Activity Of Multiple Transcription Factors Research Articles

Nuclear factor κB-dependent mechanisms coordinate the synergistic effect of PMA and cytokines on the induction of superoxide dismutase 2

Manganese superoxide dismutase (MnSOD) serves a protective role under conditions of oxidative stress mediated by such diverse agents as adriamycin, radiation, chemical hypoxia and ischaemia and might act as a newly recognized type of tumour-suppressor. MnSOD is an inducible enzyme; however, the signalling molecules and pathways involved in its induction have not been fully elucidated. Recently we reported the identification of a 342 bp enhancer within the second intron (I2E) of the human gene encoding MnSOD (SOD2), which contains sites for binding nuclear factor kappaB (NF-kappaB), CCAAT-enhancer-binding protein (C/EBP) and nuclear factor 1 (NF-1). Using a human fibroblast cell line transformed by simian virus 40, we have identified the I2E fragment as being responsive to PMA. Furthermore, simultaneous treatment with PMA and cytokines (tumour necrosis factor alpha and interleukin 1beta) synergistically increases MnSOD induction. The use of mutant constructs identified the NF-kappaB element within the enhancer fragment as being essential for the PMA and PMA/cytokine effect. Mutations in the C/EBP- and NF-1-binding sites revealed a potential co-operation between proteins that bind to these sites and the NF-kappaB element. Evaluation of inhibitory kappaB (IkappaB)-alpha and IkappaB-beta proteins reveals agent-specific differences in their turnover kinetics. Both C/EBP and NF-kappaB DNA-binding activities were increased in cells receiving a combination of cytokine and PMA. Supershift and immunoprecipitation studies suggest a physical interaction between C/EBP and NF-kappaB proteins. Taken together, these studies suggest the activation of multiple transcription factors as well as pathways leading to increased NF-kappaB activity as being the mechanisms responsible for the synergistic induction of MnSOD by PMA and cytokines.

Nuclear factor κB-dependent mechanisms coordinate the synergistic effect of PMA and cytokines on the induction of superoxide dismutase 2

Manganese superoxide dismutase (MnSOD) serves a protective role under conditions of oxidative stress mediated by such diverse agents as adriamycin, radiation, chemical hypoxia and ischaemia and might act as a newly recognized type of tumour-suppressor. MnSOD is an inducible enzyme; however, the signalling molecules and pathways involved in its induction have not been fully elucidated. Recently we reported the identification of a 342bp enhancer within the second intron (I2E) of the human gene encoding MnSOD (SOD2), which contains sites for binding nuclear factor κB (NF-κB), CCAAT-enhancer-binding protein (C/EBP) and nuclear factor 1 (NF-1). Using a human fibroblast cell line transformed by simian virus 40, we have identified the I2E fragment as being responsive to PMA. Furthermore, simultaneous treatment with PMA and cytokines (tumour necrosis factor α and interleukin 1β) synergistically increases MnSOD induction. The use of mutant constructs identified the NF-κB element within the enhancer fragment as being essential for the PMA and PMA/cytokine effect. Mutations in the C/EBP- and NF-1-binding sites revealed a potential co-operation between proteins that bind to these sites and the NF-κB element. Evaluation of inhibitory κB (IκB)-α and IκB-β proteins reveals agent-specific differences in their turnover kinetics. Both C/EBP and NF-κB DNA-binding activities were increased in cells receiving a combination of cytokine and PMA. Supershift and immunoprecipitation studies suggest a physical interaction between C/EBP and NF-κB proteins. Taken together, these studies suggest the activation of multiple transcription factors as well as pathways leading to increased NF-κB activity as being the mechanisms responsible for the synergistic induction of MnSOD by PMA and cytokines.

Isolation of target gene promoter/enhancer sequences by whole genome PCR method.

AbstractRegulation of gene expression is controlled through the combinatorial action of multiple transcription factors, which function to activate or repress transcription via binding to cis-regulatory elements. Such regulatory elements are usually present in the promoter sequences located upstream of the transcription initiation sites of a target gene. Identification of functional target gene promoters that are regulated by a specific transcription factor is one of the most critical areas in understanding the molecular mechanisms that control transcription. Furthermore, identification of target gene promoters for normal and oncogenic transcription factors provides insight into the regulation of genes that are involved in control of normal cell growth and differentiation, as well as provide information critical to understanding cancer development.KeywordsElectrophoretic Mobility Shift AssaySpecific Transcription FactorTarget Gene PromoterOncogenic Transcription FactorGenome Polymerase Chain ReactionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Retinoic Acid Stimulation of the Human Surfactant Protein B Promoter Is Thyroid Transcription Factor 1 Site-dependent

Surfactant B (SP-B) is a 79-amino acid peptide critical to postnatal respiratory adaptation. Expression of SP-B by respiratory epithelial cells is regulated by developmental and hormonal influences at the level of gene transcription. Previous studies supported the role of retinoic acids (RA) and their receptors (RARs) in SP-B gene transcription. In the present study, RARalpha was detected in mouse alveolar type II epithelial cells where SP-B is synthesized and processed. Deletion and site-specific mutagenesis analysis identified clustered retinoic acid-responsive element sites in the 5'-flanking enhancer region of the hSP-B gene that bound RARalpha proteins. RAR coactivators ACTR, SRC-1, and transcriptional intermediary factor 2 (TIF2) stimulated human (h) SP-B promoter activity in a dose-dependent fashion in pulmonary adenocarcinoma H441 cells. In addition, an RAR-associated protein, CREB-binding protein (CBP), potentiated the effects of RAR on hSP-B promoter activity in H441 cells. Importantly, RA stimulation of the hSP-B promoter depends on tissue-specific thyroid transcription factor (TTF-1) DNA-binding sites. TTF-1 protein synergistically stimulated the hSP-B promoter with RARalpha, CBP, and nuclear receptor coactivators in H441 cells. In addition, TTF-1 interacted directly with RARalpha and TIF2 in the mammalian two-hybrid system. These findings support a model in which RAR/retinoid X receptor, TTF-1, coactivators, and CBP form a transcription activation complex in the upstream enhancer region of the hSP-B gene.

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.

Proto-Oncogene mRNA Levels and Activities of Multiple Transcription Factors in C3H 10T 1/2 Murine Embryonic Fibroblasts Exposed to 835.62 and 847.74 MHz Cellular Phone Communication Frequency Radiation

This study was designed to determine whether two differently modulated radiofrequencies of the type generally used in cellular phone communications could elicit a general stress response in a biological system. The two modulations and frequencies studied were a frequency-modulated continuous wave (FMCW) with a carrier frequency of 835.62 MHz and a code division multiple-access (CDMA) modulation centered on 847.74 MHz. Changes in proto-oncogene expression, determined by measuring Fos, Jun, and Myc mRNA levels as well as by the DNA-binding activity of the AP1, AP2 and NF-kappaB transcription factors, were used as indicators of a general stress response. The effect of radiofrequency exposure on proto-oncogene expression was assessed (1) in exponentially growing C3H 10T 1/2 mouse embryo fibroblasts during their transition to plateau phase and (2) during transition of serum-deprived cells to the proliferation cycle after serum stimulation. Exposure of serum-deprived cells to 835.62 MHz FMCW or 847.74 MHz CDMA microwaves (at an average specific absorption rate, SAR, of 0.6 W/kg) did not significantly change the kinetics of proto-oncogene expression after serum stimulation. Similarly, these exposures did not affect either the Jun and Myc mRNA levels or the DNA-binding activity of AP1, AP2 and NF-kappaB in exponential cells during transit to plateau-phase growth. Therefore, these results suggest that the radiofrequency exposure is unlikely to elicit a general stress response in cells of this cell line under these conditions. However, statistically significant increases (approximately 2-fold, P = 0.001) in Fos mRNA levels were detected in exponential cells in transit to the plateau phase and in plateau-phase cells exposed to 835.62 MHz FMCW microwaves. For 847.74 MHz CDMA exposure, the increase was 1.4-fold (P = 0.04). This increase in Fos expression suggests that expression of specific genes could be affected by radiofrequency exposure.

Deciphering the early-response transcription factor networks in mast cells

External stimuli mediate signaling cascades in mast cells that lead to the activation of multiple transcription factors. Here, Hovav Nechushtan and Ehud Razin discuss the role played by some of these transcription factors in modulating gene expression and mast cell functions.

The integrated activities of IRF-2 (HiNF-M), CDP/cut (HiNF-D) and H4TF-2 (HiNF-P) regulate transcription of a cell cycle controlled human histone H4 gene: mechanistic differences between distinct H4 genes.

Maximal transcription of a prototypical cell cycle controlled histone H4 gene requires a proliferation-specific in vivo genomic protein/DNA interaction element, Site II. Three sequence-specific transcription factors interact with overlapping recognition motifs within Site II: interferon regulatory factor IRF-2 (HiNF-M), the putative H4 subtype-specific protein H4TF-2 (HiNF-P), and HiNF-D which represents a complex of the homeodomain protein CDP/cut, CDC2, cyclin A and pRB. However, natural sequence variation in the Site II sequences of different human H4 genes abolishes binding of specific trans-acting factors; the functional consequences of these variations have not been investigated. To address the precise contribution of H4 promoter factors to the level of H4 gene transcription, we performed a systematic mutational analysis of Site II transcriptional motifs. These mutants were tested for ability to bind each of the Site II cognate proteins, and subsequently evaluated for ability to confer H4 transcriptional activity using chimeric H4 promoter/CAT fusion constructs in different cell types. We also analyzed the effect of over-expressing IRF-2 on CAT reporter gene expression driven by mutant H4 promoters and assessed H4 transcriptional control in cells nullizygous for IRF-1 and IRF-2. Our results show that the recognition sequence for IRF-2 (HiNF-M) is the dominant component of Site II and modulates H4 gene transcription levels by 3 fold. However, the overlapping recognition sequences for IRF-2 (HiNF-M), H4TF-2 (HiNF-P) and CDP/cut (HiNF-D) together modulate H4 gene transcription levels by at least an order of magnitude. Thus, maximal activation of H4 gene transcription during the cell cycle in vivo requires the integrated activities of multiple transcription factors at Site II. We postulate that the composite organization of Site II supports responsiveness to multiple signalling pathways modulating the activities of H4 gene transcription factors during the cell cycle. Variations in Site II sequences among different H4 genes may accommodate differential regulation of H4 gene expression in cells and tissues with unique phenotypic properties.

Characterization of the Upstream Sequence of the Human CYP11A1 Gene for Cell Type-specific Expression

The CYP11A1 gene encodes the cholesterol side-chain cleavage enzyme P450scc, which catalyzes the synthesis of steroids from cholesterol. This gene is expressed only in steroidogenic organs such as the adrenal, gonad, placenta, and brain. We have characterized an upstream regulatory element of the human CYP11A1 gene, termed AdE, which contributed to its cell type-specific expression. The AdE sequence contains two protein binding regions, AdE1 and AdE2, which bind many proteins including NF1- and Sp1-like proteins as shown by electrophoretic mobility shift assay, footprinting, competition, antibody supershift, and mutagenesis of the binding sites. When cloned in front of the CYP11A1 promoter or the heterologous thymidine kinase promoter, AdE sequences enhanced expression of the reporter gene in steroidogenic cell lines of the adrenal, gonad, and placental origin but not in nonsteroidogenic cell lines such as COS-1 and Rat-1. The function of AdE1 and AdE2 was lower when present individually than together. The combined action of multiple transcription factors binding to the AdE sequence brings about the final activation of the CYP11A1 gene in a tissue-specific manner.

Intracellular signalling: Putting JAKs on the kinase MAP

Tyrosine kinases of the JAK family are required for activation of both STAT transcription factors and the ERK/MAP kinase pathway, suggesting a mechanism for the optimization of gene induction via the coordinate activation of multiple transcription factors.

Activation of Multiple Transcription Factors and fos and jun Gene Family Expression in Cells Exposed to a Single Electric Pulse

We report that exposure of cells to a single electric pulse (250-1250 V/cm) results in the rapid and persistent activation of the DNA binding activities of a number of transcription factors, including AP-1, SP1, AP-2, and NF-κB, and the transient expression of select members of the fos and jun gene families. Induction of gene expression occurs primarily at the level of transcription, although c-jun expression also appears to be regulated posttranscriptionally. Interestingly, maximal induction of gene expression is detected at electrical field strengths that do not result in pore formation in the plasma membrane and that do not significantly affect call viability. Exposure of cells to electric pulses does not result in the activation of HSF1 DNA binding activity, or the induction of hsp70 or p53 protein synthesis, indicating that the induction of fos and jun gene expression is not coincident with protein or DNA damage. The results of these studies suggest that electrical pulses may represent a novel mechanism for inducing the activities of multiple transcription factors and the expression of select members of the fos and jun gene families.

Synergistic activation of transcription by Escherichia coli cAMP receptor protein.

Activation of gene expression in eukaryotes generally involves the action of multiple transcription factors that function synergistically when bound near a particular target gene. Such effects have been suggested to occur because multiple activators can interact simultaneously with one or more components of the basal transcription machinery. In prokaryotes, examples of synergistic effects on transcription are much more limited and can often be explained by cooperative DNA binding. Here we show that the Escherichia coli cAMP receptor protein (CRP) functions synergistically to activate transcription from a derivative of the lac promoter that bears a second CRP-binding site upstream of the natural binding site. We present evidence indicating that cooperative DNA binding of two CRP dimers does not account for the magnitude of the observed cooperative activation. We suggest, instead, that the two dimers stimulate transcription directly by contacting two distinct surfaces of RNA polymerase simultaneously. Thus, synergistic activation by CRP may provide a relatively simple model for examining the molecular basis of such effects in higher organisms.