Development and characterization of a novel, megakaryocyte NF‐κB reporter cell line for investigating inflammatory responses

Abstract

An easily detectable readout in megakaryocyte cell lines will enhance inflammatory research in these cells. Here, we report the development and characterization of a novel megakaryocyte NF‐κB‐reporter cell line (Meg‐01R). Multiple inflammatory molecules modulate NF‐κB activity in Meg‐01R cells. Meg‐01R cells respond to small molecule inhibitors such as IMD0354 and C87 that are known to inhibit NF‐κB activity upon stimulation with TNFα. Abstract BackgroundBecause of the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. ObjectivesThe primary objective of this study is to develop and functionally characterize a novel megakaryocyte nuclear factor κB (NF‐κB) reporter cell line to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. MethodsA Meg‐01‐NF‐κB‐GFP‐Luc (Meg‐01R) cell line was developed by inserting a reporter NF‐κB‐GFP‐Luc cassette into normal Meg‐01 cells to produce luciferase following activation of NF‐κB to enable easy detection of pro‐inflammatory and reparative signalling. Results and conclusionsMeg‐01 and Meg‐01R cells have comparable characteristics, including the expression of both GPIbα and integrin β3. Meg‐01R cells responded to various inflammatory molecules as measured by NF‐κB‐dependent bioluminescence. For example, inflammatory molecules such as tumor necrosis factor‐α and Pam3CSK4 increased NF‐κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg‐01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg‐01R cells were able to respond to lipopolysaccharide (LPS; non‐ultrapure), although it was not able to react to ultrapure LPS because of the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterization of a novel megakaryocyte NF‐κB reporter cell line (Meg‐01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF‐κB activity.

The Involvement of Canonical NFκB Pathway in Megakaryocyte Differentiation Induction by Nanocurcumin

“…NFκB signaling has been known as a central mediator of inflammation and is involved in the thrombotic processes, platelet production, and platelet activity 26 . NFκB activity induces megakaryocyte progenitor cells to convert systemic or local inflammatory conditions to a transcriptional response, which may have consequences on the phenotype of megakaryocytes and released platelets 27 , 28 . Available studies showed that activation of thrombopoietin receptor (MPL) by interaction with its ligand leads to immediate induction of IκB kinase (IKK) activity during megakaryocyte differentiation in physiological conditions.…”

Polychlorinated biphenyls exposure and type 2 diabetes: Molecular mechanism that causes insulin resistance and islet damage

“…52 NF-κB is the primary signaling pathway used by proinflammatory factors to activate cells and plays a vital part in the inflammatory response. 53 As a serine/ threonine kinase, MAPKs kinase could prevent the phosphorylation of INSR and IRS tyrosine, impair their interaction, and prevent the sugar signal from being transmitted. 54 In addition, a rise in liver ROS levels may damage biomembranes' integrity and result in mitochondrial malfunction, which would cause liver lipid buildup and subsequently selective hepatic IR.…”

“…Nuclear factor kappa B (NF‐κB) and mitogen‐activated protein kinases (MAPKs), two pathways of signaling, may become active as ROS levels rise in peripheral organs 52 . NF‐κB is the primary signaling pathway used by proinflammatory factors to activate cells and plays a vital part in the inflammatory response 53 . As a serine/threonine kinase, MAPKs kinase could prevent the phosphorylation of INSR and IRS tyrosine, impair their interaction, and prevent the sugar signal from being transmitted 54 .…”