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Respiratory Research Jun 2023Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that...
BACKGROUND
Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I).
METHODS
We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E (PGE) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed.
RESULTS
DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE-mediated activation of PKA signaling by DGK inhibition.
CONCLUSIONS
Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.
Topics: Diacylglycerol Kinase; Cyclic AMP-Dependent Protein Kinases; Cells, Cultured; Cell Proliferation; Myocytes, Smooth Muscle
PubMed: 37301818
DOI: 10.1186/s12931-023-02465-8 -
Journal of Molecular Endocrinology Jun 2021Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds,...
Despite all modern advances in medicine, there are few reports of effective and safe drugs to treat obesity. Our objective was to screen anti-obesity natural compounds, and to verify whether they can reduce the body weight gain and investigate their molecular mechanisms. By using drug-screening methods, Phytohemagglutinin (PHA) was found to be the most anti-obesity candidate natural compound. Six-week-old C57BL/6J mice were fed with a high-fat diet (HFD) and intraperitoneally injected with 0.25 mg/kg PHA everyday for 8 weeks. The body weight, glucose homeostasis, oxygen consumption and physical activity were assessed. We also measured the heat intensity, body temperature and the gene expression of key regulators of energy expenditure. Prevention study results showed PHA treatment not only reduced the body weight gain but also maintained glucose homeostasis in HFD-fed mice. Further study indicated energy expenditure and uncoupling protein 1 (UCP-1) expression of brown adipose tissue (BAT) and white adipose tissue (WAT) in HFD-fed mice were significantly improved by PHA. In the therapeutic study, a similar effect was observed. PHA inhibited lipid droplet formation and upregulated mitochondrial-related gene expression during adipogenesis in vitro. UCP-1 KO mice displayed no differences in body weight, glucose homeostasis and core body temperature between PHA and control groups. Our results suggest that PHA prevent and treat obesity by increasing energy expenditure through upregulation of BAT thermogenesis.
Topics: Adipose Tissue, Brown; Adipose Tissue, White; Animals; Biological Products; Cell Differentiation; Diet, High-Fat; Energy Metabolism; Glucose; Homeostasis; Male; Mice, Inbred C57BL; Mice, Knockout; Obesity; Phytohemagglutinins; Thermogenesis; Uncoupling Protein 1; Weight Gain; Mice
PubMed: 33983894
DOI: 10.1530/JME-20-0349 -
Biochemistry Mar 2022Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on...
Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on the surface of viruses such as HIV-1. However, their therapeutic potential has been compromised by their mitogenicity that stimulates uncontrolled division of T-lymphocytes. Horcolin, a member of the jacalin family of lectins, tightly binds the HIV-1 envelope glycoprotein gp120 and neutralizes HIV-1 particles but is nonmitogenic. In this report, we combine X-ray crystallography and NMR spectroscopy to obtain atomic-resolution insights into the structure of horcolin and the molecular basis for its carbohydrate recognition. Each protomer of the horcolin dimer adopts a canonical β-prism I fold with three Greek key motifs and carries two carbohydrate-binding sites. The carbohydrate molecule binds in a negatively charged pocket and is stabilized by backbone and side chain hydrogen bonds to conserved residues in the ligand-binding loop. NMR titrations reveal a two-site binding mode and equilibrium dissociation constants for the two binding sites determined from two-dimensional (2D) lineshape modeling are 4-fold different. Single-binding-site variants of horcolin confirm the dichotomy in binding sites and suggest that there is allosteric communication between the two sites. An analysis of the horcolin structure shows a network of hydrogen bonds linking the two carbohydrate-binding sites directly and through a secondary binding site, and this coupling between the two sites is expected to assume importance in the interaction of horcolin with high-mannose glycans found on viral envelope glycoproteins.
Topics: Binding Sites; Carbohydrates; Crystallography, X-Ray; HIV-1; Lectins; Mannose
PubMed: 35225598
DOI: 10.1021/acs.biochem.1c00778 -
Toxins Aug 2022As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism,...
As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis, and immunosuppression. In this study, by constructing -deleted mutants in , it was found that the deletion of gene led to the decline of growth and development, conidia production, AFB1 toxin synthesis, and seed infection ability of . The deletion of gene also caused the disappearance of sclerotium, indicating that is required for sclerotium formation in . The sensitivity of -deficient strains responding to cell wall stress and osmotic pressure stress increased when compared to WT. The Western blot result showed that mitogen-activated serine/threonine-protein kinase Slt2 and mitogen-activated protein kinase Hog1 proteins were no longer phosphorylated in -deficient strains of , showing that Rac may be used as a molecular switch to control the Slt2-MAPK cascade pathway and regulate the osmotic Hog-MAPK cascade pathway in in response to external stress. Altogether, these results indicated that Rac was involved in regulating the growth and development, conidia formation and AFB1 synthesis, and response to cell wall stress and osmotic pressure stress in .
Topics: Aflatoxins; Aspergillus flavus; Fungal Proteins; Fungi; GTP Phosphohydrolases; Mitogen-Activated Protein Kinases; Mitogens; Serine; Threonine
PubMed: 36136519
DOI: 10.3390/toxins14090581 -
Cell Communication and Signaling : CCS Oct 2022Concanavalin A (ConA), the most studied plant lectin, has been known as a potent anti-neoplastic agent for a long time. Since initial reports on its capacity to kill... (Review)
Review
Concanavalin A (ConA), the most studied plant lectin, has been known as a potent anti-neoplastic agent for a long time. Since initial reports on its capacity to kill cancer cells, much attention has been devoted to unveiling the lectin's exact molecular mechanism. It has been revealed that ConA can bind to several receptors on cancerous and normal cells and modulate the related signaling cascades. The most studied host receptor for ConA is MT1-MMP, responsible for most of the lectin's modulations, ranging from activating immune cells to killing tumor cells. In this study, in addition to studying the effect of ConA on signaling and immune cell function, we will focus on the most up-to-date advancements that unraveled the molecular mechanisms by which ConA can induce autophagy and apoptosis in various cancer cell types, where it has been found that P73 and JAK/STAT3 are the leading players. Moreover, we further discuss the main signaling molecules causing liver injury as the most significant side effect of the lectin injection. Altogether, these findings may shed light on the complex signaling pathways controlling the diverse responses created via ConA treatment, thereby modulating these complex networks to create more potent lectin-based cancer therapy. Video Abstract.
Topics: Humans; Concanavalin A; Lectins; Matrix Metalloproteinase 14; Neoplasms; Plant Lectins
PubMed: 36289525
DOI: 10.1186/s12964-022-00972-7 -
Biochimica Et Biophysica Acta.... Jul 2020Actin dynamics regulate cell behaviour in response to physiological signals. Here we demonstrate a novel role for nuclear actin in inhibiting cell proliferation and...
Actin dynamics regulate cell behaviour in response to physiological signals. Here we demonstrate a novel role for nuclear actin in inhibiting cell proliferation and migration. We demonstrate that physiological signals that elevate cAMP, which is anti-mitogenic in vascular smooth muscle cells, increases nuclear actin monomer levels. Expression of a nuclear-targeted polymerisation-defective actin mutant (NLS-Actin) inhibited proliferation and migration. Preventing nuclear actin monomer accumulation by enhancing its nuclear export or polymerisation reversed the anti-mitogenic and anti-migratory effects of cAMP. Transcriptomic analysis identified repression of proliferation and migration associated genes regulated by serum response factor (SRF) and TEA Domain (TEAD) transcription factors. Accordingly, NLS-Actin inhibited SRF and TEAD activity and target gene expression, and these effects were reversed by constitutively-active mutants of the TEAD and SRF co-factors YAP, TAZ and MKL1. In summary, intranuclear actin inhibits proliferation and migration by inhibiting YAP-TEAD and MKL-SRF activity. This mechanism explains the anti-mitogenic and anti-migratory properties of physiological signals that elevate cAMP. SUMMARY: McNeill et al show that increased levels of intranuclear actin monomer inhibit cell proliferation and migration by inhibiting MKL1-SRF and YAP/TAZ-TEAD-dependent gene expression. This mechanism mediates the anti-mitogenic and anti-migratory effects of physiological signals that elevate cyclic-AMP.
Topics: Actins; Adaptor Proteins, Signal Transducing; Cell Movement; Cell Nucleus; Cell Proliferation; Cyclic AMP; DNA-Binding Proteins; Gene Expression Regulation; Humans; Nuclear Proteins; Serum Response Factor; TEA Domain Transcription Factors; Transcription Factors; YAP-Signaling Proteins
PubMed: 32119877
DOI: 10.1016/j.bbamcr.2020.118691 -
BMC Psychiatry Mar 2023Based on its objective characteristics, laboratory markers have always been the research direction of clinical diagnosis and assessment of mental disorders including...
BACKGROUND
Based on its objective characteristics, laboratory markers have always been the research direction of clinical diagnosis and assessment of mental disorders including Alzheimer's disease.
METHODS
MTT Colorimetric Assay, ELISA, and quantitative PCR were used to investigate the responsiveness of peripheral blood mononuclear cells (PBMCs) to mitogen Lipopolysaccharides (LPS) and Phytohemagglutinin (PHA), PBMCs genomic methylation and hydroxymethylation levels, nuclear DNA and mitochondrial DNA damage, respiratory chain enzyme activities, and circulating cell-free mitochondrial DNA levels were detected in 90 patients with Alzheimer's disease.
RESULTS
In the Alzheimer's disease group, LPS stimulated PBMCs viability, TNF-α secretion, PHA stimulated IL-10 secretion, genomic DNA methylation levels, circulating cell-free mitochondrial DNA copies, citrate synthase activity were reduced compared to the control; while the LPS stimulated PBMCs IL-1α secretion, PHA stimulated IL-1α and IFN-γ secretion, plasma IL-6 and TNF-α, mitochondrial DNA damages were increased compared to the control.
CONCLUSIONS
The reactivity of peripheral blood mononuclear cells to mitogens, mitochondrial DNA integrity characteristics, and cell-free mitochondrial DNA copies may be used as candidate laboratory biomarkers to help clinical management of Alzheimer's disease.
Topics: Humans; Mitogens; Lipopolysaccharides; Leukocytes, Mononuclear; Tumor Necrosis Factor-alpha; Cytokines; DNA, Mitochondrial; Alzheimer Disease; Phytohemagglutinins
PubMed: 36890488
DOI: 10.1186/s12888-023-04634-x -
Anatomical Record (Hoboken, N.J. : 2007) Mar 2020Loss of inner ear hair cells leads to incurable balance and hearing disorders because these sensory cells do not effectively regenerate in humans. A potential starting...
Loss of inner ear hair cells leads to incurable balance and hearing disorders because these sensory cells do not effectively regenerate in humans. A potential starting point for therapy would be the stimulation of quiescent progenitor cells within the damaged inner ear. Inner ear progenitor/stem cells, which have been described in rodent inner ears, would be principal candidates for such an approach. Despite the identification of progenitor cell populations in the human fetal cochlea and in the adult human spiral ganglion, no proliferative cell populations with the capacity to generate hair cells have been reported in vestibular and cochlear tissues of adult humans. The present study aimed at filling this gap by isolating colony-forming progenitor cells from surgery- and autopsy-derived adult human temporal bones in order to generate inner ear cell types in vitro. Sphere-forming and mitogen-responding progenitor cells were isolated from vestibular and cochlear tissues. Clonal spheres grown from adult human utricle and cochlear duct were propagated for a limited number of generations. When differentiated in absence of mitogens, the utricle-derived spheres robustly gave rise to hair cell-like cells, as well as to cells expressing supporting cell-, neuron-, and glial markers, indicating that the adult human utricle harbors multipotent progenitor cells. Spheres derived from the adult human cochlear duct did not give rise to hair cell-like or neuronal cell types, which is an indication that human cochlear cells have limited proliferative potential but lack the ability to differentiate into major inner ear cell types. Anat Rec, 303:461-470, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Topics: Adult; Adult Stem Cells; Cell Differentiation; Cochlea; Ear, Inner; Humans; Spiral Ganglion
PubMed: 31489779
DOI: 10.1002/ar.24228 -
Frontiers in Immunology 2022Autoimmune hepatitis (AIH) is mediated by a cascade of T cell-mediated events directed at liver cells and persistent inflammation within the liver can eventually result...
BACKGROUND
Autoimmune hepatitis (AIH) is mediated by a cascade of T cell-mediated events directed at liver cells and persistent inflammation within the liver can eventually result in liver cirrhosis. Targeting glutamine metabolism has an impact on T cell activation and differentiation. However, the effect of glutamine metabolism blocking upon AIH remains unknown. We use glutaminase antagonist 6-diazo-5-oxo-L-norleucine (DON) for assays and its prodrug 2-(2-amino-4-methylpentanamido)-DON (JHU083) for assays to investigate the potential therapeutic effect and molecular mechanism of glutamine metabolism blocking in an AIH murine model.
METHODS
AIH mice were treated with JHU083 or vehicle before concanavalin A (ConA) administration, and disease severity was examined. Then activation and differentiation [including Th1/Th17 cells and cytotoxic T lymphocytes (CTL)] of T cells from Vehicle-WT, JHU083-AIH and Vehicle-AIH mice were tested. Furthermore, T cell activation and differentiation were measured using separated splenocytes stimulated with ConA with or without DON. The activation and differentiation of T cells were tested using flow cytometry, qRT-PCR and ELISA. Phosphorylation level of mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase (P70S6K) were examined by western blotting.
RESULTS
JHU083 and DON significantly suppressed the activation of T cells and inhibited the differentiation of Th1/Th17 cells and CTL and . Besides, we demonstrated that glutamine metabolism blocking inhibited T cells activation and differentiation through decreasing the mRNA expression of amino acid transporter solute carrier family 7 member 5 (SLC7A5) and mitigating the activation of mTOR signaling.
CONCLUSIONS
We proved that targeting glutamine metabolism represents a potential new treatment strategy for patients with AIH and other T cell-mediated disease. Mechanistically, we demonstrated that glutamine metabolism blocking inhibits T cells activation and suppresses the differentiation of Th1/Th17 cells and CTL.
Topics: Animals; Concanavalin A; Glutamine; Hepatitis, Autoimmune; Humans; Mammals; Mice; Mice, Inbred C57BL; TOR Serine-Threonine Kinases; Th17 Cells
PubMed: 35663990
DOI: 10.3389/fimmu.2022.880262 -
International Journal of Biological... 2022Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that...
Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that receptor interacting protein kinase 3 (RIP3) is correlated with serum transaminase levels in AIH patients. However, its role and underlying mechanism in AIH are poorly understood. Here, we detected the increased expression and activation of RIP3 in livers of patients and animal models with AIH. The inhibition of RIP3 kinase by GSK872 prevented concanavalin A (ConA)-induced immune-mediated hepatitis (IMH) by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11bGr1 myeloid-derived suppressor cells (MDSCs) with immunoregulatory properties in the liver, spleen, and peripheral blood. Moreover, the depletion of Gr-1 MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Altogether, our data demonstrate that RIP3 blockade prevents ConA-induced IMH through promoting MDSCs infiltration. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment.
Topics: Animals; Concanavalin A; Disease Models, Animal; Female; Hepatitis, Autoimmune; Humans; Liver; Mice; Mice, Inbred C57BL; Myeloid-Derived Suppressor Cells; Receptor-Interacting Protein Serine-Threonine Kinases
PubMed: 34975327
DOI: 10.7150/ijbs.65402