AP-1 Binding Activity Research Articles

Basic Fibroblast Growth Factor-induced Neuronal Differentiation of Mouse Bone Marrow Stromal Cells Requires FGFR-1, MAPK/ERK, and Transcription Factor AP-1

It has been reported recently that bone marrow stromal cells (BMSCs) are able to differentiate into various neural cells both in vivo and in vitro (Egusa, H., Schweizer, F. E., Wang, C. C., Matsuka, Y., and Nishimura, I. (2005) J. Biol. Chem. 280, 23691-23697). However, the underlying mechanisms remain largely unknown. In this report, we have demonstrated that basic fibroblast growth factor (bFGF) alone effectively induces mouse BMSC neuronal differentiation. These differentiated neuronal cells exhibit characteristic electrophysiological properties and elevated levels of the neuronal differentiation marker, growth-associated protein-43 (GAP-43). To explore possible signaling pathways, we first analyzed the expression of various FGF receptors in mouse BMSCs. FGF receptor-1, -2, and -3 were detected, but only FGFR-1 was shown to be activated by bFGF. Small interfering RNA knock down of FGFR-1 in BMSCs significantly inhibited neuronal differentiation. Moreover, we have shown that the mitogen-activated protein kinase (ERK1/2) is persistently activated and blockage of ERK activity with the ERK-specific inhibitor U0126 prevents neuronal differentiation. It appears that activation of ERK cascade and neuronal differentiation of BMSCs induced by bFGF are independent of Ras activity but require functions of phospholipase C-gamma pathway. Lastly, we examined the role of the immediate-early transcription factors AP-1 and NF-kappaB and have found that phospholipase C-gamma-dependent c-Jun and ERK-dependent c-fos, but not the NF-kappaB, are strongly activated by bFGF, which in turn regulates the neuronal differentiation of BMSCs.

Homocysteine Induces Monocyte Chemoattractant Protein-1 Expression in Hepatocytes Mediated via Activator Protein-1 Activation

Hyperhomocysteinemia is characterized by abnormally high concentrations of homocysteine (Hcy) in the plasma. It is a metabolic disorder associated with dysfunction of several organs such as atherosclerosis, altered lipid metabolism, and liver injury. In this study we investigated the effect of Hcy on transcriptional regulation of monocyte chemoattractant protein-1 (MCP-1), a potent chemokine, expression in hepatocytes. Hyperhomocysteinemia was induced in rats by a high-methionine diet for 4 weeks. MCP-1 mRNA and protein levels were significantly elevated in the liver tissue homogenate and in hepatocytes of hyperhomocysteinemic rats. The role of transcription factors in MCP-1 expression was examined by electrophoretic mobility shift assay. Activation of activator protein (AP)-1 but not nuclear factor kappaB was detected in the liver tissue of those rats. Incubation of rat hepatocytes with Hcy (50-200 microm) caused a significant increase in AP-1 activation followed by an increase in intracellular MCP-1 mRNA expression and an elevation of MCP-1 protein secreted into the culture medium. Hcy markedly increased the DNA binding activity of human recombinant AP-1 (c-Fos and c-Jun proteins). The presence of a sulfhydryl group in Hcy was essential for Hcy-induced AP-1 activation. When hepatocytes were transfected with decoy AP-1 oligodeoxynucleotide to inhibit AP-1 activation, Hcy-induced MCP-1 mRNA expression was abolished. Further analysis revealed that increased hepatic MCP-1 expression was positively correlated with the serum MCP-1 level. These results suggest that Hcy-induced MCP-1 expression in the liver is mediated via AP-1 activation, which may contribute to chronic inflammation associated with hyperhomocysteinemia.

FGF‐1‐induced matrix metalloproteinase‐9 expression in breast cancer cells is mediated by increased activities of NF‐κB and activating protein‐1

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK.

Suppression of interleukin-2 gene expression by isoeugenol is mediated through down-regulation of NF-AT and NF-κB

Isoeugenol is a naturally occurring methoxyphenol found in a variety of foods and essential oils. We investigated the effect of isoeugenol on T-cell function and the regulatory mechanism underlying its effect. Isoeugenol and its structural analog eugenol suppressed the lymphoproliferative response to concanavalin A stimulation in B6C3F1 mouse splenocyte cultures. Isoeugenol inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced IL-2 mRNA expression and protein secretion in B6C3F1 mouse splenocytes, and in EL4.IL-2 mouse T-cells, as determined by real-time RT-PCR and ELISA, respectively. To further characterize the inhibitory mechanism of isoeugenol at the transcriptional level, we examined the DNA binding activity of the transcription factors for IL-2 using an electrophoretic mobility shift assay. Isoeugenol decreased the binding activity of NF-AT and NF-κB in PMA/Io-stimulated EL4.IL-2 cells, but no significant effect was observed for AP-1 or Oct binding activity. Western blot analysis showed that isoeugenol also decreased the nuclear translocation of cytoplasmic NF-AT and NF-κB. These results suggest that isoeugenol suppresses IL-2 production through a decrease of IL-2 mRNA expression and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT and NF-κB.

Transcriptional regulation of IL‐8 by iron chelator in human epithelial cells is independent from NF‐κB but involves ERK1/2‐ and p38 kinase‐dependent activation of AP‐1

We have shown that the bacterial iron chelator, deferoxamine (DFO), triggers inflammatory signals including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs) by activating the ERK1/2 and p38 kinase pathways. In this study we investigated the mechanisms involved in IL-8 generation by DFO, focusing on the transcription factors involved and the roles of both mitogen-activated protein kinases (MAPKs) in the transcription factor activation. Treatment of human epithelial HT-29 cells with DFO markedly up-regulated the expression of the essential components of the transcription factor AP-1 at a transcriptional level, while it minimally affected the expression of the NF-kappaB subunits. DFO also induced AP-1-dependent transcriptional activity in HT-29 cells, and this activity was further augmented by the wild-type c-Jun transfection. In contrast, the AP-1 activity by DFO was markedly decreased by the dominant-negative c-Jun transfection. Electrophoretic mobility shift assays revealed that DFO increases the specific binding of AP-1 but not of NF-kappaB. Such AP-1 binding and transcriptional activities were blocked by the inhibitors of the ERK1/2 and p38 kinase pathways, suggesting that both mitogen-activated protein kinases (MAPKs) lie upstream of AP-1. Besides its action on AP-1, DFO also induced the specific binding of other transcription factors such as CREB and Egr-1. In summary, our results indicate that iron chelator-induced IL-8 generation in IECs involves activation of ERK1/2 and p38 kinase and downstream activation of AP-1. A possible link between iron status and two additional transcription factors, that is, CREB and Egr-1, rather than NF-kappaB, was also suggested.

Effect of the α3β1 integrin on the IL-1 stimulated activation of c-Jun N-terminal kinase (JNK) in CACO-2 cells

Intestinal epithelial cells (IEC) are capable of responding to IL-1 stimulation by producing a variety of pro-inflammatory cytokines. Recently, we have found that binding of the α3β1 integrin may have a regulatory effect on IL-1 responses and intracellular signaling by suppressing cytokine secretion, mRNA expression and the downstream intracellular signaling events from IKK to NF-κB activation. In this study, we extend these findings by showing that treatment of the Caco-2 epithelial cells with a cross-linking anti-α3 integrin antibody resulted in a suppression in the levels of IL-1 induced AP-1 binding activity in nuclear extracts. Furthermore, suppressed levels of IL-1 induced c-Jun N-terminal kinase (JNK) phosphorylation and kinase activity were seen with the antibody treated cells. Cells cultured on purified laminin-5, the ligand for the α3β1 integrin, did not show significantly elevated levels of JNK phosphorylation after IL-1 stimulation while cells cultured on fibronectin yielded significantly elevated levels of IL-1 induced JNK phosphorylation. These results indicate that binding of the α3β1 integrin results in a suppression in the activation of the IL-1 induced intracellular signaling pathway from JNK to AP-1. This novel regulatory effect may be a potentially important mechanism to regulate IL-1 mediated responses by IEC.

TGF-β-induced transcriptional activation of MMP-2 is mediated by activating transcription factor (ATF)2 in human breast epithelial cells

We have previously shown that transforming growth factor (TGF)-β up-regulates matrix metalloproteinase (MMP)-2 leading to the induction of oncogenic signaling in preneoplastic MCF10A human breast epithelial cells. The present study investigated the mechanism of transcriptional regulation of MMP-2 by TGF-β in MCF10A cells. By using 5′ deletion constructs of MMP-2 promoter, we demonstrated that binding sites for p53, S1, AP-1 and Sp1, and to a lesser extent CREB, GCN-His and PEA3, were potential cis-acting elements for TGF-β-induced transcriptional activation of MMP-2 in MCF10A cells. Since activating transcription factor (ATF)2 was shown to mediate the TGF-β-induced cellular responses, we examined the involvement of ATF2 in TGF-β-activated MMP-2 gene transcription. TGF-β increased DNA binding activity of AP-1 in which ATF2 was involved as evidenced by electrophoretic mobility shift assay. TGF-β induced phosphorylation of ATF2 through p38 MAPK signaling. A dominant-negative (DN) ATF2 significantly inhibited the TGF-β-induced up-regulation of MMP-2, but not that of MMP-9, suggesting that ATF2 may be a transcription factor responsible for transcriptional activation of MMP-2 gene by TGF-β. Invasive and migratory phenotypes induced by TGF-β were significantly inhibited by DN ATF2, indicating a critical role of ATF2 in TGF-β-induced oncogenic progression of MCF10A cells. Taken together, this study demonstrates that ATF2 mediates the TGF-β-induced MMP-2 transcriptional activation, elucidating a molecular mechanism for the malignant progression of human breast epithelial cells exerted by TGF-β.

2-Hydroxycinnamaldehyde Inhibits SW620 Colon Cancer Cell Growth Through AP-1 Inactivation

Cinnamaldehyde derivatives isolated from Cinnamomum cassia have been widely used for treating dyspepsia, gastritis, and inflammatory disease as well as cancer. To investigate the anti-tumor activities of several cinnamaldehyde derivatives, we compared the inhibitory effect of cinnamaldehyde derivatives on cell growth and AP-1 transcriptional activity in SW620 human colon cancer cells since AP-1 is a transcriptional factor implicated to control cancer cell growth. Among the derivatives, 2’-hydroxycinnamaldehyde (HCA) most significantly inhibited cancer cell growth and AP-1 transcriptional activity in a dose-dependent manner with an IC50 value of 12.5 and 9 µg/ml, respectively. In further studies on the mechanism, we found that consistent with the inhibitory effect on cell growth, HCA dose-dependently (0 – 20 µg/ml) inhibited DNA binding activity of AP-1 accompanied with down regulation of c-Jun and c-Fos expressions. HCA also induced apoptotic cell death as well as expression of the apoptosis-regulating gene caspase-3, but inhibited the anti-apoptosis regulating gene bcl-2 in a dose-dependent manner. These results suggested that HCA has the most potent inhibitory effect against human colon cancer cell growth, and AP-1 may be an important target of HCA.

Inhibitory effects on Lipopolysaccharide‐induced cytokine production in mouse macrophage by Lithospermum erythrorhizon extracts

Inhibitory effects on Lipopolysaccharide-induced cytokine production in mouse macrophage by Lithospermum erythrorhizon extracts A variety of plant extracts have been shown to exert anti-inflammatory and chemopreventive acitivity via modulation of cytokine production. Lithospermum erythrorhizon (LE) has long been used in traditional oriental medicine for treatment of inflammation and other maladies. In the present study, we demonstrated the inhibitory effects of methanolic extracts from roots of LE on LPS-stimulated production of various inflammatory cytokines including IL-6, TNF-α, INF-γ and IL-1β. As an underlying mechanism of the inhibition, LE extracts reduced LPS-induced transactivation of transcription factors AP-1 as well as NF-κB in mouse macrophage cells. Electrophoretic mobility shift assay analysis indicated that LE extracts inhibited DNA binding activities of AP-1 and NF-κB. In addition, phosphorylation of IκB-α protein, subunit of NF-κB inhibitor was suppressed by LE extracts. Degradation of IκB-α proteins was decreased and translocation of p65 into nucleus was inhibited by LE extracts. Moreover, LE extracts inhibited c-jun N-terminal kinase and extracellular signal regulated signaling pathways. Our results suggest that the anti-inflammatory activity of LE may be mediated by signal transduction pathways leading to activation of AP-1 and NF-κB, which may be useful for chemoprevention of cancer or other inflammatory chromic diseases.

The role of sphingosine 1-phosphate in the TNF-α induction of IL-8 gene expression in lung epithelial cells

Tumor necrosis factor-α (TNF-α) is an important cytokine involved in the pathogenesis of inflammatory diseases of the lung. Interleukin-8 (IL-8), a C-X-C chemokine, is induced by TNF-α and initiates injury by acting as a chemoattractant for neutrophils and other immune cells. Although sphingolipids such as ceramide and sphingosine 1-phosphate (S1-P) have been shown to serve as signaling molecules in the TNF-α inflammatory response, their role in the TNF-α induction of IL-8 gene expression in lung epithelial cells is not known. We investigated the role of sphingolipids in the TNF-α induction of IL-8 gene expression in H441 lung epithelial cells. We found that TNF-α induced IL-8 mRNA levels by increasing gene transcription, and the stability of IL-8 mRNA was not affected. Exogenous S1-P but not ceramide or sphingosine increased IL-8 mRNA levels and IL-8 secretion. Dimethylsphingosine, an inhibitor of sphingosine kinase, partially inhibited TNF-α induction of IL-8 mRNA levels indicating the importance of intracellular increases in S1-P in the IL-8 induction. S1-P induction of IL-8 mRNA was due to an increase in gene transcription, and the stability of IL-8 mRNA was not affected. S1-P induction of IL-8 mRNA was associated with an increase in the binding activity of AP-1 but the activities of NF-κB and NF IL-6 were unchanged. S1-P induced the phosphorylation of ERK, p38 and JNK MAPKs. Pharmacological inhibitors of ERK and p38 but not JNK partly inhibited S1-P induction of IL-8 mRNA levels. These data show that increases in the intracellular S1-P partly mediate TNF-α induction of IL-8 gene expression in H441 lung epithelial cells via ERK and p38 MAPK signaling pathways and increased AP-1 DNA binding.

Mediation of aldose reductase in lipopolysaccharide-induced inflammatory signals in mouse peritoneal macrophages

Aldose reductase (AR; AKR1B1) a member of aldo-keto reductase super family, that we had shown earlier mediates cytotoxic signals induced by high glucose, cytokines and growth factors, also mediates the inflammatory signals induced by Gram-negative bacterial endotoxin, lipopolysaccharide (LPS). Inhibition of AR by three distinct AR inhibitors sorbinil, tolrestat or zopolrestat suppressed the LPS-induced production of inflammatory cytokines such as TNF-α, IL-6, IL-1β, IFN-γ, and chemokine MCP-1 in murine peritoneal macrophages. Inhibition of AR also prevented the production of nitric oxide, and prostaglandin E2 and expression of iNOS and Cox-2 proteins. The LPS-induced DNA binding activity of NF-κB and AP1 were significantly inhibited by AR inhibitors, and this effect was mediated through the inhibition of phosphorylation of IκB-α, IKK α/β and PKC. These results suggest the therapeutic use of AR inhibitors as anti-inflammatory drugs.

Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.

PROTEASOME-MEDIATED REGULATION OF CPG DNA- AND PEPTIDOGLYCAN-INDUCED CYTOKINES, INFLAMMATORY GENES, AND MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION

Our previous work demonstrated that the proteasome is central to most of genes induced by lipopolysaccharide. In this study, we evaluated the role of the proteasome in response to two other microbial stimuli, CpG DNA (bacterial DNA) and peptidoglycan (PG), by measuring the effect of proteasome inhibition on cytokine secretion, induction of inflammatory gene expression, and activation of mitogen-activated protein kinases (MAPK) in murine macrophages. Pretreatment of macrophage cultures with lactacystin, a well-established proteasome inhibitor, significantly repressed tumor necrosis factor alpha secretion and tumor necrosis factor alpha and interleukin 1 beta gene expression, blocked the degradation of IkappaB, and dysregulated phosphorylation of MAPK induced by CpG DNA or PG. With respect to MAPK, lactacystin blocked expression of PG- or CpG-induced phosphorylated ERK1 and ERK2 and increased expression of phosphorylated c-Jun amino-terminal kinase but had no significant effect on phosphorylated p38. Increased expression of phoshorylated c-Jun amino-terminal kinase did not lead to an increase in AP-1 binding activity. Collectively, these data strongly support the conclusion that the proteasome is a key regulator of the CpG DNA- and PG-induced signaling pathways.

Expression and regulation of the mu opioid peptide receptor in TPA-differentiated HL-60 promyelocytic leukemia cells

Cellular differentiation of immune cells involves an array of molecular events responsible for their commitment to cellular maturation. Treatment of HL-60 promyelocytic leukemia cells with 12- o-tetradecanoyl-phorbol-13-acetate (TPA) induces the cells to differentiate into monocyte/macrophage-like cells. In this study, following TPA treatment, there was a significant increase in mu opioid peptide receptor (MOPR) mRNA levels in the differentiated HL-60 cells as measured by quantitative-competitive RT-PCR (QC-RT-PCR) and real-time RT-PCR. Morphine′s inhibition of forskolin-induced intracellular cAMP confirmed the functionality of the MOPR. TPA-induced differentiation also significantly enhanced the binding activities of two transcriptional factors, AP-1 and NFkB. Prolonged treatment of the TPA-differentiated HL-60 cells with morphine down-regulated MOPR mRNA expression and decreased the binding activities of AP-1 and NFkB, both of which were naloxone reversible. Thus, the direct correlation between AP-1 and NFkB binding activities and MOPR expression in HL-60 cells following TPA-induced differentiation as well as in TPA-differentiated HL-60 cells given prolonged treatment with morphine suggests that transcriptional factors, such as AP-1 and NFkB, may play a role in the molecular mechanisms underlying regulation of MOPR expression in immune cells.

Involvement of JNKs and p38-MAPK/MSK1 pathways in H 2O 2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells

One of the most important challenges that cardiomyocytes experience is an increase in the levels of reactive oxygen species (ROS), i.e., during ischemia, reperfusion as well as in the failing myocardium. HOX-1 has been found to protect cells and tissues against oxidative damage; therefore, we decided to study the signalling cascades involved in its transcriptional regulation. HOX-1 mRNA levels were found to be maximally induced after 6 h of treatment with 200 μM H 2O 2 and remained elevated for at least 24 h. Inhibition of JNKs, p38-MAPK and MSK1 pathways, by pharmacological inhibitors, reduced HOX-1 mRNA levels in H 2O 2-treated H9c2 cells. In parallel, we observed that all three subfamilies of the mitogen-activated protein kinases (MAPKs) attained their maximal phosphorylation levels at 5–15 min of H 2O 2 treatment, with mitogen- and stress-activated-protein kinase 1 (MSK1) also being maximally phosphorylated at 15 min. H 2O 2-induced MSK1 phosphorylation was completely abrogated in the presence of the selective p38-MAPK inhibitor SB203580. In an effort to define possible substrates of MSK1, we found that ATF2 as well as cJun phosphorylation were equally induced after 30 min and 60 min, respectively, a response inhibited by SP600125 (JNKs inhibitor) and H89 (MSK1 inhibitor), indicating the involvement of these kinases in the observed response. This finding was further substantiated with the detection of a potential signalling complex composed of either p-MSK1 and p-cJun or p-MSK1 and p-ATF2 (co-immunoprecipitation). ATF2 and cJun are known AP1 components. Given the presence of an AP-1 site in HOX-1 promoter region, the activity of AP1 transcription factor was examined. Electrophoretic mobility shift assays performed showed a maximal upregulation of AP1 binding activity after 60 min of H 2O 2 treatment, which was significantly inhibited by SP600125 and H89. Our results show for the first time the potential role of JNKs, p38-MAPK and MSK1 in the mechanism of transducing the oxidative stress-signal to HOX-1, possibly promoting cell survival and preserving homeostasis.

Activating transcription factor‐2 (ATF2) mediates MMP‐2 transcriptional activation induced by p38 MAPK in breast epithelial cells

Mounting evidence suggests a role for matrix metalloproteinase (MMP)-2 in the malignant progression of breast cancer cells. We previously showed that H-Ras, but not N-Ras, induced invasion and migration of MCF10A human breast epithelial cells through H-Ras-specific activation of Rac-MKK3/6-p38 MAPK pathway resulted in MMP-2 up-regulation. In this study, we aimed to elucidate the transcriptional regulation of MMP-2 by p38 MAPK pathway leading to the invasive and migrative phenotypes of MCF10A breast epithelial cells. By using 5′ deletion mutant constructs of MMP-2 promoter, we showed that the AP-1 binding site is critical for the MMP-2 promoter activation in MKK6- and H-Ras-activated MCF10A cells. DNA binding and transcriptional activities of AP-1 were increased by MKK6 or H-Ras. We revealed the activating transcription factor (ATF)2 as a transcription factor for MMP-2 gene expression through binding to the functional AP-1 site. Activation of ATF2 by MKK6 or H-Ras was crucial for MMP-2 promoter activity as well as induction of invasive and migrative phenotypes in MCF10A cells. This is the first report revealing ATF2 as an essential transcription factor linking MKK3/6-p38 MAPK signaling pathway to MMP-2 up-regulation, providing evidence for a direct role of ATF2 activation in malignant phenotypic changes of human breast epithelial cells. [Supported by a grant (R01-2005-000-10596-0) from Korea Science & Engineering Foundation]

Effect of Cigarette Smoke on the Activation of Transcription Factors in Mouse Lung

The effect of cigarette smoke on in vitro and in vivo transcription factor activation in the lung was examined. We first examined if NF-κB is activated by cigarette smoke condensate (CSC) in vitro. Using two different lung cell lines (H1299 and H-838), we did not observe activation of NF-κB following CSC exposure. We next examined if cigarette smoke could activate NF-κB in vivo. Mice were exposed to cigarette smoke for 1, 2, 3, or 6 hr, and 2, 5, 10, 15, 20, 42, or 56 days (6 hr/day, 5 days/wk). For all times except 20 and 42 days, the DNA binding activity of NF-κB in the lung and liver was either not changed or decreased by exposure to cigarette smoke. For the 20- and 42-day time points, the DNA binding activity in the lung was slightly increased. We also examined two other transcription factors: AP-1 and hypoxia inducible factor (HIF). Similar to NF-κB, the DNA binding activity of AP-1 and HIF was slightly elevated at 42 days but decreased at 56 days. These studies show that cigarette smoke does not activate NF-κB, AP-1 or HIF in the lung. Cigarette smoke activation of other transcription factors in the lung is currently being studied. (Supported by Kentucky Lung Cancer Research Program)

Manganese modulates pro-inflammatory gene expression in activated glia

Redox-active metals are of paramount importance for biological functions. Their impact and cellular activities participate in the physiological and pathophysiological processes of the central nervous system (CNS), including inflammatory responses. Manganese is an essential trace element and it is required for normal biological activities and ubiquitous enzymatic reactions. However, excessive chronic exposure to manganese results in neurobehavioral deficits. Recent evidence suggests that manganese neurotoxicity involves activation of microglia or astrocytes, representative CNS immune cells. In this study, we assessed the molecular basis of the effects of manganese on the modulation of pro-inflammatory cytokines and nitric oxide (NO) production in primary rat cortical glial cells. Cultured glial cells consisted of 85% of astrocytes and 15% of microglia. Within the assayed concentrations, manganese was unable to induce tumor necrosis factor α (TNF-α) and inducible nitric oxide synthase (iNOS) expression, whereas it potentiated iNOS and TNF-α gene expression by lipopolysaccharide/interferon-γ-activated glial cells. The enhancement was accompanied by elevation of free manganese, generation of oxidative stress, activation of mitogen-activated protein kinases, and increased NF-κB and AP-1 binding activities. The potentiated degradation of inhibitory molecule IκB-α was one of underlying mechanisms for the increased activation of NF-κB by manganese. However, manganese decreased iNOS enzymatic activity possibly through the depletion of cofactor since exogenous tetrahydrobiopterin reversed manganese's action. These data indicate that manganese could modulate glial inflammation through variable strategies.

Differential regulation of the JNK/AP-1 pathway byS-adenosylmethionine and methylthioadenosine in primary rat hepatocytes versus HuH7 hepatoma cells

S-adenosylmethionine (AdoMet) and 5'-methylthioadenosine (MTA) exert a protective action on apoptosis induced by okadaic acid in primary rat hepatocytes but not in human transformed HuH7 cells. In the present work, we analyzed the role played by the JNK/activator protein (AP)-1 pathway in this differential effect. Okadaic acid induced the phosphorylation of JNK and c-Jun and the binding activity of AP-1 in primary hepatocytes, and pretreatment with either AdoMet or MTA prevented those effects. In HuH7 cells, pretreatment with either AdoMet or MTA did not affect JNK and c-Jun activation or AP-1 binding induced by okadaic acid. In both cell types, p38 was activated by okadaic acid, but neither AdoMet nor MTA presented a significant effect on its activity. Therefore, the differential effect of both AdoMet and MTA on the JNK/AP-1 pathway could explain their antiapoptotic effect on primary hepatocytes and the lack of protection they show against okadaic acid-induced apoptosis in hepatoma cells.

Inhibition of interleukin-4 production in activated T cells via the downregulation of AP-1/NF-AT activation by N-lauroyl- d-erythro-sphingosine and N-lauroyl- d-erythro-C 20-sphingosine

Allergic diseases are hypersensitivity disorders that are associated with the generation of specific immunoglobulin E (IgE) in response to environmental allergens. Interleukin (IL)-4, which is primarily produced by the CD4 + T cells, is an important stimulus for the switching of the antibody isotype to IgE in both mice and humans. In a previous study, we demonstrated that ceramide derivatives coupled with a lauroyl group exerted strong inhibitory effects on IL-4 production in T cells. In this study, we attempted to characterize the mechanisms underlying the inhibition of IL-4 production in T cells. Two ceramide derivatives, N-lauroyl- d-erythro-sphingosine and N-lauroyl- d-erythro-C 20-sphingosine (hereafter abbreviated as LES and LECS, respectively), were shown to significantly inhibit the production of IL-4 in both primary CD4 + T cells and EL4 T thymoma cells in a dose-dependent manner. LES and LECS also inhibited the activity of the IL-4 gene promoter in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells that had been transfected with an IL-4 promoter construct deleted of a P4 site harboring the AP-1 and NF-AT binding sites. Furthermore, LES and LECS inhibited the DNA binding activities of both AP-1 and NF-AT transcription factors. In addition, LES and LECS were demonstrated to suppress PMA-stimulated PKC activity, although they exerted no significant effects on the protein levels of the conventional PKCs. These results indicate that the ceramides, LES and LECS, may inhibit the production of IL-4 in the activated T cells, via the downregulation of AP-1/NF-AT activation and PKC activity.