Activating Transcription Factor Research Articles

#1656 Ezetimibe improves diabetic nephropathy by upregulating Nrf2 to alleviate endoplasmic reticulum stress

Abstract Background and Aims Diabetic nephropathy (DN), a prevalent complication of diabetes mellitus, stands as the primary contributor to chronic kidney disease and end-stage renal disease. The progression of diabetic nephropathy is facilitated by endoplasmic reticulum stress and the excessive generation of reactive oxygen species (ROS). In addition to its cholesterol-lowering properties, recent research has revealed that ezetimibe also ameliorates insulin resistance and glucose intolerance abnormalities, and is also beneficial in chronic kidney disease. However, the specific impact of ezetimibe on diabetic nephropathy and the underlying mechanism remain unknown. Therefore, the objective of this research is to investigate the effect of ezetimibe on diabetic nephropathy and elucidate its mechanism. Method In vivo experiments were conducted using C57BL/6J mice, with a total of 24 mice randomly assigned to three groups: NC, DM, and EZE (10 mg/kg/day). In vitro experiments involved the treatment of human renal proximal tubular (HK-2) cells with ezetimibe (50 μM) and high glucose (30 mmol/L) for 48 hours. Then, combined with histopathological examination, biochemical evaluation, TUNEL stain, western blot analysis, Si-RNA transfection, transmission electron microscopy and ROS probe were used to detect the renal morphology and function, oxidative stress and endoplasmic reticulum homeostasis level. Results The results demonstrated that a 10-week treatment with ezetimibe led to significant improvements in urine volume, beta-N-acetylglucosaminidase (NAG), and the urinary albumin/creatinine ratio (ACR). The administration of ezetimibe in the treatment exhibited enhancements in glomerular hypertrophy and renal tubular vacuolization, as well as mesangial matrix expansion and glomerular basement membrane thickness, in diabetic mice. Additionally, ezetimibe treatment resulted in a reduction in ROS formation by upregulating the expression of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), through the activation of NF-E2-related factor-2 (Nrf2). Furthermore, ezetimibe administration partially reversed the increased expression of endoplasmic reticulum stress-related proteins, including activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP), in DN mice. Ezetimibe demonstrated inhibitory effects on the accumulation of ROS and alleviated endoplasmic reticulum stress in HK-2 cells treated with high glucose. Furthermore, electron microscopy analysis revealed that ezetimibe effectively reduced the distension and dilation of the endoplasmic reticulum. However, the anti-endoplasmic reticulum stress effect of ezetimibe was significantly diminished following transfection with si-Nrf2. These findings suggest that ezetimibe exerts its anti-endoplasmic reticulum stress effect, at least in part, by enhancing antioxidant capacity and reducing ROS levels through the upregulation of Nrf2. Conclusion In summary, our research findings demonstrate the notable contribution of endoplasmic reticulum stress to the progression of DN. Through the up-regulation of Nrf2, ezetimibe effectively mitigates ROS generation in the context of DN, consequently ameliorating the adverse consequences of ERS, restoring equilibrium within the endoplasmic reticulum, and ultimately safeguarding against renal cell demise. Ezetimibe is an attractive potential therapy for the treatment of DN.

Regulation of eukaryotic transcription initiation in response to cellular stress

Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-β, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.

#972 A Novel Single Compound from Chinese Herbs as a Potential Candidate for Reducing obesity induced diabetic nephropathy via ATF3-RARRES1 axis pathway

Abstract Background and Aims Billions of people have obesity induced diabetic nephropathy. Promoting the browning of white adipose tissue has been suggested as a potential strategy for, but a drug still needs to be identified. Method First, WT and ATF3 knockout mice fed a high-fat diet were treated with STZ to determine that ATF3 plays an important role in obesity-induced diabetic nephropathy. Second, DBA mice fed a high-fat diet were infused with STZ, and db/db KO mice were fed a high-fat diet, and the two mouse models were injected intraperitoneally with or without the ATF3 inducer ST32da (the structure is a single drug designed based on the traditional Chinese medicine Salvia miltiorrhiza). After about one and a half months, all related kidneys function values were measured. Results After analysis the relationship between ATF3 and diabetic nephropathy in human GEO DataSet Browser indicated that the gene expression of ATF3 was lower in patients groups. Genetic deletion of activating transcription factor 3 (ATF3−/−) in mice under a high-fat diet (HFD) and STZ treatment resulted in more aggravated nephropathy phenomenon syndrome including higher urine albumin, lower creatinine clearance and high risk in death. ST32da, a synthetic ATF3 inducer isolated from Salvia miltiorrhiza, promoted ATF3 expression to downregulate inflammatory genes and ameliorated foot process effacement resulting in reduced diabetic nephropathy by suppressing the podocyte apoptosis via interleukin (IL)-6 and retinoic acid receptor responder protein 1 (RARRES1) axis. Conclusion Our study identified the ATF3 inducer ST32da and Salvia miltiorrhiza extraction, may as a promising therapeutic drug for treating diet-induced obesity induced diabetic nephropathy.

Natural compound Alternol actives multiple endoplasmic reticulum stress-responding pathways contributing to cell death.

Background: Alternol is a small molecular compound isolated from the fermentation of a mutant fungus obtained from Taxus brevifolia bark. Our previous studies showed that Alternol treatment induced reactive oxygen species (ROS)-dependent immunogenic cell death. This study conducted a comprehensive investigation to explore the mechanisms involved in Alternol-induced immunogenic cell death. Methods: Prostate cancer PC-3, C4-2, and 22RV1 were used in this study. Alternol interaction with heat shock proteins (HSP) was determined using CETSA assay. Alternol-regulated ER stress proteins were assessed with Western blot assay. Extracellular adenosine triphosphate (ATP) was measured using ATPlite Luminescence Assay System. Results: Our results showed that Alternol interacted with multiple cellular chaperone proteins and increased their expression levels, including endoplasmic reticulum (ER) chaperone hypoxia up-regulated 1 (HYOU1) and heat shock protein 90 alpha family class B member 1 (HSP90AB1), as well as cytosolic chaperone heat shock protein family A member 8 (HSPA8). These data represented a potential cause of unfolded protein response (UPR) after Alternol treatment. Further investigation revealed that Alternol treatment triggered ROS-dependent (ER) stress responses via R-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α). The double-stranded RNA-dependent protein kinase (PKR) but not activating transcription factor 6 (ATF6) cascades, leading to ATF-3/ATF-4 activation, C/EBP-homologous protein (CHOP) overexpression, and X-box binding protein XBP1 splicing induction. In addition, inhibition of these ER stress responses cascades blunted Alternol-induced extracellular adenosine triphosphate (ATP) release, one of the classical hallmarks of immunogenic cell death. Conclusion: Taken together, our data demonstrate that Alternol treatment triggered multiple ER stress cascades, leading to immunogenic cell death.

Osteocyte ferroptosis induced by ATF3/TFR1 contributes to cortical bone loss during ageing.

Cortical bone loss is intricately associated with ageing and coincides with iron accumulation. The precise role of ferroptosis, characterized by iron overload and lipid peroxidation, in senescent osteocytes remains elusive. We found that ferroptosis was a crucial mode of osteocyte death in cortical bone during ageing. Using a single-cell transcriptome analysis, we identified activating transcription factor 3 (ATF3) as a critical driver of osteocyte ferroptosis. Elevated ATF3 expression in senescent osteocytes promotes iron uptake by upregulating transferrin receptor 1 while simultaneously inhibiting solute carrier family 7-member 11-mediated cystine import. This process leads to an iron overload and lipid peroxidation, culminating in ferroptosis. Importantly, ATF3 inhibition in aged mice effectively alleviated ferroptosis in the cortical bone and mitigated cortical bone mass loss. Taken together, our findings establish a pivotal role of ferroptosis in cortical bone loss in older adults, providing promising prevention and treatment strategies for osteoporosis and fractures.

Effects of low protein feed on hepato-intestinal health and muscle quality of grass carp (Ctenopharyngodon idellus)

In this study, grass carp (33.28 ± 0.05 g) were fed three diets for 8 weeks: control (crude protein [CP] 30%, crude lipid [CL] 6%), low protein (LP; CP16%, CL6%), and low protein with high-fat (LPHF; CP16%, CL10%). The final body weight decreased in the LP and LPHF groups compared to the Control (P < 0.05). Liver triglycerides, total cholesterol, and nonesterified fatty acids were higher in the LP group than the Control, whereas these indexes in the LPHF group were higher than those in the LP group (P < 0.05). The LP group had intestinal barrier damage, while the LPHF group had a slight recovery. TNF-α, IL-8, and IL-1β content were lower in the LP group than in the Control (P < 0.05), and even higher in the LPHF group (P < 0.05). The expressions of endoplasmic reticulum stress-related genes Activating transcription factor 6 (ATF-6) and Glucose-regulated protein (GRP78) were higher in the LPHF group against the LP group (P < 0.05). The IL-1β and TNF-α content negatively correlated with intestinal Actinomycetes and Mycobacterium abundance (P < 0.05). The muscle fiber diameter was smaller in both the LP and LPHF groups than the control (P < 0.05), with the LP group showing metabolites related to protein digestion and absorption, and LPHF group exhibiting metabolites related to taste transmission. The results demonstrate reducing dietary protein affects growth, causing liver lipid accumulation, reduced enteritis response, and increased muscle tightness, while increasing fat content accelerates fat accumulation and inflammation.

Identification and Verification of Endoplasmic Reticulum Stress-Related Genes as Novel Signatures for Osteoarthritis Diagnosis and Therapy: A Bioinformatics Analysis-Oriented Pilot Study.

Endoplasmic reticulum stress (ERS) has been reported to be closely associated with the development of osteoarthritis (OA), but the underlying mechanisms are not fully delineated. The present study was designed to investigate the involvement of ERS-related genes in regulating OA progression. The expression profiles of OA patients and normal people were downloaded from the gene expression omnibus (GEO) database. The differentially expressed genes (DEGs) in datasets GSE55457 and GSE55235 were screened and identified by R software with the construction of the protein-protein interaction (PPI) networks. Through the STRING and Venn diagram analysis, hub ERS-related genes were obtained. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed. Biomarkers with high diagnostic values of osteoarthritis (OA) were studied. The hematoxylin and eosin (H&E) staining and micro-CT were applied to evaluate the establishment of the OA model. The expression levels of biomarkers were validated with the use of reverse transcription‑quantitative polymerase chain reaction (RT-qPCR) and western blot. Finally, we evaluated the correlations of hub ERS-related genes with the immune infiltration cells via the CIBERSORT algorithm. A total of 60 downregulated and 52 upregulated DEGs were identified, and the following GO and KEGG pathway analyses verified that those DEGs were mainly enriched in biological process (BP), cellular component (CC), molecular function (MF), and inflammation-associated signal pathways. Interestingly, among all the DEGs, six ER stress-associated genes, including activating transcription factor 3 (ATF3), DEAD-Box Helicase 3 X-Linked (DDX3X), AP-1 transcription factor subunit (JUN), eukaryotic initiation factor 4 (EIF4A1), KDEL endoplasmic reticulum protein retention receptor 3 (KDELR3), and vascular endothelial growth factor A (VEGFA), were found to be closely associated with OA progression, and the following RT-qPCR and Western Blot analysis confirmed that DDX3X, JUN, and VEGFA were upregulated, whereas KDELR3, EIF4A1, and ATF3 were downregulated in OA rats tissues compared to the normal tissues, which were in accordance with our bioinformatics findings. Furthermore, our receiver operating characteristic (ROC) curve analysis verified that the above six ER stress-associated genes could be used as ideal biomarkers for OA diagnosis and those genes also potentially regulated immune responses by influencing the biological functions of mast cells and macrophages. Collectively, the present study firstly identified six ER stress-associated genes (ATF3, DDX3X, JUN, EIF4A1, KDELR3, and VEGFA) that may play critical role in regulating the progression of OA.

Amphiregulin secreted by umbilical cord multipotent stromal cells protects against ferroptosis of macrophages via the activating transcription factor 3-CD36 axis to alleviate endometrial fibrosis.

Endometrium fibrosis is the leading cause of uterine infertility. Macrophages participated in the occurrence and development of endometrial fibrosis. We previously reported that human umbilical cord multipotent stromal cells (hUC-MSCs) exerted their therapeutic effect in a macrophage-dependent manner in endometrial fibrosis. However precise mechanisms by which hUC-MSCs may influence macrophages in endometrial fibrosis remain largely unexplored. Here, we demonstrated that abnormal iron and lipid metabolism occurred in patients with intrauterine adhesions (IUA) and murine models. Ferroptosis has been proven to contribute to the progression of fibrotic diseases. Our results revealed that pharmacological activation of ferroptosis by Erastin aggravated endometrial fibrosis, while inhibition of ferroptosis by Ferrostatin-1 ameliorated endometrial fibrosis in vivo. Moreover, ferroptosis of macrophages was significantly upregulated in endometria of IUA murine models. Of note, transcriptome profiles revealed that CD36 gene expression was significantly increased in patients with IUA and immunofluorescence analysis showed CD36 protein was mainly located in macrophages. Silencing CD36 in macrophages could reverse cell ferroptosis. Dual luciferase reporter assay revealed that CD36 was the direct target of activation transcription factor 3 (ATF3). Furthermore, through establishing coculture system and IUA murine models, we found that hUC-MSCs had a protective role against macrophage ferroptosis and alleviated endometrial fibrosis related to decreased CD36 and ATF3. The effect of hUC-MSCs on macrophage ferroptosis was attributed to the upregulation of amphiregulin (AREG). Our data highlighted that macrophage ferroptosis occurred in endometrial fibrosis via the ATF3-CD36 pathway and hUC-MSCs protected against macrophage ferroptosis to alleviate endometrial fibrosis via secreting AREG. These findings provided a potential target for therapeutic implications of endometrial fibrosis.

Silybin prevented avermectin-induced cardiotoxicity in carp by modulating oxidative stress, inflammation, endoplasmic reticulum stress, mitochondrial apoptosis, and autophagy

Avermectin is one of the widely used anthelmintics in aquaculture and exhibits substantial toxicity to aquatic organisms. Silybin is extensively used for its anti-inflammatory, antioxidant and anti-apoptotic biological properties. Heart is essential for the survival of fish and plays a vital role in pumping blood oxygen and nutrients. Residual avermectin in water poses harm to carp. However, there is still insufficient research on whether silybin can mitigate the toxicity of avermectin to carp heart tissues. In this research, we established a model involving carp subjected to acute avermectin exposure and administered diets containing silybin to explore the potential protective effects of silybin against avermectin-induced cardiotoxicity. The results revealed that avermectin induced oxidative stress, inflammation, endoplasmic reticulum (ER) stress, mitochondrial pathway apoptosis and autophagy in the cardiac tissues of carp. Compared with the avermectin group, silybin significantly reduced ROS accumulation in cardiac tissues, restored antioxidant enzyme activity, inhibited mRNA transcript levels of pro-inflammatory-related factors, and attenuated ER stress, mitochondrial pathway apoptosis and autophagy. Protein-protein interaction (PPI) analysis demonstrated that silybin mitigated avermectin-induced cardiac oxidative stress, inflammation, ER stress, mitochondrial pathway apoptosis and autophagy. Silybin exerted anti-inflammatory effects through the Nuclear Factor kappa B (NF-κB) pathway, antioxidant effects through the Nuclear factor erythroid 2-related factor 2 (Nrf2) - Kelch-like ECH-associated protein 1 (Keap1) pathway, alleviated cardiac ER stress through the Glucose-regulated protein 78 (GRP78)/Activating Transcription Factor 6 (ATF6)/C/EBP homologous protein (CHOP) axis, suppressed apoptosis through the mitochondrial pathway, and inhibited excessive autophagy initiation through the PTEN-induced putative kinase 1 (PINK1)/Parkin RBR E3 ubiquitin protein ligase (PARKIN) signaling pathway. This study provided evidence supporting the protective effect of silybin against avermectin-induced cardiotoxicity in carp, highlighting its potential as a dietary additive to protect fish from adverse effects caused by avermectin exposure.

Platelet-rich plasma alleviates neuropathic pain in osteoarthritis by downregulating microglial activation

BackgroundThe development of neuropathic pain (NP) is one of the reasons why the pain is difficult to treat, and microglial activation plays an important role in NP. Recently, platelet-rich plasma (PRP) has emerged as a novel therapeutic method for knee osteoarthritis (KOA). However, it’s unclarified whether PRP has analgesic effects on NP induced by KOA and the underlying mechanisms unknown.PurposeTo observe the analgesic effects of PRP on NP induced by KOA and explore the potential mechanisms of PRP in alleviating NP.MethodsKOA was induced in male rats with intra-articular injections of monosodium iodoacetate (MIA) on day 0. The rats received PRP or NS (normal saline) treatment at days 15, 17, and 19 after modeling. The Von Frey and Hargreaves tests were applied to assess the pain-related behaviors at different time points. After euthanizing the rats with deep anesthesia at days 28 and 42, the corresponding tissues were taken for subsequent experiments. The expression of activating transcription factor 3 (ATF3) in dorsal root ganglia (DRG) and ionized-calcium-binding adapter molecule-1(Iba-1) in the spinal dorsal horn (SDH) was detected by immunohistochemical staining. In addition, the knee histological assessment was performed by hematoxylin-eosin (HE) staining.ResultsThe results indicated that injection of MIA induced mechanical allodynia and thermal hyperalgesia, which could be reversed by PRP treatment. PRP downregulated the expression of ATF3 within the DRG and Iba-1 within the SDH. Furthermore, an inhibitory effect on cartilage degeneration was observed in the MIA + PRP group only on day 28.ConclusionThese results indicate that PRP intra-articular injection therapy may be a potential therapeutic agent for relieving NP induced by KOA. This effect could be attributed to downregulation of microglial activation and reduction in nerve injury.

GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage

Growth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.

Box-Behnken Design-Based Optimization of Phytochemical Extraction from Diplazium esculentum (Retz.) Sw. Associated with Its Antioxidant and Anti-Alzheimer's Properties.

A previous study reported that the ethanolic extract of the edible fern, Diplazium esculentum (Retz.) Sw. (DE), obtained from a non-optimized extraction condition exhibited anti-Alzheimer's disease (AD) properties through the inhibition of a rate-limiting enzyme in amyloid peptide formation, β-secretase-1 (BACE-1). Nevertheless, a non-optimized or suboptimal extraction may lead to several issues, such as a reduction in extraction efficiency and increased time and plant materials. In this study, extraction of the DE was optimized to obtain appropriate BACE-1 inhibition using a Box-Behnken design (BBD) and response surface methodology (RSM). Data revealed that the optimal extraction condition was 70% (v/v) aqueous ethanol, 50 min extraction time, 30 °C extraction temperature, and 1:30 g/mL solid/liquid ratio, giving BACE-1 inhibition at 56.33%. In addition, the extract also exhibited significant antioxidant activities compared to the non-optimized extraction. Metabolomic phytochemical profiles and targeted phytochemical analyses showed that kaempferol, quercetin, and their derivatives as well as rosmarinic acid were abundant in the extract. The optimized DE extract also acted synergistically with donepezil, an AD drug suppressing BACE-1 activities. Data received from Drosophila-expressing human amyloid precursor proteins (APPs) and BACE-1, representing the amyloid hypothesis, showed that the optimized DE extract penetrated the fly brains, suppressed BACE-1 activities, and improved locomotor functions. The extract quenched the expression of glutathione S transferase D1 (GSTD1), inositol-requiring enzyme (IRE-1), and molecular chaperone-binding immunoglobulin (Bip), while donepezil suppressed these genes and other genes involved in antioxidant and endoplasmic reticulum (ER) stress response, including superoxide dismutase type 1 (SOD1), activating transcription factor 6 (ATF-6), and protein kinase R-like endoplasmic reticulum kinase (PERK). To sum up, the optimized extraction condition reduced extraction time while resulting in higher phytochemicals, antioxidants, and BACE-1 inhibitors.

Phenformin activates ER stress to promote autophagic cell death via NIBAN1 and DDIT4 in oral squamous cell carcinoma independent of AMPK

The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.

Metformin induction of heat shock factor 1 activation and the mitochondrial unfolded protein response alleviate cardiac remodeling in spontaneously hypertensive rats.

Heart failure and cardiac remodeling are both characterized by mitochondrial dysfunction. Healthy mitochondria are required for adequate contractile activity and appropriate regulation of cell survival. In the mammalian heart, enhancement of the mitochondrial unfolded protein response (UPRmt) is cardioprotective under pressure overload conditions. We explored the UPRmt and the underlying regulatory mechanism in terms of hypertension-induced cardiac remodeling and the cardioprotective effect of metformin. Male spontaneously hypertensive rats and angiotensin II-treated neonatal rat cardiomyocytes were used to induce cardiac hypertrophy. The results showed that hypertension induced the formation of aberrant mitochondria, characterized by a reduced mtDNA/nDNA ratio and swelling, as well as lower levels of mitochondrial complexes I to V and inhibition of the expression of one protein subunit of each of complexes I to IV. Such changes eventually enlarged cardiomyocytes and increased cardiac fibrosis. Metformin treatment increased the mtDNA/nDNA ratio and regulated the UPRmt, as indicated by increased expression of activating transcription factor 5, Lon protease 1, and heat shock protein 60, and decreased expression of C/EBP homologous protein. Thus, metformin improved mitochondrial ultrastructure and function in spontaneously hypertensive rats. Invitro analyses revealed that metformin reduced the high levels of angiotensin II-induced mitochondrial reactive oxygen species in such animals and stimulated nuclear translocation of heat shock factor 1 (HSF1). Moreover, HSF1 small-interfering RNA reduced the metformin-mediated improvements in mitochondrial morphology and the UPRmt by suppressing hypertrophic signals and cardiomyocyte apoptosis. These results suggest that HSF1/UPRmt signaling contributes to the beneficial effects of metformin. Metformin-mediated targeting of mitochondrial protein homeostasis and modulation of HSF1 levels have potential therapeutic implications in terms of cardiac remodeling.

Activating Transcription Factor 6 (ATF6) Acts as a Rheostat To Balance UPR Signaling and The Proteostatic Response To Mutant SFTPC Substrates By The Alveolar Epithelium

The alveolar type II (AT2) epithelial cell is a metabolically active, biosynthetically robust component of the distal lung responsible for the synthesis of pulmonary surfactant. Disruption of AT2 cell quality control provides a molecular signature found in Idiopathic Pulmonary Fibrosis (IPF), a progressive interstitial lung disease. Mutations in the surfactant protein C (SP-C) gene ( SFTPC) are a high effect size etiological cause of PF in a subset of these patients. Prior in vitro studies from our lab showed that mutations located within the SFTPC BRICHOS domain of the SP-C proprotein result in production of ER retained and aggregation prone conformers. Two critical aspects of the proteostasis network are ER Associated Degradation (ERAD) and the unfolded protein response (UPR). Misfolded conformers activate the UPR via one or more of 3 ER transmembrane sensing proteins (Protein kinase R-like ER kinase (PERK), the Inositol-Requiring kinase 1 alpha (IRE1α) and Activating Transcription Factor 6 (ATF6). We have shown in vitro and in mouse models that clinical IPF associated BRICHOS SFTPC conformers produce unbalanced, exuberant UPR signaling and AT2 dysfunction. The goal of this study was to probe AT2 proteostatic repertoires using BRICHOS SFTPC mutants as substrates. For this, we generated stable doxycycline-inducible Mouse Lung Epithelial (MLE-12) cell lines expressing either Wild Type (SP-CWT) or an aggregating mutant (SP-CC121G) which were interrogated using cycloheximide chase and / or treatments with activators or inhibitors of ATF6, IRE1, and PERK as well as ERAD (VCP/p97), the proteasome, or macroautophagy. Readouts included fluorescence microscopy, qPCR, luciferase reporters, and immunoblotting to assess SFPTC aggregate formation, UPR signaling, and downstream ER stress related events. Using this model system, we found that SP-CC121G was ER retained and activated the UPR within 8 hours of Dox induction (as BIP expression). Commensurate with this, IRE1α (as p-IREα and spliced XBP1 (sXBP1)), ATF6 (by luciferase assay), and PERK (as p-Eif2α, ATF4, CHOP) signals were activated by SP-CC121G. The clearance of mutant SP-CC121G as well as a portion of proSP-CWT was dependent on ERAD and the Ubiquitin-Proteasome System (UPS) as their disruption using either inhibitors of p97 (EEY) or the proteasome (MG132) induced formation of higher molecular weight aggregates. Bafilomycin failed to induce SP-CC121G aggregates or block their clearance. The ATF6 agonist AA147 administered during ERAD inhibition accelerated aggregate protein degradation. When SP-CC121G MLE12 cells were treated with an ATF6 inhibitor (CEAPIN7), we observed marked increases in IRE1α kinase activity with downstream activation of JNK accompanied by commensurate activation of PERK signals and initiation of caspase-3 cleavage. Collectively, these data demonstrate a primary role for ERAD in the AT2 proteostatic response to mutant SFTPC substrates and identify ATF6 as a critical UPR rheostat to provide protection from SFTPC mutant expression both by limiting SP-C aggregate formation and balancing exuberant UPR signals from IRE1α and PERK. NIH R01 HL145408 (MFB), VA Merit Review 2I01BX001176-09 (MFB), Pulmonary Fibrosis Foundation (LRR), NIH 2T32 HL007586-36 (SB), NIH K08 HL150226 (JBK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The E3 ubiquitin-protein ligase synoviolin (Syvn1/Hrd1) promotes adaptive decreases in cardiac myocyte protein synthesis via eIF2α/ATF4 pathway activation

As the heart undergoes pathologic hypertrophy in response to many disease conditions, an imbalance between protein synthesis and degradation in cardiac myocytes, due to the increases in protein synthesis relative to protein degradation, happens, which challenges protein folding and endoplasmic reticulum (ER) proteostasis. This leads to ER stress and the activation of the ER stress response. Synoviolin (Syvn1), also known as HMG-CoA reductase degradation protein 1 (Hrd1), is an ER-localized protein that is upregulated in cardiac myocytes during ER stress. We previously found that Syvn1 mediates the ubiquitylation and consequent degradation of toxic misfolded proteins and preserved cardiac function in a model of pressure overload-induced cardiac pathology. The objective of this study was to examine whether Syvn1 is involved in regulating protein synthesis, and if so, what its function is during hypertrophic growth of cardiac myocytes. Given that cardiac hypertrophy is caused by increases in protein synthesis, we hypothesized that Syvn1 provides an adaptive response in part through regulation of protein synthesis and consequent decrease in cardiac hypertrophic response. We examined the effects of Syvn1 on ventricular myocyte growth in vitro and found that overexpression of Syvn1 decreased cardiac myocyte surface area as well as protein synthesis when treated with phenylephrine (PE), a treatment which normally leads to increased protein synthesis and increased cardiac myocyte size, mimicking pathological cardiac hypertrophy. We next focused on identifying the mechanism of Syvn1-mediated growth inhibition and found that increases in Syvn1 lead to activation of the PERK (protein kinase RNA (PKR)-like ER kinase) branch of the ER stress response, which leads to increased phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) at Ser51, thereby inhibiting global translational initiation. PERK activation also results in selective translation of a subset of mRNAs, including activating transcription factor 4 (ATF4), which activates the transcription of a wide range of genes involved in adaptation to stress conditions. We found increased ATF4 levels and increased ATF4 target gene expression with Syvn1 overexpression during PE treatment, compared to control PE treatment. Inhibition of the PERK pathway blocked Syvn1-mediated decrease in cardiac myocyte size and protein synthesis as well as PERK activation and ATF4 target gene induction. Additionally, we found that an adeno-associated virus encoding Syvn1, administered in a therapeutically-relevant manner after the onset of pathological cardiac hypertrophy induced by transverse-aortic constriction in mice, blunted pathological remodeling and preserved cardiac function. We posit that the Syvn1/eIF2α/ATF4 pathway constitutes a newly-discovered mechanism for balancing the proteome in cardiac myocytes. Here we found that Syvn1 upregulation blunts maladaptive increase in cardiac myocyte size during pathologic hypertrophy. In conclusion, these findings suggest that Syvn1 is a critical regulator of cardiac myocyte protein synthesis and subsequent growth and that increases in Syvn1 lead to the activation of the PERK branch of the ER stress response, which results in inhibition of global protein synthesis, and activation of ATF4. This work was supported by the National Institutes of Health (R01HL157027) and the University of Arizona Health Sciences Career Development Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Borax affects cellular viability by inducing ER stress in hepatocellular carcinoma cells by targeting SLC12A5.

Hepatocellular carcinoma (HCC) presents a persistent challenge to conventional therapeutic approaches. SLC12A5 is implicated in an oncogenic capacity and facilitates the progression of cancer. The objective of this investigation is to scrutinize the inhibitory effects of borax on endoplasmic reticulum (ER)-stress and apoptosis mediated by SLC12A5 in HepG2 cells. Initially, we evaluated the cytotoxic impact of borax on both HL-7702 and HepG2 cell lines. Subsequently, the effects of borax on cellular morphology and the cell cycle of these lines were examined. Following this, we explored the impact of borax treatment on the mRNA and protein expression levels of SLC12A5, C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78), activating transcription factor-6 (ATF6), caspase-3 (CASP3), and cytochrome c (CYC) in these cellular populations. The determined IC50 value of borax for HL-7702 cells was 40.8 mM, whereas for HepG2 cells, this value was 22.6 mM. The concentrations of IC50 (22.6 mM) and IC75 (45.7 mM) of borax in HepG2 cells did not manifest morphological aberrations in HL-7702 cells. Conversely, these concentrations in HepG2 cells induced observable morphological and nuclear abnormalities, resulting in cell cycle arrest in the G1/G0 phase. Additionally, the levels of SLC12A5, ATF6, CHOP, GRP78, CASP3, and CYC were elevated in HepG2 cells in comparison to HL-7702 cells. Moreover, SLC12A5 levels decreased following borax treatment in HepG2 cells, whereas ATF6, CHOP, GRP78, CASP3, and CYC levels exhibited a significant increase. In conclusion, our data highlight the potential therapeutic effects of borax through the regulation of ER stress in HCC by targeting SLC12A5.

Impact of Aging and Disuse on Markers of Endoplasmic Reticulum Stress and the Unfolded Protein Response

Aging and disuse can impair skeletal muscle responses to stimuli and are key drivers in dysregulated proteostasis. Such dysfunction can lead to an accumulation of misfolded proteins resulting in endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR). While the UPR blunts global translation and enhances the production of chaperones and foldases, little is known about ERS responses across the lifespan and in disuse contexts. Therefore, the purposes of this study were to examine if: i) the expression of ERS and UPR effector proteins differ across the lifespan in rats, and ii) age-associated patterns were recapitulated by 10 days of hindlimb casting in a separate cohort of rats. We hypothesized that aging would enhance the expression of ERS related proteins, and this expression pattern would be mimicked by 10 days of limb disuse. Male Fischer 344 rats were sacrificed at 3, 6, 12, 18, and 24 months (mo) of age, and plantaris (PLT) and soleus (SOL) muscles were collected for analysis. Six mo female Wistar rats were sacrificed after either 10 days of hindlimb casting or continued ambulation (age matched control), after which whole gastrocnemius muscles were collected for analysis. The effector eukaryotic initiation factor 2 alpha (eIF2a) was different across the lifespan (phospho/pan) in PLT and SOL muscles (p&lt;0.001) and was upregulated in the PLT at 24 mo as compared to 3 mo (P=0.006). The downstream UPR effector C/EBP homologous protein (CHOP) was differentially expressed across the lifespan in PLT and SOL (p&lt;0.001). CHOP was upregulated ~2.79 and 3.08-fold at 18 and 24 mo respectively in the SOL (p&lt;0.001), and 2.62 and 2.44-fold at 18 and 24 mo respectively in the PLT. Finally, principal effector Activating Transcription Factor 6 (ATF6) was differentially expressed across the lifespan (cleaved/pan) in the PLT and SOL (P≤0.024) whereby a 1.23-fold up-regulation was evident at 18 mo in the SOL (P=0.035) and 1.34-fold at 18 mo in the PLT (P=0.026). The CHOP and ATF6 proteins were similarly dysregulated in our disuse model, whereby 1.83-fold (p&lt;0.001) and 1.30-fold (P=0.002) greater values were observed in hind limb casted versus control rats. Effectors of the UPR pathway are upregulated in skeletal muscle across the lifespan in rats, and this effect is recapitulated with disuse. These findings warrant further research for potential therapeutics related to age- and disuse-related skeletal muscle atrophy. Funding for this study was provided by discretionary laboratory funds from MDR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2109: Nox1-mediated Endothelial Ros Propagates Hypoxia-induced Pulmonary Hypertension Via The Unfolded Protein Response.

Pulmonary arterial hypertension (PAH) is a devastating disease in which reactive oxygen species (ROS) play an important role. The disease is characterized by lung arterial cell proliferation (inward remodeling) leading to right ventricle (RV) failure. We postulated that NADPH oxidase 1 (NOX1)-derived ROS mediate endothelial cell (EC) proliferation and hemodynamic changes in PAH. Indeed, we found increased NOX1 (1.6±0.10-fold; n=7-9, p&lt;0.01) in human pulmonary artery ECs (HPAECs) exposed to 24 hr hypoxia (Hx) vs. normoxia (Nx) (1% v. 21% O 2 , in vitro PAH phenotype). To test an in vivo NOX1 role, C57BL/6J (WT), mice were implanted with pumps delivering a NOX1-selective peptidic inhibitor (NoxA1ds) or scrambled control (Scr) and exposed to Nx (21% O 2 ) or Hx (10% O 2 ) for 3 wks plus vehicle or VEGF receptor antagonist Sugen 5416. Sugen/Hx (Su/Hx) increased RV max pressure (RVMP) 2-fold ± 0.20-fold (n=7-9, p&lt;0.001) along with elevated mean pulmonary artery pressure (mPAP, 2-fold 0.25-fold, n=7-9, p&lt;0.001), pulmonary vascular resistance (PVR, 2-fold±0.2-fold, n=7-9, p&lt;0.001), and right ventricular max dP/dt (RVM dP/dt, 2-fold±0.2-fold, n=7-9, p&lt;0.001) vs. Nx. NoxA1ds infusion significantly attenuated these rises. In parallel, RNA-Seq of HPAECs treated with NOX1 siRNA and exposed to Nx or Hx revealed the protein kinase R-like ER kinase (PERK) branch of the unfolded protein response (UPR) as NOX1-mediated. UPR promotes tumor growth in cancer due to positive effects on cell survival and proliferation. Indeed, phospho-PERK expression measured via WB increased by 1.5-fold ± 0.3-fold (n=9, p&lt;0.01) in HPAECs exposed to Hx, which was completely reversed by siNOX1. Further, downstream members of the PERK-UPR pathway followed similar trends including phospho-eukaryotic translation initiation factor 2A (eIF2α, 3-fold ± 0.6-fold, n=9, p&lt;0.05), activating transcription factor 4 (ATF4, 2.1-fold ± 0.4-fold, n=9, p&lt;0.01), and protein disulfide-isomerase (PDI, 2.3-fold ± 0.65-fold, n=9, p&lt;0.01). We corroborated these findings in cross-sections of Su/Hx mouse lungs and in autopsied iPAH patient lungs. The findings are consistent with NOX1 mediating activation of the UPR through PERK, which, in turn, induces EC hyperproliferation in PAH.

Abstract 1039: Endothelial Deletion Of Nck1 Reduces Atherosclerosis In Apoe-/- Mice Through The Nck1-atf3 Axis

The Nck family of signaling adaptors, including both Nck1 and Nck2, classically regulate cell motility during vascular development and postnatal angiogenesis. While Nck1 and Nck2 play redundant roles in this regard with deletion of both isoforms required to affect tissue remodeling, we demonstrated that global Nck1 deletion was sufficient to reduce atherosclerotic plaque formation and selective Nck1 inhibition reduces proinflammatory endothelial activation in response to oscillatory shear stress. Therefore, we sought to determine the role of endothelial Nck1 in atherosclerosis and ischemic angiogenesis using novel endothelial-specific Nck1 knockout mice. Endothelial-specific Nck1 deletion significantly reduced Western diet-induced atherosclerotic plaque formation at multiple sites compared to mice expressing endothelial-specific Cre alone or to endothelial-specific Nck2 knockout mice. This reduced plaque formation was characterized by diminished macrophage and smooth muscle incorporation despite enhanced weight gain in endothelial Nck1 knockout mice. RNA Sequencing analysis of endothelial cells in culture showed that Nck1 depletion reduces the expression of a variety of proinflammatory pathways but enhances the expression of genes involved in central carbon metabolism and proliferation. While Nck1 deletion is sufficient to blunt atherogenic inflammation, Nck1 inhibition did not affect angiogenesis or limb perfusion in the femoral artery ligation model of hind limb ischemia. To further characterized Nck1’s proinflammatory role, we utilized affinity precipitation-mass spectrometry and identified Activating Transcription Factor 3 ATF3 as a Nck1-selective binding partner. While no studies to date have examined endothelial ATF3 signaling in atherosclerosis, our preliminary data show that Nck1 is required for ATF3 expression in response to disturbed flow in vitro and in vivo , but the mechanism by which disturbed flow stimulates ATF3 expression remains unknown. ATF3 knockdown reduces disturbed flow-induced proinflammatory gene expression, suggesting that the Nck1-ATF3 signaling axis is a novel regulator of flow-induced inflammation.