-mediated signal pathway to alleviate pulmonary fibrosis, showing its potential as a drug for treating pulmonary fibrosis. Exploring how C-PC interacts with biological molecules will help us understand the mechanism of this drug and provide valuable target genes to design new drugs.

Tetrandrine targeting SIRT5 exerts anti-melanoma properties via inducing ROS, ER stress, and blocked autophagy

Tetrandrine (TET), a natural bisbenzyl isoquinoline alkaloid extracted from Stephania tetrandra S. Moore, has diverse pharmacological effects. However, its effects on melanoma remain unclear. Cellular proliferation assays, multi-omics analyses, and xenograft models were used to determine the effect of TET on melanoma. The direct target of TET was identified using biotin-TET pull-down liquid chromatograph-mass spectrometry (LC-MS), cellular thermal shift assays, and isothermal titration calorimetry (ITC) analysis. Our findings revealed that TET treatment induced robust cellular autophagy depending on activating transcription factor 6 (ATF6)-mediated endoplasmic reticulum (ER) stress. Simultaneously, it hindered autophagic flux by inducing cytoskeletal protein depolymerization in melanoma cells. TET treatment resulted in excessive accumulation of reactive oxygen species (ROS) and simultaneously triggered mitophagy. Sirtuin 5 (SIRT5) was ultimately found to be a direct target of TET. Mechanistically, TET led to the degradation of SIRT5 via the ubiquitin (Ub)-26S proteasome system. SIRT5 knockdown induced ROS accumulation, whereas SIRT5 overexpression attenuated the TET-induced ROS accumulation and autophagy. Importantly, TET exhibited anti-cancer effects in xenograft models depending on SIRT5 expression. This study highlights the potential of TET as an antimelanoma agent that targets SIRT5. These findings provide a promising avenue for the use of TET in melanoma treatment and underscore its potential as a therapeutic candidate.

ATF5-Mediated Mitochondrial Unfolded Protein Response (UPRmt) Protects Neurons Against Oxygen-Glucose Deprivation and Cerebral Ischemia.

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved mitochondrial response that is critical for maintaining mitochondrial and energetic homeostasis under cellular stress after tissue injury and disease. Here, we ask whether UPRmt may be a potential therapeutic target for ischemic stroke. We performed the middle cerebral artery occlusion and oxygen-glucose deprivation models to mimic ischemic stroke in vivo and in vitro, respectively. Oligomycin and meclizine were used to trigger the UPRmt. We used 2,3,5-triphenyltetrazolium chloride staining, behavioral tests, and Nissl staining to evaluate cerebral injury in vivo. The Cell Counting Kit-8 assay and the Calcein AM Assay Kit were conducted to test cerebral injury in vitro. Inducing UPRmt with oligomycin protected neuronal cultures against oxygen-glucose deprivation. UPRmt could also be triggered with meclizine, and this Food and Drug Administration-approved drug also protected neurons against oxygen-glucose deprivation. Blocking UPRmt with siRNA against activating transcription factor 5 eliminated the neuroprotective effects of meclizine. In a mouse model of focal cerebral ischemia, pretreatment with meclizine was able to induce UPRmt in vivo, which reduced infarction and improved neurological outcomes. These findings suggest that the UPRmt is important in maintaining the survival of neurons facing ischemic/hypoxic stress. The UPRmt mechanism may provide a new therapeutic avenue for ischemic stroke.

Establishing the link between D-mannose and juvenile grass carp (Ctenopharyngodon idella): Improved growth and intestinal structure associated with endoplasmic reticulum stress, mitophagy, and apical junctional complexes

D-mannose, essential for protein glycosylation, has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis. In addition to evaluating growth performance, we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine. All 1800 grass carp (16.20 ± 0.01 g) were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose (0.52, 1.75, 3.02, 4.28, 5.50 and 6.78 g/kg diet) for 70 d. The study revealed that D-mannose increased feed intake (P < 0.001) but did not affect the percent weight gain (PWG), special growth rate, and feed conversion ratio (P > 0.05). D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value (P < 0.05). D-mannose supplementation at 4.28 g/kg increased intestinal length, intestinal weight and fold height of grass carp compared to the control group (P < 0.05). This improvement may be attributed to the phosphomannose isomerase (PMI)-mediated enhancement of glycolysis. This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content (P < 0.05) and improved cellular and intercellular structures for the first time. The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) signaling pathways. The alleviation of ER stress may be linked to the phosphomannomutase (PMM)-mediated enhancement of protein glycosylation. In addition, ubiquitin-dependent [PTEN-induced putative kinase 1 (PINK1)/Parkin] and ubiquitin-independent [BCL2-interacting protein 3-like (BNIP3L), BCL2-interacting protein 3 (BNIP3), and FUN14 domain containing 1 (FUNDC1)] mitophagy may play a role in maintaining cellular redox homeostasis. The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures, which may be closely associated with the small Rho GTPase protein (RhoA)/the Rho-associated protein kinase (ROCK) signaling pathway. In conclusion, D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways, and enhanced intercellular structures related to tight junctions and adherent junctions. Furthermore, quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg, respectively.

Activating transcription factor 6 contributes to cisplatin‑induced ototoxicity via regulating the unfolded proteins response

As a broad-spectrum anticancer drug, cisplatin is widely used in the treatment of tumors in various systems. Unfortunately, several serious side effects of cisplatin limit its clinical application, the most common of which are nephrotoxicity and ototoxicity. Studies have shown that cochlear hair cell degeneration is the main cause of cisplatin-induced hearing loss. However, the mechanism of cisplatin-induced hair cell death remains unclear. The present study aimed to explore the potential role of activating transcription factor 6 (ATF6), an endoplasmic reticulum (ER)-localized protein, on cisplatin-induced ototoxicity in vivo and in vitro. In this study, we observed that cisplatin exposure induced apoptosis of mouse auditory OC-1 cells, accompanied by a significant increase in the expression of ATF6 and C/EBP homologous protein (CHOP). In cell or cochlear culture models, treatment with an ATF6 agonist, an ER homeostasis regulator, significantly ameliorated cisplatin-induced cytotoxicity. Further, our in vivo experiments showed that subcutaneous injection of an ATF6 agonist almost completely prevented outer hair cell loss and significantly alleviated cisplatin-induced auditory brainstem response (ABR) threshold elevation in mice. Collectively, our results revealed the underlying mechanism by which activation of ATF6 significantly improved cisplatin-induced hair cell apoptosis, at least in part by inhibiting apoptosis signal-regulating kinase 1 expression, and demonstrated that pharmacological activation of ATF6-mediated unfolded protein response is a potential treatment for cisplatin-induced ototoxicity.

The Prognostic Biomarker RAB7A Promotes Growth and Metastasis of Liver Cancer Cells by Regulating Glycolysis and YAP1 Activation.

Activating transcription factor 6 (ATF6) and its downstream genes are involved in progression of hepatocellular carcinoma (HCC). Herein, we demonstrated that sulfhydration of Ras-related protein Rab-7a (RAB7A) was regulated by ATF6. High expression of RAB7A indicated poor prognosis of HCC patients. RAB7A overexpression contributed to proliferation, colony formation, migration, and invasion of HepG2 and Hep3B cells. Furthermore, we found that RAB7A enhanced aerobic glycolysis in HepG2 cells, indicating a higher degree of tumor malignancy. Mechanistically, RAB7A suppressed Yes-associated protein 1 (YAP1) binding to 14-3-3 and conduced to YAP1 nuclear translocation and activation, promoting its downstream gene expression, thereby promoting growth and metastasis of liver cancer cells. In addition, knocking down RAB7A attenuated the progression of orthotopic liver tumors in mice. These findings illustrate the important role of RAB7A in regulating HCC progression. Thus, RAB7A may be a potential innovative target for HCC treatment.

Beta-adrenergic stimulation promotes an endoplasmic reticulum stress-dependent inflammatory program in salivary gland epithelial cells.

The effect of beta-adrenergic stimulation on human labial minor salivary gland epithelial cells (LMSGEC) on IL-6 production and its dependency on endoplasmic reticulum (ER) stress were investigated. Primary LMSGEC from Sjögren's syndrome (SS) patients and controls in culture were stimulated with epinephrine and IL-6 expression was evaluated by qPCR and ELISA. The expression of β-ARs in cultured LMSGEC was tested by qPCR, while adrenoceptors and cAMP levels were examined in LMSGs by immunofluorescence. ER evaluation was performed by transmission electron microscopy (TEM) and ER stress by western blot. Epinephrine-induced IL-6 production by cultured LMSGEC was evaluated after alleviation of the ER stress by applying tauroursodeoxycholic acid (TUDCA) and silencing of PKR-like ER kinase (PERK) and activating transcription factor 4 (ATF4) RNAs. Expression of IL-6 by LMSGEC was upregulated after β-adrenergic stimulation, while the silencing of adrenoreceptors downregulated IL-6. The amelioration of ER stress, as well as the silencing of PERK/ATF4, prevented epinephrine-induced upregulation of IL-6. Adrenergic stimulation led to higher and sustained IL-6 levels secreted by LMSGEC of SS patients compared to controls. Adrenergic signaling was endogenously enhanced in LMSGEC of SS patients (expression of β-ARs in situ, intracellular cAMP in cultured LMSGEC). In parallel, SS-LMSGEC expressed dilated ER (TEM) and higher levels of GRP78/BiP. PERK/ATF4 pathway of the ER stress emerged as a considerable mediator of adrenergic stimulation for IL-6 production by the LMSGEC. An enhanced endogenous adrenergic activation and a stressed ER observed in SS-LMSGEC may contribute to a sustained IL-6 production by these cells after adrenergic stimulation.

ATF4 Contributes to Abdominal Aortic Aneurysm Formation via Modulating M1 Macrophage Polarization and Inflammation.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular disease primarily in the male elderly population, but there is a lack of approved medical therapies to prevent the progression and rupture of AAA. Activating Transcription Factor 4 (ATF4) has been established to be involved in cardiovascular diseases, such as heart failure and calcific aortic valve disease. However, the role of ATF4 in the pathogenesis of AAA remains unclear. We found that ATF4 expression was significantly increased in patients with AAA and mouse models of AAA and was mainly confined to macrophages in arteries. ATF4 knockdown significantly attenuated aneurysm formation in experimental mouse model of AAA, while ATF4 overexpression promoted the development of AAA. RNA sequencing suggested that ATF4 was strongly related to the biological function of acute inflammatory response. Macrophages-specific ATF4 knockout significantly reduced the incidence and development of AAA, and decreased M1 polarization of macrophages in mice. Sphingomyelin phosphodiesterase 3 (SMPD3), a regulator of inflammatory responses in monocytes/macrophages, has been identified as a target gene of ATF4 through RNA sequencing, ChIP sequencing, and standard ChIP analyses. ATF4 induces M1 polarization of macrophages through the activation of SMPD3, thereby promoting inflammatory responses. Together, these results suggest that ATF4 mediated macrophage M1 polarization by regulating the expression of target genes SMPD3, leading to an increased inflammatory response, which further promotes the formation and development of AAA. These findings suggest ATF4 may be a new therapeutic target for AAA.

Dietary threonine influences antioxidant capacity and immune status in juvenile largemouth bass (Micropterus salmoides)

As a restricted amino acid, threonine (Thr) deficiency reduces antioxidant and immune capacity in fish. In the present study, six diets containing different Thr levels (1.39 %, 1.71 %, 1.95 %, 2.28 %, 2.58 %, and 2.65 %) were fed to largemouth bass (15±0.1 g) for 60 days. The present results showed that neither growth nor whole-body composition was affected by different Thr levels. And low dietary Thr levels (1.39 %) significantly decreased serum total protein (TP) and albumin (ALB) levels, but increased alkaline phosphatase (ALP) levels. And 1.39 % Thr diet significantly decreased liver catalase (CAT) activity but increased malonaldehyde (MDA) levels, and the maximum total antioxidant capacity (T-AOC) was observed in response to the 1.95 % Thr diet. Liver proapoptotic factors (bax and caspase9) were significantly elevated in fish fed the 1.39 % Thr diet. The nuclear factor κB (nf-κb) and the NF-κB-mediated inflammatory cytokines were significantly increased in fish fed the 1.39 % Thr diet. The mRNA levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (perk), eukaryotic translation initiation factor 2 (eif2α), activating transcription factor 4 (atf4), and C/EBP-homologous protein (chop), which are factors in the PERK/ATF4/CHOP pathway, one of the unfolded protein response (UPR) signaling pathways, were significantly increased by the 1.39 % Thr diet. The present study showed that low dietary Thr levels caused oxidative damage, reduced immunity, might affected PERK-ATF4-CHOP signaling to induce apoptosis, and via NF-κB signaling to trigger an inflammatory response in largemouth bass. Based on the TP, ALB, CAT, and MDA levels, the dietary Thr requirements for juvenile largemouth bass were estimated to be 2.18 %, 2.29 %, 1.92 %, and 1.88 %. Furthermore, these findings provided a new perspective that low dietary amino acid levels may trigger apoptosis and inflammatory responses in fish through the UPR signaling pathway.

Asperuloside inhibits the activation of pancreatic cancer-associated fibroblasts via activating transcription factor 6

BackgroundPancreatic cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression and immune evasion. Asperuloside (ASP) is an iridoid glycoside with potential anti-tumor properties. This study aimed to explore the molecular mechanisms of ASP on CAFs, particularly focusing on its effects on activating transcription factor 6 (ATF6), a key regulator of endoplasmic reticulum stress.MethodCAFs were treated with different concentrations of ASP (0, 1, 3, and 5 mM), and the role of ATF6 was investigated by over-expressing it in CAFs. Subsequently, western blot was used to detect ATF6, α-smooth muscle actin (α-SMA), fibroblast activating protein (FAP), and vimentin protein levels in CAFs. The collagen gel contraction assay and Transwell assay were applied to evaluate the contraction and migration ability of CAFs. In addition, the interleukin (IL)-6, C–C motif chemokine ligand (CCL)-2, and C-X-C motif chemokine ligand (CXCL)-10 levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).ResultsCAFs had significantly higher expression levels of α-SMA, FAP, and vimentin compared to normal fibroblasts (NFs). ASP significantly inhibited the activation, contraction, and migration of CAFs in a concentration-dependent manner. ASP treatment also reduced the expression of cytokines (IL-6, CCL2, and CXCL10) and down-regulated ATF6 levels. Over-expression of ATF6 mitigated the inhibitory effects of ASP.ConclusionASP exerts its anti-tumor effects by down-regulating ATF6, thereby inhibiting the activation and function of pancreatic CAFs. These findings suggest that ASP could be a promising therapeutic agent for pancreatic cancer by modulating the tumor microenvironment.

Potential mechanism of sea cucumber gut peptides in protecting ethanol-induced gastric mucosal injury

Alcoholic gastric injury is a prevalent condition that can result in gastric ulcers. This research delves into the protective effects of sea cucumber gut peptides, Val-Thr-Pro-Tyr (VTPY) and Val-Leu-Leu-Tyr (VLLY), on ethanol-induced gastric injury. These peptides significantly alleviate the pathological symptoms of gastric tissue affected by ethanol, reducing ulcer area and index, epithelial cell loss, submucosal edema, and inflammatory infiltration. High doses of VTPY and VLLY resulted in a significant decrease in ulcer index, with reductions from 5.38 to 2.63 and 1.13, respectively (P < 0.05). Moreover, the levels of P–NF-κB/p65 expression decreased by 33.47% and 54.27%, while myeloperoxidase (MPO) activity decreased by 42.98% and 55.33%, respectively. These treatments also effectively suppressed inflammatory cytokine expression and inhibited macrophage infiltration, ultimately alleviating the inflammatory response in gastric mucosal tissue. They also enhance mitochondrial function by regulating factors associated with mitochondrial dynamics and increasing succinate dehydrogenase (SDH) activity. Furthermore, they alleviate endoplasmic reticulum stress by reducing the immunoglobulin heavy chain-binding protein (Bip), phosphorylated eukaryotic translation initiation factor 2 alpha (P-eIF2α), and activating transcription factor 4 (ATF4) expression levels. Ultimately, high doses of VTPY and VLLY increased the B-cell lymphoma-extra large to B-cell lymphoma-2 associated death promoter ratio (Bcl-XL/Bad ratio) by 138.68% and 247.99%, respectively, while the expression level of cleaved-caspase 3 decreased by 35.26% and 48.22%, thereby preventing the activation of the apoptotic pathway. Sea cucumber gut peptides VTPY and VLLY exhibit effective anti-inflammatory and anti-apoptotic properties, along with anti-endoplasmic reticulum stress effects, by inhibiting the ATF4/Bcl-2 signaling pathway, thus effectively shielding against ethanol-induced gastric damage. VLLY appears to be more potent in this aspect.

ATF3 affects osteogenic differentiation in inflammatory hPDLSCs by mediating ferroptosis via regulating the Nrf2/HO-1 signaling pathway

Activating transcription factor 3 (ATF3) has been identified as a regulator associated with osteoblast differentiation. However, the effects of ATF3 on the osteogenic differentiation and proliferation of human periodontal stem cells (hPDLSCs) in periodontitis have not been reported. With the purpose of establishing an in vitro model of periodontitis, hPDLSCs were challenged with lipopolysaccharide (LPS). The Cell Counting Kit-8 assay was applied to assess cell viability, while reverse transcription-quantitative PCR and western blotting were employed to detect ATF3 expression. Inflammatory release was assessed using ELISA, together with western blotting. Lipid peroxidation was explored using the C11 BODIPY 581/591 probe, biochemical kits, thiobarbituric acid reactive substances (TBARS) assay and DCFH-DA staining. Iron and Fe2+ levels, and the expression levels of ferroptosis-related proteins were measured using corresponding kits and western blotting. Osteogenic differentiative capability was evaluated using alkaline phosphatase staining, Alizarin red staining and western blotting. The expression levels of proteins associated with Nrf2/HO-1 signaling were identified using western blotting. The results indicated that ATF3 expression was upregulated in LPS-induced hPDLSCs. The knockdown of ATF3 alleviated the LPS-induced inflammatory response in hPDLSCs, together with increased levels of TNF-α, IL-6, IL-1β, Cox-2 and iNOS, and decreased levels of IL-10. ATF3 silencing also led to lower TBARS production rate, and reduced levels of reactive oxygen species, iron, Fe2+, ACSL4 and TFR1, whereas it elevated the levels of SLC7A11 and GPX4. In addition, ATF3 silencing promoted hPDLSC mineralization and cell differentiation, and elevated the levels of OCN2, RUNX2 and BMP2. Additionally, ATF3 depletion upregulated the expression levels of proteins related with Nrf2/HO-1 signaling. The Nrf2 inhibitor ML385 partially counteracted the effects of ATF3 interference on the LPS-challenged inflammatory response, lipid peroxidation, ferroptosis as well as osteogenic differentiative capability in hPDLSCs. In summary, the results revealed that ATF3 silencing suppressed inflammation and ferroptosis, while it improved osteogenic differentiation in LPS-induced hPDLSCs by regulating Nrf2/HO-1 signaling, which may provide promising therapeutic targets for the treatment of periodontitis.

ATF4 and mTOR regulate metabolic reprogramming in TGF-β-treated lung fibroblasts.

Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive replacement of healthy lung with scar tissue. We and others have shown that fibroblast activation is supported by metabolic reprogramming, including the upregulation of the de novo synthesis of glycine, the most abundant amino acid found in collagen protein. How fibroblast metabolic reprogramming is regulated downstream of TGF-β is incompletely understood. We and others have shown that TGF-β-mediated activation of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and downstream upregulation of Activating Transcription Factor 4 (ATF4) promote increased expression of the enzymes required for de novo glycine synthesis; however, whether mTOR and ATF4 regulate other metabolic pathways in lung fibroblasts has not been explored. Here, we used RNA sequencing to determine how both ATF4 and mTOR regulate gene expression in human lung fibroblasts following TGF-β. We found that ATF4 primarily regulates enzymes and transporters involved in amino acid homeostasis as well as aminoacyl-tRNA synthetases. mTOR inhibition resulted not only in the loss of ATF4 target gene expression, but also in the reduced expression of glycolytic enzymes and mitochondrial electron transport chain subunits. Analysis of TGF-β-induced changes in cellular metabolite levels confirmed that ATF4 regulates amino acid homeostasis in lung fibroblasts while mTOR also regulates glycolytic and TCA cycle metabolites. We further analyzed publicly available single cell RNAseq data sets and found increased expression of ATF4 and mTOR metabolic targets in pathologic fibroblast populations from the lungs of IPF patients. Our results provide insight into the mechanisms of metabolic reprogramming in lung fibroblasts and highlight novel ATF4 and mTOR-dependent pathways that may be targeted to inhibit fibrotic processes.

Fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans.

Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse models. Previous work supports that C. elegans fmo-2 promotes longevity, stress resistance, and healthspan by rewiring endogenous metabolism. However, there are five C. elegans FMOs and five mammalian FMOs, and it is not known whether promoting longevity and health benefits is a conserved role of this gene family. Here, we report that expression of C. elegans fmo-4 promotes lifespan extension and paraquat stress resistance downstream of both dietary restriction and inhibition of mTOR. We find that overexpression of fmo-4 in just the hypodermis is sufficient for these benefits, and that this expression significantly modifies the transcriptome. By analyzing changes in gene expression, we find that genes related to calcium signaling are significantly altered downstream of fmo-4 expression. Highlighting the importance of calcium homeostasis in this pathway, fmo-4 overexpressing animals are sensitive to thapsigargin, an ER stressor that inhibits calcium flux from the cytosol to the ER lumen. This calcium/ fmo-4 interaction is solidified by data showing that modulating intracellular calcium with either small molecules or genetics can change expression of fmo-4 and/or interact with fmo-4 to affect lifespan and stress resistance. Further analysis supports a pathway where fmo-4 modulates calcium homeostasis downstream of activating transcription factor-6 ( atf-6 ), whose knockdown induces and requires fmo-4 expression. Together, our data identify fmo-4 as a longevity- promoting gene whose actions interact with known longevity pathways and calcium homeostasis.

Hepatoprotective effects of zingerone on sodium arsenite-induced hepatotoxicity in rats: Modulating the levels of caspase-3/Bax/Bcl-2, NLRP3/NF-κB/TNF-α and ATF6/IRE1/PERK/GRP78 signaling pathways

ObjectiveLong-term exposure to arsenic has been linked to several illnesses, including hypertension, diabetes, hepatic and renal diseases and cardiovascular malfunction. The aim of the current investigation was to determine whether zingerone (ZN) could shield rats against the hepatotoxicity that sodium arsenite (SA) causes. MethodsThe following five groups of thirty-five male Sprague Dawley rats were created: I) Control; received normal saline, II) ZN; received ZN, III) SA; received SA, IV) SA + ZN 25; received 10 mg/kg body weight SA + 25 mg/kg body weight ZN, and V) SA + ZN 50; received 10 mg/kg body weight SA + 50 mg/kg body weight ZN. The experiment lasted 14 days, and the rats were sacrificed on the 15th day. While oxidative stress parameters were studied by spectrophotometric method, apoptosis, inflammation and endoplasmic reticulum stress parameters were measured by RT-PCR method. ResultsThe SA disrupted the histological architecture and integrity of the liver and enhanced oxidative damage by lowering antioxidant enzyme activity, such as those of glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) level and increasing malondialdehyde (MDA) level in the liver tissue. Additionally, SA increased the mRNA transcript levels of Bcl2 associated x (Bax), caspases (−3, −6, −9), apoptotic protease-activating factor 1 (Apaf-1), p53, tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), c-Jun NH2-terminal kinase (JNK), mitogen-activated protein kinase 14 (MAPK14), MAPK15, receptor for advanced glycation endproducts (RAGE) and nod-like receptor family pyrin domain-containing 3 (NLRP3) in the liver tissue. Also produced endoplasmic reticulum stress by raising the mRNA transcript levels of activating transcription factor 6 (ATF-6), protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and glucose-regulated protein 78 (GRP-78). These factors together led to inflammation, apoptosis, and endoplasmic reticulum stress. On the other hand, liver tissue treated with ZN at doses of 25 and 50 mg/kg showed significant improvement in oxidative stress, inflammation, apoptosis and endoplasmic reticulum stress. ConclusionsOverall, the study's data suggest that administering ZN may be able to lessen the liver damage caused by SA toxicity.

Acute exposure to polystyrene nanoplastics induces unfolded protein response and global protein ubiquitination in lungs of mice

Inhaling microplastics (MPs) and nanoplastics (NPs) in the air can damage lung function. Xenobiotics in the body can cause endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) activation alleviates ER stress. Degradation of unfolded or misfolded proteins is an important pathway for recovering cellular homeostasis. The UPR and protein degradation induced by MPs/NPs in lung tissues are not well understood. Here, we investigated the UPR and protein ubiquitination in the lungs of mice exposed to polystyrene (PS)-NPs and their possible molecular mechanisms leading to protein ubiquitination. Mice were intratracheally administered with 5.6, 17, and 51 mg/kg PS-NPs once for 24 h. Exposure to PS-NPs elevated protein ubiquitination in the lungs of mice in a dose-dependent manner. PS-NPs activated three branches of UPR including inositol-requiring protein 1α (IRE1α), eukaryotic translation initiator factor 2α (eIF2α), and activating transcription factor 6α (ATF6α) in the lungs of mice. However, activated IRE1α did not trigger X-box binding protein 1 (XBP1) mRNA splicing. Exposure to PS-NPs induced an increase in the levels of E3 ubiquitin ligase hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (HRD1) and carboxy terminus of Hsc70 interacting protein (CHIP) in the lungs of mice and BEAS-2B cells. ATF6α siRNA inhibited the levels of HRD1 and CHIP proteins induced by PS-NPs in BEAS-2B cells. These results suggest that ATF6α plays a critical role in increasing ubiquitination of unfolded or misfolded proteins by alleviating PS-NPs induced ER stress through UPR to achieve ER homeostasis in the lungs of mice.

LINC00894 inhibited neuron cellular apoptosis and regulated activating transcription factor 3 expression

LINC00894 may be associated with synaptic function, but its biology function in neural cells is still unknown. In this study, LINC00894 knockdown decreased the EdU incorporated into newly synthesized DNA and cell viability in MTT or CCK-8 assay in HEK-293T and BE(2)-M17 (M17) neuroblastoma cells. And LINC00894 knockdown increased cellular apoptosis in Annexin V-FITC staining, the expression of activated Caspase3 and the level of reactive oxygen species (ROS) both in HEK-293T and M17 cells. Moreover, LINC00894 also protected cells from hydrogen peroxide induced apoptosis in in vitro models. Utilizing RNA sequencing (RNA-seq) integrated with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblot, we identified that LINC00894 affected activating transcription factor 3 (ATF3) expression in HEK-293T, M17, and SH-SY5Y neuroblastoma cells. Finally, we found that ectopic expression of ATF3 restored cell proliferation and inhibited cell apoptosis in LINC00894 downregulated M17 cells. While knockdown of ATF3 also significantly increased the cell viability inhibition and apoptosis promotion induced by LINC00894 knockdown in M17 cells. Our results from in vitro models revealed that LINC00894 could promote neuronal cell proliferation and inhibit cellular apoptosis by affecting ATF3 expression.

An unexpected role for the ketogenic diet in triggering tumor metastasis by modulating BACH1-mediated transcription.

We have found that the ketogenic (Keto) diet is able to, unexpectedly, promote the metastatic potential of cancer cells in complementary mouse models. Notably, the Keto diet-induced tumor metastasis is dependent on BTB domain and CNC homolog 1 (BACH1) and its up-regulation of pro-metastatic targets, including cell migration-inducing hyaluronidase 1, in response to the Keto diet. By contrast, upon genetic knockout or pharmacological inhibition of endogenous BACH1, the Keto diet-mediated activation of those targets is largely diminished, and the effects on tumor metastasis are completely abolished. Mechanistically, upon administration of the Keto diet, the levels of activating transcription factor 4 (ATF4) are markedly induced. Through direct interaction with BACH1, ATF4 is recruited to those pro-metastatic target promoters and enhances BACH1-mediated transcriptional activation. Together, these data implicate a distinct transcription regulatory program of BACH1 for tumor metastasis induced by the Keto diet. Our study also raises a potential health risk of the Keto diet in human patients with cancer.