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LUBAC and ABIN-1 Modulate TRAIL-Based NF-?B Induction in Human Embryonic Kidney 293 Cells.

ABSTRACT: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to activate the canonical NF-?B pathway similar to TNF. The exact mechanism of the entire signaling cascade is still under investigation. The involvement of linear ubiquitylation as upregulating component has already been shown recently in some cell lines, but not in human embryonic kidney 293 (HEK293) cells. The downregulating function of the ABIN-1 (A20 binding and inhibitor of NF-?B) as linear ubiquitylation antagonist has been shown in combination with some NF-?B-inducing pathways, but not with TRAIL. We performed luciferase and western blot assays using HEK293 cells stimulated with either TRAIL (or TNF as a control) to analyze the involvement of linear ubiquitin chain assembly complex (LUBAC) components and the impact of ABIN-1 and ABIN-1-MAD (truncated form without A20 binding site) on NF-?B signaling. For overexpression experiments, we added plasmids of ABIN-1 and ABIN-1-MAD or LUBAC components HOIP, HOIL-1, or SHARPIN (single and combinations). For downregulation experiments five pairs of either SHARPIN, HOIL-1, or HOIP targeting miRNAs or one miRNA for ABIN-1 were designed and added. ABIN-1 and its truncated form ABIN-1-MAD reduced the NF-?B induction significantly indicating its involvement as antagonist (independent of deubiquitinase A20) of linear ubiquitylation in TRAIL-induced NF-?B signaling. In opposition, knockdown of ABIN-1 using a specific ABIN-1 miRNA led a clear increase of NF-?B signaling. Addition of single LUBAC components or combinations (except for SHARPIN with HOIL-1) resulted in clearly stronger NF-?B inductions. MiRNAs targeting LUBAC components significantly reduced NF-?B activation. Thus, in HEK293 cells linear ubiquitylation by LUBAC critically upregulates and ABIN-1 downregulates TRAIL-induced NF-?B signaling and may be interesting targets for future pathological therapies.

SUBMITTER: Dorn S

PROVIDER: S-EPMC5982153 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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LUBAC and ABIN-1 Modulate TRAIL-Based NF-κB Induction in Human Embryonic Kidney 293 Cells.

Dorn Sebastian S Schoergenhofer Christian C Krainer Michael M Müller Markus M Jilma Bernd B

BioResearch open access 20180501 1

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to activate the canonical NF-κB pathway similar to TNF. The exact mechanism of the entire signaling cascade is still under investigation. The involvement of linear ubiquitylation as upregulating component has already been shown recently in some cell lines, but not in human embryonic kidney 293 (HEK293) cells. The downregulating function of the ABIN-1 (A20 binding and inhibitor of NF-κB) as linear ubiquitylation antago ...[more]

PMID: 29862142

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Project description:BACKGROUND:Mycophenolic acid (MPA) is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293) after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting. RESULTS:The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1) showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I) were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%), chromatin structure/dynamics (17%) and energy production/conversion (17%). Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2. CONCLUSION:The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to understand molecular basis of its therapeutic response. The present study identifies the myosin regulatory light chain 2 and peroxiredoxin 1 along with 10 other proteins showing significant regulation by MPA. Further characterization of these proteins may help to understand the diverse cellular effects of MPA in addition to its immunosuppressive activity.

Dataset Information

LUBAC and ABIN-1 Modulate TRAIL-Based NF-?B Induction in Human Embryonic Kidney 293 Cells.

Publications

LUBAC and ABIN-1 Modulate TRAIL-Based NF-κB Induction in Human Embryonic Kidney 293 Cells.

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