Activating transcription factor 3 (ATF3) and calcitonin gene-related peptide (CGRP) increase in trigeminal ganglion neurons in female rats after photorefractive keratectomy (PRK)-like corneal abrasion.

Photorefractive keratectomy (PRK) is a type of eye surgery that involves removal of the corneal epithelium and its associated nerves, which causes intense acute pain in most people. We used a rat model of corneal epithelium removal (corneal abrasion) to examine underlying cellular and molecular mechanisms. In this study, we used immunohistochemistry of trigeminal ganglion (TG) to assess neuronal content of CGRP and ATF3, as well as orbital tightening (OT) to assess spontaneous pain behaviors. CGRP is an important neuropeptide in pain modulation and ATF3 is often used as a nerve injury marker. We found dynamic changes in CGRP and ATF3 in TG; both increased significantly at 24 h following corneal abrasion and females had a more pronounced increase at 24 h compared to males. Interestingly, there was no sex difference in OT behaviors. Additionally, the number of cells containing either CGRP or ATF3 in each animal correlate significantly with their OT behavior at the assessed timepoint. Since CGRP increased most in females, we tested the effectiveness of Olcegepant, a CGRP antagonist, at reducing OT behaviors following corneal abrasion in female rats. Olcegepant (1 mg/kg) was given prior to and again at 24 h after abrasion but did not change OT behaviors at any time over a 1-week period. Examination of CGRP and ATF3 together in TG showed that they rarely colocalized, indicating that the cells with upregulated CGRP are distinct from those responding to epithelial nerve injury. The studies also show that underlying molecular responses may be sex specific.

A comprehensive bioinformatic analysis of the role of TGF-ß1-stimulated activating transcription factor 3 by non-coding RNAs during breast cancer progression.

A potent growth inhibitor for normal mammary epithelial cells is transforming growth factor beta 1 (TGF-ß1). When breast tissues lose the anti-proliferative activity of this factor, invasion and bone metastases increase. Human breast cancer (hBC) cells express more activating transcription factor 3 (ATF3) when exposed to TGF-ß1, and this transcription factor is essential for BC development and bone metastases. Non-coding RNAs (ncRNAs), including circular RNAs (circRNAs) and microRNAs (miRNAs), have emerged as key regulators controlling several cellular processes. In hBC cells, TGF-ß1 stimulated the expression of hsa-miR-4653-5p that putatively targets ATF3. Bioinformatics analysis predicted that hsa-miR-4653-5p targets several key signaling components and transcription factors, including NFKB1, STAT1, STAT3, NOTCH1, JUN, TCF3, p300, NRF2, SUMO2, and NANOG, suggesting the diversified role of hsa-miR-4653-5p under physiological and pathological conditions. Despite the high abundance of hsa-miR-4653-5p in hBC cells, the ATF3 level remained elevated, indicating other ncRNAs could inhibit hsa-miR-4653-5p's activity. In silico analysis identified several circRNAs having the binding sites for hsa-miR-4653-5p, indicating the sponging activity of circRNAs towards hsa-miR-4653-5p. The study's findings suggest that TGF-ß1 regulates circRNAs and hsa-miR-4653-5p, which in turn affects ATF3 expression, thus influencing BC progression and bone metastasis. Therefore, focusing on the TGF-ß1/circRNAs/hsa-miR-4653-5p/ATF3 network could lead to new ways of diagnosing and treating BC.

Phenylalanine deprivation inhibits multiple myeloma progression by perturbing endoplasmic reticulum homeostasis.

Amino acid metabolic remodeling is a hallmark of cancer, driving an increased nutritional demand for amino acids. Amino acids are pivotal for energetic regulation, biosynthetic support, and homeostatic maintenance to stimulate cancer progression. However, the role of phenylalanine in multiple myeloma (MM) remains unknown. Here, we demonstrate that phenylalanine levels in MM patients are decreased in plasma but elevated in bone marrow (BM) cells. After the treatment, phenylalanine levels increase in plasma and decrease in BM. This suggests that changes in phenylalanine have diagnostic value and that phenylalanine in the BM microenvironment is an essential source of nutrients for MM progression. The requirement for phenylalanine by MM cells exhibits a similar pattern. Inhibiting phenylalanine utilization suppresses MM cell growth and provides a synergistic effect with Bortezomib (BTZ) treatment in vitro and murine models. Mechanistically, phenylalanine deprivation induces excessive endoplasmic reticulum stress and leads to MM cell apoptosis through the ATF3-CHOP-DR5 pathway. Interference with ATF3 significantly affects phenylalanine deprivation therapy. In conclusion, we have identified phenylalanine metabolism as a characteristic feature of MM metabolic remodeling. Phenylalanine is necessary for MM proliferation, and its aberrant demand highlights the importance of low-phenylalanine diets as an adjuvant treatment for MM.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

SEH1L siliencing induces ferroptosis and suppresses hepatocellular carcinoma progression via ATF3/HMOX1/GPX4 axis.

SEH1 like nucleoporin (SEH1L) is an important component of nuclear pore complex (NPC), which is crucial in the regulation of cell division. However, the interrelation between SEH1L expression and tumor progression is less studied. In this research, we performed a systematic bioinformatic analysis about SEH1L using TCGA, Timer 2.0, Cbioportal, UCLAN and CellMiner™ databases in pan-cancer. Besides, we further validated the bioinformatic results through in vitro and in vivo experiments in HCC, including transcriptome sequencing, real-time quantitative PCR (RT-qPCR), western blotting (WB), immunohistochemistry (IHC), cell proliferation assays, clone formation, EdU, transwell, flow cytometry and subcutaneous tumor model. Our results suggested that SEH1L was significantly up-regulated and related to poor prognosis in most cancers, and may serve as a potential biomarker. SEH1L could promote HCC progression in vitro and in vivo. Besides, the next generation sequencing suggested that 684 genes was significantly up-regulated and 678 genes was down-regulated after the knock down of SEH1L. SEH1L siliencing could activate ATF3/HMOX1/GPX4 axis, decrease mitochondrial membrane potential and GSH, but increase ROS and MDA, and these effects could be reversed by the knock down of ATF3. This study indicated that SEH1L siliencing could induce ferroptosis and suppresses hepatocellular carcinoma (HCC) progression via ATF3/HMOX1/GPX4 axis.

Ferroptosis-Related Genes in IgA Nephropathy: Screening for Potential Targets of the Mechanism.

Aims: Exploring key genes and potential molecular pathways of ferroptosis in immunoglobulin A nephropathy (IgAN). Methods: The IgAN datasets and ferroptosis-related genes (FRGs) were obtained in the Gene Expression Omnibus (GEO) and FerrDb database. Differentially expressed genes (DEGs) were identified using R software and intersected with FRGs to obtain differentially expressed FRGs (DE-FRGs). After that, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (PEA) and Gene Ontology (GO) functional annotation were performed on DE-FRGs. In the Search Tool for the Retrieval of Interacting Genes (STRING) website, we construct a protein-protein interaction (PPI) network. The PPI network was further investigated with screening hub genes with Cytoscape software. The core genes were then subjected to gene set enrichment analysis (GSEA). Finally, the samples were analyzed for immune infiltration in R, and the correlation between hub genes and immune cells was analyzed. Results: A total of 347 DEGs were identified. CD44, CDO1, CYBB, IL1B, RRM2, AKR1C1, activated transcription factor-3 (ATF3), CDKN1A, GDF15, JUN, MGST1, MIOX, MT1G, NR4A1, PDK4, TNFAIP3, and ZFP36 were determined as DE-FRGs. JUN, IL1B, and ATF3 were then screened as hub genes. GSEA and immune infiltration analysis revealed that the hub genes were closely associated with immune inflammatory responses such as NOD-like receptor signaling, IL-17 signaling, and TNF signaling. Conclusions: Our results show that JUN and ATF3 are possibly critical genes in the process of IgAN ferroptosis and may be related with immune cell infiltration.

Calcitriol alleviates noise-induced hearing loss by regulating the ATF3/DUSP1 signalling pathway.

BACKGROUND: Calcitriol (Cal) is the most active metabolite of vitamin D and has antioxidant and anti-inflammatory properties. The aim of this study was to investigate the role of Cal in noise-induced hearing loss (NIHL) to further elucidate the mechanism of noise-induced oxidative stress in the mouse cochlea. METHODS: C57BL/6 J mice were given six intraperitoneal injections of Cal (500 ng/kg/d). After 14 days of noise exposure, auditory brainstem response (ABR) thresholds, and the cochlear outer hair cell loss rate were analysed to evaluate auditory function. Real-time fluorescence quantitative PCR, immunofluorescence and western blotting were performed in vitro after the treatment of cochlear explants with 100 µM tert-butyl hydroperoxide (TBHP) for 2.5 h and HEI-OC1 cells with 250 µM TBHP for 1.5 h. RESULTS: In vivo experiments confirmed that Cal pretreatment mitigated NIHL and outer hair cell death. The in vitro results demonstrated that Cal significantly reduced TBHP-induced cochlear auditory nerve fibre degradation and spiral ganglion neuron damage. Moreover, treatment with Cal inhibited the expression of oxidative stress-related factors (3-NT and 4-HNE) and DNA damage-related factors (γ-H2A.X) and attenuated TBHP-induced apoptosis in cochlear explants and HEI-OC1 cells. A total of 1479 upregulated genes and 1443 downregulated genes were screened in cochlear tissue 1 h after noise exposure. The level of transcription factor 3 (ATF3) was significantly elevated in HEI-OC1 cells after TBHP stimulation. Gene Transcription Regulation Database （GTRD）and Cistrome database analyses revealed that the downstream target gene of ATF3 is dual specificity phosphatase 1 (DUSP1). Cistrome DB Toolkit database results showed that the transcription factor of DUSP1 was ATF3. In addition, the ChIP-PCR results indicated that ATF3 might be a direct transcription factor of DUSP1. CONCLUSION: The results of our study suggest that Cal attenuates NIHL and inhibits noise-induced apoptosis by regulating the ATF3/DUSP1 signalling 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.

ATF3 is involved in rSjP40-mediated inhibition of HSCs activation in Schistosoma japonicum-infected mice.

Schistosomiasis is a parasitic disease characterized by liver fibrosis, a process driven by the activation of hepatic stellate cells (HSCs) and subsequent collagen production. Previous studies from our laboratory have demonstrated the ability of Schistosoma japonicum protein P40 (SjP40) to inhibit HSCs activation and exert an antifibrotic effect. In this study, we aimed to elucidate the molecular mechanism underlying the inhibitory effect of recombinant SjP40 (rSjP40) on HSCs activation. Using a cell model in which rSjP40 inhibited LX-2 cell activation, we performed RNA-seq analyses and identified ATF3 as the most significantly altered gene. Further investigation revealed that rSjP40 inhibited HSCs activation partly by suppressing ATF3 activation. Knockdown of ATF3 in mouse liver significantly alleviated S. japonicum-induced liver fibrosis. Moreover, our results indicate that ATF3 is a direct target of microRNA-494-3p, a microRNA associated with anti-liver fibrosis effects. rSjP40 was found to downregulate ATF3 expression by upregulating microRNA-494-3p in LX-2 cells. This downregulation led to the inhibition of the expression of liver fibrosis proteins α-SMA and COL1A1, ultimately alleviating liver fibrosis caused by S. japonicum.

Short-chain fatty acids regulate erastin-induced cardiomyocyte ferroptosis and ferroptosis-related genes.

Background: Ferroptosis has been proven to contribute to the progression of myocardial ischemia/reperfusion (I/R) injury and can be inhibited or promoted by ATF3. Short-chain fatty acids (SCFAs) have shown benefits in various cardiovascular diseases with anti-inflammatory and antioxidant effects. However, the impact of SCFAs on ferroptosis in ischemic-stimulated cardiomyocytes remains unknown. This study aimed to investigate the effect of SCFAs on cardiomyocyte ferroptosis, the expression of ATF3, and its potential upstream regulators. Methods and results: The expression of ATF3, ferroptosis pathway geneset (FPG), and geneset of potential regulators for ATF3 (GPRA, predicted by the PROMO database) was explored in the public human myocardial infarction single-cell RNA-seq (sma) dataset. Cardiomyocyte data was extracted from the dataset and re-clustered to explore the FPG, ATF3, and GPRA expression patterns in cardiomyocyte subclusters. A dose-dependent toxic experiment was run to detect the suitable dose for SCFA treatment. The erastin-induced ferroptosis model and hypoxia-reoxygenation (H/R) model (10 h of hypoxia followed by 6 h of reoxygenation) were adopted to assess the effect of SCFAs via the CCK8 assay. Gene expression was examined via RT-PCR and western blot. Ferroptosis markers, including lipid peroxides and Fe2+, were detected using the liperfluo and ferroOrange probes, respectively. In the sma dataset, upregulated ferroptosis pathway genes were mainly found in the infarction-stimulated cardiac cells (border zone and fibrotic zone), particularly the cardiomyocytes and adipocytes. The ATF3 and some of its potential transcription factors (VDR, EGR3, PAX5, and SP1) can be regulated by SCFA. SCFA can attenuate erastin-induced lipid peroxidation in cardiomyocytes. SCFA treatment can also reverse erastin-induced Fe2+ increase but may strengthen the Fe2+ in the H/R model. We also precisely defined a ferroptosis subcluster of cardiomyocytes (CM09) that highly expressed FPG, ATF3, and GPRA. Conclusion: The ATF3 and the ferroptosis pathway are elevated in cardiomyocytes of injury-related cardiac regions (border zone, ischemic zone, and fibrotic zone). SCFA can attenuate cardiomyocyte ferroptosis and regulate the expression of ATF3. Our study offers novel insights into the potential targets of SCFAs in the cardiovascular system.

Activating transcription factor 3 mediates apoptosis and cell cycle arrest in TP53-mutated anaplastic thyroid cancer cells.

BACKGROUND: It is believed that loss of p53 function plays a crucial role in the progression of well to poorly differentiated thyroid cancers including anaplastic thyroid carcinoma (ATC). Given the poor prognosis of ATC due to its strong therapeutic resistance, there is a need to establish new therapeutic targets to extend the survival of ATC patients. Activating transcription factor 3 (ATF3) can inhibit the oncogenic activity of mutant p53 and, as a result, contribute to tumor suppression in several TP53-mutated cancers. Herein, we demonstrate that the ectopic overexpression of ATF3 leads to the suppression of oncogenic mutant p53 activity in chemo-resistant 8305 C thyroid cancer cells harboring R273C p53 gene mutation. METHODS: The biological behavior of 8305 C cells was assessed pre- and post-transfection with pCMV6-ATF3 plasmid using MTT assay, fluorescent microscopy, cell cycle, and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 overexpression on the cellular level of p53 was examined by western blotting assay. The mRNA expression levels of TP53, TAp63, ΔNp63, and SHARP1 were evaluated in ectopic ATF3-expressing cells compared to controls. RESULTS: The overexpression of ATF3 in 8305 C thyroid cancer cells significantly decreased cell viability and induced apoptosis and cell cycle arrest in vitro. The immunoblotting of p53 protein revealed that ATF3 overexpression significantly increased the level of mutant p53 in 8305C cells compared to mock-transfected control cells. Additionally, elevated mRNA levels of TAp63 and SHARP1 and a decreased mRNA level of ΔNp63 were observed in PCMV6-AC-ATF3-transfected 8305 C cells with significant differences compared to the mock and untreated cells. CONCLUSION: In light of our findings, it is evident that therapeutic strategies aimed at increasing ATF3 expression or enhancing the interaction between ATF3 and mutant p53 can be a promising approach for the treatment of p53-mutated metastatic thyroid cancer.

Activating transcription factor 3 is an antitumor gene synergizing with growth differentiation factor 15 to modulate cell growth in human bladder cancer.

BACKGROUND: The functions of activating transcription factor 3 (ATF3) within the human bladder remain unexplored. This study delves into the expressions, functions, and regulatory mechanisms of ATF3 in human bladder cancer. MATERIAL AND METHODS: Gene expressions were determined by immunoblot, RT-qPCR, and reporter assays. Assays of Ki67, colony formation, Matrigel invasion, and the xenograft animal study were used to assess the cell proliferation, invasion, and tumorigenesis in vitro and in vivo. Silico analysis from TCGA database examined the correlations between GDF15 and ATF3 expressions, clinicopathologic features, and progression-free survival rates. RESULTS: Silico analysis confirmed that ATF3 is an antitumor gene, and the expression positively correlates with GDF15 in bladder cancer tissues. Multivariate analysis revealed that low ATF3/GDF15 but not a single low expression of ATF3 is an independent prognostic factor for progression-free survival of bladder cancer patients. Ectopic overexpression of ATF3 downregulated cell proliferation and invasion in bladder cancer cells in vitro, while ATF3-knockdown reversed these results. Knockdown of ATF3 upregulated EMT markers to enhance cell invasion in vitro and downregulated GDF15, NDRG1, and KAI-1 to elevate tumor growth in vivo. The activation of metformin on ATF3 and GDF15 in bladder cancer cells was blocked by SB431542, a TGFß receptor inhibitor. ATF3 positively regulated GDF15 expression in bladder cancer cells through a feedback loop. CONCLUSIONS: Our results identify that ATF3 is a metformin-upregulated antitumor gene. Results of Silico analysis align with cell-based studies suggesting that low ATF3/GDF15 could be a negative prognostic marker for bladder cancer.

Identifying the protective effects of miR-874-3p/ATF3 axis in intervertebral disc degeneration by single-cell RNA sequencing and validation.

Intervertebral disc degeneration (IVDD) severely affects the work and the quality of life of people. We previously demonstrated that silencing activation transcription factor 3 (ATF3) blocked the IVDD pathological process by regulating nucleus pulposus cell (NPC) ferroptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism. Nevertheless, whether miR-874-3p mediated the IVDD pathological process by targeting ATF3 remains unclear. We performed single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis to identify ATF3 as a key ferroptosis gene in IVDD. Then, Western blotting, flow cytometry, ELISA, and animal experiments were performed to validate the roles and regulatory mechanisms of miR-874-3p/ATF3 signalling axis in IVDD. ATF3 was highly expressed in IVDD patients and multiple cell types of IVDD rat, as revealed by scRNA-seq and bioinformatics analysis. GO analysis unveiled the involvement of ATF3 in regulating cell apoptosis and ECM metabolism. Furthermore, we verified that miR-874-3p might protect against IVDD by inhibiting NPC ferroptosis, apoptosis, ECM degradation, and inflammatory response by targeting ATF3. In vivo experiments displayed the protective effect of miR-874-3p/ATF3 axis on IVDD. These findings propose the potential of miR-874-3p and ATF3 as biomarkers of IVDD and suggest that targeting the miR-874-3p/ATF3 axis may be a therapeutic target for IVDD.

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.

Brusatol induces ferroptosis to inhibit hepatocellular carcinoma progression by targeting ATF3.

Ferroptosis is a novel form of programmed cell death that is triggered by iron-dependent lipid peroxidation. Brusatol (BRU), a natural nuclear factor erythroid 2-related factor 2 inhibitor, exhibits potent anticancer effects in various types of cancer. However, the exact mechanism of BRU in the treatment of hepatocellular carcinoma (HCC) remains unknown. The anticancer effects of BRU in HCC were detected using cell counting kit-8 and colony formation assays and a xenograft model. RNA sequencing (RNA-seq) and bioinformatics analyses of HCC cells were utilized to elucidate the mechanism underlying the effects of BRU in HCC. The levels of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and Fe2+ were measured using assay kits. The expression of activating transcription factor 3 (ATF3) was tested using RT-qPCR, western blotting, and immunofluorescence staining. The role of ATF3 in BRU-induced ferroptosis was examined using siATF3. BRU significantly inhibited HCC cell proliferation, both in vitro and in vivo. BRU activated the ferroptosis signaling pathway and increased ATF3 expression. Furthermore, ATF3 knockdown impeded BRU-induced ferroptosis. BRU suppressed HCC growth through ATF3-mediated ferroptosis, supporting BRU as a promising therapeutic agent for HCC.

Identification and analysis of differently expressed transcription factors in aristolochic acid nephropathy.

BACKGROUND: Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy caused by Aristolochic acid (AA). AAN is associated with the development of nephropathy and urothelial carcinoma. It is estimated that more than 100 million people worldwide are at risk of developing AAN. However, the underlying mechanisms driving renal deterioration in AAN remain poorly understood, and the treatment options are limited. METHODS: We obtained GSE27168 and GSE136276 series matrix data from the Gene Expression Omnibus (GEO) related to AAN. Using the R Studio environment, we applied the limma package and WGCNA package to identify co-differently expressed genes (co-DEGs). By GO/KEGG/GSVA analysis, we revealed common biological pathways. Subsequently, co-DEGs were subjected to the String database to construct a protein-protein interaction (PPI) network. The MCC algorithms implemented in the Cytohubba plugin were employed to identify hub genes. The hub genes were cross-referenced with the transcription factor (TF) database to identify hub TFs. Immune infiltration analysis was performed to identify key immune cell groups by utilizing CIBERSORT. The expressions of AAN-associated hub TFs were verified in vivo and in vitro. Finally, siRNA intervention was performed on the two TFs to verify their regulatory effect in AAN. RESULTS: Our analysis identified 88 co-DEGs through the "limma" and "WGCNA" R packages. A PPI network comprising 53 nodes and 34 edges was constructed with a confidence level >0.4. ATF3 and c-JUN were identified as hub TFs potentially linked to AAN. Additionally, expressions of ATF3 and c-JUN positively correlated with monocytes, basophils, and vessels, and negatively correlated with eosinophils and endothelial cells. We observed a significant increase in protein and mRNA levels of these two hub TFs. Furthermore, it was found that siRNA intervention targeting ATF3, but not c-JUN, alleviated cell damage induced by AA. The knockdown of ATF3 protects against oxidative stress and inflammation in the AAN cell model. CONCLUSION: This study provides novel insights into the role of ATF3 in AAN. The comprehensive analysis sheds light on the molecular mechanisms and identifies potential biomarkers and drug targets for AAN treatment.

Targeting Fatty Acid Desaturase I Inhibits Renal Cancer Growth Via ATF3-mediated ER Stress Response.

Monounsaturated fatty acids (MUFAs) play a pivotal role in maintaining endoplasmic reticulum (ER) homeostasis, an emerging hallmark of cancer. However, the role of polyunsaturated fatty acid (PUFAs) desaturation in persistent ER stress driven by oncogenic abnormalities remains elusive. Fatty Acid Desaturase 1 (FADS1) is a rate-limiting enzyme controlling the bioproduction of long-chain PUFAs. Our previous research has demonstrated the significant role of FADS1 in cancer survival, especially in kidney cancers. We explored the underlying mechanism in this study. We found that pharmacological inhibition or knockdown of the expression of FADS1 effectively inhibits renal cancer cell proliferation and induces cell cycle arrest. The stable knockdown of FADS1 also significantly inhibits tumor formation in vivo. Mechanistically, we show that while FADS1 inhibition induces ER stress, its expression is also augmented by ER-stress inducers. Notably, FADS1-inhibition sensitized cellular response to ER stress inducers, providing evidence of FADS1's role in modulating the ER stress response in cancer cells. We show that, while FADS1 inhibition-induced ER stress leads to activation of ATF3, ATF3-knockdown rescues the FADS1 inhibition-induced ER stress and cell growth suppression. In addition, FADS1 inhibition results in the impaired biosynthesis of nucleotides and decreases the level of UPD-N-Acetylglucosamine, a critical mediator of the unfolded protein response. Our findings suggest that PUFA desaturation is crucial for rescuing cancer cells from persistent ER stress, supporting FADS1 as a new therapeutic target.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Revealing the mechanism of ethyl acetate extracts of Semen Impatientis against prostate cancer based on network pharmacology and transcriptomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. AIM OF THE STUDY: In this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. MATERIALS AND METHODS: The antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear-cytoplasmic separation assays. RESULTS: EAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR- PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR- PCa cells. CONCLUSIONS: EAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR- PCa cells.

HIF1α/ATF3 partake in PGK1 K191/K192 succinylation by modulating P4HA1/succinate signaling in glioblastoma.

BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSIONS: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.