Litopenaeus vannamei Notch interacts with COP9 signalosome complex subunit 1 (CNS1) to negatively regulate the NF-κB pathway

Highlights

• We identified twenty-one potential interacting partners of LvNotch in hemocytes.

• LvNotch was shown to directly bind to LvCNS1.

• LvNotch negatively regulates the NF-κB pathway via interacting with LvCSN1.

Abstract

Notch signaling pathway is a highly evolutionary conserved signaling pathway, which modulates many biological processes such as cell differentiation, tissue development and immune response. Our previous study revealed that Litopenaeus vannamei Notch (LvNotch) was involved in immune response by regulating reactive oxygen species (ROS) production in hemocytes. However, the immune regulatory networks mediated by LvNotch remain unclear in shrimp. In this study, 21 proteins that potentially interact with LvNotch were identified by GST pull-down and liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Among these proteins, COP9 signalosome complex subunit 1 (CSN1) was chosen for further studies due to its putative role in immune response. The interaction between LvNotch and LvCSN1 was confirmed by Far-Western blot and GST pull-down analyses. In vivo knockdown of LvNotch resulted in an increase in LvCSN1 expression in hemocytes, which suggest that the COP9 signalosome complex might be negatively regulated by LvNotch. In addition, in vivo silencing of LvNotch upregulated the expression of LvDorsal, LvTNFSF and LvCrustin2 (NF-κB pathway related-genes), while their expression decreased after LvCSN1 depletion. Collectively, the current results indicate that LvNotch negatively regulates the NF-κB pathway by modulating LvCSN1 in shrimp.

Significance

Although the Notch signaling pathway has been implicated in the regulation of immune response in vertebrates and invertebrates, the functions and immune-related interacting networks of Notch in shrimp immune response remain unknown. In this study, twenty-one proteins including COP9 signalosome complex subunit 1 (CSN1) were identified as potential interacting partners of LvNotch. Further analysis revealed that LvNotch negatively regulates the NF-κB pathway by binding to CSN1 and modulating its expression. These findings for the first time suggest that the Notch signaling pathway has cross-talk with the NF-κB pathway in shrimp as part of the immune response.

Introduction

The Notch gene was first identified and cloned in Drosophila, with the name Notch given because mutation of this gene caused wing tips indentation or notched wings in Drosophila [1]. The Notch signaling pathway, which mainly consists of Notch receptors, Notch ligands (such as Delta, Jagged, Serrate, Lag-2, etc.) and transcription factors (such as CBF-1/RBP-J kappa, Suppressor of Hairless (Su (H) or Lag-1 in different species, generally designated as CSL), is a highly evolutionary conserved cellular signal transduction pathway present in most vertebrates and invertebrates [2]. The pathways is activated when a Notch ligand binds to a Notch receptor of adjacent cells, which allows the release and transport of the intracellular domain of Notch (NICD) into the nucleus, where NICD binds to the transcription factor CSL and therefore regulates the expression of downstream target genes such as hairy enhancer of split (Hes), and hairy and enhancer of split with YRPW motif (Hey) [3].

For a long time, the Notch signaling pathway was thought to be mainly involved in regulating cell differentiation and tissue development [4], as well as involved in embryonic development [5], cardiomyocyte differentiation [6], Th17 cell differentiation [7] and self-renewal of stem cells [8]. However, in recent years, several studies have shown that this pathway cross-talks with many other signaling pathways in cells and therefore participates in the regulation of immune response [9]. For instance, in mouse macrophages, activation of the Notch signaling pathway could suppress Toll-like receptor (TLR)-triggered inflammatory cytokines (such as IL-6 and TNF-α) by inhibiting the nuclear factor κB (NF-κB) activity [10]. It has also been shown that activation of NF-κB in human Ntera-2 cells could up-regulate the expression of Jagged1 to promote Notch1 signaling pathway activation in adjacent cells, while the activated Notch^IC (an intracellular, constitutively active form of human Notch-1) interferes with the transcriptional activity of NF-κB by interacting with NF-κB subunit p50 to block the binding of the p50/p65 heterodimer in the nucleus, thereby down-regulating the expression of target genes [11]. In addition, in murine macrophages, after LPS stimulation, Notch signaling pathway could be activated by JNK signaling pathway, which could enhance the production of inflammatory cytokines IL-1β and IL-6 [12].

Notch pathway-related genes have been characterized and studied in most vertebrates and Drosophila, but not so in crustaceans. As part of our quest to fully delineate the signaling pathways in Litopenaeus vannamei, especially those that are involved in immunity, we recently cloned and submitted the sequence of LvNotch (KX245010.1), LvCSL (KX369026.1), LvPSE2 (KX369028.1), LvAPH-1 (KX369027.1), LvPSEN1 (KX369029.1), LvCtBP (KX236180.1) and LvTACE (KX245011.1) to the National Center for Biotechnology Information (NCBI). Based on this, LvNotch and LvCSL were shown to participate in shrimp immune defense through regulating reactive oxygen species (ROS) production and immune-related genes expression in hemocytes [13]. However, the LvNotch-mediated immune regulatory networks are still unclear in shrimp. In this study, the interacting proteins of LvNotch were determined using GST pull-down and liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. A total of 21 potential proteins was identified to interact with LvNotch. Of these, the immune-related protein COP9 signalosome complex subunit 1 (LvCSN1) was chosen for further studies. Our present data suggest that LvNotch may play negative role in the NF-κB pathway by interacting with LvCSN1 and modulating its expression in L. vannamei.