The proximal promoter of a novel interleukin-8-encoding gene in rainbow trout (Oncorhynchus mykiss) is strongly induced by CEBPA, but not NF-κB p65

Highlights

• We characterize a novel interleukin-8 gene variant (IL8-G) in rainbow trout.

• Infection increases IL8-G-encoding transcripts in secondary lymphoid tissues.

• CEBP and NF-κB binding sites in the IL-8-G promoter are functionally relevant.

• CEBPA is a strong inducer for this promoter and NF-κB p50 augments its efficacy.

• Trout and mammalian CEBPA and -B factors show the same intracellular distribution.

Abstract

Interleukin-8 (IL8) is an immediate-early chemokine that has been well characterized in several fish species. Ten IL8 gene variants have already been described in rainbow trout, but none of their promoters has structurally been defined or functionally characterized in teleost fish. To uncover key factors regulating IL8 expression, we intended to functionally characterize an IL8 promoter from rainbow trout. Incidentally, we isolated a novel IL8 gene variant (IL8-G). It is structurally highly similar to the other trout IL8 gene variants and its mRNA concentration increased significantly in secondary lymphoid tissues after infecting healthy fish with Aeromonas salmonicida. The proximal promoter sequence of the IL8-G-encoding gene features in close proximity two consensus elements for CEBP attachment. The proximal site overlaps with a NF-κB-binding site. Cotransfection of an IL8-G promoter-driven reporter gene together with vectors expressing various mammalian CEBP or NF-κB factors revealed in human HEK-293 cells that CEBPA and NF-κB p50, but not NF-κB p65 activate this promoter. The stimulatory effect of NF-κB p50 is likely conveyed by synergizing with CEBPA. Deletion or mutation of either the distal or the proximal CEBP-binding site, respectively, caused a significant decrease in IL8-G promoter activation. We confirmed the significance of the CEBPA factor for IL8-G expression by comparing the stimulatory capacity of the trout CEBPA and -B factors, thereby reducing the evolutionary distance in the inter-species expression assays. Similar promoter induction potential and intracellular localization of the mammalian and teleostean CEBPA and -B factors suggests their functional conservation throughout evolution.

Introduction

The activation of Toll-like receptors (TLRs) through adequate ligands is a key event for mounting an efficient immune response characterized by swiftly triggering the synthesis of a variety of cytokines (Szatmary, 2012), including IL8. The TLR signaling cascade is conserved among vertebrates (Rebl et al., 2010, Palti, 2011). Their pathogen-dependent activation eventually leads to the activation of, among others, members of the nuclear factor kappa B (NF-κB) family (Yan et al., 2012). These include p65/RELA, RELB, REL, p50/NFKB1, and p52/NFKB2, which all share highly conserved DNA-binding and dimerization domains mediating homo- or heterodimerization (Napetschnig and Wu, 2013). The most abundant and ubiquitous NF-κB dimer is p50:p65. Besides, NF-κB proteins are known to synergize with CCAAT/enhancer-binding proteins (CEBPs), which are also equipped with both, a DNA-binding region and a dimerization domain (Lekstrom-Himes and Xanthopoulos, 1998). CEBP factors control diverse activities and responses such as inflammation, cellular proliferation, differentiation, and metabolism (Ramji and Foka, 2002). In activated myeloid cells, NF-κB and CEBP factors cooperatively activate the strong expression of proinflammatory mediators, such as chemokines (Xia et al., 1997, Papin et al., 2003). These are implicated in the migration, maturation, and activation of immune cells. Interleukin-8 (IL8, also known as CXCL8) is among the earliest chemokines present shortly after injury and/or in the early stages of inflammation. Though IL8 has been discovered originally as a monocyte-derived neutrophil chemotactic factor (Yoshimura et al., 1987), it is nowadays known to be secreted by a wide variety of cells including epithelial and endothelial cells.

One of the key functions of IL8 is the recruitment of neutrophils from the blood stream to the site of infection (Kobayashi, 2008), not least by oppositely polarizing endothelial and epithelial cells (Krüger et al., 2000). The CXC subfamily of chemokines is divided into members either with or without a glutamic acid–leucine–arginine (ELR) tripeptide motif that is indispensable for angiogenic activities (Strieter et al., 1995) and neutrophil attraction (Thelen et al., 1988). ELR-negative CXC chemokines, on the other hand, promote T and B cell migration and inhibit angiogenesis (Strieter et al., 1995). Whereas mammalian IL8 factors are ELR-positive, most orthologs in fish lack this motif except for IL8 derived from haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua) (Corripio-Miyar et al., 2007, Seppola et al., 2008). The counterpart in trout has a homolog DLR (aspartic acid–leucine–arginine) sequence (Laing et al., 2002). This motif is suggested to retain functionality though to a lower extent (Hebert et al., 1991). However, the overall sequence conservation among piscine IL8 genes is low indicating a high degree of molecular diversification (Chen et al., 2005). All teleost species investigated so far encode in common a fish-specific IL8-encoding gene variant, CXCL8_L1 (Corripio-Miyar et al., 2007, Seppola et al., 2008, Laing et al., 2002, Lee et al., 2001). Moreover, at least some fishes possess a true IL8 ortholog of the mammalian gene, the CXCL8_L2 gene variant (Abdelkhalek et al., 2009, Van der Aa et al., 2010). Rainbow trout Oncorhynchus mykiss is reported to have at least four IL8 gene copies (Fujiki et al., 2003) and moreover some ‘IL8-like genes’ (Sangrador-Vegas et al., 2002).

The molecular regulation of IL8 promoters from fish is poorly investigated. Seppola et al. analyzed in silico the promoter of the IL8-encoding gene in Atlantic cod (EU007442; CXCL8_L1 type) detecting binding sites for NF-κB and CEBP factors within the proximal 200 nucleotides upstream of the transcriptional start (Seppola et al., 2008). On the other hand, for mammalian species it has indeed been well documented that a direct association of NF-κB and CEBPB factors on a proximal promoter fragment is crucial for the activation of the IL8 promoter (Matsusaka et al., 1993, Mukaida et al., 1990, Stein and Baldwin, 1993). CEBPB was originally identified as “nuclear factor for IL-6 expression” (Akira et al., 1990) and is nowadays also known as C/EBPβ regulating the expression of several more cytokines (Agrawal et al., 2001).

We present here a novel IL8 gene variant from rainbow trout (IL8-G) including the structure of its proximal promoter and provide evidence for its induction via CEBP and NF-κB factors in a HEK-293 reconstitution system of TLR signaling.