Quantitative proteomics and phosphoproteomics reveal TNF-alpha mediated downstream protein functions in hepatocytes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Uma Aryal
PROVIDER: MSV000087248 | MassIVE | Tue Apr 20 15:10:00 BST 2021
REPOSITORIES: MassIVE
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Project description:Glycoproteomics, TNF-alpha, membrane proteomics, insulin resistance, adipocyte, SILAC Insulin resistance (IR) is a complex process arising from both environmental and genetic perturbations and leads to a variety of diseases including type-2 diabetes (T2D). The expansion of adipose tissue during obesity results in secretion of proinflammatory cytokines which significantly impairs insulin sensitivity throughout the entire body. Inflammation modulates glycosylation in a variety of cell types however, an investigation of changes in glycosylation following inflammation-induced IR in adipocytes has not been performed. In the present study, we performed a quantitative N-glycomic analysis of TNF-alpha induced IR in adipocytes and identified the regulation of specific N-glycans including an increase in terminal di-galactose- and decrease in di-sialic acid-containing glycans with alpha-2,3 linkages. Quantitative analysis of the membrane-associated proteome in TNF-alpha treated adipocytes identified the regulation of specific glycosyltransferases and glycosidases which correlated with the regulated N-glycans. This included the up- and down-regulation of B4GalT5 and ST3Gal6, respectively at both the protein and mRNA level. To identify the protein and glycosylation sites modified with these regulated N-glycans, we performed a site-specific quantitative glycoproteomic analysis of enriched N-glycopeptides from TNF-alpha treated adipocytes with and without deglycosylation. The combined workflow provided a relative quantification of changes in protein abundance verses N-glycosylation occupancy verses site-specific N-glycans on a proteome-wide level. This revealed the modulation of a subset of N-glycan compositions on specific proteins including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane. Finally, knockdown of B4GalT5 with siRNA and analysis of released N-glycans resulted in the down-regulation of di-galactose containing glycans, confirming the involvement of this enzyme in the TNF-alpha regulated N-glycome.
2015-11-09 | PXD002435 | Pride
Project description:Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver . In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization. Results: The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately upregulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1 proposed a IL-1beta-like effect of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase-activity of the upregulated Zc3h12a have to be considered. Conclusions: Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signalling pathway. Gene expression profile suggests IL-17 a role in sustaining liver inflammatory processes most likely by RNA stabilization. Altogether, our results provide evidence that IL-17 alone and in concert with TNF-alpha may play a role in inflammatory liver diseases. Primary murine hepatocytes of three animals stimulated for 1 or 4h by TNF-alpha, IL-1beta, IL-17 or TNF-alpha followed by IL-17 were used for microarray analysis.
2010-03-07 | E-GEOD-19272 | biostudies-arrayexpress
Project description:Obesity is a growing epidemic worldwide and is a major risk factor for several chronic diseases, including diabetes, kidney disease, heart disease, and cancer. The global epidemic of obesity has been accompanied with a dramatic increase in the incidence and prevalence of type 2 diabetes mellitus (T2DM). Obesity often leads to T2D, via the increased production of proinflammatory cytokines such as tumor necrosis factor alpha. Our study combines different proteomic techniques to investigate the changes in the global proteome, secretome and phosphoproteome of adipocytes under chronic inflammation condition, as well as fundamental cross-talks between different cellular pathways regulated by chronic TNF alpha exposure. Our results show that many key regulator proteins of the canonical and non-canonical NF-kB pathways, such as Nfkb2, and its downstream effectors, including Csf-1 and Lgals3bp, directly involved in leukocyte migration and invasion, were significantly upregulated at the intra and extracellular levels, culminating in the progression of inflammation. Our data provides evidence of several key proteins that play a role in the development of insulin resistance.
2020-06-08 | MSV000085562 | MassIVE
Project description:Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver . In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization. Results: The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately upregulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1 proposed a IL-1beta-like effect of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase-activity of the upregulated Zc3h12a have to be considered. Conclusions: Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signalling pathway. Gene expression profile suggests IL-17 a role in sustaining liver inflammatory processes most likely by RNA stabilization. Altogether, our results provide evidence that IL-17 alone and in concert with TNF-alpha may play a role in inflammatory liver diseases.
2010-03-01 | GSE19272 | GEO
Project description:Insulin resistance is a sine qua non of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, aging, and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumor necrosis factor-a (TNF) or by the steroid dexamethasone (Dex) to construct detailed transcriptional profiles associated with cellular insulin resistance. Gene expression data from mouse adipocyte, with TNF or Dex treatments at different time points. Murine 3T3-L1 adipocytes were treated separately with dexamethasone (Dex; 20nM) or tumor necrosis factor-alpha. To comprehensively assess gene expression changes caused by Dex and TNF in a time-dependent manner, we profiled cells at early (2 hours), intermediate (24 hours), and late (6 days) points in the development of insulin resistance.
2014-12-23 | E-GEOD-62635 | biostudies-arrayexpress
Project description:Aberrant epithelial-mesenchymal transition (EMT) is involved in pathological processes including fibrotic disorders and cancer invasion and metastasis. Alterations of the cell-extracellular matrix (ECM) interaction also contribute to those pathological settings. However, the functional interplay between EMT and cell-ECM interaction is poorly understood. Here, we show that tumor necrosis factor (TNF)-alpha, a potent mediator of inflammation, induces EMT-associated fibrosis in retinal pigment epithelial cells, and that this is regulated by hyaluronan (HA)-CD44-Moesin interaction. TNF-alpha elicits both HA synthesis and Moesin phosphorylation through protein kinase C activation, promoting binding of CD44 to the newly synthesized HA. The HA-CD44-Moesin interaction leads to cell-cell dissociation through actin remodeling and increased cellular motility associated with mesenchymal phenotype. Furthermore, we established an in vivo model of TNF-alpha-induced fibrosis in the mouse eye, and the ocular fibrosis was completely suppressed in CD44-null mice. Therefore, HA production and its interaction with CD44 plays essential role in TNF-alpha-induced-EMT, and the interference of the complex formation can be a new strategy for the fibrotic disorders. ARPE19 cell lines were treated with TGF and TNF for 6 and 42 hour. Each experiment were repeated three times. But 1hour experiment was repeated two times. For this submission, total RNA was extracted from TGF- or TNF-treated ARPE-19 cells and differential gene expression between each time point (6 and 42 hours) was determined using genechip arrays (Affymetrix, Human Genome U133).
2010-03-23 | E-GEOD-15205 | biostudies-arrayexpress
Project description:Insulin resistance is a sine qua non of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, aging, and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumor necrosis factor-a (TNF) or by the steroid dexamethasone (Dex) to construct detailed epigenomic maps associated with cellular insulin resistance. Murine 3T3-L1 adipocytes were treated separately with dexamethasone (Dex; 20nM) or tumor necrosis factor-alpha. To comprehensively assess epigenomic changes caused by Dex and TNF in a time-dependent manner, we profiled cells at early (2 hours), intermediate (24 hours), and late (6 days) points in the development of insulin resistance.
2014-12-23 | E-GEOD-58491 | biostudies-arrayexpress
Project description:Aberrant epithelial-mesenchymal transition (EMT) is involved in pathological processes including fibrotic disorders and cancer invasion and metastasis. Alterations of the cell-extracellular matrix (ECM) interaction also contribute to those pathological settings. However, the functional interplay between EMT and cell-ECM interaction is poorly understood. Here, we show that tumor necrosis factor (TNF)-α, a potent mediator of inflammation, induces EMT-associated fibrosis in retinal pigment epithelial cells, and that this is regulated by hyaluronan (HA)-CD44-Moesin interaction. TNF-α elicits both HA synthesis and Moesin phosphorylation through protein kinase C activation, promoting binding of CD44 to the newly synthesized HA. The HA-CD44-Moesin interaction leads to cell-cell dissociation through actin remodeling and increased cellular motility associated with mesenchymal phenotype. Furthermore, we established an in vivo model of TNF-α-induced fibrosis in the mouse eye, and the ocular fibrosis was completely suppressed in CD44-null mice. Therefore, HA production and its interaction with CD44 plays essential role in TNF-α-induced-EMT, and the interference of the complex formation can be a new strategy for the fibrotic disorders.
2010-03-11 | GSE15205 | GEO
Project description:Mesenchymal stem cells (MSCs) can differentiate into endothelial cells; however, the mechanisms underlying this process in the tumor microenvironment (TME) remain elusive. This study shows that tumor necrosis factor alpha (TNF-α), a key cytokine present in the TME, promotes the endothelial differentiation of MSCs by inducing vascular endothelial growth factor receptor 2 (VEGFR2) gene expression. EGR1 is a member of the zinc-finger transcription factor family induced by TNF-α. Our findings indicate that EGR1 directly binds to the VEGFR2 promoter and transactivates VEGFR2 expression. We also demonstrate that EGR1 forms a complex with c-JUN activated by c-JUN N-terminal kinase (JNK) to promote VEGFR2 transcription and endothelial differentiation in MSCs in response to TNF-α stimulation. The shRNA-mediated silencing of EGR1 or c-JUN abrogates TNF-α-induced VEGFR2 transcription and the endothelial differentiation of MSCs. Collectively, these findings demonstrate that the JNK-EGR1 signaling axis plays a crucial role in the TNF-α-induced endothelial differentiation of MSCs in the TME, which could be a potential therapeutic target for solid tumors vasculatures.
2022-10-06 | GSE214834 | GEO
Project description:We used DNA microarray technology to investigate the cytoskeleton gene expression profile associated with the acquisition of cell resistance to TNF-alpha in breast carcinoma cell line (MCF7) versus sensitive cells (1001).
2009-10-31 | E-MEXP-1927 | biostudies-arrayexpress