Activator Protein-1 Family Of Transcription Factors Research Articles

Airborne particulate matter (PM10) induces cell invasion through Aryl Hydrocarbon Receptor and Activator Protein 1 (AP-1) pathway deregulation in A549 lung epithelial cells.

Particulate matter with an aerodynamic size ≤ 10μm (PM10) is a risk factor for lung cancer development, mainly because some components are highly toxic. Polycyclic aromatic hydrocarbons (PAHs) are present in PM10, such as benzo[a]pyrene (BaP), which is a well-known genotoxic and carcinogenic compound to humans, capable of activating AP-1 transcription factor family genes through the Aryl Hydrocarbon Receptor (AhR). Because effects of BaP include metalloprotease 9 (MMP-9) activation, cell invasion, and other pathways related to carcinogenesis, we aimed to demonstrate that PM10 (10µg/cm2) exposure induces the activation of AP-1 family members as well as cell invasion in lung epithelial cells, through AhR pathway. The role of the AhR gene in cells exposed to PM10 (10µg/cm2) and BaP (1µM) for 48h was evaluated using AhR-targeted interference siRNA. Then, the AP-1 family members (c-Jun, Jun B, Jun D, Fos B, C-Fos, and Fra-1), the levels/activity of MMP-9, and cell invasion were analyzed. We found that PM10 increased AhR levels and promoted its nuclear localization in A549 treated cells. Also, PM10 and BaP deregulated the activity of AP-1 family members. Moreover, PM10 upregulated the secretion and activity of MMP-9 through AhR, while BaP had no effect. Finally, we found that cell invasion in A549 cells exposed to PM10 and BaP is modulated by AhR. Our results demonstrated that PM10 exposure induces upregulation of the c-Jun, Jun B, and Fra-1 activity, the expression/activity of MMP-9, and the cell invasion in lung epithelial cells, effects mediated through the AhR. Also, the Fos B and C-Fos activity were downregulated. In addition, the effects induced by PM10 exposure were like those induced by BaP, which highlights the potentially toxic effects of the PM10 mixture in lung epithelial cells.

Members of the AP-1 Family of Transcription Factors Regulate the Expression of Gja1 in Mouse GC-1 Spermatogonial Cells

Gap junctions, mainly formed by Gja1 (Connexin43), play an essential role in the regulation of proliferation and differentiation of spermatogonia in the testis. Regulation of the abundance of Gja1 in spermatogonia involves various processes, including gene transcription, mRNA maturation, protein synthesis, post-translational modifications, plasma membrane integration and protein degradation. However, gene expression of Gja1 is abnormally decreased in most testicular germ cell tumors. Hence, a better understanding of the mechanisms of transcriptional regulation of Gja1 in spermatogonia is essential to understand how the loss of its expression occurs during the development of testicular cancer. As in other cell types, activator protein-1 (AP-1) transcription factors may be involved in such regulatory process. Thus, AP-1 members were overexpressed in GC-1 cells to assess their impact on Gja1 expression. We showed that Jun and Fosl2 cooperate to activate the Gja1 promoter in GC-1 cells. Furthermore, the recruitment of Jun to the proximal region (−153 to +46 bp) of the Gja1 promoter has been confirmed via chromatin immunoprecipitation. Protein kinase A and calcium-calmodulin protein kinase I also contribute to the activation of Gja1 expression by improving the cooperation between AP-1 factors. Therefore, the reduction in Gja1 expression in testicular germ cell tumors may involve a loss of cooperation between AP-1 factors.

The Ascidia Ciona robusta Provides Novel Insights on the Evolution of the AP-1 Transcriptional Complex.

The Activator Protein-1 transcription factor family (AP-1) transcriptional complex is historically defined as an early response group of transcription factors formed by dimeric complexes of the Jun, Fos, Atf, and Maf bZIP proteins that control cell proliferation and differentiation by regulating gene expression. It has been greatly investigated in many model organisms across metazoan evolution. Nevertheless, its complexity and variability of action made its multiple functions difficult to be defined. Here, we place the foundations for understanding the complexity of AP-1 transcriptional members in tunicates. We investigated the gene members of this family in the ascidian Ciona robusta and identified single copies of Jun, Fos, Atf3, Atf2/7, and Maf bZIP-related factors that could have a role in the formation of the AP-1 complex. We highlight that mesenchyme is a common cellular population where all these factors are expressed during embryonic development, and that, moreover, Fos shows a wider pattern of expression including also notochord and neural cells. By ectopic expression in transgenic embryos of Jun and Fos genes alone or in combination, we investigated the phenotypic alterations induced by these factors and highlighted a degree of functional conservation of the AP-1 complex between Ciona and vertebrates. The lack of gene redundancy and the first pieces of evidence of conserved functions in the control of cell movements and structural organization exerted by these factors open the way for using Ciona as a helpful model system to uncover the multiple potentialities of this highly complex family of bZIP transcription factors.

Emerging Role of AP-1 Transcription Factor JunB in Angiogenesis and Vascular Development.

Blood vessels are essential for the formation and maintenance of almost all functional tissues. They play fundamental roles in the supply of oxygen and nutrition, as well as development and morphogenesis. Vascular endothelial cells are the main factor in blood vessel formation. Recently, research findings showed heterogeneity in vascular endothelial cells in different tissue/organs. Endothelial cells alter their gene expressions depending on their cell fate or angiogenic states of vascular development in normal and pathological processes. Studies on gene regulation in endothelial cells demonstrated that the activator protein 1 (AP-1) transcription factors are implicated in angiogenesis and vascular development. In particular, it has been revealed that JunB (a member of the AP-1 transcription factor family) is transiently induced in endothelial cells at the angiogenic frontier and controls them on tip cells specification during vascular development. Moreover, JunB plays a role in tissue-specific vascular maturation processes during neurovascular interaction in mouse embryonic skin and retina vasculatures. Thus, JunB appears to be a new angiogenic factor that induces endothelial cell migration and sprouting particularly in neurovascular interaction during vascular development. In this review, we discuss the recently identified role of JunB in endothelial cells and blood vessel formation.

MicroRNA 199a-5p induces apoptosis by targeting JunB

MicroRNAs participate in a variety of physiological and pathophysiological processes in various organs including the heart. Our previous work revealed that the level of miR-199a-5p was significantly higher in failing hearts than in control hearts. However, whether it is associated with the progression of heart failure (HF) and mediates cardiomyocyte apoptosis remained unclear. In the present study, we used various biochemical and molecular biological approaches to investigate the changes in miR-199a-5p levels in failing hearts in a rat model induced by acute myocardial infarction. We found that miR-199a-5p levels in the heart increased with the progression of HF, and overexpression of miR-199a-5p significantly increased apoptosis in untreated H9C2 cells and potentiated angiotensin II-induced apoptosis. Thus, our results indicate that miR-199a-5p is involved in the progression of HF and mediates cardiomyocyte apoptosis. We also confirmed that JunB, a member of the activator protein-1 transcription factor family, is one of direct targets of miR-199a-5p via a dual-luciferase reporter assay and mutagenesis on the 3′ untranslated region of the JunB gene. Consistent with the above findings, overexpression of JunB in H9c2 cells suppressed cell apoptosis. Based on our findings, miR-199a-5p induces apoptosis by targeting JunB.

MEF2C Interacts With c-FOS in PTH-Stimulated Mmp13 Gene Expression in Osteoblastic Cells.

Parathyroid hormone (PTH) regulates the transcription of many genes in the osteoblast. One of these genes is Mmp13, which is involved in bone remodeling and early stages of endochondral bone formation. Previously, we reported that PTH induces Mmp13 transcription by regulating the dissociation of histone deacetylase 4 (HDAC4) from runt-related transcription factor 2 (Runx2), and the association of the HATs, p300, and p300/CREB binding protein (CBP)-associated factor. It is known that, in addition to Runx2, HDAC4 binds to the transcription factor, myocyte-specific enhancer factor 2c (MEF2C), and represses its activity. In this work, we investigated whether MEF2C participates in PTH-stimulated Mmp13 gene expression in osteoblastic cells and how it does so. Knockdown of Mef2c in UMR 106-01 cells repressed Mmp13 messenger RNA expression and promoter activity with or without PTH treatment. Chromatin immunoprecipitation (ChIP) assays showed that MEF2C associated with the Mmp13 promoter; this increased after 4 hours of PTH treatment. ChIP-reChIP results indicate that endogenous MEF2C associates with HDAC4 on the Mmp13 promoter; after PTH treatment, this association decreased. From gel shift, ChIP, and promoter-reporter assays, MEF2C was found to associate with the activator protein-1 (AP-1) site without directly binding to DNA and had its stimulatory effect through interaction with c-FOS. In conclusion, MEF2C is necessary for Mmp13 gene expression at the transcriptional level and participates in PTH-stimulated Mmp13 gene expression by increased binding to c-FOS at the AP-1 site in the Mmp13 promoter. The observation of MEF2C interacting with a member of the AP-1 transcription factor family provides knowledge of the functions of HDAC4, c-FOS, and MEF2C.

C-Abl-mediated tyrosine phosphorylation of JunB is required for Adriamycin-induced expression of p21.

The non-receptor-type tyrosine kinase c-Abl functions as a cytoplasmic signal transducer upon activation of cell-surface receptors. c-Abl is also involved in DDR (DNA-damage response), which is initiated in the nucleus, whereas its molecular functions in DDR are not fully understood. In the present study, we found that c-Abl phosphorylates JunB, a member of the AP-1 (activator protein 1) transcription factor family. Because JunB was suggested to be involved in DDR, we analysed the role of c-Abl-mediated phosphorylation of JunB in DDR. We first analysed phosphorylation sites of JunB and found that c-Abl majorly phosphorylates JunB at Tyr(173), Tyr(182) and Tyr(188). Because c-Abl promotes expression of the cyclin-dependent kinase inhibitor p21 upon stimulation with the DNA-damaging agent Adriamycin (doxorubicin), we analysed the involvement of JunB in Adriamycin-induced p21 expression. We found that JunB suppresses p21 induction through inhibition of its promoter activity. The phosphomimetic JunB, which was generated by glutamic acid substitutions at the phosphorylation sites, failed to repress p21 induction. Recruitment of JunB to the p21 promoter was promoted by Adriamycin stimulation and was further enhanced by co-treatment with the c-Abl inhibitor imatinib. The phosphomimetic glutamic acid substitutions in JunB or Adriamycin treatment impaired the JunB-c-Fos transcription factor complex formation. Taken together, these results suggest that, although JunB represses p21 promoter activity, c-Abl phosphorylates JunB and conversely inhibits its suppressive role on p21 promoter activity upon Adriamycin stimulation. Therefore JunB is likely to be a key target of c-Abl in expression of p21in Adriamycin-induced DDR.

C-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs

CpG methylation in mammalian DNA is known to interfere with gene expression by inhibiting the binding of transactivators to their cognate sequence motifs or recruiting proteins involved in gene repression. An Epstein–Barr virus-encoded transcription factor, Zta, was the first example of a sequence-specific transcription factor that preferentially recognizes and selectively binds DNA sequence motifs with methylated CpG residues, reverses epigenetic silencing and activates gene transcription. The DNA binding domain of Zta is homologous to c-Fos, a member of the cellular AP-1 (activator protein 1) transcription factor family, which regulates cell proliferation and survival, apoptosis, transformation and oncogenesis. We have identified a novel AP-1 binding site termed meAP-1, which contains a CpG dinucleotide. If methylated, meAP-1 sites are preferentially bound by the AP-1 heterodimer c-Jun/c-Fos in vitro and in cellular chromatin in vivo. In activated human primary B cells, c-Jun/c-Fos locates to these methylated elements in promoter regions of transcriptionally activated genes. Reminiscent of the viral Zta protein, c-Jun/c-Fos is the first identified cellular member of the AP-1 family of transactivators that can induce expression of genes with methylated, hence repressed promoters, reversing epigenetic silencing.

ATF4 interacts with Abro1/KIAA0157 scaffold protein and participates in a cytoprotective pathway

Abro1 (Abraxas brother 1), also known as KIAA0157, is a scaffold protein that recruits various polypeptides to assemble the BRISC (BRCC36 isopeptide) deubiquitinating enzyme (DUB) complex. The BRISC enzyme has a Lys63-linked deubiquitinating activity and is comprised of four known subunits: MERIT40 (mediator of Rap80 interactions and targeting 40kDa), BRE (brain and reproductive organ-expressed), BRCC36 (BRCA1/BRCA2-containing complex, subunit 3) and Abro1. We have previously shown that Abro1 has a cytoprotective role that involves the BRISC DUB complex acting on specific Lys63-linked polyubiquitinated substrates. In this report we identify three members of the AP-1 (activating protein-1) family, the ATF4, ATF5 (activating transcription factor) and JunD proteins, as specific interactors of Abro1. The function of ATF4–Abro1 interaction was investigated under normal conditions as well as under cellular stress. Abro1 is predominantly cytoplasmic, but during cellular stress it enters the nucleus and co-localizes with ATF4. Furthermore, this interaction with ATF4 is necessary and essential for the cytoprotective function of Abro1 following oxidative stress. The ability of Abro1 to specifically interact with a number of transcription factors suggests a new mechanism of regulation of the BRISC DUB complex. This regulation involves the participation of at least three known members of the AP-1 family of transcription factors.

Fra-1 negatively regulates lipopolysaccharide-mediated inflammatory responses

Stimulation of macrophages with a Toll-like receptor ligand, LPS, facilitates gene expression. The activator protein-1 (AP-1) family of transcription factors mediates these responses. However, c-Fos, a member of the AP-1 family, has been shown to inhibit LPS-induced gene expression in macrophages. In this study, we analyzed the role of Fos-related antigen-1 (Fra-1), another member of the AP-1 family of transcription factors, in LPS-induced responses in RAW264.7 macrophages. Fra-1 was induced in LPS-stimulated macrophages with delayed time kinetics compared with c-Fos. Lentiviral introduction of Fra-1 blocked LPS-induced expression of pro-inflammatory mediators at the protein and mRNA levels. A Fra-1 mutant, which lacks the basic leucine zipper domain required for heterodimer formation and DNA binding, did not inhibit LPS-induced responses. c-Fos bound to the AP-1-binding site early, but afterward it was replaced by Fra-1 in LPS-stimulated macrophages. Over-expression of Fra-1 induced its association with Jun proteins and stable DNA binding from an early time point following LPS stimulation. These findings indicate that Fra-1 suppresses LPS-induced mRNA expression by binding to the AP-1-binding site. RNAi-mediated knockdown of Fra-1 in RAW264.7 macrophages resulted in enhanced LPS-induced expression of a subset of genes. Thus, like c-Fos, Fra-1 negatively regulates LPS-induced responses in RAW264.7 macrophages.

Distribution of cells expressing Jun and Fos proteins and synthesizing DNA in the adrenal cortex of hypophysectomized rats: regulation by ACTH and FGF2

Protein expression of the early response genes, jun and fos, has been suggested to play an important role in the in vitro and in vivo proliferation of adrenal cells. To elucidate the immunolocalization of proliferative cells and the patterns of adrenal gland expression of members of the activating protein-1 (AP-1) family of oncogenes, we used hypophysectomized rats. The effects of adrenocorticotropic hormone (ACTH) and fibroblast growth factor 2 (FGF2) on Fos and Jun protein expression were investigated, and DNA synthesis was assessed by using bromodeoxyuridine (BrdU) incorporation. No change was detectable in the adrenal cortex at 2 days after hypophysectomy, although a reduction occurred in the number of BrdU-positive cells in the zona fasciculata. This hypophysectomy-induced early phase of adrenal cortex atrophy in the zona fasciculata was correlated with JunB protein induction, suggesting the formation of an inhibitory AP-1 complex. Accumulation of c-Jun/JunD and c-Fos/FosB, but not of JunB, in the zona fasciculata and zona reticularis implied that, after ACTH stimulation, these proteins were the principal AP-1 components in these zones. In these same zones, ACTH increased BrdU-positive cell counts, indicating that the composition of the AP-1 complex in these zones was proliferation-related. However, FGF2 induced an antagonistic modulation of the response to ACTH, by reducing the numbers of Jun-/Fos-positive cells and inhibiting DNA synthesis. Our results implicate the AP-1 family of transcription factors (in particular, the dynamics within the Jun protein family) in the regulation of cell control during ACTH-induced proliferation of the adrenal cortex.

Immunohistochemical Jun/Fos protein localization and DNA synthesis in rat adrenal cortex after treatment with ACTH or FGF2

In vitro and in vivo studies have suggested that the expression of the early response genes for Jun and Fos proteins plays an important role in adrenal cell proliferation. In order to study the expression pattern of the activating protein-1 (AP-1) family of oncogenes in the adrenal gland, we have used immunohistochemistry to localize Jun and Fos protein expression in rat adrenal cortex infused in situ with adrenocorticotropic hormone (ACTH), fibroblast growth factor 2 (FGF2), or both. The expression of AP-1 factors has been found to be correlated with in vivo ACTH and FGF2 proliferation in rats treated with dexamethasone and bromodeoxyuridine (BrdU). Induction of c-Jun and c-Fos in the zona fasciculata and of FosB in the zona reticularis suggests that, after ACTH stimulation, these proteins are the main AP-1 components in these zones. In vivo, ACTH increases BrdU-positive cells in the zona fasciculata and zona reticularis suggesting that the composition of AP-1 complexes in these zones is correlated with proliferation. Patterns of Fos and Jun induction by FGF2 do not resemble those after ACTH induction. However, in isolation, neither affects the zona glomerulosa. In the zona fasciculata, and more so in the zona reticularis, FGF2 modulates responses to ACTH, reducing the numbers of Jun-positive cells, Fos-positive cells, and DNA synthesis. This indicates that FGF2 antagonizes ACTH, and that ACTH thus controls the trophic effect independently of exogenous FGF2. Our results implicate the AP-1 family of transcription factors in the regulation of cell progression and the control of ACTH-induced proliferation in the zona fasciculata and zona reticularis.

Delayed Wound Healing and Epidermal Hyperproliferation in Mice Lacking JunB in the Skin

The cutaneous response to injury and stress comprises a temporary change in the balance between epidermal proliferation and differentiation as well as an activation of the immune system. Soluble factors play an important role in the regulation of these complex processes by coordinating the intercellular communication between keratinocytes, fibroblasts, and inflammatory cells. In this study, we demonstrate that JunB, a member of the activator protein-1 transcription factor family, is an important regulator of cytokine expression and thus critically involved in the cutaneous response to injury and stress. Mice lacking JunB in the skin develop normally, indicating that JunB is neither required for cutaneous organogenesis, nor homeostasis. However, upon wounding and treatment with the phorbol ester 12-O-decanoyl-phorbol-13-acetate, JunB-deficiency in the skin likewise resulted in pronounced epidermal hyperproliferation, disturbed differentiation, and prolonged inflammation. Furthermore, delayed tissue remodelling was observed during wound healing. These phenotypic skin abnormalities were associated with JunB-dependent alterations in expression levels and kinetics of important mediators of wound repair, such as granulocyte macrophage colony-stimulating factor, growth-regulated protein-1, macrophage inflammatory protein-2, and lipocalin-2 in both the dermal and epidermal compartment of the skin, and a reduced ability of wound contraction of mutant dermal fibroblasts in vitro.

Fos- and Jun-related transcription factors are involved in the signal transduction pathway of mechanical loading in condylar chondrocytes

The chondrocytes of the articular condylar cartilage proliferate, hypertrophy and ultimately undergo apoptosis (programmed cell death), being replaced by osteoblasts. Converging results consolidate activator protein-1 (AP-1) transcription factor as the pivotal downstream effector in the early response of stress-sensitive cells to mechanical loading, and the Fra-1, Fra-2, JunB and JunD members of the AP-1 transcription factor family, as mediators in bone remodelling and apoptotic phenomena. The aim of the present study was to examine the involvement of the Fra-1, Fra-2, JunB and JunD proteins in the biochemical response of functionally loaded mandibular condylar cartilage, and the subsequent initiation of cartilage maturation and apoptotic phenomena. Thirty, female, 14-day-old Wistar rats were assigned to two groups: one group was fed a soft diet and the other a hard diet. At day 21 after weaning, experimental animals from both groups were killed at 6, 12 and 48 hours and their condyles harvested. The condylar cartilage of both groups was immunostained using specific antibodies against Fra-1, Fra-2, JunB and JunD. Statistical analysis of the data revealed over-expression of Fra-1, Fra-2, JunB and JunD proteins in all stages of differentiation of chondrocytes derived from the mandibular condylar cartilage of animals fed on a hard diet. Moreover, the involvement of these proteins significantly increased with time in both groups. Since the aforementioned proteins play key roles in remodelling phenomena of bone and cartilage tissue, influencing pivotal cellular functions such as maturation, differentiation and apoptosis, the results of the present study suggest that mandibular condylar chondrocytes sense functional loading changes and respond by induction of proteins associated with biological phenomena that ultimately influence the growth of the condylar cartilage.

Hydrogen Peroxide Stimulates Proliferation and Migration of Human Prostate Cancer Cells through Activation of Activator Protein-1 and Up-regulation of the Heparin Affin Regulatory Peptide Gene

It is becoming increasingly recognized that hydrogen peroxide (HP) plays a role in cell proliferation and migration. In the present study we found that exogenous HP significantly induced human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) seems to be involved in the stimulatory effect of HP, because the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, HP significantly increased HARP mRNA and protein amounts in a concentration- and time-dependent manner. Curcumin and activator protein-1 (AP-1) decoy oligonucleotides abrogated both HP-induced HARP expression and LNCaP cell proliferation and migration. HP increased luciferase activity of the 5'-flanking region of the HARP gene introduced in a reporter gene vector, an effect that was abolished when even one of the two putative AP-1 binding sites of the HARP promoter was mutated. The effect of HP seems to be due to the binding of Fra-1, JunD, and phospho-c-Jun to the HARP promoter. These results support the notion that HARP is important for human prostate cancer cell proliferation and migration, establish the role of AP-1 in the up-regulation of HARP expression by low concentrations of HP, and characterize the AP-1 dimers involved.

Expression Pattern of the Activating Protein-1 Family of Transcription Factors in Gestational Trophoblastic Lesions

The human placenta is an endocrine tissue with a unique capacity for rapid, but tightly controlled, proliferation and invasion. Gestational trophoblastic diseases (GTDs) are placental pathologies with endocrine activity and partially malignant potential and include hydatidiform moles, placental site nodules, and tumors such as placental site trophoblastic tumor and choriocarcinomas. The activating protein-1 (AP-1) family of transcription factors is composed of the cellular homologs of the Jun and Fos oncoproteins, which are immediately involved in cellular proliferation, differentiation, and invasion processes and in the regulation of endocrine genes. The expression pattern of the AP-1 family in the normal human placenta has been recently described, where most of the factors were found in the extravillous (invasive) trophoblast. Their systematic expression in GTD has not been studied thus far. For this reason in this study, we investigated the expression pattern of the AP-1 family in GTDs and compared it with the expression in normal placenta using immunohistochemistry with specific polyclonal antibodies against all members of the AP-1 family (JunB, JunD, c-Jun, c-Fos, FosB, Fra-1, Fra-2). Immunohistochemistry was performed on normal human placentas (positive control) and on 28 cases of GTD including 7 choriocarcinomas and 21 hydatidiform moles. In the normal placenta and in hydatidiform molar samples, most AP-1 factors (especially c-Jun, JunD, and Fra2) were expressed in the intermediate (extravillous) trophoblast. In addition, in molar lesions, strong expression was found in trophoblasts proliferating from the surface of villi. There was only a weak expression of JunB and Fra2 in small fractions of villous cyto- and syncytiotrophoblast nuclei. In choriocarcinomas, there was a strong expression for c-Jun, JunD, Fra1, and Fra2. The specific localization to extravillous trophoblasts and their expression pattern in GTDs indicate that the AP-1 family of transcription factors might be implicated in regulating proliferation and invasion of trophoblasts and play a role in the pathogenesis of GTDs.

Protein Kinase Cζ Phosphorylates Nuclear Factor of Activated T Cells and Regulates Its Transactivating Activity

Although several isoforms of protein kinase C (PKC) have been implicated in T lymphocyte activation events, little is known about their mode of action. To address the role of PKCzeta in T cell activation, we have generated Jurkat T cell transfectants expressing either the wild type (J-PKCzeta) or "kinase-dead" mutant (J-PKCzeta(mut)) versions of this protein. Expression of PKCzeta but not PKCzeta(mut) increased transcriptional activation mediated by the NF-kappaB or nuclear factor of activated T cells (NFAT). PKCzeta cooperates with calcium ionophore and with NFAT1 or NFAT2 proteins to enhance transcriptional activation of a NFAT reporter construct. However, neither NFAT nuclear translocation nor DNA binding were in J-PKCzeta cells. Our results show that PKCzeta enhanced transcriptional activity mediated by Gal4-NFAT1 fusion proteins containing the N-terminal transactivation domain of human NFAT1. Interestingly, PKCzeta synergizes with calcineurin to induce transcriptional activation driven by the NFAT1 transactivation domain. Co-precipitation experiments showed physical interaction between PKCzeta and NFAT1 or NFAT2 isoforms. Even more, PKCzeta was able to phosphorylate recombinant glutathione S-transferase-NFAT1 (1-385) protein. These data reveal a new role of PKCzeta in T cells through the control of NFAT function by modulating the activity of its transactivation domain.

Involvement of activator protein-1 in transcriptional regulation of the human mu-opioid receptor gene.

mu-Opioid receptors mediate such opioid effects as analgesia, euphoria, and immunomodulation. Gene expression of mu-opioid receptors can be modulated by various substances, including cytokines, hormones, and drugs. Some of these stimuli (e.g., IL-1beta and cocaine) have been shown to activate members of the AP-1 transcription factor family. In addition, transcription of the mu-opioid receptor gene is induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, which in turn is an activator of AP-1 transcription factors. This indicates that signaling pathways involving protein kinase C and activator protein 1 (AP-1) transcription factors are important for the specific expression pattern of the mu-opioid receptor gene. In this report, we show that TPA activates AP-1 as well as the transcription factor nuclear factor kappaB (NFkappaB) in the mu-opioid receptor expressing neuroblastoma cell line SH SY5Y. In transfection experiments performed in these cells, both factors trans-activate expression of reporter gene constructs containing the human mu-opioid receptor gene promoter. By excluding the effects of TPA on NFkappaB with the specific NFkappaB inhibitor sulfasalazine, AP-1 regulatory elements were localized. Two AP-1 elements, which differ in one nucleotide each from the classic AP-1 binding site, were delineated to positions -2388 and -1434 of the promoter. Independent of their orientation, these elements conferred TPA responsiveness on the heterologous thymidine kinase promoter. AP-1 binding to these elements was confirmed using electrophoretic mobility shift and immunoshift assays.

Expression pattern of the AP-1 family in endometrial cancer: correlations with cell cycle regulators.

To study the expression pattern and the role of the AP-1 (activating protein-1) family of transcription factors in endometrial carcinogenesis. We performed Western blot experiments with specific antibodies for each of the AP-1 proteins (c-jun, junB, junD, c-fos, fosB, fra-1, fra-2) with 41 endometrial carcinomas. Expression levels of the AP-1 factors were correlated with clinico-pathologic tumor parameters, steroid receptor status, Ki-67 expression and the expression levels of eight cell cycle regulatory proteins (cyclin D1, cyclin E, cyclin B1, cdk2, cdk4, p16, p21, and Rb). Of the seven AP-1 factors, three (c-fos, fra2, and junB) clearly showed higher expression levels in tumors when compared to matched normal endometrial samples. These factors also correlated significantly with cell cycle promoters (c-fos with cyclin E, cyclin B1, cdk2, and cdk4; fra-2 with cyclin B1; and junB with cyclin D1). Furthermore, high c-fos expression correlated with low ER and PR immunoreactivity and high grading (G3). On the other hand, correlations with classic cell cycle inhibitors (Rb, p16, p21) have also been observed for all AP-1 factors except c-jun and junD. Our results indicate that the AP-1 family of transcription factors is probably implicated in the regulation of cell cycle progression and control in endometrial carcinomas. In particular, c-fos might be an additional negative prognostic factor and/or implicated in tumor progression in endometrial cancer.

The transcription factor activator protein-1 is activated and interleukin-6 production is increased in interleukin-1beta-stimulated human enterocytes.

The intestinal mucosa is an active participant in the inflammatory and metabolic response to sepsis, endotoxemia, and other critical illness. The genes for various cytokines, e.g., interleukin 6 (IL-6), are regulated by multiple transcription factors, including nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1). In recent studies, treatment with IL-1beta of cultured Caco-2 cells, a human intestinal epithelial cell line, resulted in increased NF-kappaB DNA binding. The effect of IL-1beta on AP-1 activity in the enterocyte and the potential role of AP-1 in enterocyte IL-6 production are not known. We treated Caco-2 cells with IL-1beta and determined AP-1 activity by electrophoretic mobility shift assay (EMSA) and IL-6 production by enzyme-linked immunosorbent assay (ELISA). Treatment of Caco-2 cells with IL-1beta resulted in a dose- and time-dependent increase in AP-1 DNA binding. Supershift analysis suggests that activated AP-1 contained c-Jun, JunD, c-Fos, FosB, and Fra1 subunits. When Caco-2 cells were transiently transfected with an AP-1 luciferase reporter plasmid, stimulation with IL-1beta resulted in increased luciferase activity, suggesting that AP-1 DNA binding increased gene activation. Additional luciferase assays were performed with a plasmid containing a wild-type or AP-1-mutated IL-6 promoter. Stimulation of these cells with IL-1beta gave rise to results supporting the role of AP-1 in the regulation of IL-6 production. Geldanamycin, which has been shown in studies to inhibit AP-1 activation, blocked IL-1beta-induced AP-1 luciferase gene activation and IL-6 production. These results suggest that the AP-1 family of transcription factors is activated by IL-1beta in human enterocytes and that AP-1 may at least in part regulate IL-6 production in these cells.