A feedback loop involving FREP and NF-κB regulates the immune response of sea cucumber Apostichopus japonicus
Introduction
Fibrinogen-related protein (FREP) acts as a pattern-recognition receptor (PRR) in vertebrates and invertebrates and contains a fibrinogen-like (FBG) domain in the carton terminal [1,2]. FREP reportedly exists in an extensive phylogenetic spectrum and plays crucial roles in defense processes in animals, such as antibacterial immunity, parasite defense, and agglutination. In Marsupenaeus japonicus (echinodermata), MjFREP2 can enhance the phagocytosis of hemocytes, which further promotes bacterial clearance [3]. Moreover, a FReD identified in bay scallop Argopecten irradians can agglutinate Gram-negative and -positive bacteria, chicken, and human erythrocytes [4]. The FREP 2 gene in snail Biomphalaria glabrata plays an important role in parasite defense and is also highly induced by LPS challenge [5]. Tenascin C, a member of the FREP family, is involved in inflammation in mice with LPS stimulation [6]. Unfortunately, the detailed activate mechanisms of FREP genes in vertebrates are poorly understood and unraveling the transcriptional regulation of these molecules may provide new ideas about immune defense and the control of disease outbreaks.
In the past decades, cloned FREP promoters reveal high homologies to binding with a mass of transcription factors, including NF-κB, CREB, C/EBP, and Oct-1 [[7], [8], [9]]. The NF-κB (nuclear factor-κB) family contains five sub-type proteins, namely, NF-κB1, NF-κB2, RelA, RelB, and C-Rel, that interact with each other to generate divergent transcriptionally activities among homo or heterodimeric complexes to regulate a wide array of genes involved in inflammatory responses, cell proliferation, and survival [10,11]. The Rel homology domain (RHD) in the NF-κB complex has approximately 300 amino acid residues, which are required for dimerization, DNA binding, inhibitor (IkB) binding, and nuclear localization [[12], [13], [14]]. Most studies revealed that NF-κB acts as a transcription factor and actively promotes the regions of various genes. For example, Vereshchagina et al. reported that NF-κB enhances the expression of the human complement receptor 2 gene [15]. Moreover, induced complement genes activate the NF-κB pathway and further induce inflammatory factors [16,17].
To date, increasing evidence has indicated that FREPs play key roles in innate immunity, especially in pathogen recognition and phagocytosis [[18], [19], [20]]. However, studies on the molecular transcription regulation of the FREP promoter in invertebrates, particularly in marine invertebrates, are few. The Apostichopus japonicus (e.g., Echinodermata and Holothuroidea) acts as an invertebrate species and turns into important aquaculture animals in China [21,22]. Regrettably, the rapid expansion and intensification of A. japonicus cultivation have led to infectious disease outbreaks and the devastating decline of production [23,24], such as skin ulceration syndrome and the highest infection and mortality rates in sea cucumber farming [25]. The amplification of the AjFREP promoter sequence and the preliminary analysis of its function would contribute to the understanding of the gene expression pattern and might provide more information to fight against bacterial pathogens. In this study, we cloned the promoter region of AjFREP and investigated its promoter activity. Furthermore, we analyzed the potential NF-κB binding sites for AjFREP transcription and the level of NF-κB/Rel transcripts in A. japonicus after AjFREP interference followed by LPS challenge. This work provides useful information about the activated mechanisms of AjFREP and might offer effective disease control strategies.
Section snippets
Experimental samples
Healthy A. japonicus (weight 115 ± 13 g) was obtained from Shandong Anyuan Aquaculture Co. Ltd. (Yantai, China) in March 2018. The sea cucumber was temporarily reared in aerated seawater (temperature of 16 ± 1 °C, and salinity of 28 ± 2) for three days before formal experiments.
Clone of the proximate promoter region of AjFREP gene
Genomic DNA was extracted from the coelomocytes of A. japonicus using the DNA kit for Marine Animals (Beijing, China). The promoter region of AjFREP was obtained using a Genome Walker Kit (Clontech, USA) following the
Analysis the promoter region of AjFREP
The FREP is a pivotal PRR in the innate immune system that has phagocytosis activities and bacterial clearance capacities. To uncover the transcriptional regulation mechanism of AjFREP, we amplified the 5′ flanking region of the gene using the genome walking method (Fig. 1). The translation start site (TSS) was predicted based on the upstream area of the signal peptide, and the Kozak sequences around the methionines were conserved [28]. Upstream of it was the 5′ UTR, and the TSS was predicted
Conclusion
AjFREP, as an important PRR, has phagocytosis activities and bacterial clearance capacities in the innate immune system. We cloned for the first time a fragment of AjFREP promoter, which possessed high promoter activity, and the transcription using NF-κB. The transcriptional AjFREP in turn affected the NF-κB pathway. These findings offered novel insights into the activation mechanism of AjFREP and the NF-κB pathway in the innate immunity.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (31802331), Natural Science Foundation of Ningbo (2018A610340), Fund from Ningbo University for Yina Shao (XYL18030), and the K.C. Wong Magna Fund in Ningbo University.
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