Upregulation of NADPH-oxidase, inducible nitric oxide synthase and apoptosis in the hippocampus following impaired insulin signaling in the rats: Development of sporadic Alzheimer's disease.

NADPH-oxidase (NOX) is a multi-subunit enzyme complex. The upregulation of NOX causes massive production of superoxide (O2¯), which avidly reacts with nitric oxide (NO) and increases cellular reactive oxygen/nitrogen species (ROS/RNS). Increased ROS/RNS plays pivotal role in the sporadic Alzheimer's disease (sAD) development and brain damage following impaired insulin signaling. Hence, this study aimed to examine early-time course of changes in NOX and NOS expression, and apoptotic proteins in the rats hippocampi following insulin signaling impairment [induced by STZ injection; intraperitoneal (IP) or in cerebral ventricles (ICV)]. Early effects (1, 3, or 6 weeks) on the NOX activity, translocation of NOX subunits from cytosol to the membrane, NO-synthases [neuronal-, inducible- and endothelial-NOS; nNOS, iNOS and eNOS], The Rac-1 protein expression, levels of NO and O2¯, cytochrome c release, caspase-3 and 9 activations (cleavage) were studied. STZ injection (in both models) increased NOX activity, O2¯ production, and enhanced cytosolic subunits translocation into membrane. The iNOS but not nNOS and eNOS expression and NO levels were increased in STZ treated rats. Finally, STZ injection increased cytochrome c release, caspase-3 and 9 activations in a manner that was significantly associated with levels of O2¯ and NO in the hippocampus. ICV-STZ administration resulted in significant profound changes over the IP route. In conclusion, impairment in insulin function induces early changes in ROS/RNS contents through NOX and iNOS upregulation and neuronal apoptosis in the hippocampus. Our results could mechanistically explain the role of impaired insulin function in the development of sAD.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Activation of AKT/mammalian target of rapamycin signaling in the peripheral blood of women with premature ovarian insufficiency and its correlation with FMR1 expression.

BACKGROUND: The protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway regulates early follicular activation and follicular pool maintenance in female germline cells. Fragile X mental retardation 1 (FMR1) regulates folliculogenesis and it is variably expressed in patients with Premature Ovary Insufficiency. FMR1 expression is supposed to be linked to AKT/mTOR signaling in an ovarian response dependent manner as demonstrated in recent in vitro and in vivo studies in the female germline in vitro and in vivo. METHODS: We evaluated changes in the expression of AKT/mTOR signaling pathway genes by real time PCR in the peripheral blood of 74 patients with Premature Ovarian Insufficiency and 56 fertile controls and correlated their expression with FMR1 expression. RESULTS: Expression of the genes AKT1, TSC2, mTOR, and S6K was significantly more abundant in patients with POI than in the controls. For AKT1, TSC2 and mTOR, gene expression was not affected by FMR1-CGG repeat number in the 5´-untranslated region. FMR1 and S6K expression levels, however, were significantly upregulated in patients with POI and an FMR1 premutation. Independent of a premutation, expression of mTOR, S6K, and TSC2 was significantly correlated with that of FMR1 in all patients. Furthermore, when grouped according to ovarian reserve, this effect remained significant only for mTOR and S6K, with higher significance note in patients with Premature Ovarian Insufficiency than in the controls. CONCLUSIONS: In Premature ovarian insufficiency patients, activation of AKT/mTOR signaling pathway is remarkable and putatively pathognomonic. Additionally, it seems to be triggered by an FMR1/mTOR/S6K linkage mechanism, most relevant in premutation carriers.

Gel-Based Proteomic Identification of Suprabasin as a Potential New Candidate Biomarker in Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.

Sp1 Plays a Key Role in Vasculogenic Mimicry of Human Prostate Cancer Cells.

Sp1 transcription factor regulates genes involved in various phenomena of tumor progression. Vasculogenic mimicry (VM) is the alternative neovascularization by aggressive tumor cells. However, there is no evidence of the relationship between Sp1 and VM. This study investigated whether and how Sp1 plays a crucial role in the process of VM in human prostate cancer (PCa) cell lines, PC-3 and DU145. A cell viability assay and three-dimensional culture VM tube formation assay were performed. Protein and mRNA expression levels were detected by Western blot and reverse transcriptase-polymerase chain reaction, respectively. The nuclear twist expression was observed by immunofluorescence assay. A co-immunoprecipitation assay was performed. Mithramycin A (MiA) and Sp1 siRNA significantly decreased serum-induced VM, whereas Sp1 overexpression caused a significant induction of VM. Serum-upregulated vascular endothelial cadherin (VE-cadherin) protein and mRNA expression levels were decreased after MiA treatment or Sp1 silencing. The protein expression and the nuclear localization of twist were increased by serum, which was effectively inhibited after MiA treatment or Sp1 silencing. The interaction between Sp1 and twist was reduced by MiA. On the contrary, Sp1 overexpression enhanced VE-cadherin and twist expressions. Serum phosphorylated AKT and raised matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) expressions. MiA or Sp1 silencing impaired these effects. However, Sp1 overexpression upregulated phosphor-AKT, MMP-2 and LAMC2 expressions. Serum-upregulated Sp1 was significantly reduced by an AKT inhibitor, wortmannin. These results demonstrate that Sp1 mediates VM formation through interacting with the twist/VE-cadherin/AKT pathway in human PCa cells.

Endothelial and systemic upregulation of miR-34a-5p fine-tunes senescence in progeria.

Endothelial defects significantly contribute to cardiovascular pathology in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Using an endothelium-specific progeria mouse model, we identify a novel, endothelium-specific microRNA (miR) signature linked to the p53-senescence pathway and a senescence-associated secretory phenotype (SASP). Progerin-expressing endothelial cells exert profound cell-non-autonomous effects initiating senescence in non-endothelial cell populations and causing immune cell infiltrates around blood vessels. Comparative miR expression analyses revealed unique upregulation of senescence-associated miR34a-5p in endothelial cells with strong accumulation at atheroprone aortic arch regions but also, in whole cardiac- and lung tissues as well as in the circulation of progeria mice. Mechanistically, miR34a-5p knockdown reduced not only p53 levels but also late-stage senescence regulator p16 with no effect on p21 levels, while p53 knockdown reduced miR34a-5p and partially rescued p21-mediated cell cycle inhibition with a moderate effect on SASP. These data demonstrate that miR34a-5p reinforces two separate senescence regulating branches in progerin-expressing endothelial cells, the p53- and p16-associated pathways, which synergistically maintain a senescence phenotype that contributes to cardiovascular pathology. Thus, the key function of circulatory miR34a-5p in endothelial dysfunction-linked cardiovascular pathology offers novel routes for diagnosis, prognosis and treatment for cardiovascular aging in HGPS and potentially geriatric patients.

TSLP up-regulates inflammatory responses through induction of autophagy in T cells.

Thymic stromal lymphopoietin (TSLP), a type I cytokine belonging to the IL-2 cytokine family, promotes Th2-mediated inflammatory responses. The aim of this study is to investigate whether TSLP increases inflammatory responses via induction of autophagy using a murine T cell lymphoma cell line, EL4 cells, and lipopolysaccharide (LPS)-injected mice. TSLP increased expression levels of autophagy-related factors, such as Beclin-1, LC3-II, p62, Atg5, and lysosome associated membrane protein 1/2, whereas these factors increased by TSLP disappeared by neutralization of TSLP in EL4 cells. TSLP activated JAK1/JAK2/STAT5/JNK/PI3K, while the blockade of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways reduced the expression levels of Beclin-1, LC3-II, and p62 in TSLP-stimulated EL4 cells. In addition, TSLP simultaneously increased levels of inflammatory cytokines via induction of autophagy by activation of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways. In an LPS-induced acute liver injury (ALI) mouse model, exogenous TSLP increased expression levels of Beclin-1 and LC3-II, whereas functional deficiency of TSLP by TSLP siRNA resulted in lower expression of Beclin-1, LC3-II, and inflammatory cytokines, impairing their ability to form autophagosomes in ALI mice. Thus, our findings show a new role of TSLP between autophagy and inflammatory responses. In conclusion, regulating TSLP-induced autophagy may be a potential therapeutic strategy for inflammatory responses.

STING up-regulates VEGF expression in oxidative stress-induced senescence of retinal pigment epithelium via NF-κB/HIF-1α pathway.

AIM: Aging-related dysfunction of retinal pigment epithelium (RPE) is the main pathogenic factors for pathological angiogenesis due to dysregulated vascular endothelial growth factor (VEGF) in retinal vascular diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). However, the molecular mechanism behind the up-regulation of VEGF in senescent RPE is still blurred. MATERIALS AND METHODS: As oxidative damage is the key cause of RPE dysfunction, we employed a model of oxidative stress-induced premature senescence of ARPE-19 to explore the effect of senescent RPE on VEGF. KEY FINDINGS: We reported that senescent ARPE-19 up-regulated VEGF expression under both short-term and prolonged H2O2 treatment, accompanying with increased HIF-1α, the key mediator of VEGF. STING signaling, which could be activated by oxidative stress-damaged DNA, was also observed to be increased in senescent ARPE-19 treated with H2O2. And the inhibition of STING significantly reduced HIF-1α expression to alleviate the up-regulation of VEGF. NF-κB was also shown to be involved in the regulation of VEGF in senescent ARPE-19 in response to STING signaling. Furthermore, oxidative stress impaired the lysosomal clearance of damaged DNA to enhance STING signaling, thereby up-regulating VEGF expression in senescent RPE. SIGNIFICANCE: Our data provide evidence that STING plays an important role in VEGF regulation in senescent RPE induced by oxidative stress.

Circular RNA circ_0000515 adsorbs miR-542-3p to accelerate bladder cancer progression via up-regulating ILK expression.

BACKGROUND: Bladder cancer (BC) is a common cause of cancer-relevant deaths globally. This study is designed to delve into expressions, biological functions and molecular mechanisms of circ_0000515 in BC. METHODS: Quantitative real-time polymerase chain reaction was accomplished to examine circ_0000515, miR-542-3p and integrin-linked kinase (ILK) mRNA expressions in BC tissues and cell lines. In RT-4 and RT-112 cells with circ_0000515 depletion and UMUC3 and BIU-87 cells with this circ RNA overexpression, a cell counting kit-8 assay was adopted to monitor the viability. Besides, transwell assay was conducted to detect cell migration and aggressiveness, and luciferase reporter gene assay was applied to probe the interplay among circ_0000515, miR-542-3p and ILK mRNA. Additionally, Besides, the regulatory function of circ_0000515 on miR-542-3p expression was under the assay of quantitative real-time polymerase chain reaction, and western blot was fulfilled to determine the regulative function of circ_0000515/miR-542-3p axis on ILK protein expressions. A xenograft animal was modeled to examine lung metastasis in vivo. RESULTS: Circ_0000515 and ILK expressions were significantly elevated in BC tissues and cell lines, while that of miR-542-3p was dramatically suppressed. Knocking down circ_0000515 could significantly repress the growth, migration and aggressiveness of BC cells while overexpression of circ_0000515 showed opposite effects. Moreover, circ_0000515 knockdown inhibited pulmonary metastasis in vivo. Circ_0000515 was validated to adsorb miR-542-3p, and ILK was testified as the downriver target of miR-542-3p. Circ_0000515 could ascend ILK expression through repressing that of miR-542-3p. CONCLUSIONS: Circ_0000515, as a tumor promoter, strengthens the viability, migration and aggressiveness of BC cells via modulating miR-542-3p/ILK axis.

Long non-coding RNA MALAT1 silencing elevates microRNA-26a-5p to ameliorate myocardial injury in sepsis by reducing regulator of calcineurin 2.

OBJECTIVE: Sepsis is a leading cause of morbidity and mortality after surgery. We aimed to explore the role of long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) sponging microRNA-26a-5p in sepsis-induced myocardial injury by regulating regulator of calcineurin 2 (Rcan2). METHODS: HL-1 cells were incubated with lipopolysaccharide (LPS) to induce in vitro cardiomyocyte injury models, which were then treated with silenced MALAT1 vector, miR-26a-5p mimic or Rcan2 overexpression vector. Next, inflammatory factor level and apoptosis of cells were determined. The in vivo mouse models were constructed by intraperitoneal injection of LPS. The modeled mice were injected with relative oligonucleotides and the pathology, apoptosis, and inflammation in mouse myocardial tissues were assessed. Expression of MALAT1, miR-26a-5p and Rcan2 in vivo and in vitro was evaluated. RESULTS: MALAT1 and Rcan2 were upregulated while miR-26a-5p was downregulated in LPS-treated HL-1 cells and mice. MALAT1 silencing or miR-26a-5p upregulation suppressed LPS-induced inflammation and apoptosis of cardiomyocytes in cellular and animal models. These effects of elevated miR-26a-5p could be reversed by upregulating Rcan2, and MALAT1 knockdown-induced ameliorative impacts could be reversed by miR-26a-5p downregulation. CONCLUSION: MALAT1 silencing elevated miR-26a-5p to ameliorate LPS-induced myocardial injury by reducing Rcan2. Our research may provide novel biomarkers for the treatment of sepsis.

MiR-340-5p inhibits Müller cell activation and pro-inflammatory cytokine production by targeting BMP4 in experimental diabetic retinopathy.

Diabetic retinopathy (DR) is a disease that can cause blindness. Bone morphogenetic protein-4 (BMP4) was reported be overexpressed in DR model. However, the specific mechanism of BMP4 in DR development has not been explored. MiR-340-5p and BMP4 levels were detected by RT-qPCR in MIO-M1 cells and retinas of mice. Western blot analysis was used to examine GFAP, BMP4 and BRB junction protein levels. Inflammatory cytokine secretion and the retina structure were examined by ELISA and H&E staining, respectively. The interaction between miR-340-5p and BMP4 was identified by luciferase reporter assay. In HG-stimulated MIO-M1 cells, BMP4 was upregulated. Mechanically, BMP4 was targeted by miR-340-5p and negatively regulated by miR-340-5p. In rescue assays, BMP4 countervailed the suppressive effects of miR-340-5p on activation of Müller cells and release of inflammatory cytokines. Additionally, miR-18a-3p overexpression alleviated BRB injury to inhibit DR progression in vivo. In conclusion, miR-340-5p inhibits DR progression by targeting BMP4, which may offer a new pathway for treatment of DR.

Cisplatin resistance of NSCLC cells involves upregulation of visfatin through activation of its transcription and stabilization of mRNA.

Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Cisplatin (CDDP) has been used as a standard adjuvant therapy for advanced NSCLC patients, while chemoresistance is one of the most challenging problems to limit its clinical application. Our data showed that the expression of visfatin was significantly increased in CDDP resistant NSCLC cells as compared with that in their parental cells, while knockdown of visfatin or its neutralization antibody can restore the CDDP sensitivity of resistant NSCLC cells. The upregulation of visfatin in CDDP resistant NSCLC cells was due to the increased mRNA stability and promoter activity. Further, we found that signal transducer and activator of transcription 3 (STAT3), which was increased in chemoresistant cells, can increase the transcription of visfatin. While tristetraprolin (TTP), which can decease mRNA stability of visfatin, was decreased in chemoresistant cells. Inhibition of STAT3 or over expression of TTP can restore CDDP sensitivity of resistant NSCLC cells. Collectively, our data showed that STAT3 and TTP-regulated expression of visfatin was involved in CDDP resistance of NSCLC cells. It indicated that targeted inhibition of visfatin should be a potential approach to overcome CDDP resistance of NSCLC treatment.

RUNX3 is up-regulated in abdominal aortic aneurysm and regulates the function of vascular smooth muscle cells by regulating TGF-ß1.

Abdominal aortic aneurysm (AAA) has been associated with the dysfunction of vascular smooth muscle cells (VSMCs) and extracellular matrix (ECM) remodelling. Runt-related transcription factor 3 (RUNX3) has been reported to be up-regulated in aneurysmal aorta samples compared with normal aorta. However, its function in VSMCs and the mechanism of function remains unknown. Therefore, our study aimed to investigate the role of RUNX3 in ECM remodelling and VSMC function, and further explore the underlying mechanism. Our results verified that RUNX3 was increased in aortic samples of AAA compared with healthy controls. Overexpression vectors of RUNX3 (ov-RUNX3) and siRNA of RUNX3 (si-RUNX3) were transfected into Human aortic smooth muscle cells (HAoSMCs). The results indicated that ov-RUNX3 promoted cell proliferation, migration, and MMP-2/3/9 secretion, and suppressed TIMP-1, collagen I/III, SM22, MYH11 and CNN1 expression in HAoSMCs. The silencing of RUNX3 has the opposite effect. Furthermore, we found that RUNX3 targets TGF-ß1 and suppressed its transcription. The silencing of TGF-ß1 increased cell proliferation, migration and MMP-2/3/9 expression, and inhibited TIMP-1, Collagen I/III, SM22, MYH11 and CNN1 expression. In addition, TGF-ß1 reversed the effect of RUNX3 overexpression on HAoSMCs. Hence, our study indicated that RUNX3 promotes cell proliferation, migration, and ECM remodelling through suppressing TGF-ß1.

Growth factor expression is enhanced, and extracellular matrix proteins are depressed in healing skin wounds in septic patients compared with healthy controls.

Sepsis manifests as a dysregulated immune response to infection, damaging organs. Skin has a critical role in protecting the body. In sepsis, skin wound healing is impaired. The mechanisms behind it have been poorly studied. In this study, suction blister wounds were induced on intact abdominal skin in 15 septic patients. A single blister wound was biopsied from each patient and from 10 healthy controls. Immunohistochemical staining of growth factors and extracellular matrix (ECM) proteins was performed. Significance (p < 0.05) of the differences was calculated. The following growth factors were overexpressed in the skin of septic patients compared with healthy controls: epithelial growth factor (intact epithelium p = 0.007, migrating epithelium p = 0.038), vascular epithelial growth factor (intact epithelium p < 0.001, migrating epithelium p = 0.011) and transforming growth factor beta (migrating epithelium p = 0.002). The expression of syndecan-1 was upregulated in the skin of septic patients compared with healthy controls (intact epithelium p = 0.048, migrating epithelium p = 0.028). The following ECM proteins had lower expression in the epithelium in septic patients than in healthy controls: tenascin-C (migrating epithelium p = 0.03) and laminin-332 (intact epithelium p = 0.036). In sepsis, growth factor and syndecan expression was enhanced, while ECM and basement membrane proteins were mostly depressed.

The spike protein of SARS-CoV-2 induces heme oxygenase-1: Pathophysiologic implications.

BACKGROUND: Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus, SARS-CoV-2. Viral entry into cells requires the viral spike protein, and both the virus and its spike protein appear in the urine of COVID-19 patients with AKI. We examined the effects of transfecting the viral spike protein of SARS-CoV-2 in kidney cell lines. METHODS: HEK293, HEK293-ACE2+ (stably overexpressing ACE2), and Vero E6 cells having endogenous ACE2 were transfected with SARS-CoV-2 spike or control plasmid. Assessment of gene and protein expression, and syncytia formation was performed, and the effects of quercetin on syncytia formation examined. FINDINGS: Spike transfection in HEK293-ACE2+ cells caused syncytia formation, cellular sloughing, and focal denudation of the cell monolayer; transfection in Vero E6 cells also caused syncytia formation. Spike expression upregulated potentially nephrotoxic genes (TNF-α, MCP-1, and ICAM1). Spike upregulated the cytoprotective gene HO-1 and relevant signaling pathways (p-Akt, p-STAT3, and p-p38). Quercetin, an HO-1 inducer, reduced syncytia formation and spike protein expression. INTERPRETATION: The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a relevant model for the study of maladaptive and adaptive responses germane to AKI in COVID-19; 2) such spike protein expression upregulates HO-1; and 3) quercetin, an HO-1 inducer, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19. FUNDING: R01-DK119167 (KAN), R01-AI100911 (JPG), P30-DK079337; R01-DK059600 (AA).

Lats2 promotes heart failure by stimulating p53-mediated apoptosis during pressure overload.

The Hippo pathway plays a wide variety of roles in response to stress in the heart. Lats2, a component of the Hippo pathway, is phosphorylated by Mst1/2 and, in turn, phosphorylates YAP, causing inactivation of YAP. Lats2 stimulates apoptosis and negatively affects hypertrophy in cardiomyocytes. However, the role of Lats2 during cardiac stress is poorly understood in vivo. Lats2 is activated in the mouse heart in response to transverse aortic constriction (TAC). We used systemic Lats2 +/- mice to elucidate the role of endogenous Lats2. Cardiac hypertrophy and dysfunction induced by 4 weeks of TAC were attenuated in Lats2 +/- mice, and interstitial fibrosis and apoptosis were suppressed. Although TAC upregulated the Bcl-2 family proapoptotic (Bax and Bak) and anti-apoptotic (Bcl-2 and Bcl-xL) molecules in non-transgenic mice, TAC-induced upregulation of Bax and Bak was alleviated and that of Bcl-2 was enhanced in Lats2 +/- mice. TAC upregulated p53, but this upregulation was abolished in Lats2 +/- mice. Lats2-induced increases in apoptosis and decreases in survival in cardiomyocytes were inhibited by Pifithrin-α, a p53 inhibitor, suggesting that Lats2 stimulates apoptosis via a p53-dependent mechanism. In summary, Lats2 is activated by pressure overload, thereby promoting heart failure by stimulating p53-dependent mechanisms of cell death.

USP1 Promotes GC Metastasis via Stabilizing ID2.

Gastric cancer (GC) is one of the most common malignant tumors all over the world. And recurrence and metastasis are still the main causes of low survival rate for advanced GC. USP1 has been shown overexpressed in multiple cancers, which indicate its important biomarker in tumorigenesis and development. Our study is aimed at defining the exact role of USP1 on GC metastasis and the underlying mechanism. USP1 was firstly found overexpressed in GC tissues and relatively high-expression levels conferred poor survival rates. Then, real-time cellular analysis (RTCA) showed that USP1 knockdown inhibited GC metastasis both in vitro and in vivo. Mechanically, we demonstrated that USP1 promoted GC metastasis via upregulating ID2 expression and further confirmed that USP1 stabilized ID2 expression through deubiquitinating ID2 in GC. In conclusion, our study showed that USP1 promoted GC metastasis via stabilizing ID2 expression, which provides a potential biomarker and therapy target for GC.

Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity.

Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte-neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein-binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte-neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.

TNF-α up-regulates Nanog by activating NF-κB pathway to induce primary rat spinal cord astrocytes dedifferentiation.

AIMS: Astrocytes re-acquire stem cell potential upon inflammation, thereby becoming a promising source of cells for regenerative medicine. Nanog is an essential transcription factor to maintain the characteristics of stem cells. We aimed to investigate the role of Nanog in astrocyte dedifferentiation. MAIN METHODS: TNF-α was used to induce the dedifferentiation of primary rat spinal cord astrocytes. The expression of immature markers CD44 and Musashi-1 was detected by qRT-PCR and immunofluorescence. The Nanog gene is knocked down by small interference RNA. Nanog expression was measured by qRT-PCR and western blotting. BAY 11-7082 was used to suppress NF-κB signals in astrocytes. NF-κB signaling was evaluated by Western blotting. KEY FINDINGS: Our results showed that TNF-α promoted the re-expression of CD44 and Musashi-1 in astrocytes. Dedifferentiated astrocytes could be induced to differentiate into oligodendrocyte lineage cells indicating that the astrocytes had pluripotency. In addition, TNF-α treatment activated NF-κB signaling pathway and up-regulated Nanog. Knockdown of Nanog reversed the increase of CD44 and Musashi-1 induced by TNF-α without affecting the activation of NF-κB signaling. Importantly, blocking NF-κB signaling by BAY 11-7082 inhibited the expression of immature markers suggesting that TNF-α induces dedifferentiation of astrocytes through the NF-κB signaling pathway. BAY 11-7082 could also inhibit the expression of Nanog, which indicated that Nanog was regulated by NF-κB signaling pathway. SIGNIFICANCE: These findings indicate that activation of the NF-κB signaling pathway through TNF-α leads to astrocytes dedifferentiation via Nanog. These results expand our understanding of the mechanism of astrocytes dedifferentiation.

NOD2-mediated P2Y12 upregulation increases platelet activation and thrombosis in sepsis.

BACKGROUND: Platelets from septic patients exhibit increased reactivity. However, the underlying mechanism of sepsis-induced platelet hyperactivity is still not completely understood. OBJECTIVE: P2Y12 is a central receptor for platelet activation. In this study, we investigated the role of platelet P2Y12 in platelet hyperactivity during sepsis. METHODS: We measured platelet P2Y12 expression and aggregation in response to ADP in septic patients and cecal ligation and puncture (CLP)-treated mice. We also detected the downstream signaling of P2Y12 in resting platelets from patients and mice with sepsis. The role of nucleotide-binding oligomerization domain 2 (NOD2)/RIP2/NF-κB/P65 pathway in sepsis-induced platelet P2Y12 high expression was also investigated. Finally, we compared the antiplatelet and antithrombotic effects of clopidogrel, prasugrel, and ticagrelor in experimental sepsis in mice and rats. RESULTS: Compared to healthy subjects, platelets from septic patients exhibit P2Y12 hyperactivity and higher P2Y12 expression. pAkt is enhanced and pVASP is impaired in resting platelets from the patients, indicating the constitutive activation of platelet P2Y12 receptor. Mouse sepsis model recapitulates the findings in septic patients. NOD2 deficiency attenuates sepsis-induced platelet P2Y12 high expression, hyperactivity, and thrombosis. Prasugrel and ticagrelor are potent P2Y12 inverse agonists, and exhibit superior antiplatelet and antithrombotic efficacy over clopidogrel in mice and rats with sepsis. CONCLUSIONS: NOD2 activation upregulates platelet P2Y12 expression, which is constitutively activated and contributes to platelet hyperactivity in septic status. Compared to clopidogrel, prasugrel and ticagrelor are potent P2Y12 inverse agonists with superior antiplatelet and antithrombotic efficacy in experimental sepsis.