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International Journal of Molecular... Oct 2021Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a...
Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a potential therapeutic target. In prostate and breast cancers, AR activation can be achieved also by a ligand-independent signaling through receptor tyrosine kinases such as epidermal growth factor receptor (EGFR). Considering its major role in glioblastoma, we explored whether EGFR is involved in AR signaling in this tumor. Analysis of mRNA expression in 28 glioblastoma samples with quantitative real-time reverse-transcription polymerase chain reaction revealed a positive and significant correlation between AR and EGFR mRNA expression levels (R = 0.47, = 0.0092), which was validated by The Cancer Genome Atlas dataset ( = 671) analysis (R = 0.3, = 0.00006). Using Western blotting and immunofluorescence staining, we showed that the transduced overexpression of EGFR or its variant EGFRvIII in the U87MG cells induced AR protein overexpression and nuclear translocation and Protein kinase B (AKT) S473 and AR S210/213 phosphorylation. The EGFR kinase inhibitor afatinib and the AKT inhibitor MK2206 reduced AR nuclear translocation. Afatinib diminished AKT phosphorylation at 30 min and 6 h in the EGFR- and EGFRvIII-overexpressing cells, respectively, and decreased AR phosphorylation in EGFR-overexpressing cells at 4 h. Afatinib or MK2206 combination therapy with the AR antagonist enzalutamide in the EGFR and EGFRvIII-overexpressing cells had synergistic efficacy. Our findings suggest that EGFR signaling is involved in AR activation in glioblastoma and buttresses the concept of combining an EGFR signaling inhibitor with AR antagonists as a potential glioblastoma treatment.
Topics: Afatinib; Androgen Receptor Antagonists; Benzamides; Brain Neoplasms; Cell Line, Tumor; ErbB Receptors; Gene Expression Regulation; Glioblastoma; Humans; Ligands; Nitriles; Phenylthiohydantoin; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction
PubMed: 34681618
DOI: 10.3390/ijms222010954 -
Plant Biology (Stuttgart, Germany) Mar 2022Soybean (Glycine max L.) is an important oil, food and economic crop in the world. High salinity severely affects the growth and yield of soybean. Overexpressing a...
Soybean (Glycine max L.) is an important oil, food and economic crop in the world. High salinity severely affects the growth and yield of soybean. Overexpressing a specific anti-retroviral transcription factor by biotechnology is an effective way to cultivate new stress-tolerant varieties of soybean. TGA transcription factor is a subfamily of bZIP and plays an important role in abiotic stress responses. A TGA subfamily gene GmTGA13 was cloned and the gene expression, subcellular localization and transcriptional activity were measured. Through the Ag. tumefaciens mediated flower dip method and the Ag. rhizogenes mediated transformation of soybean hairy roots, the transgenic Arabidopsis and the 'combination' soybean plants of overexpressing GmTGA13 were obtained. The two types of transgenic plants were treated with salt stress respectively, and the related physiological indexes were determined. Furthermore, the expression levels of five abiotic stress responsive genes were analyzed in GmTGA13 overexpression hairy roots. GmTGA13 gene was highly expressed in roots and significantly induced by saline stress in soybean. GmTGA13 encoded a nuclear localization protein and had transcriptional activation activity. Overexpression of GmTGA13 enhanced the saline stress tolerance of transgenic Arabidopsis and the 'combination' soybean plants. Furthermore, overexpression of GmTGA13 enhanced the expression of the stress responsive genes in transgenic soybean hairy roots. In conclusion, overexpression of GmTGA13 is beneficial to the absorption of K and Ca by the cell, thereby regulating the ion homeostasis in the cell balance. GmTGA13 enhanced salt resistance of plants by regulating the expression of many stress-responsive genes.
Topics: Gene Expression Regulation, Plant; Plant Proteins; Plants, Genetically Modified; Salt Stress; Glycine max; Stress, Physiological; Transcription Factors
PubMed: 34741387
DOI: 10.1111/plb.13360 -
PLoS Genetics Aug 2023Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also...
Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.
Topics: Animals; Cytoplasm; DNA-Binding Proteins; Gene Expression; Meiosis; Methyltransferases; RNA, Messenger; Saccharomyces cerevisiae Proteins; Transcription Factors; Gene Expression Regulation, Fungal
PubMed: 37639444
DOI: 10.1371/journal.pgen.1010898 -
Biochimica Et Biophysica Acta.... Aug 2020The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo...
The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.
Topics: Carboxy-Lyases; Galactokinase; Gene Expression Regulation, Fungal; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Transcriptome
PubMed: 32339526
DOI: 10.1016/j.bbamcr.2020.118727 -
The Journal of Pharmacy and Pharmacology Feb 2020The purpose of this study was to determine the effects of IGFBP-3 and GalNAc-T14 on the proliferation and cell cycle of glioblastoma cells and to explore the mechanisms...
OBJECTIVE
The purpose of this study was to determine the effects of IGFBP-3 and GalNAc-T14 on the proliferation and cell cycle of glioblastoma cells and to explore the mechanisms of action.
METHODS
U87MG and U251MG glioblastoma cells were treated with recombinant human IGFBP-3 (rhIGFBP-3). Furthermore, IGFBP-3-overexpressed cells and cells co-overexpressing IGFBP-3 and GalNAc-T14 were constructed by transfection. Cell viability, cell colony formation ability, cell cycle and protein expression were determined by MTT assay, colony formation assay, flow cytometry and Western blotting, respectively.
KEY FINDINGS
Both rhIGFBP-3 treatment and overexpression of IGFBP-3 induced the proliferation, colony formation, and G1/S phase transformation of U87MG and U251MG cells. In addition, the expression of cyclinE, CDK2 and p-ERK1/2 proteins was up-regulated in the cells. In cells co-overexpressing, IGFBP-3 and GalNAc-T14, cell proliferation, colony formation and G1/S phase transformation were inhibited, and the expression of CyclinE, CDK2 and p-ERK1/2 was significantly down-regulated, when compared with IGFBP-3-overexpressed cells.
CONCLUSIONS
IGFBP-3 can promote the proliferation, colony formation and G1/S phase transformation of U87MG and U251MG cells, which may be related to the activation of ERK signalling pathway and the up-regulation of cyclinE and CDK2 proteins. Furthermore, our study demonstrated that GalNAc-T14 can inhibit the functions of IGFBP-3.
Topics: Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 2; Down-Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Insulin-Like Growth Factor Binding Protein 3; N-Acetylgalactosaminyltransferases; Signal Transduction; Up-Regulation
PubMed: 31713889
DOI: 10.1111/jphp.13187 -
Molecular Medicine (Cambridge, Mass.) Dec 2022Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide but has no effective treatment. Amyloid beta (Aβ) protein, a primary risk factor for AD,...
BACKGROUND
Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide but has no effective treatment. Amyloid beta (Aβ) protein, a primary risk factor for AD, accumulates and aggregates in the brain of patients with AD. Paired immunoglobulin-like receptor B (PirB) has been identified as a receptor of Aβ and Aβ-PirB molecular interactions that cause synapse elimination and synaptic dysfunction. PirB deletion has been shown to suppress Aβ-induced synaptic dysfunction and behavioral deficits in AD model mice, implying that PirB mediates Aβ-induced AD pathology. Therefore, inhibiting the Aβ-PirB molecular interaction could be a successful approach for combating AD pathology. We previously showed that lateral olfactory tract usher substance (LOTUS) is an endogenous antagonist of type1 Nogo receptor and PirB and that LOTUS overexpression promotes neuronal regeneration following damage to the central nervous system, including spinal cord injury and ischemic stroke. Therefore, in this study, we investigated whether LOTUS inhibits Aβ-PirB interaction and Aβ-induced dendritic spine elimination.
METHODS
The inhibitory role of LOTUS against Aβ-PirB (or leukocyte immunoglobulin-like receptor subfamily B member 2: LilrB2) binding was assessed using a ligand-receptor binding assay in Cos7 cells overexpressing PirB and/or LOTUS. We assessed whether LOTUS inhibits Aβ-induced intracellular alterations and synaptotoxicity using immunoblots and spine imaging in a primary cultured hippocampal neuron.
RESULTS
We found that LOTUS inhibits the binding of Aβ to PirB overexpressed in Cos7 cells. In addition, we found that Aβ-induced dephosphorylation of cofilin and Aβ-induced decrease in post-synaptic density-95 expression were suppressed in cultured hippocampal neurons from LOTUS-overexpressing transgenic (LOTUS-tg) mice compared with that in wild-type mice. Moreover, primary cultured hippocampal neurons from LOTUS-tg mice improved the Aβ-induced decrease in dendritic spine density. Finally, we studied whether human LOTUS protein inhibits Aβ binding to LilrB2, a human homolog of PirB, and found that human LOTUS inhibited the binding of Aβ to LilrB2 in a similar manner.
CONCLUSIONS
This study implied that LOTUS improved Aβ-induced synapse elimination by suppressing Aβ-PirB interaction in rodents and inhibited Aβ-LilrB2 interaction in humans. Our findings revealed that LOTUS may be a promising therapeutic agent in counteracting Aβ-induced AD pathologies.
Topics: Animals; Humans; Mice; Alzheimer Disease; Amyloid beta-Peptides; Dendritic Spines; Immunoglobulins; Mice, Transgenic; Neurodegenerative Diseases; Receptors, Immunologic; Calcium-Binding Proteins
PubMed: 36510132
DOI: 10.1186/s10020-022-00581-7 -
Molecular and Cellular Biochemistry Aug 2022Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin...
Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine-choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.
Topics: Animals; Chemokines; Disease Models, Animal; Hepatic Stellate Cells; Hepatocytes; Humans; Intercellular Signaling Peptides and Proteins; Liver; Mice; Non-alcoholic Fatty Liver Disease; Protein Isoforms
PubMed: 35449483
DOI: 10.1007/s11010-022-04430-3 -
Molecular and Cellular Endocrinology Dec 2021Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or...
Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or progression. To ascertain how high GH levels could promote mammary tissue oncogenic transformation, morphological characteristics and the expression of receptors involved in mammary growth, development and cancer, and of mitogenic mediators were analyzed in the mammary gland of virgin adult transgenic mice that overexpress GH. Whole mounting and histologic analysis evidenced that transgenic mice exhibit increased epithelial ductal elongation and enlarged ducts along with deficient branching and reduced number of alveolar structures compared to wild type mice. The number of differentiated alveolar structures was diminished in transgenic mice while the amount of terminal end buds (TEBs) did not differ between both groups of mice. GH, insulin-like growth factor 1 (IGF1) and GH receptor mRNA levels were augmented in GH-overexpressing mice breast tissue, as well as IGF1 receptor protein content. However, GH receptor protein levels were decreased in transgenic mice. Fundamental receptors for breast growth and development like progesterone receptor and epidermal growth factor receptor were also increased in mammary tissue from transgenic animals. In turn, the levels of the proliferation marker Ki67, cFOS and Cyclin D1 were increased in GH-overexpressing mice, while cJUN expression was decreased and cMYC did not vary. In conclusion, prolonged exposure to high GH levels induces morphological and molecular alterations in the mammary gland that affects its normal development. While these effects would not be tumorigenic per se, they might predispose to oncogenic transformation.
Topics: Animals; Animals, Genetically Modified; Biomarkers; Carrier Proteins; Female; Growth Hormone; Insulin-Like Growth Factor I; Mammary Glands, Animal; Mice; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc
PubMed: 34597725
DOI: 10.1016/j.mce.2021.111465 -
Adipocyte Dec 2020Secreted frizzled-related protein (SFRP) 4 is an extracellular antagonist of Wnt signalling that regulates adipogenesis, and is highly in the visceral adipose tissue of...
Secreted frizzled-related protein (SFRP) 4 is an extracellular antagonist of Wnt signalling that regulates adipogenesis, and is highly in the visceral adipose tissue of obese individuals. However, it is still unclear how exactly SFRP4 regulates the secretion of adipokines in the adipose tissue , an event that is closely related to the pathogenesis of obesity and insulin resistance. In this study, we generated transgenic (Tg) mice overexpressing in the liver and investigated SFRP4 role in adipokine secretion in mice on a regular normal diet. In Tg mice, SFRP4 protein was overexpressed in the liver, as compared to wild-type littermates (non-Tg), and released into the blood. Moreover, the size of adipocytes was smaller in the visceral adipose tissue of Tg mice compared to controls. Additionally, overexpression affected the expression of genes related to adipocyte differentiation, causing the upregulation of adiponectin and glucose transporter 4, and the downregulation of CCAAT/enhancer-binding protein-β, in both visceral and subcutaneous adipose tissue. However, there was no difference in body weight or body composition between Tg and non-Tg mice. In summary, our data showed that overexpression altered adipocyte size and adipokine secretion, possibly affecting adipocyte differentiation, obesity, and glucose metabolism.
Topics: Adipocytes; Adipokines; Adipose Tissue, White; Animals; Cell Differentiation; Cell Size; Gene Expression; Gene Targeting; Humans; Intra-Abdominal Fat; Male; Mice; Mice, Transgenic; Proto-Oncogene Proteins
PubMed: 32657640
DOI: 10.1080/21623945.2020.1792614 -
Nucleic Acids Research Apr 2022Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A...
Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.
Topics: Centromere; Centromere Protein A; Chromatin; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Histones; Humans; Nucleocytoplasmic Transport Proteins; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 35234920
DOI: 10.1093/nar/gkac135