Characterization of the core elements of the NF-κB signaling pathway of the sea anemone Nematostella vectensis.

Under many circumstances, the NF-κB transcription factor signaling pathway is activated to enable animals to respond to environmental stresses, such as pathogens, chemicals, and UV light (14). Once activated, NF-κB transcription factors alter the expression of target genes to counteract these stresses. Examples of genes regulated by NF-κB include those encoding innate immune response factors (e.g., cytokines and antimicrobial peptides), antioxidizing enzymes (e.g., superoxide dismutase and nitric oxide synthase), and antiapoptosis molecules (e.g., Bcl-2, tumor necrosis factor receptor-associated factors [TRAFs], and inhibitor of apoptosis proteins [IAPs]) (14). NF-κB family members have additional roles in development. For example, the Dorsal protein controls the establishment of dorsal-ventral polarity in the Drosophila melanogaster embryo, and mammalian NF-κB proteins control various aspects of immune and liver cell growth and survival (23, 36). In Drosophila, there are three NF-κB proteins, while in humans and other mammals, there are five NF-κB proteins. All NF-κB family transcription factors have a conserved DNA-binding/dimerization domain called the Rel homology domain (RHD), and all NF-κB family proteins bind specific DNA sequences (κB sites) as homodimers or heterodimers. NF-κB proteins can be divided into two classes based on sequences C-terminal to the RHD. One class (the “NF-κB proteins”) includes Drosophila Relish and the mammalian p50/p105 and p52/p100 proteins. Proteins of this class contain C-terminal ankyrin repeat sequences, which restrict the protein to the cytosol and inhibit the DNA-binding activity of the RHD. The C-terminal regions of the Relish, p105, and p100 NF-κB proteins must be proteolytically cleaved for the proteins to become active. In contrast, members of the “Rel class” of proteins (Dorsal and Dif in Drosophila and c-Rel, RelA, and RelB in mammals) contain C-terminal transactivation domains that are not removed. The overall regulation of NF-κB activity in mammals has been extensively characterized (14). In its inactive state, NF-κB is bound to its inhibitor, IκB, in the cytoplasm. Upon activation of the pathway by an upstream signal, IκB is phosphorylated by an IκB kinase (IKK), which targets IκB for ubiquitination and degradation. Free NF-κB then translocates to the nucleus and binds to κB sites in the promoters of specific target genes to alter transcription. One well-characterized transcriptional target of NF-κB is the IκBα gene (15). Thus, NF-κB regulates its own activity by promoting the transcription of its primary inhibitor in a negative feedback loop. In mammals, there are several IκB proteins that have overlapping but nevertheless distinct functions (these proteins are IκBα, IκBβ, IκBɛ, and Bcl-3). All IκB proteins contain multiple copies of ankyrin repeat domain sequences, which mediate binding to the RHD sequences. The C-terminal regions of the mammalian NF-κB proteins also contain ankyrin repeat domains (Fig. ​(Fig.1A)1A) and consequently function as IκBs to inhibit the nuclear translocation of NF-κB. These C-terminal regions are phosphorylated and degraded in response to certain NF-κB-activating signals. FIG. 1. Cloning and expression of Nv-NF-κB, Nv-IκB, Nv-Bcl-3, and Nv-IKK proteins. (A) Structures of human (Homo sapiens [Hs]) and Nematostella vectensis (Nv) orthologs. Conserved domains are shaded in gray or black. Numbers indicate amino acid ... Nematostella vectensis is a small, burrowing sea anemone native to estuaries on the Atlantic coast of North America (29). Nematostella is a member of the phylum Cnidaria, which includes sea anemones, hydras, jellyfish, and corals. Like all anemones, it has two tissue layers, the endoderm and the ectoderm, and lacks the mesodermal layer found in triploblastic metazoans such as nematodes, insects, and vertebrates. The most recent common ancestor of Nematostella and triploblasts is thought to have lived approximately 600 million years ago (26, 27). Nematostella is emerging as the leading model system for cnidarians, in part because its complete genome sequence is known (28). Mining of genomic and expressed sequence tag (EST) databases revealed a single NF-κB gene and two IκB-like genes in Nematostella (33). The RHD of the Nematostella vectensis NF-κB protein (Nv-NF-κB) is ∼50% identical to the RHDs of human p50 and p52 but is only ∼35% identical to the Rel proteins (RelA, c-Rel, and RelB). Moreover, the RHD is immediately followed by a glycine-rich region, which is also present in mammalian p50 and p52. Surprisingly, the Nv-NF-κB protein lacks the C-terminal ankyrin repeat domain found in all other NF-κB proteins (Fig. ​(Fig.1A).1A). The ankyrin repeats of the Nv-IκB proteins, Nv-IκB and Nv-Bcl-3, are 42% and 48% identical to the ankyrin repeat domains of human p105 and Bcl-3, respectively. Additionally, Nematostella has two naturally occurring Nv-NF-κB alleles whose encoded proteins differ at 10 amino acid residues and have distinct DNA-binding and transactivating abilities (35). NF-κB homologs have been identified in a few other basal marine organisms. The coral Acropora millepora, an anthozoan cnidarian like Nematostella, has ESTs corresponding to NF-κB (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EZ002200.1","term_id":"222546010","term_text":"EZ002200.1"}}EZ002200.1) (21) and IκB (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"DY582971","term_id":"89137426","term_text":"DY582971"}}DY582971) (22). However, the genome of the cnidarian Hydra magnipapillata apparently lacks an NF-κB homolog (6). A single NF-κB homolog exists in the demisponge Amphimedon queenslandica of the phylum Porifera (13). This sponge NF-κB protein contains both the RHD and the C-terminal ankyrin repeat domain, suggesting that the independent NF-κB and IκB genes in Nematostella are the result of a gene splitting event. The biological function of the NF-κB signaling pathway in these simple marine organisms is not known; however, NF-κB has been proposed to play a role in mediating host-symbiont interactions in Cnidaria (37). In an effort to understand the evolutionary origins of NF-κB signaling, we have cloned cDNAs for Nv-NF-κB, Nv-IκB, Nv-Bcl-3, and Nv-IKK and expressed these proteins in a variety of cell-based assays. We demonstrated that the activities of these Nematostella proteins are conserved with respect to their vertebrate homologs. In addition, we showed that Nv-NF-κB is expressed primarily in a subset of ectodermal cells in both juvenile and adult Nematostella anemones.