Interferon regulatory factor 4 contributes to transformation of v-Rel-expressing fibroblasts.

v-rel, which is derived from the c-rel proto-oncogene, is the acutely transforming oncogenic member of the Rel/NF-κB family (32, 42). The Rel/NF-κB family of transcription factors regulate gene expression from promoters or enhancers containing κB binding sites (GGGRNNYYCC, where R is a purine, Y is a pyrimidine, and N is any nucleotide) (7, 85). Rel/NF-κB proteins coordinate the expression of genes involved in natural and acquired immunity (56). In addition, the Rel/NF-κB proteins participate in the regulation of other processes such as apoptosis and embryonic development (8, 15, 29, 34, 36, 51). The function of Rel/NF-κB family members has been highly conserved during vertebrate evolution, and essentially the same five transcription factors—RelA, RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52)—are present in birds and mammals (30). Altered regulation of Rel/NF-κB activity has been linked to several pathological processes, including oncogenesis (25, 90). While rearrangements and amplifications of Rel/NF-κB genes have been found in human tumors, c-Rel is the only family member for which acute oncogenesis has been demonstrated (43, 59, 78). Mutations are necessary to activate c-Rel's oncogenic potential. The most highly oncogenic c-Rel mutant, v-Rel, transforms two different cell types, fibroblasts and cells of hematopoietic origin (26, 62). v-Rel-transformed fibroblasts acquire a distinct morphology, are capable of limited growth in soft agar, have a prolonged life span, and induce solid tumors (26, 59, 72, 74). Transformed hematopoietic cells become immortalized, and most of them form aggressive lymphomas in vivo. The ability of v-Rel to transform a particular cell type depends on its ability to induce or repress a specific set of genes. Approximately two dozen genes which exhibit elevated expression in v-Rel-transformed cells have been identified (32, 42). These genes encode cytokines, cell surface receptors, chaperones, genes involved in oxidative metabolism, apoptosis, and various transcription factors. Interferon regulatory factors (IRFs) are transcription factors which bind to promoter elements that contain an AANNGAAA consensus sequence and either stimulate or repress transcription of these target genes (27). IRFs, originally identified as regulators of interferons (IFNs) and IFN-stimulated genes, are involved in the regulation of natural immunity against viruses, acquired immunity, apoptosis, and embryogenesis, functions also attributed to the Rel/NF-κB family (40, 66, 83, 88). Nine IRF family members have been described in mammals: IRF-1 through 7, IRF-8, also called IFN consensus sequence-binding protein (ICSBP), and IRF-9, originally described as IFN-stimulated gene factor 3-γ (ISGF3-γ). All IRF proteins possess a highly conserved DNA-binding domain with five regularly spaced tryptophans at their N termini. The C termini contain sequences involved in transcriptional activation and repression (66, 83). The IRF members except IRF-1 and IRF-2 have a conserved IRF association domain, which mediates interactions with both other IRF proteins and transcription factors of other families (83). IRFs transactivate multiple genes that encode proteins with diverse effects on cell proliferation and differentiation, including genes of the IFN family. IFNs are multifunctional cytokines that play an important role in the induction of antiviral responses, cell growth, differentiation, and immunomodulation (40, 103). Type I IFNs (mammalian IFN-α, -β, -τ, and -ω as well as avian IFN1 and IFN2) are produced by a variety of cell types, while type II IFNs (IFN-γ) are secreted primarily by T cells and natural killer cells (101, 102). IFN type I cytokines are the major IFNs that respond to virus infection. Viral infection activates IRF, Rel/NF-κB, and bZIP transcription factors, leading to IFN synthesis (66). Secreted IFNs exert their effect by binding to cell surface receptors and activating the JAK/STAT signal transduction pathway, leading to DNA binding by a complex composed of STAT1, STAT2, and IRF-9 (103). This results in activation of the IFN-stimulated genes, which exert an antiviral and, in many cell types, antiproliferative effect (49). Several IRF proteins have been associated with the development of cancer, and viral IRF homologues are present in oncogenic herpesviruses (28). IRF-1 and IRF-8 may function as antioncogenes, while IRF-2 and IRF-4 have some characteristics of oncogenes (39, 41, 47, 97). IRF-4 is activated by Tax in adult T-cell leukemia and is overexpressed in multiple myelomas as a result of a translocation of the IRF-4 gene into the immunoglobulin M (IgM) heavy-chain locus (47, 108, 110). In contrast to other IRF family members, IRF-4 is not induced by viral infection or IFNs but by proliferative stimuli (13, 68, 108). Furthermore, while most IRFs are expressed in a variety of cell types, IRF-4 is expressed predominantly in B cells and in activated T cells (66). Mice deficient in IRF-4 have defects principally in the development and function of B and T lymphocytes (71). However, IRF-4 is also detected in macrophages, lens cells, and melanocytes, suggesting that it may also be involved in the regulation of cells other than lymphocytes (35, 63, 64, 67, 94). IRF-4 inhibits the expression of some IFN-induced genes, presumably by interfering with the action of other IRFs (12, 68, 108). In this work, we demonstrate that IRF-4 overexpression transforms primary chicken embryonic fibroblasts (CEFs). IRF-4 also synergistically cooperates with v-Rel in transformation of CEFs, while antisense IRF-4 expression inhibits v-Rel-mediated transformation. v-Rel transformation of CEFs is associated with the induction of expression of IFN1, the IFN1 receptor, STAT1, JAK1, and the IFN target gene 2′,5′-oligo(A) synthetase (OAS). IRF-4 expression, which is induced in CEFs following infection by v-Rel- and c-Rel-expressing viruses, alters the antiviral gene expression program and likely contributes to transformation of fibroblasts by decreasing the negative antiproliferative effect of the v-Rel-induced antiviral program.