NF-κB Function in Growth Control: Regulation of Cyclin D1 Expression and G0/G1-to-S-Phase Transition

The inducible transcription factor NF-κB participates in the regulation of numerous genes, many of which are involved in inflammation and the immune response. The NF-κB/Rel family consists of five members (p50, p52, p65 [RelA], c-Rel, and RelB) which can form various homo- or heterodimeric complexes. NF-κB is activated by the release from cytoplasmic IκB proteins and subsequently translocates into the nucleus (3, 5, 34). Activation is triggered by signal-induced phosphorylation of IκB, which targets the inhibitor for rapid degradation by the proteasome (49). Several observations have suggested a role of the NF-κB and IκB gene products in cell proliferation, transformation, and tumor development (47, 53). NF-κB controls the expression of a number of growth-promoting cytokines. In fact, a nuclear NF-κB-like DNA binding activity is induced during the G0-to-G1 transition after serum stimulation in mouse fibroblasts and in regenerating liver (6, 13–15, 18, 54). Interestingly, the NF-κB transactivation potential appears to be linked to signaling that controls cell cycle progression (9, 41). The first evidence for a connection between NF-κB and cell death came from studies with mice lacking the RelA unit of NF-κB as a result of targeted mutation of the relA gene. These mice die before birth and show massive degeneration of liver cells caused by apoptosis (10). The antiapoptotic function of NF-κB is supported by several studies demonstrating that NF-κB activity prevents the induction of apoptosis by tumor necrosis factor alpha, ionizing radiation, and anticancer agents (4) and that c-Rel prevents spontaneous apoptosis of B cells (52). Recent data indicate that constitutive NF-κB activation is essential for apoptosis resistance of different types of tumor cells (7, 48). Interestingly, constitutive NF-κB is required for cell cycle progression of Hodgkin’s lymphoma cells (7). However, a direct link between NF-κB activity and cell cycle progression remains to be established. The control of mammalian cell proliferation by extracellular signals takes place in mid- to late G1 phase of the cell cycle. D-type cyclins, in association with cyclin-dependent kinases CDK4 and CDK6, promote G1-to-S-phase transition by phosphorylating the retinoblastoma protein (pRB), thereby releasing the transcription factor E2F, which is required for the activation of S-phase-specific genes (8, 11, 21, 27, 39, 44, 46, 51). The D-type cyclins are induced as part of the delayed early response to mitogenic stimulation by growth factors, form active holoenzymes with CDK4 or CDK6 by mid-G1, and are able to bind directly to pRB via their N-terminal L-X-C-X-E motifs. Furthermore, they have a substrate preference for pRB over histone H1, and they phosphorylate pRB in vitro on residues which are physiologically phosphorylated in G1 in vivo (44, 46, 51). Consistent with a major role in positive regulation of G1 progression, the D-type cyclins are required for S-phase entry, and their overexpression accelerates G1 and reduces dependency on exogenous growth factors (8). These data suggest that cyclin D-associated kinases and their pRB substrate are the central players of the G1 checkpoint control. In fact, it could be demonstrated that mitogenic signal transduction pathways from three classes of receptors converge and strictly require the cyclin D-CDK activity to induce S phase (31). In addition, members of different signal transduction pathways regulate cyclin D expression positively (e.g., the transforming mutant p21ras and p42/p44MAPK) or negatively (e.g., p38) (1, 2, 28, 40). However the transcriptional mechanisms that link mitogenic signal transduction to cyclin D expression are poorly understood. Our data indicate that NF-κB transmits growth signals directly to key regulators of the cell cycle. NF-κB activates transcription of the cyclin D1 promoter primarily through a proximal binding site. The NF-κB binding sites that were identified are required for serum induction of cyclin D1 transcription. Inhibition of NF-κB activity in mouse embryo fibroblasts (MEF), T47D mammary carcinoma cells, or HeLa cells stably expressing a dominant negative IκBα mutant led to a delayed and reduced expression of cyclin D1 during G1 phase. Furthermore, inhibition of NF-κB resulted in retarded pRB phosphorylation and in impeded G1-to-S-phase transition. The impaired G1-to-S-phase transition caused by NF-κB inhibition could be overcome by ectopic expression of cyclin D1. These observations suggest that NF-κB directly contributes to stimulation of cell cycle progression by regulating the RB pathway.