Transcriptional regulation of the cyclin D1 promoter by NF-κB. (A) Schematic map of the cyclin D1 promoter. Sequences of NF-κB binding sites are shown and are designated D1-κB1 and D1-κB2. (B) NF-κB activates the cyclin D1 promoter. COS-7 cells were cotransfected with 200 ng of pD1luc and 100 ng of either SP1, c-Jun, p50/p65 expression constructs, or pUC18 to give a total of 400 ng. Luciferase activity was measured and standardized by cotransfection of β-galactosidase expression vectors. Results represent the means and standard deviations from three individual experiments. RLU, relative light units. (C) Specific binding of NF-κB to the cyclin D1 promoter. Protein extracts from COS-7 cells, transfected with p50/p65 expression constructs, were incubated either with a specific NF-κB oligonucleotide probe (H2K) or with oligonucleotide probes containing NF-κB binding sites of the cyclin D1 promoter (D1-κB1 and D1-κB2). The identity of NF-κB-containing complexes was determined by adding anti-p50 or anti-p65 antibodies to the reaction mixture, as indicated. In lanes 4, 8, and 12, a 50-fold excess of unlabeled H2K oligonucleotide was added to the reaction mixture. ns, nonspecific.

IκBΔN expression reduces serum-induced NF-κB activity. (A) Expression of IκBΔN in primary MEF. MEF were transfected with an IκBΔN expression construct or an empty vector, and stable clones were selected with G418 (Gibco BRL). Control and IκBΔN-transfected MEF cells were lysed with extraction buffer and analyzed by Western blotting with anti-IκBα antibody. The positions of wild-type (WT) and mutant IκBα are indicated. Lanes 1 and 2, control clones (C1 and C2); lanes 3, 4, and 5, IκBΔN-expressing clones (I3, I4, and I5, respectively). (B) Serum induction of NF-κB DNA binding activity. MEF cells were synchronized in G₀ by serum starvation for 3 days, followed by stimulation with DMEM plus 10% FCS. Serum-deprived and -stimulated C1 and I3 cells were extracted at the indicated time points and analyzed by EMSA. The NF-κB–DNA complex containing p50/p65 as determined by antibody supershifting and inhibition (data not shown) is indicated.

IκBΔN expression results in a delay of pRB phosphorylation (A) NF-κB inactivation affects cyclin D1-associated kinase activity. pRB phosphorylation by cyclin D1-associated kinase activity from synchronized C1 (control) and I4 (IκBΔN) cells was analyzed as described in Materials and Methods. Similar results were obtained with C2 and I3 cells (data not shown). (B) NF-κB inactivation causes a delay in phosphorylation of endogenous pRB. Western blots of protein extracts prepared after serum induction of either control (C1 and C2) or IκBΔN-expressing (I3 and I4) cells are shown. Hypophosphorylated (pRB) and hyperphosphorylated (ppRB) RB proteins were detected with the monoclonal antibody G3-245 (Pharmingen). (C) Expression of various cell cycle-regulatory proteins in either control (C1) or IκBΔN-expressing (I3) cells. Western blots of protein extracts prepared at the indicated times after serum induction are shown. Cyclin E, cyclin D3, CDK2, p27, p21, p16, and p15 were detected as described in Materials and Methods. Similar results were obtained with C2 and I4 cells (data not shown). (D) Histone H1 kinase activity of anti-CDK2 immunoprecipitates (IP) from synchronized C1 and I3 cells. Similar results were obtained with C2 and I4 cells (data not shown).

Retardation of G₁-to-S-phase transition in T47DΔMTcycD1 cells, caused by NF-κB inactivation, was rescued by ectopic cyclin D1 expression. (A) Expression of IκBΔN in T47DΔMTcycD1 cells. Cells were transfected with an IκBΔN expression construct or an empty vector, and stable clones were selected with hygromycin (Sigma). Control and IκBΔN-transfected cells were lysed with extraction buffer and analyzed by Western blotting with anti-IκBα antibody. The positions of wild-type (WT) and mutant IκBα are indicated. Lanes 1 and 2, control clones (TC1 and TC2); lanes 3 and 4, IκBΔN-expressing clones (TI3 and TI4). (B) NF-κB-dependent cyclin D1 expression in T47DΔMTcycD1 cells. Western blots of protein extracts prepared from presynchronized TC1 and TI4 cells at the indicated time points after lovastatin removal and mevalonate addition are shown. Cyclin D1 was detected with the monoclonal antibody DCS-6. (C) FACS analysis of progression into S phase of TC1 and TI4 cells at 0, 18, or 22 h after lovastatin removal and mevalonate addition. Ectopic expression of cyclin D1 was induced by the addition of Zn²⁺ (+) or was not induced (−), as indicated. Progression through the cell cycle was monitored by detection of the DNA content. TC2 and TI3 cells (data not shown) gave similar results.

Functional characterization of NF-κB-responsive elements in the cyclin D1 promoter. (A) Reporter gene activation of wild-type (WT) and mutant constructs by p50/p65 was determined in COS-7 cells (left panel) as described for Fig. 1B. The graphs represent the means and standard deviations from three individual experiments. Serum induction of the cyclin D1 promoter was measured in NIH 3T3 cells as described previously (22) (right panel). Luciferase activity was measured as described for Fig. 1B. An EMSA was performed to show serum-induced NF-κB activation (inset). ns, nonspecific. (B) Induction of the cyclin D1 promoter by serum is reduced by IκBΔN expression. NIH 3T3 cells were transiently transfected with the cyclin D1 promoter-luciferase construct and increasing amounts of the IκBΔN expression vector, as indicated. Luciferase activity was measured after readdition of serum to the starved cells. (C) Activation of a heterologous promoter through the D1-κB2 sequence. A single copy of a cyclin D1 promoter fragment containing the D1-κB2 site was cloned in front of an HIV core promoter luciferase construct (pGL2HIV). The luciferase reporter gene assay was carried out as described for Fig. 1B. RLU, relative light units.

NF-κB regulates serum-induced cyclin D1 expression. (A) Western blots of MEF protein extracts, prepared at the indicated times after serum induction of C1 and I3 cells (upper panel). Cyclin D1 was detected with the monoclonal antibody DCS-6. The blot was stripped and reprobed with polyclonal anti-CDK4 antibody. Three individual experiments were performed, and relative intensities of the cyclin D1 bands were quantified with Quantity One software (PDI Inc.) (lower panel). Error bars indicate standard deviations. Similar results were obtained with C2 and I4 cells (data not shown). (B) NF-κB-dependent cyclin D1 expression in HeLa cells. Protein extracts of control and IκBΔN-transfected cells were prepared at the indicated times after serum induction. Cyclin D1 expression was analyzed by Western blotting (upper panel). A nonspecific band which cross-reacts with the antibody served as loading control (data not shown). Serum-induced cyclin D1 expression was quantified as described for panel A, lower panel.

NF-κB inactivation causes retardation of G₁-to-S-phase transition. FACS analysis of progression into S phase of C1 and I3 cells at 0, 16, 20, or 24 h after readdition of serum is shown. Progression through the cell cycle was monitored by detection of the DNA content by using propidium iodide staining. Similar results were obtained with C2 and I4 cells.