NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity

Abstract

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

FIG. 1.

Suppression of constitutive NF-κB activity inhibits overexpression of c-fos and AP-1 activity. (A) The expression of IκBα and IκBαM in MDAPanc-28/Puro and MDAPanc-28/IκBαM cells was determined by Western blot analysis with the cytoplasmic protein extracts using β-actin as loading controls. The nuclear protein extracts from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells were subjected to EMSA with NF-κB, AP-1, and the Oct-1 probe as a control. Expression of c-fos in MDAPanc-28/Puro and MDAPanc-28/IκBαM cells was determined by Northern blot analysis with GAPDH as the loading control. (B) Luciferase reporter gene assays for NF-κB and AP-1 activities were performed with wild-type (wt) and mutant (mt) NF-κB and AP-1 luciferase reporter gene constructs transiently transfected into MDAPanc-28/Puro and MDAPanc-28/IκBαM cells. (C and D) Competition and supershift assays were performed with EMSA to determine the specificity of the constitutive RelA/p50 NF-κB and AP-1 DNA binding activities. Ten micrograms of nuclear extract from MDAPanc-28/Puro cells was incubated with a 50× excess of unlabeled wild-type NF-κB or AP-1 probe (C and D, lane 2), mutant NF-κB or AP-1 probe (C and D, lane 3), and anti-NF-κB and anti-Fos antibodies (α-), with and without control peptides, as indicated. FP, free probe.

FIG. 2.

Induction of AP-1 activity and fos expression by doxycycline-induced ROS requires NF-κB activation, and serum-induced AP-1 activity and fos expression are partially dependent on NF-κB activation. (A) EMSAs were performed to determine NF-κB and AP-1 activity by using the nuclear extracts isolated from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with increasing doses ofdoxycycline as indicated for 24 h. An Oct-1 probe was used as a control for quality and quantity of cell extracts. RNA was isolated from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with doxycycline for the indicated dosages and analyzed by Northern blotting with a human c-fos cDNA probe, and the same blot was rehybridized to a gapdh cDNA probe. (B) Inhibition of serum-induced activation of NF-κB and AP-1 was determined by using EMSAs with the nuclear extracts from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with 20% serum for the indicated times after 48 h of serum deprivation. An Oct-1 probe was used as a control for quality and quantity of cell extracts. RNA was isolated from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with 20% serum for the indicated time and analyzed by Northern blotting with a human c-fos cDNA probe, and the same blot was rehybridized to a gapdh cDNA probe. (C) IκBαM-mediated inhibition of the doxycycline-induced activation of NF-κB and AP-1 activities was determined with wild-type (wt) and mutant (mt) NF-κB and AP-1 luciferase reporter gene constructs transiently transfected into MDAPanc-28/Puro and MDAPanc-28/IκBαM cells.

FIG. 3.

Doxycycline- and serum-inducible NF-κB and AP-1 activities were inhibited in IKK1^−/− and IKK2^−/− MEF cells. Wild-type (wt), IKK1^−/−, and IKK2^−/− MEF cells were stimulated with doxycycline as indicated for 24 h (A), and wild-type, IKK1^−/−, and IKK2^−/− MEF cellswere stimulated with 20% serum for the indicated times after 48 h of serum starvation (B). Nuclear extracts were isolated and subjected to EMSA with NF-κB and AP-1 probes. (C and D) Competition and supershift assays were performed with EMSA to determine the specificity of the doxycycline-induced RelA/p50 NF-κB and AP-1 DNA binding activities. Ten micrograms of nuclear protein from doxycycline-stimulated wild-type MEF cells with a 50× excess of unlabeled wild-type NF-κB or AP-1 probe (C and D, lane 2) or mutant NF-κB or AP-1 probe (C and D, lane 3), along with anti-NF-κB and anti-Fos antibodies (α-), with and without control peptides, as indicated. FP, free probe. (E) Wild-type (WT) and IKK2^−/− MEF cells were stimulated with PMA (50 μg/ml), IL-1α (10 ng/ml), and TNF-α (10 ng/ml) for 30 min, and EMSAs with NF-κB, AP-1, and Oct-1 probes were performed.

FIG. 4.

Doxycycline- and serum-inducible expressions of the AP-1 family members were determined in wild-type (Wt), IKK1^−/−, and IKK2^−/− MEF cells. RNA was isolated from wild-type, IKK1^−/−, and IKK2^−/− MEF cells stimulated with either doxycycline or 20% serum for the indicated doses and times. RNase protection assays were carried out with the Riboquant multiprobe protection assay system to determine doxycycline (A)- and serum stimulation (B)-induced expression of AP-1 components in wild-type, IKK1^−/−, and IKK2^−/− MEF cells as indicated.

FIG. 5.

NF-κB activity regulates elk-1 expression. (A) EMSAs were performed with the nuclear extracts isolated from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells to determine the NF-κB binding activity to the NF-κB motif on the elk-1 promoter with an Oct-1 probe as a control. (B) elk-1 mRNA expression was determined by Northern blot analysis with RNA from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells with elk-1 cDNA and gapdh probe as a loading control. (C) Elk-1 DNA binding activity on SRE from the c-fos promoter was determined by EMSA with the nuclear extracts from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells (A) and an Oct-1 probe as a control. (D) EMSAs were performed with the nuclear extracts isolatedfrom wild-type (Wt), IKK1^−/−, and IKK2^−/− MEF cells to determine the Elk-1 DNA binding activity with the SRE probe. An Oct-1 probe was used as a control for loading. The expression of elk-1 was determined by Northern blot analysis with the RNA isolated from wild-type, IKK1^−/−, and IKK2^−/− MEF cells with mouse elk-1 cDNA and gapdh probes. (E and F) Elk-1 activity in MDAPanc-28/Puro (E) and wild-type MEF (F) cells was determined by competition and supershift assay with 10 μg of nuclear extract with a 50× excess of unlabeled wild-type and mutant Elk-1 probe (lanes 2 and 3) and anti-Elk-1 antibody as indicated. FP, free probe. (G) ChIP assays were performed with MDAPanc-28/Puro and MDAPanc-28/IκBαM cells with and without anti-p65 (NF-κB) antibody as indicated.

FIG. 6.

NF-κB activity is essential for doxycycline- and serum-induced elk-1 expression. (A) Doxycycline- and (B) serum-induced NF-κB-dependent induction of elk-1 expression was determined by Northern blot analysis. RNA was isolated from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with doxycycline at the indicated doses and 20% serum for the indicated times and hybridized with a human elk-1 cDNA probe and rehybridized to a gapdh cDNA probe. (A) Doxycycline (A)- and serum (B)-induced NF-κB-dependent activation of Elk-1 was determined by EMSAs with NF-κB and SRE probes and the nuclear extracts isolated from MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with doxycycline as indicated for 24 h or with 20% serum for the indicated times after 48 h of serum withdrawal. An Oct-1 probe was used as a control for quality and quantity of cell extracts.

FIG. 7.

IKK is essential for doxycycline- and serum-induced elk-1 expression. Wild-type (wt), IKK1^−/−, and IKK2^−/− MEF cells were stimulated with doxycycline at the indicated doses for 24 h (A) or with 20% serum for the indicated times after 48 h of serum withdrawal (B). Nuclear extracts were isolated and subjected to EMSA with an SRE probe. (C) Nuclear and whole-cell extracts were isolated from the IKK2 (S177, 181E), IKK2, and p65 (NF-κB) transfectants and analyzed by EMSA with NF-κB, Elk-1, AP-1, and Oct-1 probes and Western blotting with anti-cFos, VEGF, and β-actin antibodies as indicated. (D) The protein extracts of the IKK2- or vector-transfected IKK2^−/− clone were analyzed for the expression of transfected IKK2 in Western blotting with β-actin as loading controls. (E) IKK2-reconstituted cells from panel D were stimulated with 20% serum for the indicated times after 48 h of serum starvation. (F) IKK2-reconstituted cells from panel D were stimulated with doxycycline at the indicated doses for 24 h. Nuclear extracts from panels E and F were isolated and analyzed by EMSA with NF-κB, AP-1, and Oct-1 probes, and whole-cell extracts from both panels E and F were subjected to Western blot analysis with anti-c-Fos, -VEGF, and -β-actin antibodies as indicated. (G) The nuclear extracts from an IKK2^−/− clone transfected with an expression plasmid encoding Elk-1 were analyzed by EMSA with SRE and AP-1 probes and with Oct-1 probe as a loading control.

FIG. 8.

NF-κB regulates the elk-1 promoter, and a dominant mutant Elk-1 inhibits AP-1 activity. (A) elk-1 promoter and elk-1 promoter luciferase reporter gene constructs with wild-type (wt) and mutant (MT) κB_elk-1 sequences indicated. (B) Luciferase reporter gene assays for the basal activity of elk-1 promoter constructs with wild-type (Wt) κB and mutant (mt) κB binding sites were performed with wild-type, IKK1^−/−, and IKK2^−/− MEF cells as indicated. (C) Luciferase reporter gene assays for the inducible of elk-1 promoter activity were performed with MDAPanc-28/Puro and MDAPanc-28/IκBαM cells cotransfected with elk-1 promoter reporter gene constructs with wild-type and mutant κB binding sites and p-TK Renilla luciferase and then stimulated with doxycycline as indicated. (D) MDAPanc-28/Puro and MDAPanc-28/IκBαM cells were cotransfected either with the indicated amount of pCMV expression vector encoding the mutant Elk-1 or control pCMV vector, AP-1-luciferase reporter gene construct, and p-TK Renilla luciferase in the absence or presence of doxycycline stimulation. The transient transfections in panels B, C, and D were performed by the lipotransfection method (FuGENE 6; Roche). The activities of both firefly luciferase and Renilla luciferase were determined with the Promega dual-luciferase reporter assay system. The luciferase activities were normalized to the Renilla luciferase activity of the internal control. Data represent the mean ± standard error from three different experiments performed in triplicate.

FIG. 9.

NF-κB-mediated AP-1 activity regulates VEGF expression. (A) Western blot and Northern analyses of VEGF expression in MDAPanc-28/Puro and MDAPanc-28/IκBαM cells as indicated. (B) Luciferase reporter gene assays for the activity of a VEGF promoter construct were performed with MDAPanc-28/Puro and MDAPanc-28/IκBαM cells as indicated. (C and D) Northern blot analysis of VEGF expression in MDAPanc-28/Puro and MDAPanc-28/IκBαM cells stimulated with serum at various time intervals and doxycycline at different doses. (E) Luciferase reporter gene assays for the activity of a VEGF promoter construct were performed with the expression vectors encoding IκBαM, a dominant-negative Fos mutant (ΔFos), Fos, FosB, JunB, JunD, and c-Jun as indicated. The activities of both firefly luciferase and Renilla luciferase were determined by using the Promega dual-luciferase reporter assay system (Promega). The luciferase activities were normalized to the Renilla luciferase activity of the internal control. Data represent the mean ± standard error from three different experiments performed in triplicate.