Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is regulated by complex interplay between the macrophages and surrounding cells in the liver. Here, we show that Atf3 regulates glucose-fatty acid cycle in macrophages attenuates hepatocyte steatosis, and fibrogenesis in hepatic stellate cells (HSCs). Overexpression of Atf3 in macrophages protects against the development of MASH in Western diet-fed mice, whereas Atf3 ablation has the opposite effect. Mechanistically, Atf3 improves the reduction of fatty acid oxidation induced by glucose via forkhead box O1 (FoxO1) and Cd36. Atf3 inhibits FoxO1 activity via blocking Hdac1-mediated FoxO1 deacetylation at K242, K245, and K262 and increases Zdhhc4/5-mediated CD36 palmitoylation at C3, C7, C464, and C466; furthermore, macrophage Atf3 decreases hepatocytes lipogenesis and HSCs activation via retinol binding protein 4 (Rbp4). Anti-Rbp4 can prevent MASH progression that is induced by Atf3 deficiency in macrophages. This study identifies Atf3 as a regulator of glucose-fatty acid cycle. Targeting macrophage Atf3 or Rbp4 may be a plausible therapeutic strategy for MASH.

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.

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.

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.

Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.

Glioblastoma stem cells deliver ABCB4 transcribed by ATF3 via exosomes conferring glioblastoma resistance to temozolomide.

Glioblastoma stem cells (GSCs) play a key role in glioblastoma (GBM) resistance to temozolomide (TMZ) chemotherapy. With the increase in research on the tumour microenvironment, exosomes secreted by GSCs have become a new focus in GBM research. However, the molecular mechanism by which GSCs affect drug resistance in GBM cells via exosomes remains unclear. Using bioinformatics analysis, we identified the specific expression of ABCB4 in GSCs. Subsequently, we established GSC cell lines and used ultracentrifugation to extract secreted exosomes. We conducted in vitro and in vivo investigations to validate the promoting effect of ABCB4 and ABCB4-containing exosomes on TMZ resistance. Finally, to identify the transcription factors regulating the transcription of ABCB4, we performed luciferase assays and chromatin immunoprecipitation-quantitative PCR. Our results indicated that ABCB4 is highly expressed in GSCs. Moreover, high expression of ABCB4 promoted the resistance of GSCs to TMZ. Our study found that GSCs can also transmit their highly expressed ABCB4 to differentiated glioma cells (DGCs) through exosomes, leading to high expression of ABCB4 in these cells and promoting their resistance to TMZ. Mechanistic studies have shown that the overexpression of ABCB4 in GSCs is mediated by the transcription factor ATF3. In conclusion, our results indicate that GSCs can confer resistance to TMZ in GBM by transmitting ABCB4, which is transcribed by ATF3, through exosomes. This mechanism may lead to drug resistance and recurrence of GBM. These findings contribute to a deeper understanding of the mechanisms underlying drug resistance in GBM and provide novel insights into its treatment.

m6A demethylation of FOSL1 mRNA protects hepatoma cells against necrosis under glucose deprivation.

Stress-adaptive mechanisms enabling cancer cells to survive under glucose deprivation remain elusive. N6-methyladenosine (m6A) modification plays important roles in determining cancer cell fate and cellular stress response to nutrient deficiency. However, whether m6A modification functions in the regulation of cancer cell survival under glucose deprivation is unknown. Here, we found that glucose deprivation reduced m6A modification levels. Increasing m6A modification resulted in increased hepatoma cell necrosis under glucose deprivation, whereas decreasing m6A modification had an opposite effect. Integrated m6A-seq and RNA-seq revealed potential targets of m6A modification under glucose deprivation, including the transcription factor FOSL1; further, glucose deprivation upregulated FOSL1 by inhibiting FOSL1 mRNA decay in an m6A-YTHDF2-dependent manner through reducing m6A modification in its exon1 and 5'-UTR regions. Functionally, FOSL1 protected hepatoma cells against glucose deprivation-induced necrosis in vitro and in vivo. Mechanistically, FOSL1 transcriptionally repressed ATF3 by binding to its promoter. Meanwhile, ATF3 and MAFF interacted via their leucine zipper domains to form a heterodimer, which competed with NRF2 for binding to antioxidant response elements in the promoters of NRF2 target genes, thereby inhibiting their transcription. Consequently, FOSL1 reduced the formation of the ATF3-MAFF heterodimer, thereby enhancing NRF2 transcriptional activity and the antioxidant capacity of glucose-deprived-hepatoma cells. Thus, FOSL1 alleviated the necrosis-inducing effect of glucose deprivation-induced reactive oxygen species accumulation. Collectively, our study uncovers the protective role of m6A-FOSL1-ATF3 axis in hepatoma cell necrosis under glucose deprivation, and may provide new targets for cancer therapy.

HDAC Inhibition Increases CXCL12 Secretion to Recruit Natural Killer Cells in Peripheral T-cell Lymphoma.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous and aggressive disease with a poor prognosis. Histone deacetylase (HDAC) inhibitors have shown inhibitory effects on PTCL. A better understanding of the therapeutic mechanism underlying the effects of HDAC inhibitors could help improve treatment strategies. Herein, we found that high expression of HDAC3 is associated with poor prognosis in PTCL. HDAC3 inhibition suppressed lymphoma growth in immunocompetent mice but not in immunodeficient mice. HDAC3 deletion delayed the progression of lymphoma, reduced the lymphoma burden in the thymus, spleen, and lymph nodes, and prolonged the survival of mice bearing N-methyl-N-nitrosourea-induced lymphoma. Furthermore, inhibiting HDAC3 promoted the infiltration and enhanced the function of natural killer (NK) cells. Mechanistically, HDAC3 mediated ATF3 deacetylation, enhancing its transcriptional inhibitory activity. Targeting HDAC3 enhanced CXCL12 secretion through an ATF3-dependent pathway to stimulate NK-cell recruitment and activation. Finally, HDAC3 suppression improved the response of PTCL to conventional chemotherapy. Collectively, this study provides insights into the mechanism by which HDAC3 regulates ATF3 activity and CXCL12 secretion, leading to immune infiltration and lymphoma suppression. Combining HDAC3 inhibitors with chemotherapy may be a promising strategy for treating PTCL. Significance: Targeting HDAC3 suppresses progression of T-cell lymphoma by activating ATF3 to induce secretion of CXCL12 and promote infiltration of NK cells, providing an immunostimulatory approach for treating T-cell lymphoma patients.

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.

Iridium(III)-Based PD-L1 Agonist Regulates p62 and ATF3 for Enhanced Cancer Immunotherapy.

Anti-PD-L1 immunotherapy, a new lung cancer treatment, is limited to a few patients due to low PD-L1 expression and tumor immunosuppression. To address these challenges, the upregulation of PD-L1 has the potential to elevate the response rate and efficiency of anti-PD-L1 and alleviate the immunosuppression of the tumor microenvironment. Herein, we developed a novel usnic acid-derived Iridium(III) complex, Ir-UA, that boosts PD-L1 expression and converts "cold tumors" to "hot". Subsequently, we administered Ir-UA combined with anti-PD-L1 in mice, which effectively inhibited tumor growth and promoted CD4+ and CD8+ T cell infiltration. To our knowledge, Ir-UA is the first iridium-based complex to stimulate the expression of PD-L1 by explicitly regulating its transcription factors, which not only provides a promising platform for immune checkpoint blockade but, more importantly, provides an effective treatment strategy for patients with low PD-L1 expression.

ATF3/EGR1 regulates myocardial ischemia/reperfusion injury induced autophagy and inflammation in cardiomyocytes.

Myocardial ischemia/reperfusion injury (MIRI) is an irreversible adverse event during the management of coronary heart disease that lacks effective controls. The underlying mechanism of MIRI still requires further investigation. Recent studies have suggested that overexpression of ATF3 protects against MIRI by regulating inflammatory responses, ferroptosis, and autophagy. The downstream target of ATF3, EGR1, also showed cardioprotective properties against MIRI by promoting autophagy. Therefore, further investigating the effect of ATF3/EGR1 pathway on MIRI-induced inflammation and autophagy is needed. Cardiomyocyte MIRI model was established by challenging H9C2 cells with hypoxia/reoxygenation (H/R). The ATF3 overexpression-H/R cell model by transfecting ATF3 plasmid into the H9C2 cell line. The transcription levels of ATF3 and EGR1 were determined using RT-qPCR, the levels of TNF-α and IL-6 were determined using ELISA kits, the protein expression of LC3 I, LC3 II, and P62 was determined via WB, and microstructure of H9C2 cell was observed by transmission electron microscopy (TEM). Overexpression of ATF3 significantly downregulated Egr1 levels, indicating that EGR1 might be the target of ATF3. By upregulating ATF3 levels, the extracellular levels of the inflammatory cytokines TNF-α and IL-6 significantly decreased, and the protein expression of the autophagy markers LC3 I, LC3 II, and P62 significantly increased. TEM results revealed that the cell line in the H/R-ATF3 group exhibited a higher abundance of autophagosome enclosures of mitochondria. The results indicated that ATF3/EGR1 may alleviate inflammation and improve autophagy in an H/R-induced MIRI model of cardiomyocytes.

ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC.

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

BACKGROUND: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. METHODS: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). RESULTS: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. CONCLUSION: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.

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.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. METHODS: Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. RESULTS: In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor ß) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. CONCLUSIONS: Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer.

The induction of ferroptosis is promising for cancer therapy. However, the mechanisms enabling cancer cells to evade ferroptosis, particularly in low-cystine environments, remain elusive. Our study delves into the intricate regulatory mechanisms of Activating transcription factor 3 (ATF3) on Cystathionine ß-synthase (CBS) under cystine deprivation stress, conferring resistance to ferroptosis in colorectal cancer (CRC) cells. Additionally, our findings establish a positively correlation between this signaling axis and CRC progression, suggesting its potential as a therapeutic target. Mechanistically, ATF3 positively regulates CBS to resist ferroptosis under cystine deprivation stress. In contrast, the suppression of CBS sensitizes CRC cells to ferroptosis through targeting the mitochondrial tricarboxylic acid (TCA) cycle. Notably, our study highlights that the ATF3-CBS signaling axis enhances ferroptosis-based CRC cancer therapy. Collectively, the findings reveal that the ATF3-CBS signaling axis is the primary feedback pathway in ferroptosis, and blocking this axis could be a potential therapeutic approach for colorectal cancer.

STEAP3 promotes colon cancer cell proliferation and migration via regulating histone acetylation.

Colorectal cancer (CRC) is the third most prevalent diagnosed cancer in men and second most prevalent cancer in women. H3K27ac alterations are more commonly than gene mutations in colorectal cancer. Most colorectal cancer genes have significant H3K27ac changes, which leads to an over-expression disorder in gene transcription. Over-expression of STEAP3 is involved in a variety of tumors, participating in the regulation of cancer cell proliferation and migration. The purpose of this work is to investigate the role of STEAP3 in the regulation of histone modification (H3K27ac) expression in colon cancer. Bioinformatic ChIP-seq, ChIP-qPCR and ATAC-seq were used to analyze the histone modification properties and gene accessibility of STEAP3. Western blot and qRT-PCR were used to evaluate relative protein and gene expression, respectively. CRISPR/Cas9 technology was used to knockout STEAP3 on colon cancer cells to analyze the effect of ATF3 on STEAP3. STEAP3 was over-expressed in colon cancer and associated with higher metastases and more invasive and worse stage of colon cancer. ChIP-seq and ChIP-qPCR analyses revealed significant enrichment of H3K27ac in the STEAP3 gene. In addition, knocking down STEAP3 significantly inhibits colon cancer cell proliferation and migration and down-regulates H3K27ac expression. ChIP-seq found that ATF3 is enriched in the STEAP3 gene and CRISPR/Cas9 technology used for the deletion of the ATF3 binding site suppresses the expression of STEAP3. Over-expression of STEAP3 promotes colon cancer cell proliferation and migration. Mechanical studies have indicated that H3K27ac and ATF3 are significantly enriched in the STEAP3 gene and regulate the over-expression of STEAP3.

Deubiquitinating enzyme OTUD4 stabilizes RBM47 to induce ATF3 transcription: a novel mechanism underlying the restrained malignant properties of ccRCC cells.

Dysregulation of deubiquitination contributes to various diseases, including cancer, and aberrant expression of deubiquitinating enzymes is involved in carcinoma progression. As a member of the ovarian tumor (OTU) deubiquitinases, OTUD4 is considered a tumor suppressor in many kinds of malignancies. The biological characteristics and mechanisms of OTUD4 in clear cell renal cell carcinoma (ccRCC) remain unclear. The downregulation of OTUD4 in ccRCC was confirmed based on the TCGA database and a validation cohort of 30-paired ccRCC and para-carcinoma samples. Moreover, OTUD4 expression was detected by immunohistochemistry in 50 cases of ccRCC tissues, and patients with lower levels of OTUD4 showed larger tumor size (p = 0.015). TCGA data revealed that patients with high expression of OTUD4 had a longer overall survival rate. In vitro and in vivo studies revealed that downregulation of OTUD4 was essential for tumor cell growth and metastasis in ccRCC, and OTUD4 overexpression inhibited these malignant phenotypes. We further found that OTUD4 sensitized ccRCC cells to Erastin-induced ferroptosis, and ferrostain-1 inhibited OTUD4-induced ferroptotic cell death. Mechanistic studies indicated that OTUD4 functioned as an anti-proliferative and anti-metastasic factor through the regulation of RNA-binding protein 47 (RBM47)-mediated activating transcription factor 3 (ATF3). OTUD4 directly interacted with RBM47 and promoted its stability via deubiquitination events. RBM47 was critical in ccRCC progression by regulating ATF3 mRNA stability, thereby promoting ATF3-mediated ferroptosis. RBM47 interference abolished the suppressive role of OTUD4 overexpression in ccRCC. Our findings provide mechanistic insight into OTUD4 of ccRCC progression and indicate a novel critical pathway OTUD4/RBM47/ATF3 may serve as a potential therapeutic pathway for ccRCC.

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.