PIAS1 specifically interacts with the p65 subunit of NF-κB in vivo.
STAT and NF-κB are two important families of transcription factors activated by cytokines. Upon ligand stimulation, both STAT and NF-κB translocate from the cytoplasm into the nucleus, where they bind DNA and activate transcription of specific genes. We explored the possible involvement of PIAS1 in the regulation of NF-κB signaling.
To test whether PIAS1 can interact with NF-κB in vivo, human 293T cells were transiently transfected with expression constructs encoding Flag-PIAS1 and the p65 subunit of NF-κB, alone or together. Thirty hours posttransfection, cells were either left untreated or treated with TNF-α for 15 min, and whole-cell lysates were utilized in coimmunoprecipitation assays using an anti-p65 antibody. After extensive washing, the immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting using an anti-Flag antibody. When both p65 and Flag-PIAS1 were overexpressed in 293T cells, PIAS1 was coimmunoprecipitated by anti-p65, indicating that PIAS1 and p65 interact in vivo (Fig. , top panel, lane 5). This interaction was not affected by TNF-α treatment. The proper expression of Flag-PIAS1 and p65 was confirmed by Western blot analysis of the same lysates (Fig. , middle and bottom panels). To validate the specific p65-PIAS1 interaction, coimmunoprecipitation assays were carried out with 293T lysates overexpressing both Flag-PIAS1 and p65, using rabbit IgG, anti-p65, or an antibody against E2F-1, an irrelevant transcription factor, as a negative control. As shown in Fig. , Flag-PIAS1 was immunoprecipitated only by anti-p65 but not by anti-E2F-1 or rabbit IgG. These results indicate that PIAS1 interacts with NF-κB p65 in vivo.
FIG. 1. PIAS1 specifically interacts with the p65 subunit of NF-κB in vivo. (A, top panel) Human 293T cells were transiently transfected with either Flag-PIAS1 or p65 alone or together, as indicated. Thirty hours posttransfection, cells were either untreated (more ...)
To test whether PIAS1 interacts with other subunits of NF-κB, 293T cells were transiently transfected with Flag-PIAS1, NF-κB p65, or NF-κB p50 alone or Flag-PIAS1 together with one of the NF-κB subunits. Coimmunoprecipitation assays were performed as described above using the antibodies specifically recognizing p65 or p50. PIAS1 interacts with p65 but not the p50 subunit of NF-κB (Fig. , top panel, lanes 3 and 6). The proper expression of each component was confirmed by Western blot analysis (Fig. , middle and bottom panels). These results revealed that PIAS1 specifically interacts with the p65 subunit of NF-κB in vivo.
p65 contains a transcriptional activation domain and a Rel homology domain. To examine the PIAS1 interaction region of p65, 293T cells were transiently transfected with Myc-PIAS1 together with Flag-p65(1-313) or Flag-p65(299-551) followed by coimmunoprecipitation analysis with anti-Flag. PIAS1 was found to interact with Flag-p65(299-551) but not Flag-p65(1-313). Thus, the C-terminal region of p65 containing the transcriptional activation domain is responsible for binding to PIAS1 (Fig. ).
Similar coimmunoprecipitation analysis was performed to identify the p65 interaction region of PIAS1. 293T cells were transiently transfected with Myc-p65 together with various deletion mutants of PIAS1. The region encoding amino acid residues 89 to 344, which is located between the PIAS1 SAP (SAF-A, Acinus, PIAS) domain and the RING domain, was found to be sufficient for interacting with p65 (Fig. ).
PIAS1 inhibits NF-κB-mediated gene activation.
To study the effect of PIAS1 on NF-κB-mediated gene activation, luciferase reporter assays were carried out in human 293T cells. Cells were transiently transfected with a luciferase reporter construct containing two copies of the NF-κB binding site together with increasing amounts of Flag-PIAS1. Twenty-four hours posttransfection, cells were either left untreated or treated with TNF-α for 6 h, followed by luciferase assays. As shown in Fig. , TNF-α treatment leads to an approximately 100-fold increase in the endogenous NF-κB-mediated gene activation, which was inhibited by PIAS1 in a dose-dependent manner (Fig. , left panel). Similar results were observed in human A549 lung cancer cells (Fig. , right panel). The proper expression of Flag-PIAS1 in both cell lines was confirmed by Western blot analysis (Fig. , bottom panels). These results suggest that PIAS1 is an inhibitor of the NF-κB signaling pathway.
FIG. 2. PIAS1 inhibits TNF-α-induced NF-κB-dependent gene activation. (A) Luciferase (Luc.) reporter assays. Human 293T cells (left) or lung cancer A549 cells (right) were transiently transfected with luciferase reporter construct 2×NF-κB (more ...)
To further confirm the role of PIAS1 in the regulation of the NF-κB signaling pathway, we examined the effect of PIAS1 on the activation of the endogenous NF-κB downstream genes in response to TNF-α stimulation. We established a TET-off PIAS1 cell line (Utf-PIAS1) from human osteosarcoma U2OS cells, where the expression of PIAS1 is induced in the absence of DOX, an analog of TET (24
) (Fig. ). To examine the effect of PIAS1 on the transcriptional activation of endogenous NF-κB-dependent genes, Utf-PIAS1 cells growing in the presence or absence of DOX for 12 h were either left untreated or treated with TNF-α for 1 or 3 h. Total RNA was collected and subjected to Northern blot analysis using a cDNA probe of Bfl-1
, two known NF-κB downstream genes (17
). Quantitative analysis indicated that the induction of PIAS1 by DOX removal in Utf-PIAS1 cells inhibited the transcriptional activation of Bfl-1
by approximately 60% in response to 3 h of TNF-α treatment, while DOX treatment had no significant effect on Bfl-1
induction in Utf control cells (Fig. ). Similarly, the TNF-α-mediated transcriptional activation of IκBα
was significantly repressed by PIAS1. These results are consistent with those of the luciferase reporter assays and suggest that PIAS1 acts as an inhibitor of NF-κB-mediated gene activation.
Enhanced NF-κB-mediated gene activation in Pias1 null cells.
To validate a role of PIAS1 in the regulation of NF-κB, we examined whether NF-κB-mediated gene activation is altered in Pias1
null cells. BMMs from Pias1
null mice and their wild-type littermates were untreated or treated with TNF-α for various time periods (0 h, 30 min, 1 h, and 3 h). Total RNA isolated from these cells was used for the analysis of transcriptional activation of several known NF-κB-dependent genes by Q-PCR (38
). The induction of IκBα
(macrophage inflammatory protein 2), Irf1
(interferon regulatory factor 1), and Junb
by TNF-α was significantly increased in Pias1
null cells compared with wild-type controls (Fig. ). Interestingly, the induction of other NF-κB target genes, including Cxcl10
, and Mcp1
, was not significantly affected in the absence of PIAS1 (Fig. ). These data suggest that PIAS1 displays specificity in the regulation of NF-κB target genes. Similarly, the TNF-α-induced activation of the IL-1β
genes, but not Mcp1
, was significantly enhanced in Pias1
null primary embryonic fibroblasts (Fig. ).
FIG. 3. Enhanced expression of NF-κB-regulated genes in response to TNF-α and LPS in Pias1 null cells. (A) Q-PCR analyses of BMMs from wild-type (black bars, +/+) or Pias1 null (gray bars, −/−) littermates, untreated (more ...)
To further examine the specificity of PIAS1 in the regulation of NF-κB signaling, BMMs from Pias1 null cells and their wild-type control cells were either untreated or treated with TNF-α for 30 min or LPS for 1 h. Total RNA isolated from these cells was subjected to microarray analysis. Under these conditions, 65 genes were induced at least twofold after TNF-α stimulation for 30 min, 48% of which showed at least 1.3-fold more induction in Pias1 null cells than wild-type cells. Similarly, 98 genes were induced at least twofold after 1 h of LPS stimulation, but only 22% of them were affected by PIAS1 (Fig. and Table S1 in the supplemental material). These data indicate that PIAS1 selectively affects the induction of a subgroup of TNF-α or LPS-induced genes.
Elevated proinflammatory cytokine production in Pias1 null mice.
The induction of IL-1β and TNF-α genes by TNF-α treatment was significantly enhanced in Pias1 null cells (Fig. ), consistent with a known important role of NF-κB in the regulation of proinflammatory cytokine gene expression. To directly determine the cytokine levels in mice under physiological conditions, serum samples were prepared from Pias1 null mice and their wild-type littermates and subjected to ELISAs. The levels of two proinflammatory cytokines, IL-1β and TNF-α, were elevated in Pias1 null mice 6-fold and 3.5-fold, respectively (Fig. ). In contrast, serum levels of IFN-γ and IL-4, two cytokines not directly regulated by the NF-κB pathway, were not significantly altered in Pias1 null mice.
FIG. 4. Elevated serum cytokine levels in Pias1 null mice compared to their wild-type littermates. Serum samples were collected from 4- to 15-week-old Pias1 null mice (open circles) and their wild-type littermates (filled circles). Serum IL-1β (n = (more ...) Normal T- and B-lymphocyte development and enhanced granulopoiesis in Pias1 null mice.
Given a potential role of PIAS1 in cytokine-activated signaling pathways, we analyzed whether Pias1 null mice have defects in lymphocyte development. PIAS1 protein is normally expressed in the spleen and thymus. Flow cytometric analyses were carried out with cells from spleens and thymuses of 4- to 8-week-old Pias1 null mice and their wild-type littermates. The CD4/CD8 profiles of the thymocytes and the B220/IgM profiles of the splenocytes were similar for these mice (Fig. ), suggesting that both T and B lymphocytes developed normally in the absence of PIAS1.
FIG. 5. Flow cytometric analysis of the hematopoietic cells from Pias1 null mice and their wild-type littermates. (A) Normal T- and B-lymphoid development in Pias1 null mice. Cells from the thymus and spleen of 6-week-old mice were stained with anti-CD4 and anti-CD8 (more ...)
The NF-κB pathway regulates the proliferation and differentiation of granulocytes. In fact, IκBα
null mice exhibited enhanced granulopoiesis (5
). When splenocytes were stained with the granulocyte-specific marker Gr-1, an increased population of mature granulocytes was reproducibly observed in Pias1
null mice compared to that in the wild-type controls (Fig. ). It has been implicated that granulocyte colony-stimulating factor (G-CSF), a key cytokine regulated by the NF-κB pathway, plays an important role in the production of granulocytes (5
). Therefore, we determined the RNA levels of G-CSF
in wild-type and Pias1
null mice. Total RNA isolated from the thymus of the wild-type and Pias1
null mice was subjected to Q-PCR analysis using specific primers for murine G-CSF
. As shown in Fig. , the relative expression of G-CSF
was increased in Pias1
null thymus compared to that of the wild-type littermates. In contrast, the RNA levels of GM-CSF
were not altered in Pias1
null thymus. Similar observations have been described for IκBα
null mice (5
). These results further support the role of PIAS1 in the negative regulation of NF-κB in vivo.
PIAS1 has no effect on the activation and nuclear translocation of NF-κB.
To understand how PIAS1 regulates NF-κB signaling, we examined whether PIAS1 affects the activation and nuclear translocation of NF-κB p65. The localization of the endogenous PIAS1 and p65 was examined by immunofluorescence analyses. Wild-type (6+/+) and Pias1 null (7−/−) mouse embryo fibroblasts (MEFs) were either untreated or treated with TNF-α for 15 min and then stained with anti-p65 and anti-PIAS1 antibodies simultaneously. In wild-type MEFs, NF-κB p65 resided in the cytoplasm of untreated cells and translocated into the nucleus upon TNF-α stimulation, which is consistent with published results. In contrast, PIAS1 remained in the nucleus with or without TNF-α stimulation. The colocalization of p65 and PIAS1 was observed in the nucleus after TNF-α stimulation (Fig. ). In Pias1 null MEFs, anti-PIAS1 did not reveal specific nuclear staining as observed in Pias1+/+ cells, which validates the specificity of the anti-PIAS1 antibody (Fig. ). Most importantly, the nuclear translocation of p65 appeared normal in Pias1 null MEFs, suggesting that PIAS1 does not affect the nuclear translocation of NF-κB p65 in response to TNF-α. Similar results were also obtained in wild-type and Pias1 null BMMs untreated or treated with LPS (data not shown).
FIG. 6. PIAS1 does not affect the activation and nuclear translocation of NF-κB. (A) Immunofluorescence analysis of PIAS1 and NF-κB p65 in wild-type (6+/+) and Pias1 null (7−/−) MEFs. Cells were untreated or treated (more ...)
To further examine the effect of PIAS1 on the activation and nuclear translocation of NF-κB, cytoplasmic and nuclear extracts of Pias1+/+ and Pias1−/− BMMs untreated or treated with TNF-α or LPS for various times were prepared, followed by Western blot analysis using anti-IκBα or anti-p65. In Pias1+/+ BMMs, IκBα was degraded in the cytoplasm with the concurrent translocation of p65 into the nucleus upon stimulation (Fig. ). In Pias1−/− BMMs, IκBα degradation and p65 translocation were normal compared to the wild-type controls (Fig. , compare lanes 2 to 6 and 8 to 12). Thus, PIAS1 does not affect the signaling events leading to the nuclear translocation of p65.
PIAS1 blocks the DNA binding activity of NF-κB.
We examined the effect of PIAS1 on the DNA binding activity of NF-κB in vitro by EMSA. Nuclear extracts from MCF-7 cells untreated or treated with TNF-α were analyzed by EMSA using an NF-κB binding site as the probe. TNF-α treatment induced the formation of a specific shift band, which represents the NF-κB p50-p65 heterodimer, since it was specifically supershifted by anti-p50 or anti-p65 antibody but not by anti-E2F or rabbit IgG (Fig. ). To test the effect of PIAS1 on the DNA binding activity of NF-κB, GST-PIAS1 protein was prepared and used in EMSA. The addition of purified GST-PIAS1 protein inhibited the DNA binding activity of NF-κB p50-p65 in a dose-dependent manner (Fig. , lanes 6 to 8). As a control, the addition of the same amounts of GST protein had no effect on the DNA binding activity of NF-κB (Fig. , lanes 3 to 5). Under similar conditions, GST-PIAS1 does not affect the DNA binding activity of SP-1 (Fig. ), supporting the conclusion that the effect of GST-PIAS1 on the DNA binding of NF-κB is specific.
FIG. 7. PIAS1 can block the DNA binding activity of NF-κB. (A) Nuclear extracts from MCF-7 cells untreated or treated with TNF-α for 15 min were analyzed by EMSA using an oligonucleotide containing the NF-κB binding site as a probe. The (more ...)
To test the hypothesis that PIAS1 inhibits the NF-κB-mediated gene activation by blocking the DNA binding activity of p65 in vivo, we used a Gal4-p65 fusion protein, which can activate the luciferase reporter constructs carrying either the Gal4-binding site or the NF-κB binding site. When human 293T cells were transiently transfected with the Gal4-p65 expression construct, the 5×Gal4 reporter containing five copies of the Gal4 binding site and increasing amounts of PIAS1, PIAS1 showed no inhibition on Gal4-p65-mediated gene activation (Fig. ). In contrast, when the 2×NF-κB reporter construct was used in the luciferase assays, PIAS1 inhibited the transcriptional activity of the Gal4-p65 fusion protein (Fig. ). These results support the conclusion that PIAS1 inhibits NF-κB-mediated transcription by blocking the DNA binding activity of NF-κB p65.
To further examine whether PIAS1 has the ability to regulate the DNA binding activity of p65 in vivo, we performed ChIP assays to examine the binding of p65 to the promoters of endogenous NF-κB-regulated genes. Protein extracts from wild-type and Pias1 null BMMs untreated or treated with LPS for 20 min or 1 h were immunoprecipitated with anti-p65 or anti-IgG. The bound DNA was quantified by Q-PCR analysis using specific primers (Fig. ). The binding of p65 to the endogenous IκBα promoter upon LPS stimulation was significantly enhanced in Pias1 null cells. Similar analysis was also performed with protein extracts prepared from the TET-off PIAS1 cell line (Utf-PIAS1). The induction of PIAS1 in the absence of DOX significantly repressed the binding of p65 to the promoter of IκBα in response to TNF-α treatment (Fig. ). These results further support the conclusion that PIAS1 inhibits the activity of NF-κB by interfering with the recruitment of p65 to the promoters of NF-κB-regulated genes.
FIG. 8. PIAS1 affects the binding of p65 to the endogenous promoters revealed by ChIP assays. (A) The binding of p65 to the IκBα promoter is increased in Pias1 null cells. ChIP assays were performed with BMMs from wild-type (+/+) (more ...)