Dominant-negative Mutant Of c-Jun Research Articles

JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells.

Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-κB and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src-transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumor suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumor suppressor function of DAPK1.

Abstract 4641: Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the Cheliensisin A (Chel A) inhibition of cell transformation

Abstract Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the present study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP down-regulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A. Citation Format: Chuanshu Huang, Junlan Zhu, Jingxia Li. Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the Cheliensisin A (Chel A) inhibition of cell transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4641. doi:10.1158/1538-7445.AM2015-4641

Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the cheliensisin a inhibition of cell transformation.

Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the current study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A.

Differentiation-associated genes regulated by c-Jun and decreased in the progression of esophageal squamous cell carcinoma.

Transcription factor c-Jun plays a key role in controlling epithelium cell proliferation, apoptosis and differentiation. However, molecular mechanism and biological functions of c-Jun in squamous differentiation and the progression of esophageal squamous cell carcinoma (ESCC) remain elusive. In this study, we found that c-Jun bound directly to the promoter region, and activated the transcription of differentiation-associated genes including cystatin A, involucrin and SPRR3 in vivo. Ectopic expression of c-Jun enhanced SPRR3 transactivation in KYSE450 cells. Conversely, TAM67, a dominant negative mutant of c-Jun, inhibited SPRR3 transactivation. c-Jun increased expression of SPPR3 mainly via a PKC/JNK pathway in response to TPA in KYSE450 cells. Furthermore, c-Jun was remarkably reduced in esophageal cancer. Interestingly, cystatin A, involucrin and SPRR3 were significantly downregulated as well, and associated with differentiation grade. Expression of c-Jun was correlated with the expression of these genes in normal epithelium and ESCC. Importantly, the expression of these genes was remarkably decreased during the malignant transformation from normal epithelium to low-grade intraepithelial neoplasia (LGIN) or high-grade intraepithelial neoplasia (HGIN). The expression of cystatin A and involucrin was significantly reduced from LGIN to HGIN. These results suggest c-Jun was involved in the regulation of differentiation-associated genes in ESCC. These genes might serve as the potential markers in distinguishing normal epithelium from esophageal squamous intraepithelial neoplasia.

Transcriptional Response to Calcium-Sensing Receptor Stimulation

Elevated extracellular Ca(2+) concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.

Cyclooxygenase-2 Expression Is Up-regulated by 2-Aminobiphenyl in a ROS and MAPK-Dependent Signaling Pathway in a Bladder Cancer Cell Line

Overexposure to biphenyl amine compounds, which are found in smoke and azo-dyes, is linked to the occurrence of bladder cancer. However, the molecular mechanisms of biphenyl amine compound-induced bladder cancer are still unclear. Many studies have demonstrated that overexpression of cyclooxygenase-2 (COX-2) in neoplastic lesions is associated with carcinogenesis. In this study, we have demonstrated that 2-aminobiphenyl (2-ABP) up-regulated the expression of COX-2 in a dose- and time-dependent manner in TSGH-8301 bladder cancer cells. This 2-ABP-induced COX-2 expression was attenuated by ROS scavenger NAC and NADPH oxidase inhibitors apocynin and DPI. The p22phox subunit of NADPH oxidase, but not p67, and Nox2 was up-regulated by 2-ABP. Knocking down p22phox by siRNA significantly reduced 2-ABP-induced COX-2 expression. Furthermore, 2-ABP also activated the ERK/JNK-AP1 pathways, and this effect was also abolished by NADPH oxidase inhibitors. Blocking the ERK/JNK-AP1 signaling pathways by pharmacological inhibitors attenuated 2-ABP-induced COX-2 expression. Overexpression of the upstream ERK activator MEK1 significantly and consistently increased 2-ABP-mediated COX-2 expression. Transfection of a dominant negative c-Jun mutant, TAM-67, blocked 2-ABP-mediated COX-2 expression, demonstrating that c-Jun was responsible for the transcriptional activation. Taken together, these results demonstrate that 2-ABP induces the carcinogenic factor COX-2 and that this induction is mediated through NADPH oxidase-derived ROS-dependent JNK/ERK-AP-1 pathways.

Arsenite-induced apoptosis of human neuroblastoma cells requires p53 but occurs independently of c-Jun

Arsenite treatment of human SH-SY5Y neuroblastoma cells leads to an upregulation of caspase-3/7 activity and to the fragmentation of chromatin that is accompanied by elevated p53 and c-Jun levels. Expression of a truncated mutant of p53, p53DD, which interfered with the oligomerization of p53, suppressed the arsenite-induced upregulation of caspase-3/7 activity and the fragmentation of chromatin, indicating that p53 is required for arsenite-induced cell death. These data were corroborated by knockdown experiments of p53 following expression of a p53-specific short hairpin RNA. Likewise, expression of either p53DD or knockdown of p53 prevented caspase-3/7 activation and chromatin fragmentation induced by nutlin-3, a compound that prevents the interaction between p53 and the E3 ubiquitin ligase MDM2. Transcriptional upregulation of a chromatin-embedded p53-responsive reporter gene in either arsenite or nutlin-3 stimulated neuroblastoma cells revealed that the transcriptional activity of p53 was increased under these conditions. Expression of a c-Jun–specific short hairpin RNA failed to impair arsenite-induced caspase-3/7 activation and fragmentation of chromatin. Likewise, inhibition of c-Jun target gene expression by expression of a dominant-negative mutant of c-Jun did not interfere with arsenite-induced caspase-3/7 activation and chromatin fragmentation. However, this approach successfully reduced caspase-3/7 activity induced as a result of forced expression of a constitutively active mutant of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK)-1. Together, these data show that the upregulation of p53 is causally linked with arsenite-induced cell death in neuroblastoma cells, whereas the upregulation of c-Jun is not part of this apoptotic signaling cascade.

Hexavalent Chromium Cr(VI) Up-Regulates COX-2 Expression through an NFκB/c-Jun/AP-1–Dependent Pathway

Background: Hexavalent chromium [Cr(VI)] is recognized as a human carcinogen via inhalation. However, the molecular mechanisms by which Cr(VI) causes cancers are not well understood.Objectives: We evaluated cyclooxygenase-2 (COX-2) expression and the signaling pathway leading to this induction due to Cr(VI) exposure in cultured cells.Methods: We used the luciferase reporter assay and Western blotting to determine COX-2 induction by Cr(VI). We used dominant negative mutant, genetic knockout, gene knockdown, and chromatin immunoprecipitation approaches to elucidate the signaling pathway leading to COX-2 induction.Results: We found that Cr(VI) exposure induced COX-2 expression in both normal human bronchial epithelial cells and mouse embryonic fibroblasts in a concentration- and time-dependent manner. Deletion of IKKβ [inhibitor of transcription factor NFκB (IκB) kinase β; an upstream kinase responsible for nuclear factor κB (NFκB) activation] or overexpression of TAM67 (a dominant-negative mutant of c-Jun) dramatically inhibited the COX-2 induction due to Cr(VI), suggesting that both NFκB and c-Jun/AP-1 pathways were required for Cr(VI)-induced COX-2 expression. Our results show that p65 and c-Jun are two major components involved in NFκB and AP-1 activation, respectively. Moreover, our studies suggest crosstalk between NFκB and c-Jun/AP-1 pathways in cellular response to Cr(VI) exposure for COX-2 induction.Conclusion: We demonstrate for the first time that Cr(VI) is able to induce COX-2 expression via an NFκB/c-Jun/AP-1–dependent pathway. Our results provide novel insight into the molecular mechanisms linking Cr(VI) exposure to lung inflammation and carcinogenesis.

Caspase-3 is a target gene of c-Jun:ATF2 heterodimers during apoptosis induced by activity deprivation in cerebellar granule neurons

Caspase-3, a key executor of neuronal apoptosis, is up-regulated and activated during apoptosis induced by activity deprivation in cerebellar granule neurons (CGNs). However, the transcriptional mechanism regulating caspase-3 during CGN apoptosis remains unknown. Here, we show that the caspase-3 gene is transactivated and its induction is preceded by c-Jun NH2-terminal kinase (JNK)/c-Jun:ATF2 pathway activation following activity deprivation in CGNs. We observed that caspase-3 induction is abolished by pharmacological inhibition of the JNK/c-Jun:ATF2 pathway. Destroying c-Jun:ATF2 heterodimers with dominant negative mutants of c-Jun and ATF2 or knockdown by small RNA interference reduced caspase-3 promoter activity and mRNA level. Furthermore, chromatin immunoprecipitation showed increased binding of c-Jun:ATF2 heterodimers to the caspase-3 promoter in response to activity deprivation in vivo. Site-directed mutagenesis of the caspase-3 promoter revealed that caspase-3 transcriptional activation depends primarily on an ATF site −233 to −225 nucleotides upstream of the start site. Taken together, these data demonstrate that caspase-3 is a target gene of c-Jun:ATF2 heterodimers during apoptosis induced by activity deprivation in CGNs.

Immediate-Early Transcriptional Response to Angiotensin II in Human Adrenocortical Cells

Angiotensin II binds to the angiotensin II receptors type 1 (AT1 receptors) in adrenocortical cells and triggers an intracellular signaling cascade leading to changes in the gene expression pattern. Here, we show that stimulation with angiotensin II induces the expression of biologically active early growth response (Egr)-1, a zinc finger transcription factor, in human H295R adrenocortical cells. Expression of a dominant-negative mutant of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression in angiotensin II-stimulated H295R cells, indicating that Ets-like protein-1 (Elk-1) or related ternary complex factors connect the intracellular signaling cascade elicited by activation of AT1 receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that angiotensin II stimulation increased the transcription activation potential of Elk-1. In addition, activator protein-1 transcriptional activity was significantly elevated in angiotensin II-treated H295R cells. Expression of c-Jun and c-Fos was increased as well as the transcription activation potential of c-Fos. Expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression in angiotensin II-stimulated adrenocortical cells, suggesting that the serum response element within the c-Fos promoter functions as an angiotensin II-response element. Expression of a dominant-negative mutant of c-Jun reduced activator protein-1 activity in angiotensin II-stimulated adrenocortical cells and reduced the up-regulation of c-Jun after angiotensin II stimulation. Thus, c-Jun regulates its own expression in adrenocortical cells. Together, the data show that angiotensin II stimulation activates the transcription factors Egr-1, Elk-1, c-Jun, and c-Fos in adrenocortical cells, leading to stimulus-dependent changes in the gene expression pattern.

Expression of interleukin-24 and its receptor in human pancreatic myofibroblasts

Interleukin (IL)-24 is a member of the IL-10 family of cytokines. In this study, we investigated IL-24 expression in chronic pancreatitis tissue and characterized the molecular mechanisms responsible for IL-24 expression in human pancreatic myofibroblasts. IL-24 expression in the tissues was evaluated by immunohistochemical methods. IL-24 mRNA and protein expression in the pancreatic myofibroblasts was determined by real-time-PCR and ELISA, respectively. IL-24 was expressed by α-smooth muscle actin-positive myofibroblasts in the chronic pancreatitis tissues. In isolated human pancreatic myofibroblasts, IL-1β significantly enhanced IL-24 mRNA and protein expression. The p38 MAPK inhibitor SB203580 and the PI3K inhibitor LY294002 significantly reduced the IL-β-induced IL-24 mRNA expression. An adenovirus containing a dominant-negative mutant of c-Jun significantly inhibited the effects of IL-1β on IL-24 mRNA expression, indicating that the IL-1β-induced IL-24 mRNA expression was mediated by the activation of transcription factor AP-1. Pancreatic myofibroblasts expressed IL-22R1, IL-20R1 and IL-20R2, and recombinant IL-24 induced the phosphorylation of p42/44, p38, JNK and STAT1/3. IL-24 is expressed in chronic pancreatitis tissue, and may play a role in the pathophysiology of chronic pancreatitis via autocrine pathways.

Pleiotropic Action of AP-1 in v-Src-Transformed Cells

The activation of AP-1 is a hallmark of cell transformation by tyrosine kinases. In this study, we characterize the role of AP-1 proteins in the transformation of chicken embryo fibroblasts (CEF) by v-Src. In normal CEF, the expression of a dominant negative mutant of c-Jun (TAM67) induced senescence. In contrast, three distinct phenotypes were observed when TAM67 was expressed in v-Src-transformed CEF. While senescent cells were also present, the inhibition of AP-1 caused apoptosis in a fraction of the v-Src-transformed cells. In addition, cells containing lipid-rich vesicles accumulated, suggesting that a subpopulation of the v-Src-transformed cells underwent differentiation in response to the inhibition of AP-1. JunD and Fra-2 were the main components of this factor, while c-Jun accounted for a minor fraction of AP-1 in v-Src-transformed CEF. The downregulation of c-Jun expression by short hairpin RNA (shRNA) induced senescence in normal and v-Src-transformed cells. In contrast, a high incidence of apoptosis was caused by the downregulation of JunD, suggesting that it is required for the survival of v-Src-transformed CEF. Levels of the p53 tumor suppressor were elevated under conditions of JunD inhibition. Repression of p53 by shRNA enhanced the survival and anchorage-independent proliferation of v-Src-transformed CEF with JunD/AP-1 inhibition. The inhibition of Fra-2 had no visible phenotype in normal CEF but caused the appearance of lipid-rich vesicles in v-Src-transformed CEF. Therefore, AP-1 facilitated transformation by acting as a survival factor, by inhibiting premature entry into senescence, and by blocking the differentiation of v-Src-transformed CEF.

A feedback loop between BEX2 and ErbB2 mediated by c‐Jun signaling in breast cancer

BEX2 is a member of brain expressed X-linked gene family that is differentially expressed in primary breast tumors. We have previously demonstrated that BEX2 expression protects breast cancer cells against mitochondrial apoptosis and G1 cell cycle arrest. In addition, we have shown that BEX2 is a c-Jun target gene and, in turn, regulates the phosphorylation of c-Jun in breast cancer cells. In our study, we investigated BEX2 protein expression in a tissue microarray cohort of 225 breast tissue samples with known clinical, pathological and biomarker information. We observed that BEX2 protein was overexpressed in ∼50% of malignant breast tumors compared to only 7% of benign breast samples. Notably, BEX2 positive tumors identified a subset of breast cancers with the overexpression of ErbB2 and phosphorylated c-Jun proteins. Furthermore, using in vitro models, we demonstrated that the mechanism of this association is a functional feedback loop involving ErbB2, c-Jun and BEX2 in breast cancer cells. In this feedback loop, ErbB2 overexpression results in an induction of c-Jun and BEX2 expression. Importantly, ErbB2 induction of BEX2 expression was abrogated by a dominant-negative mutant of c-Jun, suggesting that this effect was mediated through the regulation of c-Jun signaling. In turn, the overexpression of BEX2 led to an increase in both c-Jun-mediated induction of ErbB2 and c-Jun binding to the ErbB2 promoter in MCF-7 cells. Our study suggests that BEX2 protein is overexpressed in approximately half of breast cancers and has a positive feedback loop with ErbB2 mediated by c-Jun signaling in breast cancer cells.

Abstract 2497: Induction of AP-1 activity by ceramide results in ASAH1 upregulation: A mechanism of prostate cancer cell radioresistance

Abstract Background: Although radiation therapy is a mainstay of treatment for localized prostatic disease, the escape of a subset of tumor cells from ionizing radiation (IR)-induced killing may lead to disease relapse and progression. The metabolic balance between the pro-apoptotic sphingolipid, ceramide, and its anti-apoptotic metabolites has been characterized as a critical mediator of cell death under conditions of cell stress, including IR exposure. While the lysosomal ceramide-metabolizing enzyme, acid ceramidase (ASAH1), is constitutively expressed in all human tissues, ASAH1 has been found to be over-expressed in cancer cells exposed to IR. The goal of this study was to examine the biological impact of and possible mechanism for altered ASAH1 expression observed in irradiated prostate cancer cells. Methods: Sphingolipid expression was measured by LC-MS. Ceramidase activities were assessed by thin layer chromatography. ASAH1 mRNA and protein expression in cell lines were measured by qPCR and western blot, respectively. Transcription activity was assessed using a luciferase reporter driven by the 1.5kb proximal promoter region of ASAH1. AP-1 promoter occupancy was determined by chromatin immunoprecipitation (ChIP)-qPCR. Post-irradiative cell growth was determined by clonogenic survival assay. Results: IR induced nearly two-fold expression of ceramide, and between three- and four-fold expression of its metabolites, sphingosine and sphingosine-1-phosphate. Enzyme activity assays demonstrated specific increase of acid, but not neutral or alkaline, ceramidase in the irradiated prostate cancer cells, suggesting a prominent role for ASAH1 in IR-induced sphingolipid metabolism. Consistent with increased mRNA and protein levels, luciferase-promoter-reporter assay confirmed that ASAH1 regulation occurs at least in part at the transcriptional level. Interestingly, inhibition of de novo and salvage pathways of ceramide biogenesis abrogated IR-induced ASAH1 upregulation, implicating the involvement of ceramide in IR-induced ASAH1 overexpression. Through studies using protein-DNA binding arrays and ChIP-qPCR, we identified AP-1 as a direct, ceramide-sensitive transcription factor of ASAH1 in irradiated cells. Expression of a dominant negative mutant of c-Jun, a central component of the AP-1 complex, was able to abrogate IR-induced ASAH1 overexpression. Since ASAH1 silencing by shRNA sensitized prostate cancer cells to IR-induced cell killing, here we also observed radiosensitization by expression of the c-Jun dominant negative construct, which was rescued in part by forced overexpression of an ASAH1 transgene. Conclusion: The reported studies demonstrate that IR programs an unexpected phenotype of radiation resistance through ASAH1 upregulation mediated by ceramide-sensitive AP-1 activation. This study was supported in part by NIH/NCI P01 CA097132. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2497. doi:10.1158/1538-7445.AM2011-2497

Inhibition of AP-1 suppresses cervical cancer cell proliferation and is associated with p21 expression

AP-1, a transcription factor comprised primarily of Jun and Fos family proteins, regulates genes involved in proliferation, differentiation and oncogenesis. Previous studies demonstrated that elevated expression of Jun and Fos family member proteins is associated with numerous human cancers and in cancer-relevant biological processes. In this study we used a dominant-negative mutant of c-Jun, Tam67, which interferes with the functional activity of all AP-1 complexes, to investigate the requirement of AP-1 in the proliferation and cell cycle progression of cervical cancer cells. Transient and stable expression of Tam67 in CaSki cervical cancer cells resulted in decreased AP-1 activity that correlated with a significant inhibition of cell proliferation and anchorage-independent colony formation. Inhibiting AP-1 activity resulted in a two-fold increase in cells located in the G(2)/M phase of the cell cycle and an accompanying increase in the expression of the cell cycle regulatory protein, p21. The increase in p21 was associated with a decrease in HPV E6 expression and an increase in p53. Importantly, blocking the induction of p21 in CaSki-Tam67-expressing cells accelerated their proliferation rate to that of CaSki, implicating p21 as a key player in the growth arrest induced by Tam67. Our results suggest a role for AP-1 in the proliferation, G(2)/M progression and inhibition of p21 expression in cervical cancer.

Regulatory mechanism of activator protein-1 on the expression of MUC5AC induced by cigarette smoke extract

To investigate the mechanism of activator protein-1 (AP-1) on cigarette smoke-induced airway mucous hypersecretion and to explore the possible signal transduction pathway that activates AP-1. The airway epithelial cell line (BEAS-2B) was cultured in vivo and treated with cigarette smoke extract (CSE). The DNA binding activity of AP-1 was blocked by the transfection of c-Jun dominant negative mutant TAM67 into the cells. SP600125 and PD98059 were used to block the activation of c-Jun terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) respectively. MUC5AC protein was detected by enzyme-linked immunosorbent assay, MUC5AC mRNA level was analyzed by RT-PCR, while the protein contents of p-JNK, p-ERK and p-P38 were detected by Western blot, and the DNA binding activity of AP-1 was determined by electrophoretic mobility shift assay. The MUC5AC protein production and mRNA expression in the CSE group were significantly higher than those in the control group, and the DNA binding activity of AP-1 was also higher than that in the control group (P<0.01). The protein contents of p-ERK and p-JNK in the CSE group were higher than those in the control group (P<0.01), but the p-P38 level was not significantly different from that in the control group (P>0.05). After the transfection of TAM67 into the cells, the expression levels of MUC5AC protein and mRNA and the binding activity of AP-1 decreased significantly (P<0.01). The DNA binding activity of AP-1 and the expression levels of MUC5AC protein and mRNA were lower in the SP600125 group and in the PD98059 group than those in the CSE group (P<0.05). After being activated by JNK and ERK which are phosphorylated by cigarette smoke, AP-1 binds to its DNA binding elements on the promoter of MUC5AC gene and up-regulates the MUC5AC expression at the transcriptional level.

Minichromosome maintenance (MCM) protein 4 as a marker for proliferation and its clinical and clinicopathological significance in non-small cell lung cancer

Background Minichromosome maintenance (MCM) proteins 2–7 form a complex essential for the initiation of DNA replication. In the process to screen expression changes related to growth suppression of non-small cell lung cancer (NSCLC) cells by a cJun dominant-negative mutant, we found that reduced expression of MCM4 was correlated with this growth suppression. Method We determined the relevance of MCM4 in proliferation of NSCLC by downregulating its expression with small-interfering RNA in three NSCLC cell lines. We then immunohistochemically analyzed MCM4 expression in 156 surgically resected NSCLCs to correlate clinicopathologic characteristics. Results MCM4 downregulation reduced proliferation in two cell lines. MCM4 expression was higher in cancer cells than in adjacent normal bronchial epithelial cells ( p < 0.001). High MCM4 expression was correlated with male gender, heavy smoking, poorer differentiation and non-adenocarcinoma histology ( p < 0.001, respectively). High MCM4 expression was also correlated with proliferation markers, Ki-67 and cyclin E expression ( p < 0.001, respectively). MCM4 expression was not associated with survival. Conclusion MCM4 may play an essential role in the proliferation of some NSCLC cells. Taken together with higher expression in NSCLCs and its correlation with clinicopathologic characteristics such as non-adenocarcinoma histology, MCM4 may have potential as a therapeutic target in certain population with NSCLCs.

A Dominant-Negative c-jun Mutant Inhibits Lung Carcinogenesis in Mice

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. The identification of key regulatory and molecular mechanisms involved in lung tumorigenesis is therefore critical to increase our understanding of this disease and could ultimately lead to targeted therapies to improve prevention and treatment. Induction of members of the activator protein-1 (AP-1) transcription factor family has been described in human non-small cell lung carcinoma. Activation of AP-1 can either stimulate or repress transcription of multiple gene targets, ultimately leading to increased cell proliferation and inhibition of apoptosis. In the present study, we show induction of AP-1 in carcinogen-induced mouse lung tumors compared with surrounding normal lung tissue. We then used a transgenic mouse model directing conditional expression of the dominant-negative c-jun mutant TAM67 in lung epithelial cells to determine the effect of AP-1 inhibition on mouse lung tumorigenesis. Consistent with low AP-1 activity in normal lung tissue, TAM67 expression had no observed effects in adult mouse lung. TAM67 decreased tumor number and overall lung tumor burden in chemically induced mouse lung tumor models. The most significant inhibitory effect was observed on carcinoma burden compared with lower-grade lesions. Our results support the concept that AP-1 is a key regulator of mouse lung tumorigenesis, and identify AP-1-dependent transcription as a potential target to prevent lung tumor progression.

Regulation of AP‐1 Activity in Glucose‐Stimulated Insulinoma Cells

The transcriptional activity of AP-1 has been analyzed in glucose-stimulated INS-1 insulinoma cells using a chromosomally embedded AP-1-responsive reporter gene. We show that AP-1 activity was significantly elevated in glucose-treated INS-1 cells. Preincubation of the cells with nifedipine or expression of the Ca(2+) binding protein parvalbumin in the cytoplasm of INS-1 cells reduced AP-1 activity. Thus, activation of L-type Ca(2+) channels and an elevated cytoplasmic Ca(2+) concentration are crucial to connecting glucose stimulation with enhanced AP-1 activity. Expression of dominant negative forms of A-Raf, MKK4 or MKK6 and pharmacological inhibition of MEK and p38 revealed that extracellular signal-regulated protein kinase, p38 and c-Jun NH(2)-terminal protein kinase participate in the upregulation of AP-1 activity. Expression of dominant-negative mutants of c-Jun and Elk-1 reduced AP-1 transcriptional activity in INS-1 cells indicating that c-Jun and ternary complex factors are involved in the regulation of AP-1 activity in glucose-stimulated insulinoma cells.

Involvement of aryl hydrocarbon receptor nuclear translocator in EGF-induced c-Jun/Sp1-mediated gene expression.

Aryl hydrocarbon receptor nuclear translocator (ARNT) binds to other basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins to form functional transcriptional complexes in order to regulate specific biological pathways. Here, we report a novel mechanism that upon EGF treatment, ARNT associated with non-bHLH-PAS transcription factors, c-Jun/Sp1, and regulated gene expression, through forming a c-Jun/ARNT/Sp1 complex and binding to the Sp1 site of the gene promoter. EGF-induced promoter activity and the mRNA level of 12(S)-lipoxygenase as well as the association between c-Jun and Sp1 were reduced by ARNT knockdown. Notably, dominant negative c-Jun mutant, TAM-67, blocked ARNT-mediated 12(S)-lipoxygenase expression, demonstrating that c-Jun was responsible for the transcriptional activation. Moreover, ARNT knockdown also inhibited other EGF-induced c-Jun/Sp1 mediated gene expression, such as p21( WAF1/CIP1 ). Our results reveal a novel mechanism by which ARNT acts as a modulator to bridge the c-Jun/Sp1 interaction and plays a role in EGF-mediated gene expression under normoxic conditions.