Induction Of Transactivation Research Articles

INHIBITION OF REDD1 EXPRESSION FOR THE REDUCTION OF GLUCOCORTICOID-INDUCED SIDE EFFECTS

Glucocorticoids (GC ) have been an integral component of the treatment of leukemias and lymphomas for several decades. Specific cytotoxic effect of GC on transformed lymphoblasts mediates their use at the stage of the remission induction as well as consolidation of treatment. However, the main problem of the long-term GC use is the development of atrophic and metabolic side effects as well as GC resistance. The biological effects of GC are realized via activation of the glucocorticoid receptor (GR) by two mechanisms: transrepression (TR) associated with the therapeutic effects of GC , and transactivation (TA ), which mediates the development of metabolic and atrophic complications. It was demonstrated that an increase in the expression of the GC - dependent gene REDD1 associated with GC -induced skin, muscle and bone atrophy of the skin, muscle and bone tissue was realized via the induction of transactivation. Therefore, identification of potential inhibitors of REDD1 expression and study of their biological effects in combination with GC in models of leukemia and lymphoma is of particular interest. In our recent study we have selected a number of drugs from the class of PI 3K/Akt/mTO R modulators using bioinformatic screening. These drugs effectively inhibited REDD1 expression, modulated GR activity and shifted it towards transrepression, and prevented the development of GC -induced side effects in mice. Here we aimed to study the effects of potential inhibitors of REDD1 expression from different pharmacological groups, the compounds Emetine and CGP -60474, on leukemia and lymphoma cells in combination with GC . We demonstrated antitumor effect of the compounds in vitro, a decrease in the expression of TA -associated genes and an increase in TR induction. Further studies of the antitumor effects of REDD1 expression inhibitors (Emetine and CGP -60474 is a promising area of research.

COMPARATIVE ANALYSIS OF BIOLOGICAL EFFECTS OF SELECTIVE ACTIVATOR OF THE GLUCOCORTICOID RECEPTOR CPDA ON DIFFERENT SUBTYPES OF BREAST CANCER CELL LINES

Glucocorticoids (GCs) are often used as an adjuvant therapy to reduce the adverse effects of chemotherapy in breast cancer patients. Moreover, GCs can display pro-proliferative or anti-proliferative effects on BC cells depending on their molecular subtype. In addition, long-term use of GCs can induce drug resistance and tumor progression. The biological activity of GCs is mediated by glucocorticoid receptor (GR) via either transrepression or transactivation. The anti-inflammatory effects of GCs are thought to be due to transrepression, while side effects, drug resistance and tumor progression/metastasis are associated with transactivation. We have previously demonstrated that Compound A, a selective GR agonist (SEGRA), has a GR-dependent antitumor effect on blood cancer cells in vitro, not triggering the GR transactivation. This study was focused on the analysis of the CpdA activity in BC models in vitro. We demonstrated the antiproliferative effect of CpdA on BC cells and its ability to induce transrepression of GR-depended genes such as CCND1-3, COX-2, iNOS without the induction of transactivation. A comparative analysis showed that CpdA was an effective and safe alternative to dexamethasone in adjuvant chemotherapy for breast cancer.

Regulation of different human NFAT isoforms by neuronal activity.

Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear translocation extent and kinetics, and transcription activation capacities of alternative NFAT proteins vary.

MKL1 is an epigenetic modulator of TGF-β induced fibrogenesis

Transforming growth factor (TGF-β) induced activation of portal fibroblast cells serves as a primary cause for liver fibrosis following cholestatic injury. The underlying epigenetic mechanism is not clear. We studied the role of a transcriptional modulator, megakaryoblastic leukemia 1 (MKL1) in this process. We report here that MKL1 deficiency ameliorated BDL-induced liver fibrosis in mice as assessed by histological stainings and expression levels of pro-fibrogenic genes. MKL1 silencing by small interfering RNA (siRNA) abrogated TGF-β induced transactivation of pro-fibrogenic genes in portal fibroblast cells. TGF-β stimulated the binding of MKL1 on the promoters of pro-fibrogenic genes and promoted the interaction between MKL1 and SMAD3. While SMAD3 was necessary for MKL1 occupancy on the gene promoters, MKL1 depletion impaired SMAD3 binding reciprocally. TGF-β treatment induced the accumulation of trimethylated histone H3K4 on the gene promoters by recruiting a methyltransferase complex. Knockdown of individual members of this complex significantly weakened the binding of SMAD3 and down-regulated the activation of portal fibroblast cells. In conclusion, we have identified an epigenetic pathway that dictates TGF-β induced pro-fibrogenic transcription in portal fibroblast thereby providing novel insights for the development of therapeutic solutions to treat liver fibrosis.

Interactions of Chromatin Context, Binding Site Sequence Content, and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10−7) and correlated with stronger p53RE sequences (p<10−110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.

Olfactomedin 4 Mediates All-Trans Retinoic Acid Induced Growth Inhibition, Differentiation and Apoptosis in Myeloid Leukemia Cells and Inhibits 4E-BP1 Phosphorylation.

Abstract 2395Poster Board II-372 Introduction:All-trans retinoic acid (ATRA) has been shown to induce cellular differentiation and growth inhibition of acute promyelocytic leukemia cells. Clinical application of ATRA has proved extremely successful in inducing clinical remission in most acute promyelocytic leukemia patients. Although the mechanisms of retinoid-dependent gene transcription regulation are well understood, the target genes that mediate retinoid-induced biological responses still remain to be defined. Olfactomedin 4 (OLFM4, also called hGC-1 and GW112) is a member of olfactomedin-related glycoprotein family. It is constitutively expressed in myeloid cells and gastrointestinal tract. It has been recently reported that OLFM4 expression is up-regulated in stomach and colon cancer patients. The purpose of this study is to examine its expression in myeloid leukemia patients, its regulation by ATRA and potential biological functions in myeloid leukemia. Results:1) In this study, we found that OLFM4 expression was up-regulated in the peripheral leukocytes of chronic myeloid leukemia patients (91%, 22 cases) and acute myeloid leukemia patients (30%, 10 cases). OLFM4 expression in accelerated phase of chronic myeloid leukemia patients was significantly higher than that in chronic phase. 2) We identified that OLFM4 is a novel target gene of retinoic acids in myeloid leukemia cells. Treatment of HL-60 cells with ATRA and 9-cis-RA induced OLFM4 expression. The expression level of OLFM4 is correlated with the myeloid cell differentiation stage. Deletion analysis led to the identification of a positive retinoic acid response element (DR5) and a negative response element (DR1) within OLFM4 promoter. Furthermore, electrophoretic mobility-shift assays and transfection study in COS-7 cells demonstrated that RARα/RXRα binds to the DR5 site and mediates ATRA induced transactivation of OLFM4 promoter. 3) We showed that OLFM4 over-expression in HL-60 cells lead to growth inhibition, differentiation and apoptosis and potentates ATRA mediated these effects. Conversely, silencing of endogenous OLFM4 by lentiviral shRNA against OLFM4 in AML-193 cells reduces ATRA induced growth inhibition, differentiation and apoptosis. 4) We further investigated the molecular mechanism that OLFM4 is involved in leukemia cell growth and differentiation. We found that over-expression of OLFM4 in HL60 cells inhibited ATRA induced phosphorylation of translation repressor 4E-BP1. This inhibitory effect was further confirmed in 293T cells, in which over-expression of OLFM4 inhibits 4E-BP1 phosphorylation at all four phosphorylation sites (Thr37/46, Thr70 and Ser65). Application of lentiviral shRNA against OLFM4 in AML-193 increased phosphorylation of 4E-BP1 compared with control shRNA. Over-expression of OLFM4 in HL60 cells does not affect phosphorylation of Akt, p70S6 kinase, GSK3β and ERK1/2 . Conclusion:1) OLFM4 expression is up-regulated in chronic and acute myeloid leukemia patients and is correlated with the stage of chronic myeloid leukemia. 2) OLFM4 is a novel target gene of retinoic acids. RARα/RXRα binds to the DR5 site of OLFM4 promoter and mediates the ATRA induced transactivation of OLFM4. 3) OLFM4 mediates ATRA induced growth inhibition, differentiation and apoptosis of myeloid leukemia cells. 4) OLFM4 acts an inhibitor of 4E-BP1 phosphorylation down stream of Akt and mTOR, suggesting OLFM4 may inhibit protein synthesis that is observed with the differentiation of myeloid leukemia cells. Our results suggest that OLFM4 up-regulation in the leukocytes of leukemia patients might have a feedback effect to restrain cell growth, and induce differentiation and apoptosis rather than act as an initiator of leukemiagenesis. As OLFM4 is a secreted glycoprotein, it could represent a promising therapeutic agent in the treatment of myeloid leukemia patients. The effect of purified OLFM4 on leukemia cells is currently under investigation. Disclosures:No relevant conflicts of interest to declare.

Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay

The present study was aimed to develop a sensitive, fast and user friendly progesterone receptor transactivation assay using recombinant yeast cells, Saccharomyces cerevisiae, modified to express human progesterone receptor (PR) and progesterone response element (PRE) driving the expression of green fluorescent protein. Stimulation of cells with increasing concentrations of progesterone resulted in significant elevation in fluorescence activity, with the minimum effective dose of progesterone being 0.1nM. RU486, significantly inhibited progesterone induced transactivation and non-progesterogenic steroids failed to transactivate PR till 10μM concentrations. About 7 different chemicals (mostly pesticides or their metabolites) like DDT and its metabolites, nonylphenol, endosulfan were screened in this assay system for their role in transactivation and they were all found to be anti-progestative and IC50 values within the range of 3–20μM. Further, the assay was used to analyze the endocrine disrupting activity of extracted water samples from leather industries known for their high content of various chemicals and it was found to be rich in anti-progestative compounds. It resulted in about 30% reduction in transactivation. In conclusion, we demonstrated that this yeast based bioassay provides a rapid and robust assay for high throughput screening of (anti)progestative compounds from various sources.

Prohibitin Is a Novel Target Gene of Vitamin D Involved in Its Antiproliferative Action in Breast Cancer Cells

Previously, we showed that N-methyl-N-nitrosourea-transformed MCF12F breast epithelial cells exhibited differential expression of several genes, including up-regulation of prohibitin and elevated sensitivity to a relatively noncalcemic vitamin D analogue, 1alpha-hydroxyvitamin D5 [1alpha(OH)D5]. In this report, we evaluated the functional significance of prohibitin in relation to the cellular response to vitamin D. The in silico screening for putative transcription factor binding sites identified two vitamin D receptor (VDR)/retinoid X receptor binding sites in the 1-kb promoter region of prohibitin. Prohibitin up-regulation by 1alpha(OH)D5 treatment at both transcriptional and translational levels was confirmed by real-time reverse transcription-PCR and Western blot analysis in breast cancer cells, identifying prohibitin as a vitamin D target gene. Confocal microscopic analysis showed that prohibitin was localized in the nuclei of MCF-7 cells and a portion of prohibitin was colocalized with VDR, but direct physical interaction between VDR and prohibitin in cell lysates was not detectable. In MCF-7 cells expressing tetracycline-inducible prohibitin (Tet-On model), the overexpression of prohibitin inhibited cell proliferation and enhanced vitamin D-induced antiproliferative activity. Knockdown of prohibitin was accompanied by increased number of cells incorporating bromodeoxyuridine in the whole population and increased cell distribution in the S phase of cell cycle. In addition, prohibitin level had no significant effect on the vitamin D-induced transactivation of CYP24, a VDR target gene. This is the first report to suggest that prohibitin serves as a novel vitamin D target gene, which is involved in the antiproliferative action of vitamin D without affecting CYP24 transactivation in breast cancer cells.

Mae inhibits Pointed-P2 transcriptional activity by blocking its MAPK docking site

During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of a mitogen activated protein tyrosine kinase, called Rolled. Key nuclear targets of Rolled are two antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2), an activator. A critical regulator of this process, Mae, can interact with both Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the mechanism of Pnt-P2 regulation by Mae remained undefined. We find that efficient phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2 activity. Docking site blocking provides a new mechanism whereby the cell can precisely modulate kinase signaling at specific targets, providing another layer of regulation beyond the more global changes effected by alterations in the activity of the kinase itself.

AUTOCRINE OVER EXPRESSION OF FIBRONECTIN BY HUMAN TRANSITIONAL CARCINOMA CELLS IMPAIRS BACILLUS CALMETTE-GUERIN ADHERENCE AND SIGNALING

Bacillus Calmette-Guerin (BCG) binds to the tumor cell as a result of mycobacterial receptors for fibronectin (FN). Cell surface bound FN serves as a bridge through which BCG attaches to the tumor cell. Despite the importance of FN studies have demonstrated an idiosyncratic decrease in BCG adherence in response to exogenous FN. We evaluated the effect of exogenous and autocrine FN on the ability of BCG to adhere to the tumor cell surface and initiate cellular signaling. BCG adherence to parental 253J and FN over expressing 253JTGFbeta1-8 cells as well as to the intrinsic FN expressing cell line 647V was quantified using green fluorescent protein-BCG. Experiments were performed to assess the effect of FN on BCG initiated signal transduction through nuclear factor kappaB and AP1. Finally, the integrity of the BCG activated signaling pathway in transforming growth factor-beta1/FN over expressors was assessed using antibody mediated cross-linking of the FN receptor. BCG adherence was decreased in cell lines with high autocrine expression of FN. Exogenous FN prevented BCG induced transactivation of nuclear factor kappaB and AP1 reporter constructs. No BCG stimulated signaling to these reporters could be detected in FN over expressing 253J cells. NonFN dependent alpha5beta1 cross-linking initiated signal transduction in FN over expressing cells. We propose that by saturating cellular and BCG receptors excess FN expression decreases the ability of cellular or mycobacterial bound FN to bind vacant receptors on BCG or on the cell. Excess FN inhibits BCG adherence and BCG initiated signal transduction.

Coactivator ASC-2 mediates heat shock factor 1-mediated transactivation dependent on heat shock

Upon exposure to elevated temperatures, mammalian cells increase the expression of the heat shock proteins (HSP) through activation of the heat shock factor 1 (HSF1). Since most transcription factors require coactivators for efficient transcriptional activity, we tried to identify the coactivator(s) that interacts with and modulates the activities of HSF1. In vitro glutathione S-transferase (GST) pull-down assay revealed that HSF1 strongly interacts with activating signal cointegrator (ASC)-2 and weakly with cyclic adenosine monophosphate responsive element binding protein (CBP). We also show that cotransfection of ASC-2, but not CBP, potentiates HSF1-mediated transactivation based on its cognate element (heat shock element, HSE) linked to luciferase reporter. The molecular interaction of HSF1 and ASC-2 was stimulated by heat shock in cells and the overexpression of HSF1-interacting domain of ASC-2 inhibited the specific induced protein association and HSF1-mediated transactivation. Taking these results together, we suggest that ASC-2 in a novel coactivator for HSF1 and heat shock stress may contribute the strong active transcription complex through sequential recruitment of HSF1 and ASC-2.

No role of protected region b of human cytochrome P4501A2 gene (CYP1A2) as an AP-1 response element

Cytochrome P4501A2 (CYP1A2) is a member of the cytochrome P450 family of isozymes involved in the phase I drug metabolism of vertebrates. CYP1A2 is responsible for the activation of a number of aromatic amines to mutagenic and carcinogenic forms. Thus, the level of CYP1A2, which varies among different populations, may determine an individual's susceptibility to these chemicals. We have previously reported on the importance of a cis element named PRB (protected region B) in the regulation of human Cytochrome P4501A2 (CYP1A2) gene, which appeared to act as a positive regulatory element. Closer examination of the PRB sequence (-2218 to -2187 bp) revealed a putative AP-1 binding site, TGACTAA, at -2212 bp (Chung and Bresnick, 1997). To elucidate the role of AP-1 in CYP1A2 regulation, we transiently overexpressed c-Jun and c-Fos transcription factors in human hepatoma HepG2 cells, and examined their influence on the CYP1A2 promoter activity by reporter gene assays. Cotransfection of the c-Jun and the c-Fos expression vectors increased the induced transactivation by five to six fold from the CYP1A2 promoter constructs. However, deletion of the PRB element did not affect the degree of activation by the c-Jun and the c-Fos. Therefore, it is unlikely that the c-Jun and the c-Fos activate the CYP1A2 promoter through this AP-1 consensus-like sequence in the PRB region.

CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.

Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells

BACKGROUND AND AIMSHelicobacter pylori infection induces expression of proinflammatory cytokines such as interleukin (IL)-8 and tumour necrosis factor α (TNF-α) in gastric mucosa, and their genes have AP-1 binding sites...

Carboxylic ester antagonists of 1α,25-dihydroxyvitamin D 3 show cell-specific actions

Background: The nuclear hormone 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2D 3) acts through the transcription factor vitamin D receptor (1α,25(OH) 2D 3 receptor, VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins and specific DNA binding sites (1α,25(OH) 2D 3 response elements, VDREs). Ligand-mediated conformational changes of the VDR are the basis of the molecular mechanisms of nuclear 1α,25(OH) 2D 3 signaling. Cell-specific VDR antagonists would allow to dissect and fine regulate the pleiotropic 1α,25(OH) 2D 3 endocrine system affecting the regulation of calcium homeostasis, bone mineralization and other cellular functions. Results: Two carboxylic ester analogues of 1α,25(OH) 2D 3, ZK159222 and ZK168281, which have additional cyclopropyl rings and allylic alcohol substructures in their side chain, were characterized in different 1α,25(OH) 2D 3 target tissues as functional antagonists of 1α,25(OH) 2D 3 signaling. In all tested systems, ZK168281 showed lower residual agonistic activity and higher antagonistic effects than ZK159222, but the strength of these effects was cell-specific. Both antagonists were shown to act via the same mechanisms: they selectively stabilize an antagonistic conformation of the ligand-binding domain of the VDR within VDR–RXR–VDRE complexes, which then inhibits the interaction of the VDR with coactivator proteins and an induction of transactivation. Interestingly, cells that have been treated with antagonists were found to contain VDR–RXR heterodimers in a different conformation than cells that were stimulated with an agonist. Moreover, the strength of the functional antagonism of ZK159222 and ZK168281 appears to depend on the VDR/RXR expression ratio and high RXR levels were found to reduce the antagonistic effect of both compounds. In support of this observation, the overexpression of an transactivation function 2 (AF-2) deletion mutant of RXR resulted for both ZK159222 and ZK168281 in a reduced agonistic activity and an increased antagonistic effect. Conclusions: A novel, more potent VDR antagonist, ZK168281, was identified, which stabilizes VDR–RXR heterodimers in living cells in a different conformation than agonists. In addition, the VDR/RXR ratio was found as the major discriminating factor for understanding cell-specific effects of VDR antagonists.

Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor.

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.

Plasma membrane-targeted Raf kinase activates NF-kappaB and human immunodeficiency virus type 1 replication in T lymphocytes.

Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappaB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(delta26-303)) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(delta26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappaB, as judged by kappaB-dependent reporter assays and enhanced NF-kappaB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV(NL4-3) LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV(NL4-3) DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-kappaB activation and transactivation of the HIV(NL4-3) LTR but also synthesis and release of HIV particles.