Activator Protein-1 Function Research Articles

Bidirectional regulation between AP-1 and SUMOylation pathway genes modulates inflammatory signaling duringSalmonellainfection

Post-translational modifications (PTMs), such as SUMOylation, are known to modulate fundamental processes of a cell. Infectious agents such as Salmonella Typhimurium (STm), which causes gastroenteritis, utilize the PTM mechanism SUMOylation to hijack the host cell. STm suppresses host SUMO pathway genes UBC9 (also known as UBE2I) and PIAS1 to perturb SUMOylation for an efficient infection. In the present study, the regulation of SUMO pathway genes during STm infection was investigated. A direct binding of c-Fos (encoded by FOS), a component of activator protein-1 (AP-1), to promoters of both UBC9 and PIAS1 was observed. Experimental perturbation of c-Fos led to changes in the expression of both UBC9 and PIAS1. STm infection of fibroblasts with SUMOylation-deficient c-Fos (c-FOS-KOSUMO-def-FOS) resulted in uncontrolled activation of target genes, leading to massive immune activation. Infection of c-FOS-KOSUMO-def-FOS cells favored STm replication, indicating misdirected immune mechanisms. Finally, chromatin immunoprecipitation assays confirmed a context-dependent differential binding and release of AP-1 to and from target genes due to its phosphorylation and SUMOylation, respectively. Overall, our data point towards the existence of a bidirectional cross-talk between c-Fos and the SUMO pathway and highlight their importance in AP-1 function in STm infection and beyond. This article has an associated First Person interview with the first author of the paper.

Regulation and function of AP-1 in insulinoma cells and pancreatic β-cells

Cav1.2 L-type voltage-gated Ca2+ channels play a central role in pancreatic β-cells by integrating extracellular signals with intracellular signaling events leading to insulin secretion and altered gene transcription. Here, we investigated the intracellular signaling pathway following stimulation of Cav1.2 Ca2+ channels and addressed the function of the transcription factor activator protein-1 (AP-1) in pancreatic β-cells of transgenic mice. Stimulation of Cav1.2 Ca2+ channels activates AP-1 in insulinoma cells. Pharmacological and genetic experiments identified c-Jun N-terminal protein kinase as a signal transducer connecting Cav1.2 Ca2+ channel activation with gene transcription. Moreover, the basic region-leucine zipper proteins ATF2 and c-Jun or c-Jun-related proteins were involved in stimulus-transcription coupling. We addressed the functions of AP-1 in pancreatic β-cells analyzing a newly generated transgenic mouse model. These transgenic mice expressed A-Fos, a mutant of c-Fos that attenuates DNA binding of c-Fos dimerization partners. In insulinoma cells, A-Fos completely blocked AP-1 activation following stimulation of Cav1.2 Ca2+ channels. The analysis of transgenic A-Fos-expressing mice revealed that the animals displayed impaired glucose tolerance. Thus, we show here for the first time that AP-1 controls an important function of pancreatic β-cells in vivo, the regulation of glucose homeostasis.

AP-1 controls the p11-dependent antidepressant response

Selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed drugs for mood disorders. While the mechanism of SSRI action is still unknown, SSRIs are thought to exert therapeutic effects by elevating extracellular serotonin levels in the brain, and remodel the structural and functional alterations dysregulated during depression. To determine their precise mode of action, we tested whether such neuroadaptive processes are modulated by regulation of specific gene expression programs. Here we identify a transcriptional program regulated by activator protein-1 (AP-1) complex, formed by c-Fos and c-Jun that is selectively activated prior to the onset of the chronic SSRI response. The AP-1 transcriptional program modulates the expression of key neuronal remodeling genes, including S100a10 (p11), linking neuronal plasticity to the antidepressant response. We find that AP-1 function is required for the antidepressant effect in vivo. Furthermore, we demonstrate how neurochemical pathways of BDNF and FGF2, through the MAPK, PI3K, and JNK cascades, regulate AP-1 function to mediate the beneficial effects of the antidepressant response. Here we put forth a sequential molecular network to track the antidepressant response and provide a new avenue that could be used to accelerate or potentiate antidepressant responses by triggering neuroplasticity.

AP-1 Contributes to Chromatin Accessibility to Promote Sarcomere Disassembly and Cardiomyocyte Protrusion During Zebrafish Heart Regeneration.

The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur. To identify transcription factor regulators of the chromatin landscape, which promote cardiomyocyte regeneration in zebrafish, and investigate their function. Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that activator protein-1 (AP-1) binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the fbxl22 and ilk loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the AP-1 family members Junb and Fosl1 can promote changes in mammalian cardiomyocyte behavior in vitro. AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.

Identification of FRA-1 as a novel player in pancreatic cancer in cooperation with a MUC1: ERK signaling axis.

The MUC1 glycoprotein is overexpressed and aberrantly glycosylated in >90% of pancreatic ductal adenocarcinoma cases and impacts tumor progression by initiating downstream signaling through phosphorylation of its cytoplasmic tail. Previous studies have demonstrated that MUC1 alters expression of known targets of activator protein 1 (AP-1); however, no studies have evaluated the precise impact of MUC1 signaling on the activity and formation of AP-1. Given the known role of these proteins in modulating migration, invasion, and tumor progression, we explored the effects of MUC1 on AP-1 dimer formation and function. We determined that MUC1 increased the protein levels of c-Jun, the major component of AP-1, and promoted dimerization of c-Jun with the Fos-protein FRA-1. We demonstrate that FRA-1 acts as a potent mediator of migration and invasion in a manner that is modulated by signals through MUC1, which acts as a dominant regulator of specific AP-1 and FRA-1 target genes. Our results provide the first in vivo evidence of a FRA-1 mediated expression profile that impacts pancreatic tumor growth properties. In summary, we show that MUC1 enhancement of ERK activation influences FRA-1 activity to modulate tumor migration, invasion and metastasis in a subset of pancreatic cancer cases.

Host JDP2 expression in the bone marrow contributes to metastatic spread.

The c-Jun Dimerization Protein 2, JDP2, is a basic leucine zipper protein member of the activator protein-1 (AP-1) family of transcription factors. JDP2 typically suppresses gene transcription through multiple mechanisms and plays a dual role in multiple cellular processes, including cell differentiation and proliferation which is dependent on AP-1 function. Whereas the role of JDP2 expression within cancer cells has been studied, its role in stromal cells at the tumor microenvironment is largely unknown. Here we show that mice lacking JDP2 (JDP2-/-) display a reduced rate of metastasis in Lewis lung carcinoma (LLC) and polyoma middle T-antigen (PyMT) breast carcinoma mouse models. The replacement of wild-type bone marrow derived cells (BMDCs) with JDP2-deficient BMDCs recapitulates the metastatic phenotype of JDP2-/- tumor-bearing mice. In vitro, conditioned medium of wild-type BMDCs significantly potentiates the migration and invasion capacity of LLC cells as compared to that of JDP2-/- BMDCs. Furthermore, wild-type BMDCs secrete CCL5, a chemokine known to contribute to metastasis, to a greater extent than JDP2-/- BMDCs. The supplementation of CCL5 in JDP2-/- BMDC conditioned medium was sufficient to potentiate the invasion capacity of LLC. Overall, this study suggests that JDP2-expressing BMDCs within the tumor microenvironment contribute to metastatic spread.

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown. Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin9s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive. First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis. In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown.Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin’s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive.First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity.To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p < 0.01) compared with MM/ SCR control cells, when co-cultured with BMSCs in particular. Moreover, our preliminary data show that knockdown of JunB overcomes resistance of MM cells against doxorubicin as well as melphalan.Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p < 0.01) as well as MM cell proliferation. In ongoing experiments, the in vivo relevance of our in vitro data is evaluated in a xenograft mouse model inoculated with MM /JunB-ER-IRES-GFP and MM/ IRES-GFP cells. Finally, gene expression profiles on > 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis.In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. DisclosuresNo relevant conflicts of interest to declare.

Small molecule inhibitors targeting activator protein 1 (AP-1).

Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases.

The Role of Heterodimeric AP-1 Protein Comprised of JunD and c-Fos Proteins in Hematopoiesis

Activator protein-1 (AP-1) regulates a wide range of cellular processes including proliferation, differentiation, and apoptosis. As a transcription factor, AP-1 is commonly found as a heterodimer comprised of c-Jun and c-Fos proteins. However, other heterodimers may also be formed. The function of these dimers, specifically the heterodimeric AP-1 comprised of JunD and c-Fos (AP-1(JunD/c-Fos)), has not been elucidated. Here, we identified a function of AP-1(JunD/c-Fos) in Xenopus hematopoiesis. A gain-of-function study performed by overexpressing junD and c-fos and a loss-of-function study using morpholino junD demonstrate a critical role for AP-1(JunD/c-Fos) in hematopoiesis during Xenopus embryogenesis. Additionally, we confirmed that JunD of AP-1(JunD/c-Fos) is required for BMP-4-induced hematopoiesis. We also demonstrated that BMP-4 regulated JunD activity at the transcriptional regulation and post-translational modification levels. Collectively, our findings identify AP-1(JunD/c-Fos) as a novel hematopoietic transcription factor and the requirement of AP-1(JunD/c-Fos) in BMP-4-induced hematopoiesis during Xenopus hematopoiesis.

Dietary energy restriction, in part through glucocorticoid hormones, mediates the impact of 12‐O‐tetradecanoylphorbol‐13‐acetate on jun D and fra‐1 in sencar mouse epidermis

Dietary energy restriction (DER, 40% calorie reduction from fat and carbohydrate) inhibited mouse skin carcinogenesis and decreased 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced activator protein-1 (AP-1):DNA binding previously. This study measured protein levels of c-jun, jun B, jun D, c-fos, fra-1, and fra-2 and examined their contribution to AP-1:DNA binding by electrophoretic mobility shift assay (EMSA) with supershift analysis in the epidermis of control and DER Sencar mice exposed to TPA. TPA significantly increased c-jun, jun B, c-fos, fra-1, and fra-2 and decreased jun D within 3-6 h after treatment. AP-1:DNA binding reached a maximum 2.5-fold induction over controls 4 h after TPA treatment and antibodies to jun B, jun D, and fra-2 in the EMSA binding reaction resulted in supershifts in both acetone- and TPA-treated mice 1-6 h after treatment. The effect of corticosterone (CCS) and DER on the AP-1 proteins and on the composition of the AP-1:DNA complex was measured in adrenalectomized (adx) mice. DER reduced the TPA impact on jun D and enhanced the induction of fra-1. In addition, CCS-supplemented groups had significantly lower jun D and higher fra-2 than adx groups and sham groups. While sham animals treated with either acetone or TPA contained jun B, jun D, and fra-2 proteins in the AP-1:DNA complex by supershift analysis, fra-2 was no longer seen in adx DER animals. In summary, our study supports potential roles for jun D, jun B, and fra-1 in the DER regulation of AP-1 function in the Sencar mouse skin carcinogenesis model.

Inactivation of AP1 Proteins by a Nuclear Serine Protease Precedes the Onset of Radiation-Induced Fibrosing Alveolitis1

Radiation-induced lung damage comprises inflammation (alveolitis) as well as disturbed regulation of cell differentiation and proliferation (fibrosis). The transcriptional regulation of this process is poorly understood. One key transcription factor involved in the regulation of proliferation and differentiation is AP1 (activator protein 1). The present study examined changes in the DNA-binding activity of AP1 after irradiation and defined the underlying molecular mechanisms in an animal model. The right lungs of Fischer rats received a single radiation dose of 20 Gy. Lung tissue was tested for AP1 DNA-binding activity, AP1 mRNA, and levels of AP1 proteins as well as for c-Jun specific proteolytic activity. After an initial increase, the AP1 DNA-binding activity was completely lost starting at 5.5 weeks after irradiation, which is 2.5 weeks before the onset of fibrosing alveolitis. This was not caused by reduction of mRNA levels or size. Instead, a selective nuclear cleavage of c-Jun by a serine protease caused the loss of AP1 activity. Considering the central role of AP1 in cell proliferation and differentiation and the strict timely correlation to the onset of the disease, the complete loss of AP1 function is likely to play a critical role in radiation-induced fibrosing alveolitis.

Effect of Beclomethasone Dipropionate and Dexamethasone Isonicotinate on Lung Function, Bronchoalveolar Lavage Fluid Cytology, and Transcription Factor Expression in Airways of Horses with Recurrent Airway Obstruction

Glucocorticoid (GC) therapy is recognized to be effective for the treatment of recurrent airway obstruction (RAO) in horses. Anti-inflammatory properties of GC are thought to be mediated by suppression of inflammatory gene expression via inhibition of transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). The purpose of this study was to evaluate the effect of low-dose inhaled beclomethasone dipropionate and injectable dexamethasone 21-isonicotinate on clinical signs, pulmonary function, airway cytology, and activity of NF-kappaB and AP-1 in bronchial cells of RAO-affected horses. Seven horses with RAO were exposed to moldy hay until they developed airway obstruction on 3 separate occasions. In a crossover design, they were then treated with a placebo (injection on day 1), inhaled beclomethasone (500 microg q12h for 10 days), or dexamethasone (0.06 mg/kg, IM on day 1) and monitored for 10 days. Pulmonary function, bronchoalveolar lavage fluid cytology, and NF-kappaB and AP-1 activity in bronchial brushing cells were measured before (day 1) and after treatment (day 10). Treatment with beclomethasone resulted in significantly improved pulmonary function of RAO-affected horses compared with placebo and dexamethasone treatments. However, none of the treatments had an effect on bronchoalveolar lavage fluid cytology or NF-kappaB and AP-1 activity. These findings reveal that, in a model of severe RAO, the benefits of low-dose inhaled beclomethasone on pulmonary function are not accompanied by a decrease in airway inflammatory cells or a suppression of transcription factors NF-kappaB and AP-1 DNA-binding activity.

Function of AP-1 in Transcription of the Telomerase Reverse Transcriptase Gene (TERT) in Human and Mouse Cells

The transcriptional regulation of the human telomerase catalytic subunit (hTERT) plays a critical role in telomerase activity. Approximately 200 bp of the proximal core promoter is responsible for basic hTERT expression; however, the function of the distal regulatory elements remains unclear. The transcription factor activator protein 1 (AP-1) is involved in cellular proliferation, differentiation, carcinogenesis, and apoptosis and is expressed broadly in both cancer and normal cells. There are several putative AP-1 sites in the hTERT promoter, but their functions are unknown. The present study examined the regulatory role of AP-1 in hTERT gene transcription. Overexpression of AP-1 leads to transcriptional suppression of hTERT in cancer cells. The combination of c-Fos and c-Jun or c-Fos and JunD strongly suppresses hTERT promoter activity in transient-expression analyses. The hTERT promoter region between -2000 and -378 is responsible for this function. Gel shift and supershift analyses, as well as ChIP, show binding of JunD and c-Jun on two putative AP-1 sites within this region. Mutations in the AP-1 binding sites rescued suppressions caused by AP-1, suggesting this is a direct regulation of the hTERT promoter. In contrast, there was no effect on mTERT expression or mTERT promoter activity by AP-1 overexpression in mouse fibroblasts. The species-specific function of AP-1 in TERT expression may in part help explain the difference in telomerase activity between normal human and mouse cells.

Mechanism of glucocorticoid resistance

Many patients have a form of glucocorticoid resistance, yet the mechanism of this serious medical problem has not been established. Previous studies have shown that glucocorticoid resistance is associated with impairment of transcriptional factors, such as activator protein 1 (AP-1), but an integrated view that allows for significant variability in individual patient responses has been unclear. In the current study, the investigators examined murine and human T cells for their responsiveness to glucocorticoids in vitro, aiming to determine whether the degree of signaling during T cell activation, affected responsiveness. Notably, dexamethasone (Dex) did not promote substantial T-cell apoptosis. Rather, Dex suppressed T-cell receptor (TCR)–induced proliferation of naive CD4+ T cells through a mechanism involving downregulation of c-Fos expression and inhibition of AP-1, nuclear factor of activated T cells (NF-AT), and NF-kB transcriptional activity. Importantly, enhancement of TCR signaling by CD28- or IL-2–mediated costimulation blocked the suppressive effect of Dex on c-Fos expression and AP-1 function and restored cellular proliferation. In contrast, NF-kB remained inhibited by Dex. Using specific signaling inhibitors, the investigators demonstrated that costimulatory signaling via MAPK kinase (MEK) and extracellular signal–regulated kinase (ERK) was essential for the development of T-cell resistance to Dex. Selective blockade of MEK/ERK signal transduction abolished the costimulation-induced resistance. Thus, the amount of signaling through the MAPK pathway was critical in determining the effect of Dex on T-cell activation. These results imply that the degree of local costimulation through MEK/ERK, affected by variable processes (including antigenic exposure in individual patients), can modify the effect of glucocorticoids on T cells. These findings raise hope that MAPK inhibitors might have therapeutic utility for glucocorticoid resistance.

Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD4+ T cells.

Glucocorticoids have potent immunosuppressive properties, but their effects are often modulated by the conditions prevailing in the local immune milieu. In this study we determined whether the action of glucocorticoids is influenced by the degree of signaling during T cell activation. We found that dexamethasone (Dex) effectively suppressed T cell receptor-induced (TCR-induced) proliferation of naive CD4+ T cells, through a mechanism involving downregulation of c-Fos expression and inhibition of activator protein-1 (AP-1), nuclear factor of activated T cells (NF-AT), and NF-kappaB transcriptional activity. However, enhancement of TCR signaling by CD28- or IL-2-mediated costimulation abrogated the suppressive effect of Dex on c-Fos expression and AP-1 function and restored cellular proliferation. The amount of signaling through the MAPK pathway was critical in determining the effect of Dex on T cell activation. In particular, costimulatory signaling via MAPK kinase (MEK) and extracellular signal-regulated kinase (ERK) was essential for the development of T cell resistance to Dex. Selective blockade of MEK/ERK signal transduction abolished the costimulation-induced resistance. In contrast, transmission of IL-2 signals via STAT5 and CD28 signals via NF-kappaB remained inhibited by Dex. These results imply that the immune system, by regulating the degree of local costimulation through MEK/ERK, can modify the effect of glucocorticoids on T cells. Moreover, these findings suggest that MAPK inhibitors may offer a therapeutic solution for glucocorticoid resistance.

Schnurri-3 (KRC) interacts with c-Jun to regulate the IL-2 gene in T cells.

The activator protein 1 (AP-1) transcription factor is a key participant in the control of T cell proliferation, cytokine production, and effector function. In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell–specific coactivator proteins exist to selectively potentiate AP-1 function. Here, we describe that the expression of Schnurri-3, also known as κ recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene. Overexpression of KRC in transformed and primary T cells leads to increased IL-2 production, whereas dominant-negative KRC, or loss of KRC protein in KRC-null mice, results in diminished IL-2 production. KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1–dependent IL-2 gene transcription.

Suppression of insulin-induced AP-1 transactivation by menin accompanies inhibition of c-Fos induction.

The translation product of the MEN1 gene, menin, has been reported to suppress JunD-mediated activator protein-1 (AP-1) transactivation and inhibit Ras-mediated tumor formation, but its molecular mechanisms and physiologic significance have been poorly elucidated. To better understand the function of menin as a tumor suppressor, we examined the effect of menin on physiologically induced AP-1 activity. Overexpression of menin strongly suppressed insulin-induced AP-1 activity in CHO-IR cells, which express high levels of insulin receptor. We found that menin suppressed c-Fos induction at the transcriptional level, although that cannot explain the entire mechanism of AP-1 suppression by menin. Menin did not alter the expression levels of AP-1 proteins except c-Fos, phosphorylation of c-Jun and JunD and DNA binding properties of AP-1 proteins. Suppression of AP-1 activation by menin may be exerted through 2 independent mechanisms, direct inhibition on AP-1-mediated transcription and suppression of c-Fos induction. The molecular mechanism of inhibition of AP-1 function by menin needs further elucidation.

Effect of protein kinase inhibitors on the stretch-elicited c-Fos and c-Jun up-regulation in human PDL osteoblast-like cells.

Osteoblastic cells transduce signals of mechanical loading that plays a key role in maintaining bone formation. In an attempt to elucidate the biochemical events associated with the conversion of mechanical stress to biological outcome, we examined cultured human periodontal ligament (hPDL) osteoblastic cells exposed to continuous stretch, in terms of cellular parameters correlating known signaling cascades to the initial phase of osteoblast-specific transcriptional control. Time-course experiments revealed that mechanical stretch-loaded hPDL cells exhibit a very rapid and relatively sustained increase in the abundance of the immediate-early gene products, c-Fos and c-Jun, components of the activator protein-1 (AP-1) transcription factor. Moreover, this increase in protein levels was accompanied by hyperphosphorylation and thereby potentiation of c-Jun, the principal modulator of AP-1 activity. Importantly, these inductive effects were partly or completely abolished by pre-incubating the cells with SB 203580, PD 098059, and the novel compound Y-27632, inhibitors of p38 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), and Rho-associated protein kinase (RhoK), respectively. These results consolidate AP-1 as the pivotal downstream effector in the early response of hPDL cells to continuous mechanical stretching, via the coordinate stimulation of de novo synthesis and post-translational regulation of AP-1 proteins. This "integrating" function of AP-1 is mediated through a mechanotransduction circuit that incorporates elements of well-defined upstream signaling protein kinase systems.

Green Tea Polyphenol Stimulates a Ras, MEKK1, MEK3, and p38 Cascade to Increase Activator Protein 1 Factor-dependent Involucrin Gene Expression in Normal Human Keratinocytes

(-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea that efficiently reduces epidermal cancer cell proliferation. This inhibition is associated with a reduction in activator protein 1 (AP1) transcription factor level and activity. However, its effects on AP1 function in normal epidermal cells have not been extensively explored. Our present studies show that EGCG regulates normal keratinocyte function. To understand the mechanism of action, we examined the effects of EGCG on AP1 factor activity, MAPK signal transduction, and expression of the AP1 factor-regulated human involucrin (hINV) gene. EGCG increases hINV promoter activity in a concentration-dependent manner that requires the presence of an intact hINV promoter AP1 factor binding site. This response appears to be physiologic, as endogenous hINV gene expression is also increased. Fra-1, Fra-2, FosB, JunB, JunD, c-Jun, and c-Fos levels are increased by EGCG treatment, as is AP1 factor binding to hINV promoter AP1 site. Gel mobility shift studies show that this complex contains Fra-1 and JunD. Signal transduction analysis indicates that the EGCG response requires Ras, MEKK1, MEK3, and p38 kinases. Kinase assays and inhibitor studies suggest that p38delta is the p38 isoform responsible for the regulation. These changes are also associated with a cessation of cell proliferation and enhanced cornified envelope formation. These studies show that in normal human keratinocytes EGCG markedly increases, via a MAPK signaling mechanism, AP1 factor-associated responses.