The present study provides evidence that the pleiotropic transcription factor NF-κB transmits growth signals directly to key regulators of the cell cycle. Our results suggest that NF-κB stimulates cyclin D1 transcription in G1 phase and thereby subsequently affects both pRB phosphorylation and G1-to-S-phase transition. The impaired cell cycle progression following NF-κB inhibition could be rescued by ectopic cyclin D1 expression, indicating that either cyclin D1 or another component of the RB pathway is controlled by NF-κB.
Interestingly, we observed that cyclin D1-associated kinase activity was even more affected upon NF-κB inactivation than would be expected from the modest modulation of cyclin D1 expression. However, narrow individual threshold levels are critical for key cell cycle regulators to exert their effects (46
). Hence, subtle changes in their expression level can interfere with cell cycle progression. Alternatively, our results may indicate that NF-κB regulates cyclin D1-associated kinase activity through another pathway. So far we cannot identify any further cell cycle regulators whose expression levels were affected by NF-κB inactivation (Fig. C). However, we do not rule out that NF-κB could control expression or activity of additional components. The observation that ectopic expression of cyclin D1 can overcome the delay of G1
-to-S-phase transition caused by NF-κB inactivation makes it unlikely that NF-κB controls an event late in G1
or S phase. In this respect, the observed delay in CDK2 activity (Fig. D) could be due to delayed pRB phosphorylation.
NF-κB activates transcription of the cyclin D1 promoter primarily through a proximal binding site. Weak residual NF-κB responsiveness after mutation of two identified major binding sites indicates the presence of further cryptic NF-κB elements. NF-κB binding sites as well as cellular NF-κB activation are required for serum induction of cyclin D1 transcription (Fig. ). Even though the appropriate expression of cyclin D1 depended on NF-κB, activation of NF-κB, e.g., by tumor necrosis factor alpha, in serum-deprived cells was not sufficient to induce cyclin D1 (not shown). Thus, further growth factor-activated regulators must contribute in parallel to ensure efficient cyclin D1 induction in G1
phase. Recent data indicate that NF-κB can functionally interact with other transcription factors, such as c-Fos/c-Jun, SP1, or E2F-1 (26
). Since the cyclin D1 promoter has been shown to be regulated by these transcription factors (2
), maximal activation might result from multiple functional interactions. The fact that the cyclin D1 promoter contains binding sites for all of these transcription factors indicates the possibility of multiple cooperative interactions. Such cooperativity could form the basis for the suggested function of cyclin D1 to integrate diverse mitogenic stimuli (31
In agreement with previous findings, we observed growth factor activation of NF-κB DNA binding activity in early G1
). The activation level varied between different cell types analyzed (Fig. A and B and data not shown). A relatively weak induction of NF-κB DNA binding activity in response to serum may indicate that growth factor signaling additionally leads to RelA phosphorylation, ultimately increasing the transactivation potential of NF-κB. Recently, it has been demonstrated that phosphorylation of the RelA subunit stimulates NF-κB transcriptional activity by promoting an interaction with CBP/p300 (55
). The observation that even cyclin-dependent kinases may regulate RelA through interaction with the coactivator CBP/p300 indicates a possible further link between NF-κB and cell cycle control (41
The data presented here raise the question of how NF-κB is linked to mitogenic signal transduction. Activation of NF-κB involves the phosphorylation of IκBα at its regulatory N terminus, subsequent conjugation with ubiquitin, and degradation of the inhibitor mediated by the proteasome (3
). Recently an IκBα-specific kinase activity was identified as part of a 700-kDa complex (12
), which can be activated by MEKK1 (29
). Interestingly, MEKK1 can interact with Ras (42
), a component of one major mitogenic signaling cascade, the Ras–Raf–mitogen-activated protein kinase (MAPK) pathway (20
). Another interesting link is provided by the observation that the ribosomal S6 kinase pp90rsk
, a downstream target of the Ras-Raf-MAPK pathway, phosphorylates IκBα (19
). Furthermore, it has been shown that the transforming mutant p21ras
can activate cyclin D1 expression (1
). Consistently, Ras inactivation causes a decline in cyclin D1 protein levels, accumulation of hypophosphorylated pRB, and G1
). Finally, Raf kinase activates NF-κB (16
), and NF-κB activity is required for Ras-mediated oncogenesis (17
). Taken together, these observations provide a connection between the mitogenic Ras-Raf-MAPK pathway, NF-κB activation, and cell cycle progression.
Recent data have indicated a role of the NF-κB and IκB gene products in cell proliferation, transformation, and tumor development (47
). Constitutive NF-κB activation is essential for survival and progression of Hodgkin’s lymphoma and breast cancer cells (7
). The direct link between NF-κB activity and the central pathway of G1
checkpoint control presented here provides a basis for understanding how NF-κB/Rel deregulation may result in tumorigenesis.