PRMT6-FOXO3A ATTENUATES APOPTOSIS BY UPREGULATING PARKIN EXPRESSION IN INTESTINAL ISCHEMIA-REPERFUSION INJURY.

ABSTRACT: Intestinal ischemia-reperfusion injury (IIRI) is a serious disease with high morbidity and mortality. This study aims to investigate the potential regulatory mechanisms involving protein arginine methyltransferase 6 (PRMT6), Forkhead box O3a (FoxO3a), and Parkin in IIRI and elucidate their roles in mediating cell apoptosis. The IIRI animal model was established and confirmed using hematoxylin and eosin staining. Oxygen-glucose deprivation and reperfusion (OGD/R) cell model was established to mimic ischemic injury in vitro . Transient transfection was used to overexpress or knock down genes. Cell death or apoptosis was assessed by propidium iodide staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and flow cytometry. The expression of proteins was detected by western blot. The histopathology observed by hematoxylin and eosin staining suggested that the IIRI animal model was successfully established. Our findings revealed that IIRI resulted in increased Bax and decreased Bcl-2 levels. In vitro experiments showed that overexpression of Parkin decreased OGD/R injury and suppressed elevation of Bax/Bcl-2. PRMT6 regulated the methylation level of FoxO3a. Moreover, FoxO3a directly binds to Parkin, and FoxO3a overexpression reduced OGD/R-induced cell death and regulation of Parkin. Overexpression of PRMT6 can attenuate the downregulation of Parkin and elevation of Bax/Bcl-2 caused by OGD/R. Knockdown of PRMT6 promoted apoptosis in intestinal epithelial cells of OGD/R group, while PRMT6 overexpression exhibited the opposite effect. Notably, the levels of PRMT6, FoxO3a, and Parkin were decreased in IIRI mouse intestinal tissue. Knocking out PRMT6 causes a significant decrease in the lifespan of mice. Altogether, our results demonstrated that PRMT6 upregulated the expression of Parkin by regulating FoxO3a methylation level, attenuating the apoptosis induced by IIRI.

Alpha-linolenic acid pretreatment alleviates NETs-induced alveolar macrophage pyroptosis by inhibiting pyrin inflammasome activation in a mouse model of sepsis-induced ALI/ARDS.

Background: Neutrophil extracellular traps (NETs) can cause acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) by inducing macrophage pyroptosis. The purpose of this study was to find out whether pretreatment of alpha-linolenic acid (ALA) could inhibit NETs-induced macrophage pyroptosis in sepsis-induced ALI/ARDS, as well as to identify which inflammasome is involved in this process. Methods: LPS was instilled into the trachea to establish sepsis-induced ALI/ARDS in a mouse model. ​Lung injury was assessed by microscopic examination of lung tissue after hematoxylin and eosin staining, pathology score, and bronchoalveolar lavage fluid (BALF) total protein concentration. The level of NETs in lung tissue was detected by MPO-DNA ELISA. Purified NETs, extracted from peritoneal neutrophils, induced macrophage pyroptosis in vitro. Expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1ß in the lung tissue and bone marrow-derived macrophages (BMDMs) were determined by western blotting or ELISA. Specks of Pyrin/ASC were examined by confocal immunofluorescence microscopy. Mefv (Pyrin)-/- mice were used to study the role of Pyrin in the process of sepsis-induced ALI/ARDS. Results: ALA alleviated LPS-induced lung injury. ALA reduced the level of NETs, pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC), and IL-1ß in the lung tissue of sepsis mice. In vitro, NETs increased the expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1ß significantly in BMDMs. Pyrin protein was found to be higher and form the inflammasome with ASC in NETs challenged-BMDMs. Knockout of Mefv (Pyrin) gene fully restored the increased expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1ß in vitro and in vivo. Lung injury was alleviated significantly in Mefv (Pyrin)-/- mice as well.​ ALA suppresses all the NETs-induced changes as mentioned above. Conclusion: Our study is the first to demonstrate Pyrin inflammasome driving NETs-induced macrophage pyroptosis, and ALA may reduce ALI/ARDS by inhibiting the activation of the Pyrin inflammasome-driven macrophage pyroptosis.

Gα13-Mediated Signaling Cascade Is Related to the Tau Pathology Caused by Anesthesia and Surgery in 5XFAD Transgenic Mice.

BACKGROUND: Our previous studies indicated that anesthesia and surgery could aggravate cognitive impairment of 5XFAD transgenic (Tg) mice, and this aggravation was associated with tau hyperphosphorylation. We previously identified that GNA13 (the gene encoding Gα13) was a hub gene with tau hyperphosphorylation. OBJECTIVE: This study aims to further investigate the mechanism that whether the Gα13-mediated signaling pathway acts as an instigator to regulate cofilin activation and autophagy impairment in this process. METHODS: 5XFAD Tg mice and their littermate (LM) mice were randomly allocated into four groups: LM Control group, LM Anesthesia/Surgery group, AD Control group, and AD Anesthesia/Surgery group. For mice in the Anesthesia/Surgery groups, abdominal surgery was performed under 1.4% isoflurane anesthesia followed by sustaining anesthetic inhalation for up to 2 h. RESULTS: Compared with the AD Control group, protein levels of Gα13, ROCK2, LPAR5, and p-tau/tau46 ratio were increased, while p-cofilin/cofilin protein expression ratio was decreased in the AD Anesthesia/Surgery group. However, the differences in these protein levels were not significant among LM groups. CONCLUSION: This study demonstrated that anesthesia and surgery might exacerbate p-tau accumulation in 5XFAD Tg mice but not in LM mice. And this might be closely related to cofilin activation via Gα13-mediated signaling cascade.

The transcription factor KLF14 attenuates LPS-induced acute lung injury by ameliorating apoptosis of alveolar epithelial cells in mice.

Lipopolysaccharide (LPS)-induced lung injury (ALI) is characterized by severe lung inflammation with high mortality. Pulmonary inflammation and apoptosis of alveolar epithelial cells cause the disruption of alveolar epithelial cell integrity, resulting in the failure of gas exchange, which is correlated with poor prognosis. However, the underlying regulatory mechanism remains unclear. In this study, we found that the Krüppel-like transcription factor KLF14 can suppress the apoptosis of alveolar epithelial cells in a LPS-mediated murine acute lung injury model by western blot and TUNEL assays. First, we found that the expression of KLF14 in alveolar epithelial cells of a murine ALI model was upregulated, and KLF14 deletion exacerbated lung injury, increased mortality, and promoted the apoptosis of alveolar epithelial cells. Furthermore, we identified that KLF14 protects lung tissue from LPS-mediated damage and suppresses the apoptosis of alveolar epithelial cells by inhibiting the NF-κB pathway. Our study provides further research insight and helps to explore the protective mechanism of KLF14 in inflammation and cell death. Our results suggested that the transcription factor KLF14 may act as a new target for the prevention and treatment of acute lung injury.

Mitochondrial DNA mediates immunoparalysis of dendritic cells in sepsis via STING signalling.

BACKGROUND: Mitochondrial DNA (mtDNA) is a potent activator for pro-inflammatory response. Dendritic cells (DCs) are immunosuppressed in sepsis, whether mtDNA mediates immunoparalysis in sepsis remains unknown. METHODS: The mRNAs were assessed by qPCR. Flow cytometry was used to measure the expression of costimulatory molecules and the proliferation of CD4+ T cells. Western blot and immunofluorescence staining were used to analyse the expression of proteins. Cytokine secretion was detected by ELISA. Histology of lung tissue was used to assess the inflammatory injury. RESULTS: Lipopolysaccharide-induced endotoxemia increased plasma mtDNA levels and immunoparalysis of spleen DCs, while hydrolysing mtDNA reversed immunoparalysis of spleen DCs in vivo. Moreover, cytoplasmic mtDNA of DCs was accumulated in endotoxemia and sepsis. mtDNA transfection into bone marrow-derived DCs (BMDCs) inhibited the expression of costimulatory molecules (e.g., CD40, CD80 and CD86) and the release of IL-12p70, while increasing the secretion of IL-10. Cytoplasmic mtDNA also inhibited the ability of BMDCs to promote the proliferation of CD4+ T cells. Mechanistic analysis revealed that STING signalling was required for mtDNA-mediated immunoparalysis of DCs in vivo and in vitro. Further studies showed deletion of STING reversed mtDNA-mediated immunoparalysis of DCs and improved the prognosis of endotoxemia and sepsis. CONCLUSION: Our results demonstrated that mtDNA promotes immunoparalysis of DCs, and contributes to sepsis-associated immunosuppression by activating STING signalling. Our study may provide new insights to elucidate the molecular pathogenesis of immunosuppressive DCs in sepsis.

The transcription factor KLF14 regulates macrophage glycolysis and immune function by inhibiting HK2 in sepsis.

Sepsis is a heterogeneous syndrome induced by a dysregulated host response to infection. Glycolysis plays a role in maintaining the immune function of macrophages, which is crucial for severely septic patients. However, how the pathways that link glycolysis and macrophages are regulated is still largely unknown. Here, we provide evidence to support the function of KLF14, a novel Krüppel-like transcription factor, in the regulation of glycolysis and the immune function of macrophages during sepsis. KLF14 deletion led to significantly increased mortality in lethal models of murine endotoxemia and sepsis. Mechanistically, KLF14 decreased glycolysis and the secretion of inflammatory cytokines by macrophages by inhibiting the transcription of HK2. In addition, we confirmed that the expression of KLF14 was upregulated in septic patients. Furthermore, pharmacological activation of KLF14 conferred protection against sepsis in mice. These findings uncover a key role of KLF14 in modulating the inflammatory signaling pathway and shed light on the development of KLF14-targeted therapeutics for sepsis.

Gypenosides Inhibit Inflammatory Response and Apoptosis of Endothelial and Epithelial Cells in LPS-Induced ALI: A Study Based on Bioinformatic Analysis and in vivo/vitro Experiments.

INTRODUCTION: Severe inflammatory response leads to poor prognosis of acute lung injury (ALI), the role of gypenosides (GPs) on ALI is not fully clear. The study aimed at investigating the effects of GPs on ALI. METHODS: We firstly established LPS-induced ALI mice model. Then, we tested whether GPs contributed to alleviate inflammatory response and lung injury of ALI in vivo. In order to identify specific mechanisms of the phenomenon, we conducted a bioinformatic analysis of LPS-induced ALI mice based on GEO database to identify hub differentially expressed genes (DEGs). PPI network of the DEGs was used to find hub-genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted based on the DAVID database to identify which pathways the genes enriched. Then, we tested whether GPs inhibited lung injury and inflammatory response via the enriched pathways. We also tested whether GPs inhibited the apoptosis of endothelial and epithelial cells secondary to severe inflammation. RESULTS: We found GPs significantly alleviated lung injury and improved the survival rate of LPS-induced ALI mice in vivo. Bioinformatic analysis identified 20 hub-genes from DEGs, they were mainly enriched in NF-κB and TNF-α pathways. GPs could reduce the lung injury and inflammatory response via inhibiting NF-κB and TNF-α pathways in vivo. Our results indicated that GPs also inhibited inflammatory response of epithelial and endothelial cells via NF-κB and TNF-α pathways in vitro. Severe inflammatory response could also lead to apoptosis of endothelial and epithelial cells. Our results indicated that GPs effectively inhibited the apoptosis of endothelial and epithelial cells. CONCLUSION: Our study suggested GPs contributed to alleviated lung injury in vivo and inhibited inflammation and apoptosis of endothelial and epithelial cells in vitro, providing novel strategies for the prevention and therapy for ALI.

Reciprocal REGγ-mTORC1 regulation promotes glycolytic metabolism in hepatocellular carcinoma.

Despite significant progression in the study of hepatocellular carcinoma (HCC), the role of the proteasome in regulating cross talk between mTOR signaling and glycolysis in liver cancer progression is not fully understood. Here, we demonstrate that deficiency of REGγ, a proteasome activator, in mice significantly attenuates DEN-induced liver tumor formation. Ablation of REGγ increases the stability of PP2Ac (protein phosphatase 2 catalytic subunit) in vitro and in vivo, which dephosphorylates PRAS40 (AKT1 substrate 1) and stabilizes the interaction between PRAS40 and Raptor to inactive mTORC1-mediated hyper-glycolytic metabolism. In the DEN-induced animal model and clinical hepato-carcinoma samples, high levels of REGγ in HCC tumor regions contribute to reduced expression of PP2Ac, leading to accumulation of phosphorylated PRAS40 and mTORC1-mediated activation of HIF1α. Interestingly, mTORC1 enhances REGγ activity in HCC, forming a positive feedback regulatory loop. In conclusion, our study identifies REGγ-PP2Ac-PRAS40 axis as a new layer in regulating mTORC1 activity and downstream glycolytic alterations during HCC development, highlighting the REGγ-proteasome as a potential target for personalized HCC therapy.

REGγ controls Th17 cell differentiation and autoimmune inflammation by regulating dendritic cells.

Interleukin-17A (IL-17A)-producing helper T (Th17) cells are a subset of CD4+ T cells that play important pathological roles in autoimmune diseases. Although the intrinsic pathways of Th17 cell differentiation have been well described, how instructive signals derived from the innate immune system trigger the Th17 response and inflammation remains poorly understood. Here, we report that mice deficient in REGγ, a proteasome activator belonging to the 11S family, exhibit significantly deteriorated autoimmune neuroinflammation in an experimental autoimmune encephalomyelitis (EAE) model with augmented Th17 cell polarization in vivo. The results of the adoptive transfer of CD4+ T cells or dendritic cells (DCs) suggest that this phenotype is driven by DCs rather than T cells. Furthermore, REGγ deficiency promotes the expression of integrin αvß8 on DCs, which activates the maturation of TGF-ß1 to enhance Th17 cell development. Mechanistically, this process is mediated by the REGγ-proteasome-dependent degradation of IRF8, a transcription factor for αvß8. Collectively, our findings delineate a previously unknown mechanism by which REGγ-mediated protein degradation in DCs controls the differentiation of Th17 cells and the onset of an experimental autoimmune disease.

The proteasome activator REGγ counteracts immunoproteasome expression and autoimmunity.

For quite a long time, the 11S proteasome activator REGɑ and REGß, but not REGγ, are known to control immunoproteasome and promote antigen processing. Here, we demonstrate that REGγ functions as an inhibitor for immunoproteasome and autoimmune disease. Depletion of REGγ promotes MHC class I-restricted presentation to prime CD8+ T cells in vitro and in vivo. Mice deficient for REGγ have elevation of CD8+ T cells and DCs, and develop age-related spontaneous autoimmune symptoms. Mechanistically, REGγ specifically interacts with phosphorylated STAT3 and promotes its degradation in vitro and in cells. Inhibition of STAT3 dramatically attenuates levels of LMP2/LMP7 and antigen presentation in cells lacking REGγ. Importantly, treatment with STAT3 or LMP2/7 inhibitor prevented accumulation of immune complex in REGγ-/- kidney. Moreover, REGγ-/- mice also expedites Pristane-induced lupus. Bioinformatics and immunohistological analyses of clinical samples have correlated lower expression of REGγ with enhanced expression of phosphorylated STAT3, LMP2 and LMP7 in human Lupus Nephritis. Collectively, our results support the concept that REGγ is a new regulator of immunoproteasome to balance autoimmunity.

REGγ accelerates melanoma formation by regulating Wnt/ß-catenin signalling pathway.

It has been reported that the proteasome activator REGγ is associated with multiple oncogenic pathways in human cancers. However, the role of REGγ in the development of melanoma and the underlying mechanisms remain unclear. In this study, we attempted to investigate the effects of REGγ on human melanoma cell proliferation in vitro and in vivo. We demonstrated that knockdown of REGγ inhibited melanoma cell growth and arrested melanoma cell at G1 phase. Furthermore, depletion of REGγ also inhibited the xenograft growth of human melanoma. Mechanistically, REGγ activates Wnt/ß-catenin signal pathway by degrading GSK-3ß in melanoma cell lines and mouse models. Transient knockdown of ß-catenin effectively blocked cell proliferation in REGγ wild-type melanoma cells. In human melanoma samples, REGγ was overexpressed and positively correlated with ß-catenin levels. This study demonstrates that REGγ is a central molecule in the development of melanoma by regulating Wnt/ß-catenin pathway. This suggests that targeting REGγ could be an alternative therapeutic approach for melanoma.

Preferentially Expressed Antigen of Melanoma Prevents Lung Cancer Metastasis.

Lung cancer is the most common cause of cancer death worldwide. The poor survival rate is largely due to the extensive local invasion and metastasis. However, the mechanisms underlying the invasion and metastasis of lung cancer cells remain largely elusive. In this study, we examined the role of preferentially expressed antigen of melanoma (PRAME) in lung cancer metastasis. Our results show that PRAME is downregulated in lung adenocarcinoma and lung bone metastasis compared with normal human lung. Knockdown of PRAME decreases the expression of E-Cadherin and promotes the proliferation, invasion, and metastasis of lung cancer cells by regulating multiple critical genes, most of which are related to cell migration, including MMP1, CCL2, CTGF, and PLAU. Clinical data analysis reveals that the expression of MMP1 correlates with the clinical features and outcome of lung adenocarcinoma. Taken together, our data demonstrate that PRAME plays a role in preventing the invasion and metastasis of lung adenocarcinoma and novel diagnostic or therapeutic strategies can be developed by targeting PRAME.

Loss of KLF14 triggers centrosome amplification and tumorigenesis.

Centrosome amplification is frequent in cancer, but the underlying mechanisms remain unclear. Here we report that disruption of the Kruppel-like factor 14 (KLF14) gene in mice causes centrosome amplification, aneuploidy and spontaneous tumorigenesis. Molecularly, KLF14 functions as a transcriptional repressor of Plk4, a polo-like kinase whose overexpression induces centrosome overduplication. Transient knockdown of KLF14 is sufficient to induce Plk4-directed centrosome amplification. Clinically, KLF14 transcription is significantly downregulated, whereas Plk4 transcription is upregulated in multiple types of cancers, and there exists an inverse correlation between KLF14 and Plk4 protein expression in human breast and colon cancers. Moreover, KLF14 depletion promotes AOM/DSS-induced colon tumorigenesis. Our findings reveal that KLF14 reduction serves as a mechanism leading to centrosome amplification and tumorigenesis. On the other hand, forced expression of KLF14 leads to mitotic catastrophe. Collectively, our findings identify KLF14 as a tumour suppressor and highlight its potential as biomarker and therapeutic target for cancer.

REGγ is critical for skin carcinogenesis by modulating the Wnt/ß-catenin pathway.

Here we report that mice deficient for the proteasome activator, REGγ, exhibit a marked resistance to TPA (12-O-tetradecanoyl-phorbol-13-acetate)-induced keratinocyte proliferation, epidermal hyperplasia and onset of papillomas compared with wild-type counterparts. Interestingly, a massive increase of REGγ in skin tissues or cells resulting from TPA induces activation of p38 mitogen-activated protein kinase (MAPK/p38). Blocking p38 MAPK activation prevents REGγ elevation in HaCaT cells with TPA treatment. AP-1, the downstream effector of MAPK/p38, directly binds to the REGγ promoter and activates its transcription in response to TPA stimulation. Furthermore, we find that REGγ activates Wnt/ß-catenin signalling by degrading GSK-3ß in vitro and in cells, increasing levels of CyclinD1 and c-Myc, the downstream targets of ß-catenin. Conversely, MAPK/p38 inactivation or REGγ deletion prevents the increase of cyclinD1 and c-Myc by TPA. This study demonstrates that REGγ acts in skin tumorigenesis mediating MAPK/p38 activation of the Wnt/ß-catenin pathway.

REGγ deficiency promotes premature aging via the casein kinase 1 pathway.

Our recent studies suggest a role for the proteasome activator REG (11S regulatory particles, 28-kDa proteasome activator)γ in the regulation of tumor protein 53 (p53). However, the molecular details and in vivo biological significance of REGγ-p53 interplay remain elusive. Here, we demonstrate that REGγ-deficient mice develop premature aging phenotypes that are associated with abnormal accumulation of casein kinase (CK) 1δ and p53. Antibody array analysis led us to identify CK1δ as a direct target of REGγ. Silencing CK1δ or inhibition of CK1δ activity prevented decay of murine double minute (Mdm)2. Interestingly, a massive increase of p53 in REGγ(-/-) tissues is associated with reduced Mdm2 protein levels despite that Mdm2 transcription is enhanced. Allelic p53 haplodeficiency in REGγ-deficient mice attenuated premature aging features. Furthermore, introducing exogenous Mdm2 to REGγ(-/-) MEFs significantly rescues the phenotype of cellular senescence, thereby establishing a REGγ-CK1-Mdm2-p53 regulatory pathway. Given the conflicting evidence regarding the "antiaging" and "proaging" effects of p53, our results indicate a key role for CK1δ-Mdm2-p53 regulation in the cellular aging process. These findings reveal a unique model that mimics acquired aging in mammals and indicates that modulating the activity of the REGγ-proteasome may be an approach for intervention in aging-associated disorders.