Activating Transcription Factor Research Articles

Pharmacologic activation of activating transcription factor 6 contributes to neuronal survival after spinal cord injury in mice.

The impact of primary and secondary injuries of spinal cord injury (SCI) results in the demise of numerous neurons, and there is still no efficacious pharmacological intervention for it. Recently, studies have shown that endoplasmic reticulum stress (ERS) plays a pivotal role in recovery of neurological function after spinal cord injury. As a process to cope with intracellular accumulation of misfolded and unfolded proteins which triggers ERS, the unfolded protein response (UPR) plays an important role in maintaining protein homeostasis. And, a recently disclosed small molecule AA147, which selectively activates activating transcription factor 6 (ATF6), has shown promising pharmacological effects in several disease models. Thus, it seems feasible to protect the neurons after spinal cord injury by modulating UPR. In this study, primary neurons were isolated from E17-19 C57BL/6J mouse embryos and we observed that AA147 effectively promoted the survival of neurons and alleviated neuronal apoptosis after oxygen-glucose deprivation/reoxygenation (OGD/R) invitro. This was evident through a decrease in the proportion of PI-positive and TUNEL-positive cells, an increase in BCL-2 expression, and a decrease in the expression of BAX and C-caspase3. In in-vivo experiments, these findings were corroborated by TUNEL staining and immunohistochemistry. It was also found that AA147 enhanced three arms of the unfolded protein response with reduced CHOP expression. Besides, AA147 mitigated the accumulation of ROS in neurons probably by upregulating catalase expression. Furthermore, spinal cord injury models of C57BL/6J mice were established and behavioral experiments revealed that AA147 facilitated the recovery of motor function following SCI. Thus, pharmacologic activation of ATF6 represents a promise therapeutic approach to ameliorate the prognosis of SCI.

Varicella Zoster Virus disrupts MAIT cell polyfunctional effector responses.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that respond to riboflavin biosynthesis and cytokines through TCR-dependent and -independent pathways, respectively. MAIT cell activation plays an immunoprotective role against several pathogens, however the functional capacity of MAIT cells following direct infection or exposure to infectious agents remains poorly defined. We investigated the impact of Varicella Zoster Virus (VZV) on blood-derived MAIT cells and report virus-mediated impairment of activation, cytokine production, and altered transcription factor expression by VZV infected (antigen+) and VZV exposed (antigen-) MAIT cells in response to TCR-dependent and -independent stimulation. Furthermore, we reveal that suppression of VZV exposed (antigen-) MAIT cells is not mediated by a soluble factor from neighbouring VZV infected (antigen+) MAIT cells. Finally, we demonstrate that VZV impairs the cytolytic potential of MAIT cells in response to riboflavin synthesising bacteria. In summary, we report a virus-mediated immune-evasion strategy that disarms MAIT cell responses.

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

BackgroundIt 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.MethodsThe 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.ResultsThe 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.ConclusionIn 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.

Differential effects of montelukast and zafirlukast on MDA‑MB‑231 triple‑negative breast cancer cells: Cell cycle regulation, apoptosis, autophagy, DNA damage and endoplasmic reticulum stress.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple‑negative breast cancer MDA‑MB‑231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription‑quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki‑67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti‑apoptotic protein Bcl‑2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3‑II and DNA damage markers, including cleaved PARP‑1, phosphorylated (p)‑ATM and p‑histone H2AX. The number of caspase 3/7‑positive cells was greater in montelukast‑treated cells compared with zafirlukast‑treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol‑requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast‑induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

ATF2/BAP1 Axis Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage via P53 Pathway.

Neuronal apoptosis plays an essential role in the pathogenesis of brain injury after subarachnoid hemorrhage (SAH). BAP1 (BRCA1-associated protein 1) is considered to exert pro-apoptotic effects in multiple diseases. However, evidence supporting the effect of BAP1 on the apoptotic response to SAH is lacking. Therefore, we aimed to confirm the role of BAP1 in SAH-induced apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to detect BAP1 expression in the cerebrospinal fluid. Endovascular perforation was performed in mice to induce SAH. Lentiviral short hairpin RNA targeting BAP1 mRNA was transduced into the ipsilateral cortex of mice with SAH to investigate the role of BAP1 in neuronal damage. Luciferase and coimmunoprecipitation assays were performed to investigate the mechanism through which BAP1 participates in hemin-induced SAH. First, BAP1 expression was upregulated in the cerebrospinal fluid of patients with SAH and positively associated with unfavorable outcomes. ATF2 (activating transcription factor-2) then regulated BAP1 expression by binding to the BAP1 promoter. In addition, BAP1 overexpression enhanced P53 activity and stability by reducing P53 proteasome-mediated degradation. Subsequently, elevated P53 promoted neuronal apoptosis via the P53 pathway. Inhibition of the neuronal BAP1/P53 axis significantly reduced neurological deficits and neuronal apoptosis and improved neurological dysfunction in mice after SAH. Our results suggest that the neuronal ATF2/BAP1 axis exerts a brain-damaging effect by modulating P53 activity and stability and may be a novel therapeutic target for SAH.

Activation of PERK/eIF2α/ATF4/CHOP branch of endoplasmic reticulum stress response and cooperation between HIF-1α and ATF4 promotes Daprodustat-induced vascular calcification

Introduction: Vascular calcification is accelerated in patients with chronic kidney disease (CKD) and increases the risk of cardiovascular events. CKD is frequently associated with anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor for the treatment of CKD-associated anemia that enhances erythropoiesis through the activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Studies showed that DPD promotes osteogenic differentiation of human aortic smooth muscle cells (HAoSMCs) and increases aorta calcification in mice with CKD. HIF-1 activation has been linked with endoplasmic reticulum (ER) stress; therefore, here we investigated the potential contribution of ER stress, particularly activating transcription factor 4 (ATF4), to the pro-calcification effect of DPD.Methods: Here, we used an adenine-induced CKD mouse model and HAoSMCs as an in vitro vascular calcification model to study the effect of DPD.Results: DPD treatment (15 mg/kg/day) corrects anemia but increases the expression of hypoxia (Glut1, VEGFA), ER stress (ATF4, CHOP, and GRP78), and osteo-/chondrogenic (Runx2, Sox9, BMP2, and Msx2) markers and accelerates aorta and kidney calcification in CKD mice. DPD activates the PERK/eIF2α/ATF4/CHOP pathway and promotes high phosphate-induced osteo-/chondrogenic differentiation of HAoSMCs. Inhibition of ER stress with 4-PBA or silencing of ATF4 attenuates HAoSMC calcification. DPD-induced ATF4 expression is abolished in the absence of HIF-1α; however, knockdown of ATF4 does not affect HIF-1α expression.Conclusion: We concluded that DPD induces ER stress in vitro and in vivo, in which ATF4 serves as a downstream effector of HIF-1 activation. Targeting ATF4 could be a potential therapeutic approach to attenuate the pro-calcific effect of DPD.

Ferroptosis is involved in Staphylococcus aureus-induced mastitis through autophagy activation by endoplasmic reticulum stress

Cell death caused by severe Staphylococcus aureus (S. aureus) infection is a fatal threat to humans and animals. However, whether ferroptosis, an iron-dependent form of cell death, is involved in S. aureus-induced cell death and its role in S. aureus-induced diseases are unclear. Using a mouse mastitis model and mammary epithelial cells (MMECs), we investigated the role of ferroptosis in the pathogenesis of S. aureus infection. The results revealed that S. aureus-induced ferroptosis in vivo and in vitro as demonstrated by dose-dependent increases in cell death; the level of malondialdehyde (MDA), the final product of lipid peroxidation; and dose-dependent decrease the production of the antioxidant glutathione (GSH). Treatment with typical inhibitors of ferroptosis, including ferrostatin-1 (Fer-1) and deferiprone (DFO), significantly inhibited S. aureus-induced death in MMECs. Mechanistically, treatment with S. aureus activated the protein kinase RNA-like ER kinase (PERK)-eukaryotic initiation factor 2, α subunit (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) pathway, which subsequently upregulated autophagy and promoted S. aureus-induced ferroptosis. The activation of autophagy degraded ferritin, resulting in iron dysregulation and ferroptosis. In addition, we found that excessive reactive oxygen species (ROS) production induced ferroptosis and activated endoplasmic reticulum (ER) stress, manifesting as elevated p-PERK-p-eIF2α-ATF4-CHOP pathway protein levels. Collectively, our findings indicate that ferroptosis is involved in S. aureus-induced mastitis via ER stress-mediated autophagy activation, implying a potential strategy for the prevention of S. aureus-associated diseases by targeting ferroptosis. In conclusion, the ROS-ER stress-autophagy axis is involved in regulating S. aureus-induced ferroptosis in MMECs. These findings not only provide a new potential mechanism for mastitis induced by S. aureus but also provide a basis for the treatment of other ferroptotic-related diseases.

Idiopathic juvenile osteoporosis - a polygenic disorder?

Abstract Idiopathic juvenile osteoporosis (IJO) is a rare condition presenting with vertebral and metaphyseal fractures that affects otherwise healthy prepubertal children. Bone mineral density (BMD) measurements are very low. The primary problem appears to be deficient bone formation, with a failure to accrue bone normally during growth. The onset in childhood suggests IJO is a genetic disorder, and a number of reports indicate that some children carry heterozygous pathogenic variants in genes known to be associated with defective osteoblast function and low bone mass, most commonly LRP5 or PLS3. However, a positive family history is unusual in IJO, suggesting the genetic background can be complex. We describe a young man with classical IJO who was investigated with a bone fragility gene panel and whole genome sequencing. The proband was found to carry four variants in three different genes potentially affecting osteoblast function. From his mother he had inherited mutations in ALPL (p.Asn417Ser) and LRP5 (p.Arg1036Gln), and from his father mutations in LRP5 (p.Asp1551Alsfs*13) and ATF4 (p.Leu306Ile). His sister had also inherited the LRP5 (p.Asp1551Alsfs*13) from her father, but not the ATF4 mutation. Their spinal BMD z-scores differed substantially (sister -1.6, father -3.2) pointing to the potential importance of the ATF4 mutation. Activating transcription factor 4 (ATF4) acts downstream from RUNX2 and osterix and plays an important role in osteoblast differentiation and function. This case, together with others recently published, support the view that IJO can result from clustering of mutations in genes related to osteoblast development and function. Novel genes in these pathways may be involved. Our case also emphasises the value of detailed study of other family members. After a bone biopsy had excluded a mineralization defect due to hypophosphatasia, the proband was treated with zoledronate infusions with good clinical effect.

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

NNMT switches the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2/VEGFA axis.

Cancer-associated fibroblasts (CAFs) are known to promote angiogenesis in oral squamous cell carcinoma (OSCC). However, the epigenetic mechanisms through which CAFs facilitate angiogenesis within the tumor microenvironment are still poorly characterized. Nicotinamide N'-methyltransferase (NNMT), a member of the N-methyltransferase family, was found to be a key molecule in the activation of CAFs. This study shows that NNMT in fibroblasts contributes to angiogenesis and tumor growth through an epigenetic reprogramming-ETS2-VEGFA signaling axis in OSCC. Single-cell RNA Sequencing (scRNA-seq) analysis suggests that NNMT is mainly highly expressed in fibroblasts of head and neck squamous cell carcinoma (HNSCC). Moreover, analysis of the TCGA database and multiple immunohistochemical staining of clinical samples also identified a positive correlation between NNMT and tumor angiogenesis. This research further employed an assembled organoid model and a fibroblast-endothelial cell co-culture model to authenticate the proangiogenic ability of NNMT. At the molecular level, high expression of NNMT in CAFs was found to promote ETS2 expression by regulating H3K27 methylation level through mediating methylation deposition. Furthermore, ETS2 was verified to be an activating transcription factor of VEGFA in this study. Collectively, our findings delineate an epigenetic molecular regulatory network of angiogenesis and provide a theoretical basis for exploring new targets and clinical strategy in OSCC.

Disturbance of Fetal Growth by Azithromycin Through Induction of ER Stress in the Placenta.

Aim: Azithromycin (AZM) is widely used to treat mycoplasma infection in pregnancy. However, there is no adequate evaluation of its side effect on the placenta. In this study, using human placental syncytiotrophoblasts and a mouse model, we investigated whether AZM use in pregnancy might adversely affect placental function and pregnancy outcome. Results: Transcriptomic analysis of AZM-treated human placental syncytiotrophoblasts showed increased expression of endoplasmic reticulum (ER) stress-related genes and decreased expression of genes for hormone production and growth factor processing. Verification studies showed that AZM increased the abundance of ER stress mediators (phosphorylated eIF2α, activating transcription factor 4 [ATF4], and C/EBP Homologous Protein [CHOP]) and decreased the abundance of enzymes involved in progesterone and estradiol synthesis (STS, CYP11A1, and CYP19A1) and insulin-like growth factor binding protein (IGFBP) cleavage (PAPPA and ADAM12) in human placental syncytiotrophoblasts. Inhibition of ER stress blocked AZM-induced decreases in the expression of CYP19A1, CYP11A1, PAPPA, and ADAM12, suggesting that the inhibition of AZM on those genes' expression was secondary to AZM-induced ER stress. Further mechanism study showed that increased ATF4 in ER stress might repressively interact with C/EBPα to suppress the expression of those genes, including CEBPA itself. Mouse studies showed that AZM administration decreased fetal weights along with increased ER stress mediators and decreased levels of insulin-like growth factor, estrogen, and progesterone in the maternal blood, which could be alleviated by inhibition of ER stress. Innovation and Conclusion: These findings first support the fact that AZM, often used during pregnancy, may affect fetal growth by inhibiting crucial enzymes for estrogen and progesterone synthesis and disrupting crucial proteases for IGFBP cleavage via inducing ER stress in placental syncytiotrophoblasts.

The ISR downstream target ATF4 represses long-term memory in a cell type–specific manner

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.

Nitidine chloride inhibits mTORC1 signaling through ATF4-mediated Sestrin2 induction and targets IGF2R for lysosomal degradation

AimsNitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined. MethodsWe explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model. Key findingsNC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts. SignificanceNC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo.

To explore the effect of silent information regulator factor 2-related enzyme 1 (SIRT1) on modulating apoptosis of human lens epithelial cells (HLECs) and alleviating lens opacification of rats through suppressing endoplasmic reticulum (ER) stress. HLECs (SRA01/04) were treated with varying concentrations of tunicamycin (TM) for 24h, and the expression of SIRT1 and C/EBP homologous protein (CHOP) was assessed using real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8 (CCK-8) assay, respectively. In the SRA01/04 cell apoptosis model, which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation, the expression levels of SIRT1, CHOP, glucose regulated protein 78 (GRP78), and activating transcription factor 4 (ATF4) were examined. The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid (4-PBA; an ER stress inhibitor) was investigated. In vivo, age-related cataract (ARC) rat models were induced by sodium selenite injection, and the protective role of SIRT1, activated by SRT1720 intraperitoneal injections, was evaluated through morphology observation, hematoxylin and eosin (H&E) staining, Western blotting, and RT-PCR. SIRT1 expression was downregulated in TM-induced SRA01/04 cells. Besides, in SRA01/04 cells, both cell apoptosis and CHOP expression increased with the rising doses of TM. ER stress was stimulated by TM, as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model. Inhibition of SIRT1 by siRNA knockdown increased ER stress activation, whereas SRT1720 treatment had opposite results. 4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis. In vivo, SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models. SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress. These findings suggest a novel strategy for cataract treatment focused on targeting ER stress, highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Tyrosine Kinase Inhibitor Lenvatinib Causes Cardiotoxicity by Inducing Endoplasmic Reticulum Stress and Apoptosis through Activating ATF6, IRE1α and PERK Signaling Pathways.

Lenvatinib is a tyrosine kinase inhibitor that can improve progression-free survival in patients with thyroid cancer and hepatocellular carcinoma. However, it is limited by adverse cardiovascular events, including hypertension and cardiac dysfunction. Activation of endoplasmic reticulum stress is involved in cardiomyocyte apoptosis. This study aimed to confirm whether the cardiotoxicity of lenvatinib is associated with endoplasmic reticulum stress by targeting the activating transcription factor 6 (ATF6), inositol- requiring enzyme 1α (IRE1α) and protein kinase RNA-like ER kinase (PERK) signaling pathways. Male C57/BL6 mice were intragastric administration with 30 mg/kg/day lenvatinib. Electrocardiography (ECG) and echocardiography were used to detect arrhythmias and cardiac function. Neonatal rat cardiomyocytes were treated with lenvatinib for 48h. Cell counting kit (CCK8), 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFHDA), Hoechst 33258 and dihydrorhodamine 123 were respectively used for evaluating cell viability, the level of reactive oxygen species (ROS), nuclear morphological changes and mitochondrial membrane potential (MMP) level. Lenvatinib remarkably decreased the posterior wall thickness of left ventricle during diastole and systole but caused little decrease to the left ventricular ejection fraction (LVEF, %). Furthermore, lenvatinib greatly prolonged the corrected QT interval (QTc) and altered the morphology of cardiomyocytes. No dramatic difference in fibrosis was found in mouse cardiac slices. Lenvatinib upregulates apoptosis-related protein expression. In addition, lenvatinib increased ERS-related protein expression (GRP78, CHOP, and ATF6) and enhanced PERK phosphorylation. In neonatal rat cardiac myocytes, lenvatinib markedly decreased the viability of cardiomyocytes and induced apoptosis. Furthermore, ROS production increased and MMP decreased. Similar to the mice experiment, lenvatinib caused upregulation of apoptosis-related and ERS-related proteins and increased the phosphorylation levels of PERK and IRE1α. Lenvatinib-induced cardiotoxicity is associated with ERS-induced apoptosis by targeting the ATF6, IRE1α, and PERK signaling pathways.

Hepatic IL22RA1 deficiency promotes hepatic steatosis by modulating oxysterol in the liver.

Imbalance in lipid metabolism is the main cause of nonalcoholic fatty liver disease (NAFLD). While the pathogenesis of lipid accumulation mediated by extrahepatic regulators has been extensively studied, the intrahepatic regulators modulating lipid homeostasis remain unclear. Previous studies have shown that systemic administration of interleukin-22 (IL-22) protects against NAFLD; however, the role of IL-22/IL22RA1 signaling in modulating hepatic lipid metabolism remains uncertain. This study shows hepatic IL22RA1 is vital in hepatic lipid regulation. IL22RA1 is downregulated in palmitic acid-treated mouse primary hepatocytes, as well as in the livers of NAFLD model mice and patients. Hepatocyte-specific Il22ra1 knockout (HKO) mice display diet-induced hepatic steatosis, insulin resistance, impaired glucose tolerance, increased inflammation, and fibrosis compared with flox/flox mice. This is attributed to increased lipogenesis mediated by the accumulation of hepatic oxysterols, particularly, 3 beta-hydroxy-5-cholestenoic acid (3β HCA). Mechanistically, hepatic IL22RA1 deficiency facilitates 3β HCA deposition via the activating transcription factor 3 (ATF3)/oxysterol 7 alpha-hydroxylase (CYP7B1) axis. Notably, 3β HCA facilitates lipogenesis in MPHs and human liver organoids (HLOs) by activating LXR-alpha signaling, but IL-22 treatment attenuates this effect. Additionally, restoring CYP7B1 or silencing hepatic ATF3 reduces both hepatic 3β HCA and lipid contents in HKO mice. These findings indicate that IL22RA1 plays a crucial role in maintaining hepatic lipid homeostasis in an ATF3/CYP7B1-dependent manner, and establish a link between 3β HCA and hepatic lipid homeostasis.

Neuromedin S regulates goat ovarian granulosa cell proliferation and steroidogenesis via endoplasmic reticulum Ca2+-YAP1-ATF4-c-Jun pathway.

Neuromedin S (NMS) plays key roles in reproductive regulation, while its function and mechanism in follicular development remain unclear. The current study aims to investigate the specific role and mechanisms of NMS and its receptors in regulating the proliferation and steroidogenesis of ovarian granulosa cells (GCs). Phenotypically, a certain concentration of NMS addition promoted the proliferation and estrogen production of goat GCs, accompanied by an increase in the G1/S cell population and upregulation of the expression levels of cyclin D1, cyclin dependent kinase 6, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450, family 11, subfamily A, polypeptide 1, while the effects of NMS treatment were effectively hindered by knockdown of neuromedin U receptor type 2 (NMUR2). Mechanistically, activation of NMUR2 with NMS maintained endoplasmic reticulum (ER) calcium (Ca2+) homeostasis by triggering the PLCG1-IP3R pathway, which helped preserve ER morphology, sustained an appropriate level of endoplasmic reticulum unfoldedprotein response (UPRer), and suppressed the nuclear translocation of activating transcription factor 4. Moreover, NMS maintained intracellular Ca2+ homeostasis to activate the calmodulin 1-large tumor suppressor kinase 1pathway, ultimately orchestrating the regulation of goat GC proliferation and estrogen production through the Yes1 associated transcriptional regulator-ATF4-c-Jun pathway. Crucially, the effects of NMS were mitigated by concurrent knockdown of the NMUR2 gene. Collectively, these data suggest that activation of NMUR2 by NMS enhances cell proliferation and estrogen production in goat GCs through modulating the ER and intracellular Ca2+ homeostasis, leading to activation of the YAP1-ATF4-c-Jun pathway. These findings offer valuable insights into the regulatory mechanisms involved in follicular growth and development, providing a novel perspective for future research.

Differential Impairment Mechanism of Sperm Production via Induction of miR-34c-Activated Apoptosis and Spermatogenesis Pathway in Diet-Induced Obesity and Resistant Mice and GC-1 Spg Cells.

Male reproductive dysfunction is a clinical disease, with a large number of cases being idiopathic. Reproductive disorders have been found in obese (diet-induced obesity and diet-induced obesity-resistant) mice, but the mechanism behind the male reproductive dysfunction between them may be different. The purpose of this study was to explore the possible role and mechanism of miR-34c on sperm production in high-fat-diet-induced obesity-resistant (DIO-R) mice and GC-1 spg cells, which may differ from those in high-fat-diet-induced obesity (DIO) mice. In vivo and in vitro experiments were performed. C57BL/6J mice were fed a high-fat diet for 10 weeks to establish the DIO and DIO-R mouse model. GC-1 spg cells were used to verify the mechanism of miR-34c on sperm production. During in vivo experiments, sperm production damage was found in both DIO and DIO-R male mice. Compared to the control mice, significantly decreased levels of testosterone, LH, activities of acrosome enzyme (ACE), HAse, and activating transcription factor 1 (ATF1) were found in both DIO and DIO-R male mice (p < 0.05). Compared with the control group, the ratio of B-cell lymphoma-2 (Bcl-2)/bcl-2-associated X protein (Bax) in the DIO group was significantly decreased, and the expression level of cleaved caspase-3 was significantly increased (p < 0.05). Compared with the control group, the Bcl-2 protein expression level in the testes of the DIO-R group significantly decreased (p < 0.05). However, the Bax expression level increased. Thus, the Bcl-2/Bax ratio significantly decreased (p < 0.01); however, the factor-related apoptosis (Fas), Fas ligand (FasLG), cleaved caspase-8, caspase-8, cleaved caspase-3, and caspase-3 protein expression levels significantly increased (p < 0.05). Compared with the DIO group, in DIO-R mice, the activities of ACE, ATF1, Bcl-2, and Bcl-2/Bax's spermatogenesis protein expression decreased, while the apoptosis-promoting protein expression significantly increased (p < 0.05). During the in vitro experiment, the late and early apoptotic ratio in the miR-34c over-expression group increased. MiR-34c over-expression enhanced the expression of apoptosis-related proteins Fas/FasLG and Bax/Bcl-2 while inhibiting the expression of ATF1 and the sperm-associated protein in GC-1 spg cells. DIO and DIO-R could harm sperm production. DIO-R could impair sperm production by inducing the miR-34c-activated apoptosis and spermatogenesis pathway, which may be different from that of DIO.

Piperine inhibits the proliferation of colorectal adenocarcinoma by regulating ARL3-mediated endoplasmic reticulum stress.

Colorectal adenocarcinoma (COAD) is a significant cause of cancer-related mortality worldwide, necessitating the identification of novel therapeutic targets and treatments. This research aimed to investigate the role of ARL3 in COAD progression and to explore the effects of Piperine on ARL3 expression, cell proliferation, epithelial-mesenchymal transition (EMT), and endoplasmic reticulum (ER) stress. Bioinformatics analysis of The Cancer Genome Atlas (TCGA)-COAD, GSE39582, and GSE44861 datasets assessed ARL3 expression levels. Immunohistochemical data from the Human Protein Atlas (HPA) database confirmed ARL3 overexpression in COAD. The association of ARL3 with COAD clinical parameters and prognosis was also examined. COAD cells were treated with Piperine, and in vitro assays evaluated cell proliferation, apoptosis, EMT marker expression, and ER stress responses. ARL3 overexpression in COAD correlated with poor prognosis and varied across pathological stages. Piperine treatment inhibited COAD cell proliferation in a concentration- and time-dependent manner, as indicated by reduced Ki-67 levels and decreased colony-forming ability. Piperine induced S-phase cell cycle arrest and facilitated apoptosis in COAD cells, evidenced by changes in Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) levels. Moreover, Piperine downregulated ARL3 expression in COAD cells, thereby suppressing transforming growth factor beta (TGF-β)-induced EMT. Additionally, Piperine attenuated the ARL3-mediated ER stress response, significantly reducing binding immunoglobulin protein (BiP), inositol-requiring enzyme 1 alpha (p-IRE1α), activating transcription factor 6 (ATF6), and C/EBP homologousprotein (CHOP) levels. Piperine exerted anti-cancer effects in COAD by modulating ARL3 expression, disrupting cell cycle progression, inhibiting the EMT pathway, and regulating ER stress. These findings suggest that Piperine holds promise as a therapeutic agent for COAD through its targeting of ARL3.

Proteome profiling of cutaneous leishmaniasis lesions due to dermotropic Leishmania donovani in Sri Lanka

BackgroundCharacterization of the host response in cutaneous leishmaniasis (CL) through proteome profiling has gained limited insights into leishmaniasis research compared to that of the parasite. The primary objective of this study was to comprehensively analyze the proteomic profile of the skin lesions tissues in patients with CL, by mass spectrometry, and subsequent validation of these findings through immunohistochemical methods.MethodsEight lesion specimens from leishmaniasis-confirmed patients and eight control skin biopsies were processed for proteomic profiling by mass spectrometry. Formalin-fixed paraffin-embedded lesion specimens from thirty patients and six control skin specimens were used for Immunohistochemistry (IHC) staining. Statistical analyses were carried out using SPSS software. The chi-square test was used to assess the association between the degree of staining for each marker and the clinical and pathological features.ResultsSixty-seven proteins exhibited significant differential expression between tissues of CL lesions and healthy controls (p < 0.01), representing numerous enriched biological processes within the lesion tissue, as evident by both the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. Among these, the integrated endoplasmic reticulum stress response (IERSR) emerges as a pathway characterized by the up-regulated proteins in CL tissues compared to healthy skin. Expression of endoplasmic reticulum (ER) stress sensors, inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6) in lesion tissue was validated by immunohistochemistry.ConclusionsIn conclusion, proteomic profiling of skin lesions carried out as a discovery phase study revealed a multitude of probable immunological and pathological mechanisms operating in patients with CL in Sri Lanka, which needs to be further elaborated using more in-depth and targeted investigations. Further research exploring the intricate interplay between ER stress and CL pathophysiology may offer promising avenues for the development of novel diagnostic tools and therapeutic strategies in combating this disease.