RelB Modulation of IκBα Stability as a Mechanism of Transcription Suppression of Interleukin-1α (IL-1α), IL-1β, and Tumor Necrosis Factor Alpha in Fibroblasts

Members of the Rel/NF-κB family of transcription factors play a central role in the regulation of inflammatory and immune responses (for recent reviews, please see (1, 3, 4, 25, 46, 54). In vertebrates, NF-κB consists of homo- or heterodimers of Rel (c-Rel), p65 (RelA), RelB, p50 (NFKB1), and p52 (NFKB2), all of which contain a conserved N-terminal Rel homology domain that contains the DNA-binding and dimerization domains and the nuclear localization signal. In most unstimulated cells, a large portion of NF-κB is retained in the cytoplasm as inactive complexes by a family of inhibitory proteins called IκB that bind to the Rel homology domain and mask the nuclear localization signal. There are at least five distinct IκB proteins, IκBα, IκBβ, IκBɛ, IκBγ, and bcl-3; both the p105 precursor of p50 and the p100 precursor of p52 possess domains that function as IκBs as well. Upon cell stimulation by a wide variety of stimuli, signal-responsive IκB kinases (IKK) α and β are activated and phosphorylate two serine residues in the IκB proteins (14, 19, 36, 42, 43, 61, 63, 64). For IκBα and IκBβ, the inducible phosphorylation sites are serines 32 and 36 and serines 19 and 23, respectively (9, 10, 18, 51). The phosphorylated IκBs are subsequently ubiquitinated and targeted for degradation by the 26S proteosome, releasing the NF-κB dimers to translocate to the nucleus to activate the transcription of genes containing the so-called κB-binding site (2, 26, 48). Among the NF-κB-inducible genes are IκB members, and the newly synthesized IκBs quickly interact with and inactivate NF-κB, leading to an autoregulation of the NF-κB system (11, 45, 50). Both IκBα and IκBβ function not only in the cytoplasm but also in the nucleus to inhibit NF-κB activity; however, only IκBα is required for postinduction repression of NF-κB (5, 52). RelB shares many common features of the NF-κB family, and is a strong transcriptional activator (7, 8, 21, 44). Unlike other NF-κB members, however, RelB cannot form homodimers and only associates efficiently with p50 and p52 (22). The RelB heterodimers have a much lower affinity for IκBα than other NF-κB complexes do and are less susceptible to inhibition by IκBα (22, 32). As a result, it is predicted that RelB will be located in the nucleus and represent the constitutive NF-κB activity. Indeed, RelB heterodimers represent the major constitutive NF-κB activity in lymphoid tissues and are expressed at high levels in the nuclei of interdigitating dendritic cells, suggesting an important role for RelB in the constitutive expression of κB-regulated genes in lymphoid tissues (13, 32, 33, 40, 56). The implicated in vivo function of RelB in lymphoid tissues is supported by studies of relb−/− mice (12, 17, 34, 57–60, 62). Mice deficient in RelB have a dramatic reduction in constitutive κB-binding activity and specific defects in lymphoid tissues, including the absence of mature lymphoid dendritic cells, myeloid hyperplasia, and splenomegaly (12, 57). relb−/− mice also have multifocal defects in immune responses and fail to mount inflammatory reactions against a number of pathogens (17, 60). Surprisingly, relb−/− mice spontaneously develop a persistent noninfectious multiorgan inflammatory syndrome (12, 34, 57). This apparent discrepancy suggests additional defects in nonlymphoid tissues in relb−/− mice. In this regard, we showed in our previous report that the multifocal inflammation is due to non-bone-marrow-derived cells, that lipopolysaccharide (LPS)-stimulated relb−/− fibroblasts overexpress chemokines and induce leukocyte recruitment into tissues, and that RelB, while being a transcriptional activator for κB-regulated genes in macrophages, acts as a transcription suppressor in fibroblasts (62). Our findings may provide hints about the role that fibroblasts might play in the initial leukocyte infiltration and how RelB might be involved in the regulation of this process (49, 62). Additional pathological changes, however, must be involved in the development of multiorgan inflammation in relb−/− mice. Cytokines which promote inflammatory effector functions, including tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), IL-1β, and gamma interferon, are expressed at increased levels in the nonlymphoid organs of relb−/− mice but have either normal or reduced expression in lymphoid tissues; in particular, isolated relb−/− macrophages are impaired in the production of TNF-α (60, 62). Since macrophages are normally the major source of TNF-α production, one wonders what the probable cellular source of TNF-α and other cytokines in the nonlymphoid organs of the relb−/− mice might be, what molecular mechanisms are involved, and, especially, how RelB may function in this process. In this report, we show that RelB is a key transcriptional suppressor of cytokine expression in fibroblasts and that its absence leads to the dysregulation of IL-1α, IL-1β, and TNF-α expression in relb−/− fibroblasts. We further demonstrate that RelB exerts its transcriptional suppressor function through the stabilization of IκBα protein. These data suggest new physiological roles for the NF-κB/Rel factors in the regulation of inflammatory and immune responses and may provide insights to uncover novel intra- and interfamily interactions of the NF-κB/Rel and IκB regulatory molecules.