Modulation of proinflammatory bacteria- and lipid–coupled intracellular signaling pathways in a transwell triple co-culture model by commensal Bifidobacterium animalis R101-8

2020 ◽  
Vol 19 ◽  
Author(s):  
Darab Ghadimi ◽  
Annegret Nielsen ◽  
Mohamed Farghaly Yoness Hassan ◽  
Regina Fölster-Holst ◽  
Michael Ebsen ◽  
...  
Keyword(s):  
Protein Expression ◽  
Palmitic Acid ◽  
Signaling Pathways ◽  
Cell Line ◽  
Intracellular Signaling ◽  
Expression Levels ◽  
Bifidobacterium Animalis ◽  
Intracellular Signaling Pathways ◽  
Culture Model ◽  
Tnf Α

Background and aims: Following a fat-rich diet, alterations in gut microbiota contribute to enhanced gut permeability, metabolic endotoxemia, and low grade inflammation–associated metabolic disorders. To better understand whether commensal bifidobacteria influence the expression of key metaflammation-related biomarkers (chemerin, MCP-1, PEDF) and modulate the pro-inflammatory bacteria- and lipid–coupled intracellular signaling pathways, we aimed at i) investigating the influence of the establishment of microbial signaling molecules- based cell-cell contacts on the involved intercellular communication between enterocytes, immune cells, and adipocytes, and ii) assessing their inflammatory mediators’ expression profiles within an inflamed adipose tissue model. Material and Methods: Bifidobacterium animalis R101-8 and Escherichia coli TG1, respectively, were added to the apical side of a triple co-culture model consisting of intestinal epithelial HT-29/B6 cell line, human monocyte-derived macrophage cells, and adipose-derived stem cell line in the absence or presence of LPS or palmitic acid. mRNA expression levels of key lipid metabolism genes HILPDA, MCP- 1/CCL2, RARRES2, SCD, SFRP2 and of TLR4 were determined using TaqMan qRT-PCR. Protein expression levels of cytokines (IL-1β, IL-6, and TNF-α), key metaflammation-related biomarkers including adipokines (chemerin and PEDF), chemokine (MCP-1) as well as cellular triglycerides were assessed by cell-based ELISA, while those of p-ERK, p-JNK, p-p38, NF-κB, p-IκBα, pc-Fos, pc-Jun, and TLR4 were assessed by Western blotting. Results: B. animalis R101-8 inhibited LPS- and palmitic acid-induced protein expression of inflammatory cytokines IL-1β, IL-6, TNF-α concomitant with decreases in chemerin, MCP-1, PEDF, and cellular triglycerides, and blocked NF-kB and AP-1 activation pathway through inhibition of p-IκBα, pc-Jun, and pc-Fos phosphorylation. B. animalis R101-8 downregulated mRNA and protein levels of HILPDA, MCP-1/CCL2, RARRES2, SCD and SFRP2 and TLR4 following exposure to LPS and palmitic acid. Conclusion: B. animalis R101-8 improves biomarkers of metaflammation through at least two molecular/signaling mechanisms that are triggered by pro-inflammatory bacteria/lipids. First, B. animalis R101-8 modulates the coupled intracellular signaling pathways via metabolizing saturated fatty acids and reducing available bioactive palmitic acid. Second, it inhibits NF-kB’s and AP-1’s transcriptional activities, resulting in reduction of pro-inflammatory markers. Thus, the molecular basis may be formed by which commensal bifidobacteria improve intrinsic cellular tolerance against excess pro-inflammatory lipids, and participate in homeostatic regulation of metabolic processes in vivo.

Analysis of Differentially Activated Signaling Pathways in Myeloproliferative Disease Using Kinomics Chip Profiling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3723-3723
Author(s):  
Daniel B Lipka ◽  
Marie-Christine Blum ◽  
Florian H Heidel ◽  
Thomas Kindler ◽  
Thomas Fischer
Keyword(s):  
Signaling Pathways ◽  
Cell Line ◽  
Intracellular Signaling ◽  
Jak2v617f Mutation ◽  
Myeloproliferative Disease ◽  
Differential Activation ◽  
Cell Lysate ◽  
Intracellular Signaling Pathways ◽  
Chip Analysis

Abstract In a multitude of cases, oncogenic mutations are gain of function mutations that confer a constitutively activated gene product. Currently, evidence from a large body of experimental studies suggests that oncogenic transformation induced by activating kinase mutations is not sufficiently explained by constitutive kinase activation alone but is a result of aberrantly activated signaling pathways in affected cells. The JAK2V617F-mutation is a highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, Ba/F3-cells expressing both erythropoietin receptor (EpoR) and the JAK2V617F-mutation show constitutive activation of the JAK-STAT pathway and cytokine independent growth. Multiple in-vitro and in-vivo studies demonstrate that the JAK2V617F-mutation mediates many of the phenotypic characteristics of the MPDs. Nevertheless, until now it is largely unclear, which signaling pathways in particular are involved in the process of malignant transformation in JAK2V617F-positive cells. Therefore, we applied a kinomics chip approach to screen for activated intracellular signaling pathways. We used a commercially available peptide chip containing 960 synthetic kinase substrate peptides spotted in triplicates and covering peptide substrates for approximately 50% of the human kinome. Peptides have been selected for their biologic relevance in physiologic processes such as stress response, growth and cell differentiation. With this approach, a broad spectrum of intracellular signaling pathways and kinases can be investigated simultaneously in a single experiment. As a proof of principle, we performed kinomics chip analysis of Ba/F3-cells stably transfected with EpoR and either the JAK2V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT). In brief, cells were seeded and treated with erythropoietin. One chip per cell lysate was incubated with an activation buffer containing the cell lysate and radioactively labelled ATP for a defined time period and washed several times afterwards. The chip was then analyzed by means of autoradiography using a phospho-storage-screen and a phospho-imager. Chip analysis was performed using standard microarray software and Microsoft Excel software. Chip experiments were performed simultaneously for Ba/F3-EpoR-VF and Ba/F3-EpoR-WT and in duplicate. Analysis revealed differential activation of known pathways such as Ras/Raf/MEK/ERK, JAK/STAT, and PI3-Kinase with pronounced activation seen in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT cells. This was not a surprising result but strongly underlines the feasibility and validity of this approach and therefore served as an internal control. Differential regulation of a number of other signaling nodes that have not yet been described in the context of mutant JAK2 signaltransduction have been detected. To select for relevant hits among these potential targets, we first excluded all substrates from further analysis that are known to be involved in lymphocyte-specific pathways. For the remaining hits we performed a literature search to learn more about their known functions and their potential impact in JAK2V617F-positive MPD. Validation of selected signaling molecules by means of Western blotting analysis and functional investigations such as siRNA knock-down experiments are currently under way. In addition to the widely used lymphoid Ba/F3-model, we also established a novel cell culture model with simultaneous expression of EpoR and either mutant or wildtype JAK2 in a myeloid 32D-cell background. This model will be helpful to us to determine false-positive results due to cell-line specific changes. We conclude, that kinomic profiling using the above mentioned chip-technology is a valid method to comprehensively investigate differential activation of signaling pathways in cell lysates. In our cell line model, we were able to detect activation of well known signaling pathways in JAK2V617F-positive cells. Furthermore, we were able to identify candidate proteins that appear to be specifically involved in JAK2V617F-signaling.

p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression

2005 ◽  
Vol 289 (5) ◽  
pp. L883-L889 ◽  
Author(s):  
Weidong Wu ◽  
Robert A. Silbajoris ◽  
Young E. Whang ◽  
Lee M. Graves ◽  
Philip A. Bromberg ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Egf Receptor ◽  
Cyclooxygenase 2 ◽  
Dominant Negative ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Intracellular Signaling Pathways ◽  
Cox 2

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.

Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor

2011 ◽  
Vol 51 (4) ◽  
pp. 463-471 ◽  
Author(s):  
Sara Carbajo-Pescador ◽  
Andrés García-Palomo ◽  
Javier Martín-Renedo ◽  
Maiara Piva ◽  
Javier González-Gallego ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Line ◽  
Hepg2 Cell ◽  
Intracellular Signaling ◽  
Hepg2 Cell Line ◽  
Intracellular Signaling Pathways
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