Sandra Moshonov

Mechanism of Rapid Transcriptional Induction of Tumor Necrosis Factor Alpha-Responsive Genes by NF-κB

ABSTRACT NF-κB induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-κB-responsive genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism by which these genes are activated is not entirely understood. Here we report that even though NF-κB interacts directly with TAFIIs, induction of NF-κB by tumor necrosis factor alpha (TNF-α) does not enhance TFIID recruitment and preinitiation complex formation on some NF-κB-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-α stimulus. Using the immediate-early TNF-α-responsive gene A20 as a prototype promoter, we found that the constitutive association of the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-κB. In vitro transcription assays confirmed that NF-κB plays a postinitiation role since it enhances the transcription reinitiation rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-κB on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.

Core Promoter Binding by Histone-Like TAF Complexes

ABSTRACT A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.

Links between core promoter and basic gene features influence gene expression.

BackgroundDiversity in rates of gene expression is essential for basic cell functions and is controlled by a variety of intricate mechanisms. Revealing general mechanisms that control gene expression is important for understanding normal and pathological cell functions and for improving the design of expression systems. Here we analyzed the relationship between general features of genes and their contribution to expression levels.ResultsGenes were divided into four groups according to their core promoter type and their characteristics analyzed statistically. Surprisingly we found that small variations in the TATA box are linked to large differences in gene length. Genes containing canonical TATA are generally short whereas long genes are associated with either non-canonical TATA or TATA-less promoters. These differences in gene length are primarily determined by the size and number of introns. Generally, gene expression was found to be tightly correlated with the strength of the TATA-box. However significant reduction in gene expression levels were linked with long TATA-containing genes (canonical and non-canonical) whereas intron length hardly affected the expression of TATA-less genes. Interestingly, features associated with high translation are prevalent in TATA-containing genes suggesting that their protein production is also more efficient.ConclusionOur results suggest that interplay between core promoter type and gene size can generate significant diversity in gene expression.

TAF4/4b·TAF12 Displays a Unique Mode of DNA Binding and Is Required for Core Promoter Function of a Subset of Genes

The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b·TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is ∼70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes.

Calcium-dependent PAF-stimulated generation of reactive oxygen species in a human keratinocyte cell line

During inflammation and other pathological states, the lipid mediator platelet-activating factor (PAF) and reactive oxygen species (ROS) are both generated. We have been investigating the effect of exogenous PAF on ROS formation in the human keratinocyte cell line (HaCaT). ROS production, measured using luminol-enhanced chemiluminescence (CL), proved to be rapid, transient, PAF receptor-mediated, and totally dependent on an increase in intracellular Ca2+ ([Ca2+]i) and on the presence of extracellular Ca2+. Repeated administration of PAF resulted in refractoriness to the agonist in terms of both capacities to increase [Ca2+]i and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growth factor, thapsigargin). The PAF-induced increases in [Ca2+]i (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paralleled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the ROS producing system(s). Inhibitors of xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF evoked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondrial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making NAD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation.

Crosstalk Between Elevation of [Ca2+]i, Reactive Oxygen Species Generation and Phospholipase A2 Stimulation in a Human Keratinocyte Cell Line

The aim of the study was to explore the possible interrelationship between reactive oxygen species (ROS) formation and cPLA2 activation and the mediator role that [Ca2+]i may play in these processes in the human keratinocyte cell line, HaCaT. HaCaT cells can be invoked to transiently produce ROS by epidermal growth factor (EGF), thapsigargin (TPG) and the Ca(2+)-ionophore, A23187. These 3 agonists transiently increase [Ca2+]i with characteristic kinetics and magnitude. TPG and A23187 each activates on its own [3H]AA release from prelabeled cells, whereas EGF on its own has no effect on [3H]AA release. However, EGF augments [3H]AA release invoked by TPG or A23187 several fold. EGF activates MAP kinase cascades in HaCaT cells, leads to ROS formation and induces relatively small (1.6 fold) elevation in [Ca2+]i, whereas A23187 and TPG lead to a substantial elevation in [Ca2+]i (2.5 to 5 fold) and to ROS formation. Both have a minor effect on MAP kinase activation. The synergism in PLA2 activation by EGF and TPG or A23187, and the sensitivity of [3H]AA release to N-acetylcysteine (NAC) and dithiothreitol (DTT) (potent reducing agents) or to DPI (an inhibitor of FAD-dependent oxidases) lead to the suggestion that ROS formation, elevation of [Ca2+]i and PLA2 activation are causally related. Since we show that elevation of [Ca2+]i is a prerequisite for both ROS and PLA2 activation, it is possible that these processes contribute to the toxicity (apoptosis) exerted by chronic elevation of [Ca2+]i.

Nitric oxide: A mediator in anaphylactic shock in guinea-pigs

In this study we show that the pathophysiology of anaphylaxis includes generation of nitric oxide (NO), a very powerful, short-acting vasodilator. Guinea-pigs sensitized to ovalbumin were treated with 200 microgram/kg diphenylene iodonium (DPI), and NO synthase inhibitor, prior to antigen challenge. Mortality following the challenge fell from 71 to 39% (p < 0.001, n = 59). In the Langendorff preparation perfused isolated hearts from sensitized guinea-pigs were challenged to initiate cardiac anaphylaxis. The coronary flow rate (CFR), a direct reflection of coronary arterial resistance, was reduced by antigen challenge to 56 +/- 4% (n = 16) of the basal rate. DPI (2 micrograms/ml) intensified the antigen-induced fall in CFR to 13 +/- 3% of control (p < 0.005, n = 5), and the false substrate for NO, L-N-methylarginine, to 37 +/- 3% (p < 0.05, n = 4). Sodium nitroprusside (SNP), a NO generator, raised the basal CFR by 46% (from 11.2 +/- 1.7 ml/min to 16.3 +/- 1.9 ml/min) and blunted the antigen-induced fall in CFR. Paradoxically, DPI, which can inhibit flavoprotein enzymes other than NO synthase, potentiated the vasodilator effect of SNP, raising the basal CFR by 116%. Together these results strongly indicate that the vasodilator NO is generated in anaphylaxis. However, whereas in the heart it may function as a counterweight to the vasospasm of the coronary arteries, in the intact animal it appears to be a major contributor to the potentially lethal hypotension of anaphylactic shock.

Activation of Map Kinases, cPLA2 and Reactive Oxygen Species Formation by EGF and Calcium Mobilizing Agonists in a Human Keratinocyte Cell Line

Our results corroborate the notion that cPLA2 activation requires both a phosphorylation step (by a MAP kinase (ERK2) and possibly another kinase) and elevation of intracellular Ca2+ concentration. Ca2+ mobilizing agonists (Ca2+-ionophore and thapsigargin) on their own do not activate (or lead to a very modest activation) ERK2 but lead to the release of [3H]arachidonic acid ([3H]AA) from prelabeled HaCaT cells (a human keratinocyte cell line). EGF, on the other hand, activates ERK2 but on its own does not lead to the release of [3H]AA. EGF potentiates the activity of cPLA2 in the presence of the Ca2+- mobilizing agonists. Both the Ca2+- mobilizing agonists and EGF invoke reactive oxygen species formation. VOOH is a potent activator of both ERK2 and cPLA2. Both VOOH and EGF activate also the N-terminal jun kinase (JNK) which is an important factor in the activation of the transcription factor AP1.

Lipoxygenase inhibitor and colchicine as anti-arthritic agents in the rat

The non-steroidal anti-inflammatory agents do not have identical activities on the various pathways of arachidonic acid metabolism. The purpose of this study is to examine and compare the activities of colchicine, an anti-arthritic agent, indomethacin, a known prostaglandin synthesis inhibitor and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, in an experimental model of arthritis. Acute arthritis of the knee was induced in rats by injection of lipopolysaccharide (LPS) into the joints. Arthritis was characterized by an increase in joint diameter (18%), increased synovial weight (34%) and an increase in synovial prostaglandin E (PGE) production (56%). While administration of all of the agents examined abolished LPS-induced joint diameter and synovial weight increase, only indomethacin reduced increased PGE content. NDGA and colchicine had no inhibitory effect on LPS-induced PGE production, and moreover they actually stimulated PGE production when compared to control values. It is concluded that: Among the mediators of the inflammatory process are factors sensitive to colchicine and NDGA which are not PGs. Lipoxygenase products of arachidonic acid including leukotrienes may have an important role in inflammation. Leukotrienes and prostaglandins may act in concert in mediating the inflammatory process.

Lipid Mediators Stimulate Reactive Oxygen Species Formation in Immortalized Human Keratinocytes

Cell signaling is accompanied by rapid remodeling of membrane lipids by activated phospholipases (PLA2, PLC, PLD and sphingomyelinase) with the generation of bioactive lipids that can serve as intra- and/or extracellular mediators (Serhan, et al., 1996). These lipid mediators include eicosanoids, platelet activating factor (PAF), diacyl glycerides, lyso-phosphatidylcholine (LPC), phosphatidic acid (PA), lyso-PA, ceramide and other newly-discovered autacoids. They are important in a wide range of cell-cell communication processes, such as host defense, inflammation, ischemia reperfusion and homeostasis.