Factor AP-1 Research Articles

IL-1β Activates the Transcription Factors NF-IL6, NF-IL6α, and AP-1 in Human Enterocytes

IL-1β Activates the Transcription Factors NF-IL6, NF-IL6α, and AP-1 in Human Enterocytes

Retinoic Acid and Its Receptors Repress the Expression and Transactivation Functions of Nur77: A Possible Mechanism for the Inhibition of Apoptosis by Retinoic Acid

Nur77 (NGFI-B) is an orphan nuclear receptor that has been implicated in activation-induced T-cell apoptosis. Retinoids, potent immune modulators, were shown to inhibit the activation-induced apoptosis of immature thymocytes and T-cell hybridomas. To illustrate the mechanism of the inhibition, we examined the effects of retinoic acid (RA) on the expression and transactivation functions of Nur77 in the human peripheral blood mononuclear cells and the human T-cell leukemia, Jurkat. All-trans-RA remarkably repressed the DNA binding and transcriptional induction of Nur77. Among the two potential trans-acting factors that activate Nur77 gene promoter, i.e., AP-1 and related serum response factor (RSRF), all-trans-RA repressed DNA binding and reporter gene activity of AP-1 but not that of RSRF, suggesting that the inhibition may be mediated through AP-1. We also demonstrated a posttranscriptional regulation of Nur77 function by retinoid receptors by showing that transactivation activity of Nur77 was significantly inhibited by cotransfection of RARα or RXRα. Nur77 bound RARα or RXRα in both yeast and mammalian two-hybrid tests, suggesting that direct protein–protein interaction between these receptors may mediate the inhibition. Taken all together, we demonstrated that RA repressed Nur77 function through multiple mechanisms that may provide the basis for RA inhibition on the apoptosis of activated T-lymphocytes.

Signaling angiogenesis via p42/p44 MAP kinase cascade.

Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the p42/p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp-1/AP-2 factors bind. Activation of p42/p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2 tension, hypoxia inducible factor-1 alpha (HIF-1 alpha), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that p42/p44MAPKs stoichiometrically phosphorylate HIF-1 alpha in vitro and that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of p42/p44MAPKs. Finally, we demonstrated that the regulation of p42/p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where p42/p44 MAP kinases exert a determinant action.

Acetaldehyde production from ethanol regulates activation of transcription factors NF-KB and AP-1 in pancreatic acinar cells

Acetaldehyde production from ethanol regulates activation of transcription factors NF-KB and AP-1 in pancreatic acinar cells

Regulation of collagenase-3 gene expression in osteoblastic and non-osteoblastic cell lines.

Collagenase-3 expression in osteoblastic (UMR 106-01, ROS 17/2.8) and non-osteoblastic cell lines (BC1, NIH3T3) was examined. We observed that parathyroid hormone (PTH) induces collagenase-3 expression only in UMR cells but not in BC1 (which express collagenase-3 constitutively) or ROS and NIH3T3 cells. Since we know from UMR cells that the AP-1 factors and Cbfa1 are required for collagenase-3 expression, we analyzed the expression and PTH regulation of these factors by gel shift and Northern blot analysis in all cell lines. Gel mobility shift with a [(32)P]-labeled collagenase-3 AP-1 site probe indicated the induction of c-Fos in osteoblastic cells upon PTH treatment. While c-fos was induced in UMR cells, both c-fos and jun B were induced in ROS cells. Since Jun B is inhibitory of Fos and Jun in the regulation of the rat collagenase-3 gene in UMR cells, it is likely that high levels of Jun B prevent PTH stimulation of collagenase-3 in ROS cells. When we carried out gel shift analysis with a [(32)P]-labeled collagenase-3 RD (runt domain) site probe and Northern blot analysis with a Cbfa1 specific probe, we have observed the presence of Cbfa1 in both osteoblastic and non-osteoblastic cell lines, but there was no change in the levels of Cbfa1 RNA or protein in these cells under either control conditions or PTH treatment. From our studies above, it is evident that the expression of collagenase-3 and its regulation by PTH in osteoblastic and non-osteoblastic cells may be influenced by differential temporal stimulation of the AP-1 family members, especially c-Fos and Jun B along with the potential for posttranslational modification(s) of Cbfa1.

Progesterone Receptor Isoforms, PR-B and PR-A, in Breast Cancer: Correlations with Clinicopathologic Tumor Parameters and Expression of AP-1 Factors

In the present study, we used Western blot analysis to determine the expression of the progesterone receptor (PR) isoforms, PR-B and PR-A, in breast tumors (n = 53), and correlated the expression patterns of the two isoforms with the clinicopathological parameters of these tumors and with expression of the AP-1 family of transcription factors. Expression of the two PR isoforms correlated significantly with each other, indicating that the expression of the two isoforms is probably regulated in a correlated fashion. Expression of both isoforms correlated significantly with expression of the estrogen receptor (ER). Furthermore, expression of PR-B was found to correlate significantly with the absence of ErbB2/neu. For the AP-1 factors, Fra-1 expression showed an inverse correlation with PR-B expression. In contrast, expression of FosB correlated significantly with expression of both isoforms, and the association was stronger with PR-B expression. An analysis of the ratio of expression of the two isoforms showed that most of the tumors expressed PR-A levels which were equal or higher than the corresponding PR-B expression levels (together 94% of the analyzed tumors) indicating that, in mammary carcinomas, a predominance of the PR-A isoform over the PR-B isoform seems to be the case. While there was no statistically significant correlation with age, staging and histological type, expression of both isoforms correlated with a more differentiated phenotype (G1/G2 grading). However, this association was stronger for PR-B. Also, a PR-A ≤ PR-B expression level was associated with G1/G2 grading, while a PR-A > PR-B expression level showed an association with a more undifferentiated phenotype (G3 grading). The expression level of the two PR isoforms might prove to be of prognostic and/or predictive value, especially since the two isoforms have been shown to be functionally different and to modulate the response of tumor cells to progestins and antiprogestins differently.

Progesterone Receptor Isoforms, PR-B and PR-A, in Breast Cancer: Correlations with Clinicopathologic Tumor Parameters and Expression of AP-1 Factors

In the present study, we used Western blot analysis to determine the expression of the progesterone receptor (PR) isoforms, PR-B and PR-A, in breast tumors (n = 53), and correlated the expression patterns of the two isoforms with the clinicopathological parameters of these tumors and with expression of the AP-1 family of transcription factors. Expression of the two PR isoforms correlated significantly with each other, indicating that the expression of the two isoforms is probably regulated in a correlated fashion. Expression of both isoforms correlated significantly with expression of the estrogen receptor (ER). Furthermore, expression of PR-B was found to correlate significantly with the absence of ErbB2/neu. For the AP-1 factors, Fra-1 expression showed an inverse correlation with PR-B expression. In contrast, expression of FosB correlated significantly with expression of both isoforms, and the association was stronger with PR-B expression. An analysis of the ratio of expression of the two isoforms showed that most of the tumors expressed PR-A levels which were equal or higher than the corresponding PR-B expression levels (together 94% of the analyzed tumors) indicating that, in mammary carcinomas, a predominance of the PR-A isoform over the PR-B isoform seems to be the case. While there was no statistically significant correlation with age, staging and histological type, expression of both isoforms correlated with a more differentiated phenotype (G1/G2 grading). However, this association was stronger for PR-B. Also, a PR-A ≤ PR-B expression level was associated with G1/G2 grading, while a PR-A > PR-B expression level showed an association with a more undifferentiated phenotype (G3 grading). The expression level of the two PR isoforms might prove to be of prognostic and/or predictive value, especially since the two isoforms have been shown to be functionally different and to modulate the response of tumor cells to progestins and antiprogestins differently.

Involvement of AP-1 and steroidogenic factor (SF)-1 in the cAMP-dependent induction of human adrenocorticotropic hormone receptor (ACTHR) promoter.

Adrenocorticotropic hormone receptor (ACTHR) is expressed predominantly in the adrenal glands, and its expression is upregulated by its own ligand, ACTH, via a cAMP-dependent pathway. In the present study, we characterized the 5'-regulatory region of human ACTHR gene to elucidate the molecular mechanisms underlying its adrenal-specific and ACTH/cAMP-dependent expression. The promoter region (-1017/+47 when the transcription start site is regarded as + 1) and its serial 5'-deletions (-764/+47, -503/+-47, -214/+47 and -56/+47) were ligated into the upstream of a luciferase (luc) reporter gene. These constructs were transfected into adrenocortical Y1 cells or non-adrenal JEG3 and Cos-1 cells. In all the cell lines, the luc activity gradually increased with serial 5'-deletions and the maximum activity was conferred by - 56/+ 47. However, the magnitude of luc activity of each deletion construct in non-adrenal cells was much less than that in Y1 cells, suggesting that the promoter functions in an adrenal-specific manner. We identified two Steroidogenic Factor (SF)-1-binding sites at -209 and -35. Electrophoretic mobility shift assay (EMSA) demonstrated that both sites bind to SF-1. Mutation of both sites significantly decreased the activity of -214/+47 promoter in Y1 cells. Transfection of SF-1-expressing plasmid into non-adrenal cells significantly increased the promoter activity, suggesting that SF-1 plays a role in the tissue-specific expression of human ACTHR gene. We identified the region, -764 to -503, that was required for the forskolin/cAMP responsiveness of the promoter. This region contains one AP-1 site. EMSA revealed that the binding of AP-1 to this site increased significantly upon treatment of Y1 cells with forskolin. Mutation of the site abolished the forskolin-responsiveness. In non-adrenal cells, the forskolin-responsiveness was observed only when SF-1-expressing plasmid was cotransfected. This is the first demonstration that both AP-1 and SF-1 are required for the cAMP-dependent induction of human ACTHR gene.

Tissue factor expression in vital organs during murine traumatic shock: role of transcription factors AP-1 and NF-kappaB.

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation that has been shown to have a role in the pathophysiology of sepsis and reperfusion injury. The purpose of this study was to investigate TF expression in vital organs and to determine possible regulatory mechanisms of TF expression in the lung during traumatic shock in rats. Noble-Collip drum trauma was induced in anesthetized Sprague-Dawley rats. Anesthetized rats without trauma served as controls. TF activity was measured in plasma and lung tissue. TF messenger RNA (mRNA) was measured in the lung, liver, and small intestine using ribonuclease protection assays. Electromobility shift assays were used to quantify binding of nuclear extracts from lung to TF-specific consensus domains for transcription factors NF-kappaB and AP-1. TF activity in plasma increased up to 14-fold and +232% in the lung (P < 0.001 for plasma and lung) 2 h after trauma. TF mRNA level was significantly increased in the lungs (P < 0.01), small intestine (P < 0.01), and liver (P < 0.05) 1 h after trauma compared to sham-operated control rats. TF mRNA expression continued to increase in the lungs and the liver (both, P < 0.001) 2 h after trauma TF sequence-specific complex binding to AP-1 and NF-kappaB domains was enhanced in the lungs of trauma rats (+395%, P < 0.001 and +168%, P < 0.001, respectively). These results suggest that TF may play an important role in the pathophysiology of severe trauma and that regulatory elements AP-1 and NF-kappaB may be involved in the regulation of TF mRNA expression in traumatic shock.

Lack of negative influence on the cellular transcription factors NF-kappaB and AP-1 by the nef protein of human immunodeficiency virus type 1.

In order to investigate the molecular mechanism of the reported negative effect of the Nef protein of human immunodeficiency virus type 1 (HIV-1) on the cellular transcription factors NF-kappaB and AP-1, human T cell lines (both populations and subclones) expressing the nef gene from HIV-1 clone pNL432 were constructed. Functional expression of the nef gene was confirmed by downregulation of CD4 and MHC class I proteins on the cell surface as measured by fluorescence-activated cell sorter analysis. However, contrary to previous reports, no significant difference was found in the induced level of NF-kappaB and AP-1 activity between nef(+) and nef(-) cell lines upon stimulation by phorbol 12-myristate 13-acetate and phytohaemagglutinin, as measured by transient transfection and electromobility shift assays. These data indicate that the Nef protein does not have a negative effect on the induction of NF-kappaB and AP-1.

CHOP enhancement of gene transcription by interactions with Jun/Fos AP-1 complex proteins.

The transcription factor CHOP (C/EBP homologous protein 10) is a bZIP protein induced by a variety of stimuli that evoke cellular stress responses and has been shown to arrest cell growth and to promote programmed cell death. CHOP cannot form homodimers but forms stable heterodimers with the C/EBP family of activating transcription factors. Although initially characterized as a dominant negative inhibitor of C/EBPs in the activation of gene transcription, CHOP-C/EBP can activate certain target genes. Here we show that CHOP interacts with members of the immediate-early response, growth-promoting AP-1 transcription factor family, JunD, c-Jun, and c-Fos, to activate promoter elements in the somatostatin, JunD, and collagenase genes. The leucine zipper dimerization domain is required for interactions with AP-1 proteins and transactivation of transcription. Analyses by electrophoretic mobility shift assays and by an in vivo mammalian two-hybrid system for protein-protein interactions indicate that CHOP interacts with AP-1 proteins inside cells and suggest that it is recruited to the AP-1 complex by a tethering mechanism rather than by direct binding of DNA. Thus, CHOP not only is a negative or a positive regulator of C/EBP target genes but also, when tethered to AP-1 factors, can activate AP-1 target genes. These findings establish the existence of a new mechanism by which CHOP regulates gene expression when cells are exposed to cellular stress.

Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway.

The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stress-activated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway.

Re-expression of SPR1 in Breast Cancer Cells by Phorbol 12-Myristate 13-Acetate (PMA) or UV Irradiation Is Mediated by the AP-1 Binding Site in the SPR1 Promoter

Invasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype accompanying normalization of the expression of such genes may be exploited therapeutically if gene expression can be specifically modulated by drugs or other treatments. Considering that genes are coordinately regulated in complex networks, it is likely that the expression of multiple genes can be simultaneously modulated in tumor cells by drugs acting on the signal transduction pathway that regulates their expression. The SPR1 gene is associated with differentiation and its expression is down-regulated or inactivated in malignant cells. Analysis of the SPR1 promoter showed that down-regulation of SPR1 expression in breast tumor cells occurs at the level of transcription. SPR1 presents an example of class II genes, since its expression was up-regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ultraviolet (UV) irradiation. The SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cell lines compared to normal mammary epithelial cell strains. Differential expression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region -619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. Up-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays. The mechanism of down-regulation of SPR1 expression in breast tumor cells was investigated. It was found that the -619 to +15 upstream promoter region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion analysis and mutagenesis, two upstream cis-acting promoter elements were identified. Our data indicate that the AP-1 element located between -139 and -133 acts as a major enhancer of SPR1 transcription only in normal mammary epithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In addition, a transcriptional repressor was identified that binds unknown factor(s) and is active in both normal and tumor breast cells. Inhibitor function was mapped to a 35-bp element located from -178 to -139 upstream of the human SPR1 mRNA start site. The expression of SPR1 could be induced in the 21MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irradiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expression of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA. Our results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory element active in both normal and tumor cells. Furthermore, our results provide a basis for therapeutic manipulation of down-regulated genes, such as SPR1, in human cancers.

Identification of a functional transcriptional factor AP-1 site in the sheep interferon τ gene that mediates a response to PMA in JEG3 cells

To examine regulatory mechanisms of sheep interferon tau (oIFNtau) gene expression, potential enhancer/silencer elements of the oIFNtau gene were examined using a transient transfection system with oIFNtau gene-chloramphenicol acetyltransferase (oIFNtau-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5'-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNtau gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNtau were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNtau-SV40-CAT transactivation. A subsequent study with the oIFNtau-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNtau-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNtau gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNtau-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNtau gene transactivation.

Identification of a functional transcriptional factor AP-1 site in the sheep interferon τ gene that mediates a response to PMA in JEG3 cells

To examine regulatory mechanisms of sheep interferon τ (oIFNτ) gene expression, potential enhancer/silencer elements of the oIFNτ gene were examined using a transient transfection system with oIFNτ gene-chloramphenicol acetyltransferase (oIFNτ-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5ʹ-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNτ gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNτ were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNτ-SV40-CAT transactivation. A subsequent study with the oIFNτ-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNτ-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNτ gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNτ-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNτ gene transactivation.

Physiology and molecular genetics of multidrug resistance in Entamoeba histolytica.

Entamoeba histolytica presents the evolutionarily conserved multidrug-resistance (MDR) phenotype, discovered in mammalian cells. MDR cells overexpress the membrane P-glycoprotein, which excludes unrelated drugs from the cytoplasm. E. histolytica mutants exhibit cross-resistance to unrelated drugs, which are pumped out from the cytoplasm. In drug-resistant trophozoites, the constitutively expressed EhPg1 gene appears to be up-regulated by a C/EBP-like factor and a multiprotein complex that were not found in drug-sensitive trophozoites. The drug-induced EhPgp5 gene, on the other hand, appears to be up-regulated by AP-1 and HOX factors. Here we review the main physiological and molecular facts of the MDR phenotype in E. histolytica. Copyright 1999 Harcourt Publishers Ltd.

Attenuation of NF-κB Signaling Response to UVB Light during Cellular Senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-κB-mediated signaling is a major defensive system against various environmental challenges. The aim of this study was to find out whether replicative senescence affects the response of the NF-κB signaling pathway to UVB light in human WI-38 and IMR-90 fibroblasts. The exposure of early passage fibroblasts to UVB light inhibited the proliferation and induced a flat phenotype similar to that observed in replicatively senescent fibroblasts not exposed to UVB light. The UVB radiation dose used (153 mJ/cm2) did not induce apoptosis in either early or late passage WI-38 fibroblasts. UVB exposure induced a prominent activation of the NF-κB signaling pathway both in early and in late passage WI-38 and IMR-90 fibroblasts. Interestingly, the response to UVB light was significantly attenuated in late passage fibroblasts. This attenuation was most prominent in DNA binding activities of nuclear NF-κB complexes. Similar senescence-related attenuation was also observed in the DNA binding activities of nuclear AP-1 and Sp-1 factors after UVB treatment. Immunoblotting and -cytochemistry showed an increase in nuclear localization of p50 and p65 components of NF-κB complexes. Supershift experiments showed that the specific NF-κB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IκBα showed a marked decrease at protein level but an increase in phosphorylation after UVB treatment. Transient transfection assays with TK5-CAT and TK10-CAT plasmids carrying NF-κB-responsive sites of the TNFα promoter were used to analyze the functional activity of the NF-κB complexes. Results showed that UVB exposure induced an increase in NF-κB-driven CAT expression both in early and in late passage fibroblasts though the response was significantly stronger in early passage fibroblasts. Our results show that the induction of NF-κB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.

Activation of a low pH-dependent nuclease by apoptotic agents.

Intracellular acidification caused by agents such as UV(C), etoposide or ceramide accompanies the progression of apoptosis. It is suggested that cellular acidosis may set favorable conditions for a dormant, low pH-dependent (acidic) nuclease, which could be involved in intranucleosomal genome degradation, a hallmark of programmed cell death. Here we show that exposure of HL-60 cells to acidotic/apoptotic agents results in the several-fold activation of a novel low pH-dependent (acidic) nuclease activity, as revealed by zymography. Its activity, which resides in nuclei, is associated with four polypeptides with apparent Mr of 56, 48, 45 and 40 kDa. Treatment of HeLa cells with UV(C) or ceramide causes also the up-regulation of an acidic nuclease activity which is represented by 70 and 62 kDa polypeptides. These observations suggest that acidic nuclease activation can be induced by the same apoptotic agents in different cell types. In HL-60 cells, acidic nuclease up-regulation triggered by acidotic agents follows the induction of AP-1 transcription factor active complexes and accompanies the progression of apoptosis. Inhibition of AP-1 factor activity caused by either anti-caspase/anti-acidotic agent Zn2+ or curcumin, an inhibitor of AP-1 binding to DNA and c-jun synthesis, protects cells from genome destruction. Acidic nuclease activation, however, is only partially inhibited by these factors. We propose that (i) the up-regulation of an acidic nuclease activity is governed by a regulatory pathway different from that responsible for AP-1 factor induction, caspases activation and intracellular acidification, and (ii) activation of an acidic nuclease does not cause any deleterious effects when AP-1 transcription factor induction, caspases activation and intracellular acidification are down-regulated. Thus, the acidic nuclease up-regulation alone is not a sufficient prerequisite for apoptosis.

Differential expression of AP-2alpha and AP-2beta in the developing chick retina: repression of R-FABP promoter activity by AP-2.

Retinal fatty acid binding protein (R-FABP) is the avian counterpart of murine brain FABP implicated in glial cell differentiation and neuronal cell migration. R-FABP is highly expressed in the undifferentiated retina and brain of chick embryos. We have previously shown by in vitro studies that the AP-2 transcription factor binds to a consensus AP-2 binding site in the R-FABP promoter region. Based on the expression pattern of AP-2 in the developing retina and on mutational analysis of the AP-2 binding site in DNA transfection experiments, we proposed that AP-2 could be involved in the down-regulation of R-FABP transcription. Here, we describe the cDNA isolation of two members of the AP-2 family expressed in the chick retina, AP-2alpha and AP-2beta. We show that R-FABP mRNA and the AP-2 factors are expressed in mutually exclusive patterns in the differentiating retina: whereas AP-2alpha and AP-2beta are selectively expressed either in amacrine, or in amacrine and horizontal cells, respectively, R-FABP mRNAis found in Muller glial cells and/or bipolar cells. Furthermore, a decrease in R-FABP-dependent expression is obtained upon cotransfection of primary retinal cultures with AP-2 expression vectors and a CAT reporter construct. The early and cell-specific expression of AP-2alpha and AP-2beta in the developing retina suggest a role for this transcription factor family in the early steps of amacrine and horizontal cell differentiation. Repression of the R-FABP gene in these cells may be an important component of their developmental program.

NF-κB or AP-1-Dependent Reporter Gene Expression Is Not Altered in Human U937 Cells Exposed to Power-Line Frequency Magnetic Fields

A number of studies have reported that human leukemia cells respond to exposure to power-line frequency electromagnetic fields (EMFs), providing evidence for an EMF-induced signaling pathway involving activation of protein tyrosine kinases (PTKs), phospholipase-Cy and protein kinase C (PKC). Because activation of PKC is also important in the signaling pathways that regulate the transcription factors NF-kappaB and AP-1, we evaluated the effect of exposure to a 60 Hz EMF on NF-kappaB or AP-1-dependent reporter gene expression in cells of the human promonocytic U937 leukemia cell line. Reporter genes were electroporated into U937 cells and activation of the NF-kappaB or AP-1 signaling pathway was evaluated by measuring chloramphenicol acetyltransferase (CAT) protein by CAT ELISA. In contrast to the effects of well-understood chemical or biological agents, the exposure to magnetic-field intensities of 0.08, 0.1, 1.0 or 1.3 mT had no effect on the NF-kappaB or AP-1 signaling pathways.