Raelene J. Grumont

Genetic approaches in mice to understand Rel/NF-κB and IκB function : transgenics and knockouts

Rel/NF-κB transcription factors have been implicated in regulating a wide variety of genes important in cellular processes that include cell division, cell survival, differentiation and immunity. Here genetic models in which various Rel/NF-κB and IκB proteins have either been over-expressed or deleted in mice will be reviewed. Although expressed fairly ubiquitously, homozygous disruption of individual Rel/NF-κB genes generally affects the development of proper immune cell function. One exception is rela, which is essential for embryonic liver development. The disruption of genes encoding the individual subunits of the IκB kinase, namely IKKα and IKKβ, has demonstrated that IKKβ transmits the response to most common NF-κB inducing agents, whereas IKKα has an unexpected role in keratinocyte differentiation. Future studies will no doubt focus on the effect of multiple gene disruptions of members of this signaling pathway, on tissue-specific disruptions of these genes, and on the use of these mice as models for human diseases.

Unravelling the complexities of the NF-κB signalling pathway using mouse knockout and transgenic models

The nuclear factor-κB (NF-κB) signalling pathway serves a crucial role in regulating the transcriptional responses of physiological processes that include cell division, cell survival, differentiation, immunity and inflammation. Here we outline studies using mouse models in which the core components of the NF-κB pathway, namely the IκB kinase subunits (IKKα, IKKβ and NEMO), the IκB proteins (IκBα, IκBβ, IκBɛ and Bcl-3) and the five NF-κB transcription factors (NF-κB1, NF-κB2, c-Rel, RelA and RelB), have been genetically manipulated using transgenic and knockout technology.

The regulation and roles of Rel/NF-kappa B transcription factors during lymphocyte activation.

The activation of B and T cells by a wide range of stimuli can rapidly induce specific gene expression via a mechanism that promotes the nuclear translocation of different Rel/nuclear factor-kappa B (NF-kappa B) transcription factors which are normally resident in the cytoplasm. Recent findings highlight the crucial roles of specific Rel/NF-kappa B family members in the processes of cell division, apoptosis and differentiation that accompany lymphocyte activation.

c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-κB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel−/− mice, thymic T reg cell numbers are markedly reduced as a result of a T cell–intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-β conversion of peripheral CD4+CD25− T cells into CD4+Foxp3+ cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel−/− mice, the residual peripheral c-rel−/− T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

B Cell Growth Is Controlled by Phosphatidylinosotol 3-Kinase-Dependent Induction of Rel/NF-κB Regulated c-myc Transcription

Rel/NF-kappaB transcription factors regulate the division and survival of B lymphocytes. Here we show that B cells lacking NF-kappaB1 and c-Rel fail to increase in size upon mitogenic stimulation due to a reduction in induced c-myc expression. Mitogen-induced B cell growth, although not markedly impaired by FRAP/mTOR or MEK inhibitors, required phosphatidylinositol 3-kinase (PI3K) activity. Inhibition of PI3K-dependent growth coincided with a block in the nuclear import of NF-kappaB1/c-Rel dimers and a failure to upregulate c-myc. In addition, PI3K was shown to be necessary for a transcription-independent increase in c-Myc protein levels that accompanies mitogenic activation. Collectively, these findings establish a role for Rel/NF-kappaB signaling in the mitogen-induced growth of mammalian cells, which in B lymphocytes requires a PI3K/c-myc-dependent pathway.

The Rel subunit of NF-kappaB-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations.

The role of Rel in the monocyte/macrophage lineage was examined in mice with an inactivated c‐rel gene. Although the frequency of monocytic cells was normal in Rel−/− mice, we show that Rel serves distinct roles in regulating gene expression and immune effector function in different mature macrophage populations. Stimulated Rel−/− resident peritoneal macrophages produced higher than normal levels of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF) and interleukin‐6 (IL‐6), but tumour necrosis factor‐alpha (TNF‐alpha) production was not induced. Diminished cytotoxic activity exhibited by resident Rel−/− macrophages was consistent with reduced nitric oxide production resulting from impaired up‐regulation of inducible nitric oxide synthase expression. While a similar altered pattern of IL‐6 and TNF‐alpha expression was observed in stimulated Rel−/− peritoneal effusion macrophages, cytotoxic activity, nitric oxide, GM‐CSF and G‐CSF production by these cells was normal. The alternate regulation of certain genes in the two macrophage populations coincided with different patterns of nuclear Rel/NF‐kappaB complexes expressed in normal resident and elicited cells. Collectively, these results establish that Rel is a positive or negative regulator of transcription in macrophages and that Rel has distinct roles in different macrophage populations.

The combined absence of NF-κB1 and c-Rel reveals that overlapping roles for these transcription factors in the B cell lineage are restricted to the activation and function of mature cells

Transcription factors NF-κB1 and c-Rel, individually dispensable during embryogenesis, serve similar, yet distinct, roles in the function of mature hemopoietic cells. Redundancy among Rel/NF-κB family members prompted an examination of the combined roles of c-Rel and NF-κB1 by using mice that lack both proteins. Embryonic development and the maturation of hemopoietic progenitors were unaffected in nfkb1−/−c-rel−/− mice. Peripheral T cell populations developed normally, but follicular, marginal zone, and CD5+ peritoneal B cell populations all were reduced. In culture, a failure of mitogen-stimulated nfkb1−/−c-rel−/− B cells to proliferate was caused by a cell cycle defect in early G1 that prevented growth. In vivo, defects in humoral immunity and splenic architecture seen in nfkb1−/− and c-rel−/− mice were exacerbated in the double mutant mice. These findings demonstrate that in the B lineage overlapping roles for NF-κB1 and c-Rel appear to be restricted to regulating the activation and function of mature cells.

NF-κB control of T cell development.

The NF-κB signal transduction pathway is best known as a major regulator of innate and adaptive immune responses, yet there is a growing appreciation of its importance in immune cell development, particularly of T lineage cells. In this Review, we discuss how the temporal regulation of NF-κB controls the stepwise differentiation and antigen-dependent selection of conventional and specialized subsets of T cells in response to T cell receptor and costimulatory, cytokine and growth factor signals.

New insights into the roles of ReL/NF-kappa B transcription factors in immune function, hemopoiesis and human disease.

In mammals, Rel/NF-kappa B proteins are a small family of transcription factors which serve as pivotal regulators of immune, inflammatory and acute phase responses. Pathways leading to the activation of Rel/NF-kappa B have recently been dissected in some detail and shown to converge on a unique high molecular weight cytoplasmic complex that includes several kinases and regulatory molecules. Moreover, gene targeting experiments have identified novel roles for Rel/NF-kappa B proteins in the development and maturation of hemopoietic precursors as well as in the function of mature cells in the immune system. These include regulating the cell cycle, controlling cell survival and providing a link between the innate and adaptive immune systems. Since the dysregulation of Rel/NF-kappa B function is associated with various pathologies including inflammatory and neoplastic disease, new insights into the role of Rel/NF-kappa B in human disease may provide a basis for therapeutic strategies in the treatment of chronic inflammatory diseases and certain malignancies.

The transcription factors c-rel and RelA control epidermal development and homeostasis in embryonic and adult skin via distinct mechanisms.

ABSTRACT Determining the roles of Rel/NF-κB transcription factors in mouse skin development with loss-of-function mutants has been limited by redundancy among these proteins and by embryonic lethality associated with the absence of RelA. Using mice lacking RelA and c-rel, which survive throughout embryogenesis on a tumor necrosis factor alpha (TNF-α)-deficient background (rela −/− c-rel −/− tnfα−/−), we show that c-rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G1 progression, and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed that the mutant epidermis develops a TNF-α-dependent hyperproliferative condition. Collectively, our findings indicate that RelA and c-rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a postnatal environment through the suppression of innate immune-mediated inflammation.