Non-alcoholic fatty liver disease (NAFLD) has emerged as the leading cause of chronic liver disease worldwide. Caspase 8 and FADD-like apoptosis regulator (CFLAR) has been identified as a potent factor in mitigating non-alcoholic steatohepatitis (NASH) by inhibiting the N-terminal dimerization of apoptosis signal-regulating kinase 1 (ASK1). While arginine methyltransferase 1 (PRMT1) was previously reported to be associated with increased hepatic glucose production, its involvement in hepatic lipid metabolism remains largely unexplored. The interaction between PRMT1 and CFLAR and the methylation of CFLAR were verified by Co-IP and immunoblotting assays. Recombinant adenoviruses were generated for overexpression or knockdown of PRMT1 in hepatocytes. The role of PRMT1 in NAFLD was investigated in normal and high-fat diet-induced obese mice. In this study, we found a significant upregulation of PRMT1 and downregulation of CFLAR after 48h of fasting, while the latter significantly rebounded after 12h of refeeding. The expression of PRMT1 increased in the livers of mice fed a methionine choline-deficient (MCD) diet and in hepatocytes challenged with oleic acid (OA)/palmitic acid (PA). Overexpression of PRMT1 not only inhibited the expression of genes involved in fatty acid oxidation (FAO) and promoted the expression of genes involved in fatty acid synthesis (FAS), resulting in increased triglyceride accumulation in primary hepatocytes, but also enhanced the gluconeogenesis of primary hepatocytes. Conversely, knockdown of hepatic PRMT1 significantly alleviated MCD diet-induced hepatic lipid metabolism abnormalities and liver injury in vivo, possibly through the upregulation of CFLAR protein levels. Knockdown of PRMT1 suppressed the expression of genes related to FAS and enhanced the expression of genes involved in FAO, causing decreased triglyceride accumulation in OA/PA-treated primary hepatocytes in vitro. Although short-term overexpression of PRMT1 had no significant effect on hepatic triglyceride levels under physiological conditions, it resulted in increased serum triglyceride and fasting blood glucose levels in normal C57BL/6J mice. More importantly, PRMT1 was observed to interact with and methylate CFLAR, ultimately leading to its ubiquitination-mediated protein degradation. This process subsequently triggered the activation of c-Jun N-terminal kinase 1 (JNK1) and lipid deposition in primary hepatocytes. Together, these results suggested that PRMT1-mediated methylation of CFLAR plays a critical role in hepatic lipid metabolism. Targeting PRMT1 for drug design may represent a promising strategy for the treatment of NAFLD.

Mutations in STAMBP have been well-established to cause congenital human microcephaly-capillary malformation (MIC-CAP) syndrome, a rare genetic disorder characterized by global developmental delay, severe microcephaly, capillary malformations, etc. Previous biochemical investigations and loss-of-function studies in mice have provided insights into the mechanism of STAMBP, however, it remains controversial how STAMBP deficiency leads to malformation of those affected tissues in patients. In this study, we investigated the function and underlying mechanism of STAMBP during neural differentiation of human embryonic stem cells (hESCs). We found that STAMBP is dispensable for the pluripotency maintenance or neural differentiation of hESCs. However, neural progenitor cells (NPCs) derived from STAMBP-deficient hESCs fail to be long-term maintained/expanded in vitro. We identified the anti-apoptotic protein CFLAR is down-regulated in those affected NPCs and ectopic expression of CFLAR rescues NPC defects induced by STAMBP-deficiency. Our study not only provides novel insight into the mechanism of neural defects in STAMBP mutant patients, it also indicates that the death receptor mediated apoptosis is an obstacle for long-term maintenance/expansion of NPCs in vitro thus counteracting this cell death pathway could be beneficial to the generation of NPCs in vitro.

Anoikis is highly associated with tumor cell apoptosis and tumor prognosis; however, the specific role of anoikis-related genes (ARGs) in soft tissue sarcoma (STS) remains to be fully elucidated. The present study aimed to use a variety of bioinformatics methods to determine differentially expressed anoikis-related genes in STS and healthy tissues. Subsequently, three machine learning algorithms, Least Absolute Shrinkage and Selection Operator, Support Vector Machine and Random Forest, were used to screen genes with the highest importance score. The results of the bioinformatics analyses demonstrated that CASP8 and FADD-like apoptosis regulator (CFLAR) exhibited the highest importance score. Subsequently, the diagnostic and prognostic value of CFLAR in STS development was determined using multiple public and in-house cohorts. The results of the present study demonstrated that CFLAR may be considered a diagnostic and prognostic marker of STS, which acts as an independent prognostic factor of STS development. The present study also aimed to explore the potential role of CFLAR in the STS tumor microenvironment, and the results demonstrated that CFLAR significantly enhanced the immune response of STS, and exerted a positive effect on the infiltration of CD8+ T cells and M1 macrophages in the STS immune microenvironment. Notably, the aforementioned results were verified using multiplex immunofluorescence analysis. Collectively, the results of the present study demonstrated that CFLAR may act as a novel diagnostic and prognostic marker for STS, and may positively regulate the immune response of STS. Thus, the present study provided a novel theoretical basis for the use of CFLAR in STS diagnosis, in predicting clinical outcomes and in tailoring individualized treatment options.

Immunogenic cell death (ICD), a type of cell death that activates the tumor-specific immune response and thus exerts anti-tumor effects, is an emerging target in tumor therapy, but research on ICD-related genes (ICDGs) in colorectal cancer (CRC) remains limited. This study aimed to identify the CRC-specific ICDGs and explore their potential roles. Through RNA sequencing for tissue samples from CRC patients and integration with The Cancer Genome Atlas (TCGA) data, we identified 33 differentially expressed ICDGs in CRC. We defined the ICD score based on these genes in single-cell data, where a high score indicated an immune-active microenvironment. Additionally, molecular subtypes identified in bulk RNA data showed distinct immune landscapes. The ICD-related signature constructed with machine learning effectively distinguished patients\' prognosis. The summary data-based Mendelian randomization (SMR) and colocalization analysis prioritized CFLAR for its positive association with CRC risk. Molecular docking revealed its stable binding with chemotherapeutic drugs like irinotecan. Furthermore, experimental validation confirmed CFLAR overexpression in CRC samples, and its knockdown inhibited tumor cell proliferation. Overall, this study expands the understanding of the potential roles and mechanisms of ICDGs in CRC and highlights CFLAR as a promising target for CRC.

CASP8 and FADD Like Apoptosis Regulator (CFLAR) is a key anti-apoptotic regulator for resistance to apoptosis mediated by Fas and TRAIL. In addition to its anti-apoptotic function, CFLAR is also an important mediator of tumor growth. High level of CFLAR expression correlates with a more aggressive tumor. However, the mechanism of CFLAR signaling in malignant progression is not clear. Here we report a novel CFLAR-associated protein p130Cas, which is a general regulator of cell growth and cell migration. CFLAR-p130Cas association is mediated by the DED domain of CFLAR and the SD domain of p130Cas. Immunofluorescence observation showed that CFLAR had the colocalization with p130Cas at the focal adhesion of cell membrane. CFLAR overexpression promoted p130Cas phosphorylation and the formation of focal adhesion complex. Moreover, the enhancement of cell migration induced by CFLAR overexpression was obviously inhibited by p130Cas siRNA. In silico analysis on human database suggests high expressions of CFLAR or/and p130Cas are associated with poor prognosis of patients with lung cancer. Together, our results suggest a new mechanism for CFLAR involved in tumor development via association with p130Cas.

Previous literature has indicated that cyclin-dependent kinase inhibitor 2 A (CDKN2A) is upregulated, while the Protein Inhibitor of Activated STAT1 (PIAS1) is downregulated in the liver tissues of obese mice. The current study aimed to investigate the relationship between CDKN2A and PIAS1 in the lipogenesis of fatty liver disease. In the C57BL/6J db/db mouse model and hepatocyte model of fatty liver, the expression pattern of CDKN2A, PIAS1, Protein arginine methyltransferase 1 (PRMT1) and CASP8 and FADD-like apoptosis regulator (CFLAR) was characterized by RNA quantitative and Western blot analysis. The lipogenesis-related genes (Srebp1c and Fas) in the liver tissues and cells were employed in the assessment of lipogenesis in response to gain- or loss-of-function of CDKN2A, PIAS1, PRMT1, and CFLAR, while triglyceride and fat content were evaluated in relation to fat accumulation. Western blot analysis was conducted to determine c-Jun amino-terminal kinase (JNK) phosphorylation, while the ubiquitination of CFLAR and SUMOylation of PIAS1 was examined by immunoprecipitation. PIAS1 and CFLAR were downregulated, while CDKN2A, PRMT1, and phosphorylation of JNK was elevated in the tissues and cells of the fatty liver models. Our results suggested that CDKN2A enhanced the SUMOylation of PIAS1 to reduce the expression of PIAS1. PRMT1 downregulated CFLAR by triggering its ubiquitination, while CFLAR repressed phosphorylation of JNK. The in vitro and in vivo results indicated that CDKN2A silencing prevented lipogenesis and fat accumulation by impairing the PRMT1-dependent ubiquitination of CFLAR and blocking the phosphorylation of JNK. Taken together, the central observations of our study demonstrate that targeting CDKN2A contributes to the suppression of lipogenesis and fat accumulation in fatty liver disease. The findings of our study highlight the potential of CDKN2A as a promising target against fatty liver.

Stroke is a leading cause of mortality and disability globally. Cerebral ischaemia-reperfusion (I/R) injury is characterized by significant inflammation and extensive cell death. Multiple signaling pathways play essential roles in the process, and identifying the unclear crucial regulators of these pathways may provide promising targets for treatment. CASP8 and FADD-like apoptosis regulator (CFLAR) is expressed in multiple organs to regulate inflammation. Here, we reported that CFLAR expression was markedly reduced in brain samples of mice with middle cerebral artery occlusion (MCAO) stroke. Furthermore, CFLAR knockdown markedly elevated the neurological deficit, brain water content and the infarct volume. In addition, significantly promoted inflammation and endoplasmic reticulum (ER) stress was detected in brain tissues of mice after MCAO, as evidenced by the promoted expression of p-IκBα, p-nuclear factor (NF)-κB (p65), glucose-regulated protein 78 (GRP78), PKR-like ER kinase (PERK), activating transcription factor-6 (ATF-6) and cleaved Caspase-12. Notably, MCAO-induced cerebral I/R injury was markedly alleviated in mice over-expressing CFLAR through suppressing inflammation and ER stress. Furthermore, our in vitro results indicated that oxygen-glucose deprivation (OGD)-induced cell death was evidently ameliorated by CFLAR over-expression. In contrast, the cell death triggered by OGD was accelerated by CFLAR knockdown in vitro through enhancing Caspase-3 cleavage, and this effect was obviously ameliorated by the blockage of ER stress using 4-phenyl butyric acid (4-PBA). Collectively, these results demonstrated that CFLAR could be considered as a novel candidate to develop effective therapeutic treatment against cerebral I/R injury.

Non-alcoholic steatohepatitis (NASH) is a chronic metabolic syndrome and the CFLAR-JNK pathway can reverse the process of NASH. Although silibinin is used for the treatment of NASH in clinical, its effect on CFLAR-JNK pathway in NASH remains unclear. This study aimed to investigate the effect of silibinin on CFLAR-JNK pathway in NASH models both in vivo and in vitro. The in vivo study was performed using male C57BL/6 mice fed with methionine- choline-deficient diet and simultaneously treated with silibinin for 6 weeks. The in vitro study was performed by using mouse NCTC-1469 cells which were respectively pretreated with oleic acid plus palmitic acid, and adenovirus-down Cflar for 24 h, then treated with silibinin for 24 h. After the drug treatment, the key indicators involved in CFLAR-JNK pathway including hepatic injury, lipid metabolism and oxidative stress were determined. Silibinin significantly activated CFLAR and inhibited the phosphorylation of JNK, up-regulated the mRNA expression of Pparα, Fabp5, Cpt1α, Acox, Scd-1, Gpat and Mttp, reduced the activities of serum ALT and AST and the contents of hepatic TG, TC and MDA, increased the expression of NRF2 and the activities of CAT, GSH-Px and HO-1, and decreased the activities and expression of CYP2E1 and CYP4A in vivo. These effects were confirmed by the in vitro experiments. Silibinin prevented NASH by regulating CFLAR-JNK pathway, and thereby on one hand promoting the β-oxidation and efflux of fatty acids in liver to relieve lipid accumulation, and on the other hand inducing antioxidase activity (CAT, GSH-Px and HO-1) and inhibiting pro-oxidase activity (CYP2E1 and CYP4A) to relieve oxidative stress.

BACKGROUND: CFLARL, also known as c-FLIPL, is a critical anti-apoptotic protein that inhibits activation of caspase 8 in mammalian cells. Previous studies have shown that arginine 122 of CFLARL can be mono-methylated. However, the precise role of arginine methyltransferase of CFLARL remains unknown. PRMT5 and PRMT1, which are important members of the PRMT family, catalyze the transfer of methyl groups to the arginine of substrate proteins. PRMT5 can monomethylate or symmetrically dimethylate arginine residues, while PRMT1 can monomethylate or asymmetrically dimethylate arginine residues.
METHODS: Lung cancer cells were cultured following the standard protocol and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the protein interaction was assayed by co-immunoprecipitation (Co-IP) or GST pull-down assay. CFLARL ubiquitination level was evaluated by proteasomal inhibitor treatment combined with HA-Ub transfection and WB assay. PRMT1 and PRMT5 genes were knocked down by siRNA technique.
RESULTS: We show that PRMT5 up-regulated the protein levels of CFLARL by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLARL by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLARL and ITCH, which has been identified as an E3 ubiquitin ligase of CFLARL, while overexpressed PRMT1 enhances the interaction between CFLARL and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLARL as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity. Finally, we showed that PRMT5 and PRMT1 could suppress or facilitate apoptosis induced by doxorubicin or pemetrexed by affecting CFLARL in NSCLC cells.
CONCLUSIONS: PRMT5 and PRMT1 mediate the distinct effects on CFLARL degradation by regulating the binding of E3 ligase ITCH in NSCLC cells. This study identifies a cell death mechanism that is fine-tuned by PRMT1/5 that modulate CFLARL degradation in human NSCLC cells.

OBJECTIVE: Silibinin is a flavonolignan from milk thistle with many pharmacological activities including lipid-lowering and antioxidant. Caspase 8 and Fas-associated protein with death domain-like apoptosis regulator (CFLAR) is an important target gene in regulating non-alcoholic steatohepatitis (NASH). At present, the effect of silibinin on CFLAR-JNK pathway related to NASH was unknown. Here the effect of silibinin on CFLAR-JNK pathway and its downstream target genes involved in lipid metabolism, glucose uptake, oxidative stress and inflammatory response were studied in oleic acid (OA)-treated HepG2 cells.
METHODS: OA-treated HepG2 cells were employed as a in vitro model of steatosis, insulin resistance and oxidative stress. The model cells were then treated by silibinin (5, 20, 50, and 100 μM) for 24 h and detected for the related indicators as follows: (1) cellular triglycerides (TG), nitric oxide (NO) and glucose uptake; (2) the mRNA levels of the sterol regulatory element binding protein-1C (SREBP-1C), patatin-like phospholipase domain containing 3 (PNPLA3) and peroxisome proliferator activated receptor-α (PPARα); (3) the protein levels of PPARα, SREBP-1C, PNPLA3, CFLAR, phosphorylated c-Jun N-terminal kinase (pJNK), phosphatidylinositol 3-kinase (PI3K), phosphorylated serine-threonine protein kinase (pAKT), nuclear factor E2-related factor 2 (NRF2), cytochrome P450 2E1 (CYP2E1) and 4A (CYP4A).
RESULTS: Compared to the control, OA-treatment led to a result as follows: (1) increased the intracellular levels of TG and NO; (2) up-regulated the protein expression of SREBP-1C, PNPLA3, pJNK, CYP 2E1 and CYP 4A; (3) decreased the uptake of 2-NBDG; (4) down-regulated the protein expression of CFLAR, PPARα, PI3K, pAKT and NRF2. Compared to OA-treated HepG2 cells, silibinin treatment could improve the indicators as follows: (1) decreased the intracellular levels of TG and NO; (2) down-regulated the protein expression of SREBP-1C, PNPLA3, pJNK, CYP 2E1 and CYP 4A; (3) increased the uptake of 2-NBDG; (4) up-regulated the protein expression of CFLAR, PPARα, PI3K, pAKT and NRF2.
CONCLUSIONS: Silibinin can ameliorate some metabolic alterations and induce some molecular changes by activating the CFLAR-JNK pathway and thereby regulating its downstream target genes involved in lipid metabolism (PPARα, SREBP-1C and PNPLA3), glucose uptake (PI3K-AKT), oxidative stress (NRF2, CYP2E1, CYP4A) and inflammatory response(NO) in OA-treated HepG2 cells demonstrating its possible use in ameliorating various symptoms of NASH.