Shuichi Fujioka

NF-κB and AP-1 Connection: Mechanism of NF-κB-Dependent Regulation of AP-1 Activity

ABSTRACT Nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-κB is regulated by the inducible phosphorylation of NF-κB inhibitor IκB by IκB 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-κB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-κB 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/IκBαM 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-κB downstream target genes. Inhibition of NF-κB 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-κB in participating in the regulation of elk-1, c-fos, and VEGF expression.

The function of multiple IκB: NF-κB complexes in the resistance of cancer cells to Taxol-induced apoptosis

The Rel/NF-κB transcription factors play a key role in the regulation of apoptosis and in tumorigenesis by controlling the expressions of specific genes. To determine the role of the constitutive activity of RelA in tumorigenesis, we generated pancreatic tumor cell lines that express a dominant negative mutant of IκBα (IκBαM). In this report, we show that the inhibition of constitutive NF-κB activity, either by ectopic expression of IκBαM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IκBα proteins, downregulates the expression of bcl-xl. We identified two putative NF-κB binding sites (κB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-κB proteins. p65/p50 heterodimer interacts with κB/A site whereas p50/p50 homodimer interacts with κB/B. The bcl-xl promoter reporter gene assays reveal that NF-κB dependent transcriptional activation is mainly mediated by κB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IκBαM and PS-341 completely abolish NF-κB DNA binding activity; however, PS-341, but not ectopic expression of IκBαM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IκBβ and the re-expression of NF-κB regulated bcl-xl gene in these cancer cells as ectopic expression of the bcl-xl gene confers resistance to Taxol-induced apoptosis in PS-341 sensitized cells. These results demonstrate the important function of various NF-κB/IκB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-κB : IκBα and NF-κB : IκBβ complexes are regulated by different upstream activators, and that NF-κB plays a key role in pancreatic tumorigenesis.

Mechanisms of Proinflammatory Cytokine-Induced Biphasic NF-κB Activation

Abstract The transcription factor NF-κB regulates genes involved in innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Proinflammatory cytokines induce the activation of NF-κB in both transient and persistent phases. We investigated the mechanism for this biphasic NF-κB activation. Our results show that MEKK3 is essential in the regulation of rapid activation of NF-κB, whereas MEKK2 is important in controlling the delayed activation of NF-κB in response to stimulation with the cytokines TNF-α and IL-1α. MEKK3 is involved in the formation of the IκBα:NF-κB/IKK complex, whereas MEKK2 participates in assembling the IκBβ:NF-κB/IKK complex; these two distinct complexes regulate the proinflammatory cytokine-induced biphasic NF-κB activation. Thus, our study reveals a novel mechanism in which different MAP3K and IκB isoforms are involved in specific complex formation with IKK and NF-κB for regulating the biphasic NF-κB activation. These findings provide further insight into the regulation of cytokine-induced specific and temporal gene expression.

Inhibition of constitutive NF-kappa B activity by I kappa B alpha M suppresses tumorigenesis.

We have demonstrated that nuclear factor-κB (NF-κB) is constitutively activated in human pancreatic adenocarcinoma and human pancreatic cancer cell lines but not in normal pancreatic tissues or in immortalized, nontumorigenic pancreatic epithelial cells, suggesting that NF-κB plays a critical role in the development of pancreatic adenocarcinoma. To elucidate the role of constitutive NF-κB activity in human pancreatic cancer cells, we generated pancreatic tumor cell lines that express a phosphorylation defective IκBα (S32, 36A) (IκBαM) that blocks NF-κB activity. In this study, we showed that inhibiting constitutive NF-κB activity by expressing IκBαM suppressed the tumorigenicity of a nonmetastatic human pancreatic cancer cell line, PANC-1, in an orthotopic nude mouse model. Immunohistochemical analysis showed that PANC-1-derived tumors expressed vascular endothelial growth factor (VEGF) and induced angiogenesis. Inhibiting NF-κB signaling by expressing IκBαM significantly reduced expression of Bcl-xL and Bcl-2. The cytokine-induced expression of VEGF and Interleukin-8 in PANC-1 cells is also decreased. Taken together, these results suggest that the inhibition of NF-κB signaling can suppress tumorigenesis of pancreatic cancer cells and that the NF-κB signaling pathway is a potential target for anticancer agents.

Restoring apoptosis in pancreatic cancer cells by targeting the nuclear factor-κb signaling pathway with the anti-epidermal growth factor antibody IMC-C225

We have previously demonstrated that RelA is constitutively activated in the majority of human pancreatic cancers and plays an important role in tumorigenesis and metastasis. The antiapoptotic gene bcl-xl is a downstream target of RelA, and regulation of bcl-xl transcription is mediated directly by the nuclear factor κB (NF-κB) binding sites present in the upstream promoter element of the bcl-xl gene. In this study we investigated the effects of inhibition of epidermal growth factor receptor (EGFR) signaling pathway with the anti-EGFR monoclonal antibody IMC-C225 on constitutive NF-κB activation and regulation of apoptosis-related genes in human pancreatic cancer cells. We found that activation of EGFR can be blocked with the anti-EGFR antibody IMC-C225 in the human pancreatic cancer cell line MDA Panc-28, leading to a marked decrease in constitutive NF-κB DNA binding activity. Our data also suggest that downregulation of NF-κB DNA binding activity by IMC-C225 leads to a decrease in bcl-xl and bfl-1 expression. Therefore, targeting the NF-κB signaling pathway with an anti-EGFR antibody may be one strategy to restore apoptosis in human pancreatic cancer cells, thereby enhancing the effect of chemotherapy and radiation therapy.

NF-κB in pancreatic cancer

Although the genetic profile of pancreatic cancer is emerging as a result of much research, the role of specific genetic alterations that initiate tumorigenesis and produce its cardinal clinical features of locally aggressive growth, metastasis, and chemotherapy resistance remains unresolved. Recently, a number of studies have shown that the inhibition of constitutive NF-κB activation, one of the frequent molecular alterations in pancreatic cancer, inhibits tumorigenesis and metastasis. It also sensitizes pancreatic cancer cell lines to anticancer agent-induced apoptosis. Therefore because of the crucial role of NF-κB in pancreatic cancer, it is a potential target for developing novel therapeutic strategies for the disease. In vivo and in vitro models that mimic the tumorigenic phenotypes in the appropriate histological and molecular concert would be very useful for confirming the suspected role of the pancreatic cancer signature genetic lesions and better understanding the molecular basis of this disease.

Inhibition of constitutive NF- κ B activity by I κ B α M suppresses tumorigenesis

We have demonstrated that nuclear factor-κB (NF-κB) is constitutively activated in human pancreatic adenocarcinoma and human pancreatic cancer cell lines but not in normal pancreatic tissues or in immortalized, nontumorigenic pancreatic epithelial cells, suggesting that NF-κB plays a critical role in the development of pancreatic adenocarcinoma. To elucidate the role of constitutive NF-κB activity in human pancreatic cancer cells, we generated pancreatic tumor cell lines that express a phosphorylation defective IκBα (S32, 36A) (IκBαM) that blocks NF-κB activity. In this study, we showed that inhibiting constitutive NF-κB activity by expressing IκBαM suppressed the tumorigenicity of a nonmetastatic human pancreatic cancer cell line, PANC-1, in an orthotopic nude mouse model. Immunohistochemical analysis showed that PANC-1-derived tumors expressed vascular endothelial growth factor (VEGF) and induced angiogenesis. Inhibiting NF-κB signaling by expressing IκBαM significantly reduced expression of Bcl-xL and Bcl-2. The cytokine-induced expression of VEGF and Interleukin-8 in PANC-1 cells is also decreased. Taken together, these results suggest that the inhibition of NF-κB signaling can suppress tumorigenesis of pancreatic cancer cells and that the NF-κB signaling pathway is a potential target for anticancer agents.