Nuclear factor-kappaB (NF-kappaB) is a novel positive transcriptional regulator of the oncogenic Wip1 phosphatase

Abstract

The nuclear factor-kappaB (NF-kappaB) family of transcription factors plays a key role in inflammation and augments the initiation, promotion, and progression of cancer. NF-kappaB activation generally leads to transcriptional enhancement of genes important in cell survival and cell growth, which is exploited in cancer cells. In this study, we identify an additional oncogene, PPM1D, which encodes for Wip1, as a transcriptional target of NF-kappaB in breast cancer cells. Inhibition of NF-kappaB or activation of NF-kappaB resulted in decreased or increased Wip1 expression, respectively, at both the mRNA and protein levels. PPM1D promoter activity was positively regulated by NF-kappaB, and this regulation was dependent on the presence of the conserved kappaB site in the PPM1D promoter region. Chromatin immunoprecipitation analysis showed basal binding of the p65 NF-kappaB subunit to the PPM1D promoter region encompassing the kappaB site, which is enhanced after NF-kappaB activation by tumor necrosis factor-alpha. Finally, we show that Wip1 expression is induced in lipopolysaccharide-stimulated mouse splenic B-cells and is required for maximum proliferation. Taken together, these data suggest an additional mechanism by which NF-kappaB may promote tumorigenesis, support the selective use of NF-kappaB inhibitors as chemotherapeutic agents for the treatment of human cancers, and further define a function for Wip1 in inflammation.

FIGURE 1.

PPM1D promoter region includes an evolutionarily conserved κB site. A, conserved κB site was found at position −793 bp in the PPM1D promoter region using the Alibaba 2.1 software. B, κB site is highly homologous in sequence and in position with respect to the translational start site among the various organisms indicated.

FIGURE 2.

Inhibition of NF-κB decreases Wip1 expression. A, nuclear extracts were immunoblotted for p65 and β-actin (loading control). Wip1 mRNA levels were quantified (B), or whole cell lysates were immunoblotted for Wip1 and β-actin (loading control) from MCF-7 cells incubated with IMD-0354 for the time periods indicated (C). D and E, MCF-7 cells were transfected with either control siRNA or p65 siRNA for 24 h. Wip1 mRNA (D) and total Wip1 protein from these cells were analyzed (E). Wip1 mRNA levels were quantified by qRT-PCR and normalized to GAPDH. E, whole cell lysates were immunoblotted for Wip1, p65, and β-actin (loading control).

FIGURE 3.

Activation of NF-κB increases Wip1 expression. Wip1 mRNA levels were quantified by qRT-PCR and normalized to GAPDH after NF-κB activation. A, MCF-7 cells were incubated with either IMD-0354 alone, TNFα alone, or TNFα with pretreatment of IMD-0354 for the indicated time periods. B, primary splenic lymphocytes from wild type mice were incubated with LPS with or without pretreatment of IMD-0354 for the indicated time periods. Dotted lines represent the value of the untreated control samples, which are set to 1.

FIGURE 4.

p65 activity regulates PPM1D promoter activity and directly binds to the PPM1D promoter region. A, schematic of the construction of the luciferase reporter vectors used to measure PPM1D promoter activity. pWip1-luc includes the complete 855-bp sequence directly upstream from the PPM1D translational start site, and pWip1_ΔκB-luc includes the same sequence with the exception of the putative κB site. B, PPM1D promoter activity assay. MCF-7 cells were transfected with either pWip1-luc or pWip1_ΔκB-luc alone or cotransfected with p65 siRNA. Luciferase activity was measured 48 h later. C, ChIP analysis of NF-κB promoter binding. MCF-7 cells were either untreated, treated with TNFα alone, or treated with TNFα after a 5-h pretreatment with IMD-0354. The cells were harvested for ChIP assays using either IgG (negative control) or a p65-specific antibody. The primers used were specific for either the region of the PPM1D promoter, including the κB site, the promoter region of the NFKBIA gene (positive control), or the region between the GAPDH and CNAP1 genes (Negative, negative control).

FIGURE 5.

Wip1 expression in LPS-stimulated splenic B-cells inversely correlates with p38 activity. A, primary splenic lymphocytes from wild type mice were incubated with LPS for the indicated time periods. Whole cell lysates were subject to immunoblot analysis, and the fold change in Wip1 mRNA levels (normalized to GAPDH) was measured by qRT-PCR. B, primary splenic lymphocytes from Wip1^−/− mice were stimulated with LPS (or not, −), and whole cell lysates were subject to immunoblot analysis. C, proliferation was measured in LPS-stimulated wild type lymphocytes at 48 h without SB203580 (−) or with SB203580 added 8 or 24 h after LPS addition. D, primary splenic lymphocytes from wild type mice were incubated with LPS (or not), with or without IMD-0354 pretreatment, for the indicated time periods. Whole cell lysates were subject to immunoblot analysis. E, fold change in Wip1 mRNA levels was measured at 48 h after LPS stimulation without IMD-0354 (−) or with IMD-0354 added at the indicated time points after LPS stimulation. A black horizontal line indicates the value of “1” on the x axis and highlights that all IMD-0354-treated cells show no proliferation in response to LPS (specific values: 0, 0.3 ± 0.0015; 8, 0.08 ± 0.0005; 15, 0.1 ± 0.001; 24, 0.522 ± 0.007). All immunoblotting was analyzed using specific antibodies for phospho-p38 (Thr-180/Tyr-182) and p38. For the analysis of Wip1 mRNA fold changes, values were normalized to those of nonstimulated controls, which are set to 1 (indicated by a black horizontal line).

FIGURE 6.

NF-κB-induced Wip1 promotes LPS-stimulated B-cell proliferation. A, relative proliferation was calculated for primary splenic lymphocytes at the indicated times of LPS incubation (wt, lymphocytes harvested from wild type mice; wt, IMD-0354, lymphocytes harvested from wild type mice treated with IMD-0354; Wip1^−/−, lymphocytes harvested from Wip1^−/− mice). B, proliferation was measured at 24 and 48 h after LPS stimulation in wild type splenic lymphocytes without IMD-0354 (−) or with IMD-0354 added at the indicated time periods after LPS addition.

FIGURE 7.

Diagram of Wip1 cross-talk with stress signaling. Asterisk designates finding from our studies.