BACH1 regulates erythrophagocytosis and iron-recycling in β-thalassemia

Macrophages play essential roles in erythrophagocytosis and iron recycling. β-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of β-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in β-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with β-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.     

PROP1 and CTNNB1 expression in adamantinomatous craniopharyngiomas with or without β-catenin mutations

INTRODUCTION:

Activating mutations in exon 3 of the β-catenin gene are involved in the pathogenesis of adamantinomatous craniopharyngiomas. Recently, the interaction between β-catenin and PROP1 has been shown to be responsible for pituitary cell lineage determination. We hypothesized that dysregulated PROP1 expression could also be involved in the pathogenesis of craniopharyngiomas.

OBJECTIVES:

To determine whether dysregulated gene expression was responsible for tumor pathogenesis in adamantinomatous craniopharyngiomas, the β-catenin gene was screened for mutations, and the expression of the β-catenin gene and PROP1 was evaluated.

METHODS:

The β-catenin gene was amplified and sequenced from 14 samples of adamantinomatous craniopharyngiomas. PROP1 and β-catenin gene expression was assessed by real-time RT-PCR from 12 samples, and β-catenin immunohistochemistry was performed on 11 samples.

RESULTS:

Mutations in the β-catenin gene were identified in 64% of the adamantinomatous craniopharyngiomas samples. Evidence of β-catenin gene overexpression was found in 71% of the tumors with β-catenin mutations and in 40% of the tumors without mutations, and β-catenin immunohistochemistry revealed a nuclear staining pattern for each of the analyzed samples. PROP1 expression was undetectable in all of the tumor samples.

CONCLUSION:

We found evidence of β-catenin gene overexpression in the majority of adamantinomatous craniopharyngiomas, and we also detected a nuclear β-catenin staining pattern regardless of the presence of a β-catenin gene mutation. These results suggest that WNT signaling activation plays an important role in the pathogenesis of adamantinomatous craniopharyngiomas. Additionally, this study was the first to evaluate PROP1 expression in adamantinomatous craniopharyngiomas, and the absence of PROP1 expression indicates that this gene is not involved in the pathogenesis of this tumor, at least in this cohort.     

PTEN regulates PDGF ligand switch for β-PDGFR signaling in prostate cancer

Platelet-derived growth factor (PDGF) family members are potent growth factors that regulate cell proliferation, migration, and transformation. Clinical studies have shown that both PDGF receptor β (β-PDGFR) and its ligand PDGF D are up-regulated in primary prostate cancers and bone metastases, whereas PDGF B, a classic ligand for β-PDGFR, is not frequently detected in clinical samples. In this study, we examined the role of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of PDGF expression levels using both a prostate-specific, conditional PTEN-knockout mouse model and mouse prostate epithelial cell lines established from these mice. We found an increase in PDGF D and β-PDGFR expression levels in PTEN-null tumor cells, accompanied by a decrease in PDGF B expression. Among Akt isoforms, increased Akt3 expression was most prominent in mouse PTEN-null cells, and phosphatidylinositol 3-kinase/Akt activity was essential for the maintenance of increased PDGF D and β-PDGFR expression. In vitro deletion of PTEN resulted in a PDGF ligand switch from PDGF B to PDGF D in normal mouse prostate epithelial cells, further demonstrating that PTEN regulates this ligand switch. Similar associations between PTEN status and PDGF isoforms were noted in human prostate cancer cell lines. Taken together, these results suggest a mechanism by which loss of PTEN may promote prostate cancer progression via PDGF D/β-PDGFR signal transduction.     

Recombinant WNTs differentially activate β-catenin-dependent and -independent signalling in mouse microglia-like cells

Aim: The objective of this study was to compare the efficacy of different recombinant, commercially available Wingless/Int‐1 (WNTs) with regard to WNT/β‐catenin signalling, dishevelled (DVL) and G protein activation and the induction of cell proliferation in a microglia‐like cell line called N13. Methods: For detection of activated signalling molecules, cell lysates are analysed by immunoblotting. Furthermore, we used a [γ³⁵S] GTP binding assay to monitor the exchange of GDP for GTP in heterotrimeric G proteins in N13 membrane preparations. Cell proliferation was assessed by the 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay measuring mitochondrial function, which is proportional to the amount of viable cells. Results: Of the WNTs tested (WNT‐3A, ‐4, ‐5A, ‐5B, ‐7A,‐9B), only WNT‐3A activated WNT/β‐catenin signalling in N13 cells. All WNTs induced the formation of P hosphorylated and S hifted DVL (PS‐DVL) and the activation of heterotrimeric G proteins with variable efficacies. WNT‐5A and WNT‐9B, which had the highest efficacy in the G protein assay, also induced N13 cell proliferation. Conclusion: WNTs show significant differences in their efficacy to activate β‐catenin‐dependent and ‐independent signalling. The WNTs tested are present during maturation of the central nervous system and/or in the adult brain and are thus potential regulators of microglia‐mediated neuroinflammation.     

Pyruvate kinase,muscle isoform 2 promotes proliferation and insulin secretion of pancreatic β-cells via activating Wnt/CTNNB1 signaling

Failure of pancreatic β-cells is closely associated with type 2 diabetes mellitus (T2DM), an intractable disease affecting numerous patients. Pyruvate kinase, muscle isoform 2 (PKM2) is a potential modulator of insulin secretion in β-cells. This study aims at revealing roles and possible mechanisms of PKM2 in pancreatic β-cells. Mouse pancreatic β-cell line NIT-1 was used for high glucose treatment and PKM2 overexpression by its specific expression vector. Cell proliferation by Thiazolyl blue assay, cell apoptosis by annexin V-fluorescein isothiocyanate/prodium iodide staining and insulin secretion assay by ELISA were performed in each group. The mRNA and protein levels of related factors were analyzed by real-time quantitative PCR and western blot. Results showed that Pkm2 was inhibited under high glucose conditions compared to the untreated cells (P < 0.01). Its overexpression significantly suppressed NIT-1 cell apoptosis (P < 0.01), and induced cell proliferation (P < 0.05) and insulin secretion (P < 0.05). Related factors showed consistent mRNA expression changes. Protein levels of β-catenin (CTNNB1), insulin receptor substrate 1 (IRS1) and IRS2 were all promoted by PKM2 overexpression (P < 0.01), indicating the activated Wnt/CTNNB1 signaling. These results indicated the inductive roles of PKM2 in pancreatic β-cell NIT-1, including promoting cell proliferation and insulin secretion, and inhibiting cell apoptosis, which might be achieved via activating the Wnt/CTNNB1 signaling and downstream factors. This study offers basic information on the role and mechanism of PKM2 in pancreatic β-cells, and lays the foundation for using PKM2 as a potential therapeutic target in T2DM.     

Cytohesin-1 regulates fMLF-mediated activation and functions of the β2 integrin Mac-1 in human neutrophils

The nucleotide exchange factor cytohesin-1 was previously reported to interact with the cytoplasmic domains of the integrin β-chain common to all β(2) integrins such as LFA-1 and Mac-1. We show here that cytohesin-1, which contributes to fMLF-induced functional responses in PMNs through activation of Arf6, restrains the activation of the β(2) integrin Mac-1 (αMβ(2)) in PMNs or dcAMP-differentiated PLB-985 cells. We found that the cytohesin-1 inhibitor SecinH3 or siRNA increased cell adhesion to immobilized fibrinogen and fMLF-mediated conformational changes of Mac-1, monitored using mAb CBRM1/5, specific for the activation epitope of the αM subunit. In contrast, PLB-985 cells overexpressing cytohesin-1 showed little adhesion to fibrinogen. The use of SecinH3 and siRNA also revealed that interference with cytohesin-1 signaling also enhanced phagocytosis of zymosan particles and chemotaxis toward fMLF in transwell migration assays. These increments of phagocytosis and chemotaxis in cells treated with SecinH3 and cytohesin-1 siRNA were reversed by a blocking mAb to the integrin-αM subunit. We provide evidence for increased polymerized cortical actin in cells treated with SecinH3 and that altered signaling through cytohesin-1 increased cell surface expression of FPRL-1 and impairs the late calcium mobilization response elicited by fMLF. The data provide evidence that stimulation with fMLF initiates a signaling cascade that restrains Mac-1 activation in PMNs. Such crosstalk between FPRL-1 and Mac-1 involves cytohesin-1. We suggest that cytohesin-1 may coordinate activation of the β(2) integrins to regulate PMN adhesion, phagocytosis, and chemotaxis.     

Chicken TBK1 interacts with STING and is involved in IFN-β signaling regulation

TANK-binding kinase 1 (TBK1) is an essential serine/threonine-protein kinase required for the Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I) -mediated induction of type I IFN. Through endogenous Co-IP and LC-MS/MS, we identified chicken TBK1 (chTBK1) as a chSTING-interactive protein. Through exogenous Co-IP assay in transfected cells, we confirmed the interaction between chSTING and chTBK1. To better understand the biological role of chTBK1 in the chSTING-mediated IFN pathway, we cloned the chTBK1 and investigated its biological functions. Quantitative RT-PCR showed that chTBK1 mRNA was widely expressed in different tissues. The overexpression of chTBK1 in DF-1 cells induced the expression of IFN-β and ISGs and inhibited AIV viral replication. We identified indispensable domains of chTBK1 on IFN-β production via the generation of various chTBK1 mutant forms. Together, we identified the chTBK1 as a chSTING interactive protein and concluded that chTBK1 is involved in chSTING-triggered IFN-β signaling in chicken cells.     

Jab1 expression is associated with TGF-β1 signaling in chronic rhinosinusitis and nasal polyposis

Jab1, which is a fifth component of COP9 signalosome, plays an essential role in cell growth and proliferation. Jab1 is also shown to regulate transforming growth factor-beta (TGF-β) signaling in carcinoma cells. The aim of the present study was to investigate the expression and the correlation of Jab1 and TGF-β1 in chronic rhinosinusitis and nasal polyposis. Here, we show the elevated expression of Jab1 and TGF-β1 in diseased mucosa without nasal polyps and a correlation between Jab1 and TGF-β1 expression. Forty-six samples (26 patients with nasal polyps, 10 patients with chronic rhinosinusitis and 10 control subjects) were included to this study. Immunohistochemistry and Western blotting were performed for the assessment of Jab1 and TGF-β1 localization and the expression of proteins. Double staining of both proteins showed that Jab1 and TGF-β1 were colocalized in the epithelium, inflammatory cells and the vascular endothelium of nasal mucosa. There was a significant increase in the expression of TGF-β1 and Jab1 in patients without nasal polyps and a significant decrease in patients with nasal polyps compared to controls. Moreover, correlation was detected between the expression of Jab1 and TGF-β1 in chronic rhinosinusitis and nasal polyposis. Our results demonstrate that chronic rhinosinusitis is characterized by elevated expression of Jab1 and TGF-β1 compared to nasal polyposis and Jab1 may play a vital role in the pathogenesis of both chronic rhinosinusitis and nasal polyposis.     

Prosaposin is a biomarker of mesenchymal glioblastoma and regulates mesenchymal transition through the TGF-β1/Smad signaling pathway

Mesenchymal glioblastoma (GBM) is the most aggressive subtype of GBM. Our previous study found that neurotrophic factor prosaposin (PSAP) is highly expressed and secreted in glioma and can promote the growth of glioma. The role of PSAP in mesenchymal GBM is still unclear. In this study, bioinformatic analysis, western blotting and RT-qPCR were used to detect the expression of PSAP in different GBM subtypes. Human glioma cell lines and patient-derived glioma stem cells were studied in vitro and in vivo, revealing that mesenchymal GBM expressed and secreted the highest level of PSAP among four subtypes of GBM, and PSAP could promote GBM invasion and epithelial–mesenchymal transition (EMT)-like processes in vivo and in vitro. Bioinformatic analysis and western blotting showed that PSAP mainly played a regulatory role in GBM invasion and EMT-like processes via the TGF-β1/Smad signaling pathway. In conclusion, the overexpression and secretion of PSAP may be an important factor causing the high invasiveness of mesenchymal GBM. PSAP is therefore a potential target for the treatment of mesenchymal GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Sodium/calcium exchanger is upregulated by sulfide signaling,forms complex with the β1 and β3 but not β2 adrenergic receptors,and induces apoptosis

Hydrogen sulfide (H₂S) as a novel gasotransmitter regulates variety of processes, including calcium transport systems. Sodium calcium exchanger (NCX) is one of the key players in a regulation calcium homeostasis. Thus, the aims of our work were to determine effect of sulfide signaling on the NCX type 1 (NCX1) expression and function in HeLa cells, to investigate the relationship of β-adrenergic receptors with the NCX1 in the presence and/or absence of H₂S, and to determine physiological importance of this potential communication. As a H₂S donor, we used morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate—GYY4137. We observed increased levels of the NCX1 mRNA, protein, and activity after 24 h of GYY4137 treatment. This increase was accompanied by elevated cAMP due to the GYY4137 treatment, which was completely abolished, when NCX1 was silenced. Increased cAMP levels would point to upregulation of β-adrenergic receptors. Indeed, GYY4137 increased expression of β1 and β3 (but not β2) adrenergic receptors. These receptors co-precipitated, co-localized with the NCX1, and induced apoptosis in the presence of H₂S. Our results suggest that sulfide signaling plays a role in regulation of the NCX1, β1 and β3 adrenergic receptors, their co-localization, and stimulation of apoptosis, which might be of a potential importance in cancer treatment.     

Increased energy expenditure and activated β3-AR-cAMP-PKA signaling pathway in the interscapular brown adipose tissue of 6-OHDA-induced Parkinson's disease model rats

To explore the possible mechanism of weight loss in Parkinson's disease (PD). Bilateral injections of 6-hydroxydopamine (6-OHDA) into substantia nigra (SN) were performed to induce the PD model rats. The rotarod test, food intake, body weight, and interscapular brown adipose tissue (IBAT) weight were recorded 6 weeks postoperation. HE staining was performed to observe the morphology of multilocular adipose cells in IBAT. Immunohistochemistry and western blot were used to determine the protein levels of tyrosine hydroxylase (TH) in the SN, and the levels of uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), phosphorylated-hormone sensitive lipase (p-HSL), HSL, TH, β3-adrenergic receptor (β3-AR), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) in IBAT. After treatment with 6-OHDA for 6 weeks, 6-OHDA rats exhibited decreased TH expression in SN accompanied with shortened staying time on the rotating rod. This motor impairment paralleled with no significant alteration in body mass, IBAT weight, and food intake until the end of the experimental protocol. However, the decreasing diameter of the single fat vesicle in IBAT was observed in the 6-OHDA group. Meanwhile, compared with the control group, the protein expression of UCP1, PGC-1α, p-HSL, TH, β3-AR, cAMP, and PKA in IBAT were increased significantly in the 6-OHDA group, whereas no obvious change in the expression of HSL. The present study suggested an increased energy expenditure and activation of the β3-AR-cAMP-PKA signaling pathway in the IBAT after the destruction of the dopamine system in the SN of the rat.     

Treadmill training regulates β-catenin signaling through phosphorylation of GSK-3β in lumbar vertebrae of ovariectomized rats

Postmenopausal osteoporosis is associated with high level of adipogenesis within the bone marrow at the expense of osteoblast population. The mechanical effect on β-catenin through phosphorylation of glycogen synthase kinase-3β (GSK-3β) is critical for inhibition of adipogenesis in mesenchymal stem cells in vitro. In present study, we hypothesized that treadmill training could regulate the β-catenin signaling through phosphorylation of GSK-3β in the lumbar vertebrae of ovariectomized (OVX) rats. 3-month-old female Sprague-Dawley rats were divided randomly into the following four groups: (a) Sham, (b) OVX, (c) OVX exercised (EX), and (d) OVX estrogen replacement (E(2)). At the end of the experiment, the serum levels of estradiol (E(2)) and luteinizing hormone (LH), the ultimate lumbar vertebra strength, as well as the protein expression for peroxisome proliferators-activated receptor γ (PPARγ), β-catenin, P-GSK-3β, and osterix (Osx) in lumbar vertebrae were analyzed. Moreover, the protein expression for β-catenin and P-GSK-3β were also examined in the uterus. The EX group had lower protein level of PPARγ, higher ultimate lumbar vertebral strength, and higher protein levels of β-catenin, and P-GSK-3β in lumbar vertebral bodies compared with sedentary OVX group. The effects of EX treatment on the protein levels of β-catenin and P-GSK-3β in bones were not reproducible in the uterus. Moreover, exercise treatment produced no estrogenic effect as evidenced by serum level of LH. In conclusion, this study suggested that treadmill training could activate the GSK-3β/β-catenin signaling and inhibit the production of PPARγ in lumbar vertebrae of OVX rats, which may contribute to the prevention of bone loss in OVX rats.     

NLRX1 negatively regulates TLR-induced NF-κB signaling by targeting TRAF6 and IKK

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Highlights? NLRX1 negatively regulates Toll-like receptor-mediated NF-κB activation ? NLRX1 disassociates from TRAF6 and then binds to the IKK complex after stimulation ? NLRX1 knockdown enhances NF-κB activation and cytokine production after stimulation ? NLRX1 knockdown in mice enhances susceptibility to septic shock and plasma IL-6     

Reduced expression of peroxisome-proliferator activated receptor gamma coactivator-1α enhances α-synuclein oligomerization and down regulates AKT/GSK3β signaling pathway in human neuronal cells that inducibly express α-synuclein

Intracellular accumulation of filamentous α-synuclein (α-Syn) aggregates to form Lewy bodies is a pathologic hallmark of Parkinson's disease. To determine whether mitochondrial impairment plays a role in the accumulation of α-Syn oligomer, we used 3D5 cell culture model of human neuronal type whereby conditional overexpression of wild-type α-Syn via the tetracycline-off (TetOff) induction mechanism results in formation of inclusions that exhibit many characteristics of Lewy bodies. In the present study, we compromised mitochondrial function in 3D5 cells by using shRNA to knockdown peroxisome-proliferator activated receptor gamma coactivator-1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism and found that PGC-1α suppression at both protein and mRNA levels results in α-Syn accumulation (i.e. monomeric and oligomeric species in the TetOff-induced cells and monomeric only in the non-induced). These changes were accompanied with reduced mitochondrial potential as well as decreased levels of AKT, GSK3β (total and Ser⁹-phosphorylated) and p53 that are important for cell survival. The extent to which these proteins decreased following PGC-1α knockdown, in contrast to what was demonstrable with the viability assay, is greater in the induced than the non-induced. Together these findings indicate that such knockdown increases the propensity to accumulate α-Syn oligomers, but the accumulation appears to have very little toxic impact to the neuronal cells.