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T Collins

Publications and source records attributed to T Collins.

At least 55 records · Page 3Linked to original sources
PubMed1998

cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1).

Egr-1 (early-growth response factor-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important for cell growth, development and the pathogenesis of disease. The transcriptional co-activators CBP (cAMP-response-element-binding-protein-binding protein) and p300 interact with sequence-specific transcription factors as well as components of the basal transcription machinery to facilitate RNA polymerase II recruitment and transcriptional initiation. Here we demonstrate a unique way in which Egr-1 physically and functionally interacts with CBP/p300 to modulate gene transcription. CBP/p300 potentiated Egr-1 mediated expression of 5-lipoxygenase (5-LO) promoter-reporter constructs, and the degree of trans-activation was proportional to the number of Egr-1 consensus binding sites present in wild-type and naturally occurring mutants of the 5-LO promoter. The N- and C-terminal domains of CBP interact with the transcriptional activation domain of Egr-1, as demonstrated by a mammalian two-hybrid assay. Direct protein-protein interactions between CBP/p300 and Egr-1 were demonstrated by glutathione S-transferase fusion-protein binding and co-immunoprecipitation/Western-blot studies. These data suggest that CBP and p300 act as transcriptional co-activators for Egr-1-mediated gene expression and that variations between individuals in such co-activation could serve as a genetic basis for variability in gene expression.

Animals↗
PubMed1998

Nuclear integration of glucocorticoid receptor and nuclear factor-kappaB signaling by CREB-binding protein and steroid receptor coactivator-1.

The p65 (RelA) component of nuclear factor-kappaB (NF-kappaB) and the glucocorticoid receptor (GR) mutually repress each other's ability to activate transcription. Both of these transcriptional activators depend upon the coactivators CREB-binding protein (CBP) and steroid receptor coactivator-1 (SRC-1) for maximal activity. Here we show that increased levels of CBP relieves the inhibition of glucocorticoid-mediated repression of NF-kappaB activity and the NF-kappaB-mediated repression of GR activity. SRC-1 can relieve the NF-kappaB-mediated repression of GR activity. We propose that cross-talk between the p65 component of NF-kappaB and glucocorticoid receptors is due, at least in part, to nuclear competition for limiting amounts of the coactivators CBP and SRC-1, thus providing a novel mechanism for decreasing expression of genes involved in the inflammatory response.

Animals↗
PubMed1998

CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor beta transcriptional responses in endothelial cells.

The transforming growth factor-beta (TGF-beta) superfamily of growth factors and cytokines has been implicated in a variety of physiological and developmental processes within the cardiovascular system. Smad proteins are a recently described family of intracellular signaling proteins that transduce signals in response to TGF-beta superfamily ligands. We demonstrate by both a mammalian two-hybrid and a biochemical approach that human Smad2 and Smad4, two essential Smad proteins involved in mediating TGF-beta transcriptional responses in endothelial and other cell types, can functionally interact with the transcriptional coactivator CREB binding protein (CBP). This interaction is specific in that it requires ligand (TGF-beta) activation and is mediated by the transcriptional activation domains of the Smad proteins. A closely related, but distinct endothelial-expressed Smad protein, Smad7, which does not activate transcription in endothelial cells, does not interact with CBP. Furthermore, Smad2,4-CBP interactions involve the COOH terminus of CBP, a region that interacts with other regulated transcription factors such as certain signal transduction and transcription proteins and nuclear receptors. Smad-CBP interactions are required for Smad-dependent TGF-beta-induced transcriptional responses in endothelial cells, as evidenced by inhibition with overexpressed 12S E1A protein and reversal of this inhibition with exogenous CBP. This report demonstrates a functional interaction between Smad proteins and an essential component of the mammalian transcriptional apparatus (CBP) and extends our insight into how Smad proteins may regulate transcriptional responses in many cell types. Thus, functional Smad-coactivator interactions may be an important locus of signal integration in endothelial cells.

Animals↗
PubMed1998

Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription.

Transcriptional activation of the IFN beta gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappa B, IRF1, ATF2/c-Jun, and the architectural protein HMG I(Y). The level of transcription generated by all of these activators is greater than the sum of the levels generated by individual factors, a phenomenon designated transcriptional synergy. We demonstrate that this synergy, in the context of the enhanceosome, requires a new protein-protein interaction domain in the p65 subunit of NF-kappa B. Transcriptional synergy requires recruitment of the CBP/p300 coactivator to the enhanceosome, via a new activating surface assembled from the novel p65 domain and the activation domains of all of the activators. Deletion, substitution, or rearrangement of any one of the activation domains in the context of the enhanceosome decreases both recruitment of CBP and transcriptional synergy.

Activating Transcription Factor 2↗
PubMed1998

The nuclear factor-kappa B/inhibitor of kappa B autoregulatory system and atherosclerosis.

Nuclear factor-kappa B is a ubiquitous transcription factor that can be activated by diverse proatherogenic stimuli such as inflammatory cytokines, lipopolysaccharide, oxidant stress and physical forces. Recently, there have been major advances in understanding signal transduction from the tumor necrosis factor receptor, a model activator of the nuclear factor-kappa B system. One set of signals from the receptor initiates a phosphorylation cascade resulting in the activation of a kinase complex which phosphorylates an inhibitor of nuclear factor-kappa B, or inhibitor of kappa B. Degradation of the inhibitor occurs in parallel with activation and nuclear accumulation of the transcription factor. Subsequent changes in gene expression induce the production of multiple cytokines and adhesion molecules, which are important in early atherosclerotic lesion formation, and generation of survival signals, which could be important in lesion progression. A second set of signals from the tumor necrosis factor receptor leads to cell death. Understanding these competing pathways in vascular cells may help to clarify the role of this transcription factor in the proliferative lesions of atherogenesis.

Arteriosclerosis↗
PubMed1998

The role of nitric oxide in the control by the vagal nerves of the heart of the ferret.

This study focuses on the potential role for nitric oxide on the actions of the parasympathetic innervation to the heart. Earlier, we showed that the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) reduced the bradycardia induced by stimulation of vagal efferent motor fibres and that these effects are reversible through administration of the NOS substrate L-arginine. In the present study, we show that D-arginine does not reverse the effects of the inhibitors and confirm that they are reversed by L-arginine. Another NOS inhibitor, NG-nitro-L-arginine (L-NOARG), produced similar effects which were not reversed by L-arginine. In an examination of the effect of increasing NO availability with the NO donor sodium nitroprusside the vagally induced bradycardia was enhanced at all frequencies tested. In a separate series, the effects of NOS inhibitors and NO donors on the dromotropic actions of the vagus were examined. The NOS inhibitor L-NAME, reduced the increase in atrio-ventricular conduction delay normally induced by efferent vagal stimulation at all frequencies tested both in the 'paced' and 'unpaced' heart. Further, sodium nitroprusside enhanced this delay. Overall the study indicates that NO has an important facilitatory role on both the chronotropic and dromotropic actions of the vagus nerve on the heart and that NO may be a rate-limiting factor in the cardiac responses to vagal stimulation.

Animals↗
PubMed1998

Regulation of PDGF-B in endothelial cells exposed to cyclic strain.

The present study was designed to examine the regulation by cyclic strain of endothelial cell (EC) platelet-derived growth factor-B chain (PDGF-B) expression. We demonstrate in this study that bovine aortic ECs subjected to 10% (but not 6%) average strain resulted in a 2.6-fold increase in PDGF-B steady state mRNA and immunoreactive protein. Nuclear runoff transcription assays confirmed the induction of PDGF-B transcripts. To address the regulation of PDGF-B gene expression by cyclic strain, we transfected bovine aortic ECs with a construct containing 450 bp of human PDGF-B promoter sequence coupled to chloramphenicol acetyltransferase (CAT), and found that subjecting these cells to 10% average strain resulted in a twofold increase in CAT activity by 4 hours. Analysis of nested 5' deletions of the promoter transfected into ECs demonstrated a 55% drop-off in activity between position -313 and -153, with no induction of activity with the - 101-bp minimal promoter. Since a shear stress response element (SSRE) is located at position -125, we tested the hypothesis that the SSRE site was necessary and/or sufficient for induction of PDGF-B activity with strain. Electromobility shift assays revealed that nuclear proteins from ECs exposed to strain for short intervals (30 minutes) bound to the PDGF-B SSRE. However, transfection of ECs with hybrid promoter constructs containing the SV40 sequence promoter downstream of the SSRE or the -153 PDGF-B promoter sequence bearing a mutation in the SSRE demonstrated that the SSRE was not necessary for inducible reporter gene expression in ECs exposed to cyclic strain.

Animals↗
PubMed1998

Strain-responsive regions in the platelet-derived growth factor-A gene promoter.

Proliferation of cultured neonatal vascular smooth muscle (VSM) cells is enhanced by exposure to cyclic mechanical strain, in part through autocrine action of secreted platelet-derived growth factor (PDGF). We examined transcription factors and DNA response elements that may participate in the induction of PDGF-A gene transcription by mechanical strain. PDGF-A mRNA increased gradually over 4 to 24 hours exposure to cyclic (1 Hz) strain. This was due, at least in part, to increased transcription since a full length (890 bp) PDGF-A promoter reporter construct was induced 3.5-fold in transfected VSM cells exposed to strain for 24 hours. A series of PDGF-A promoter truncation reporter constructs was used to identify potential regions of the promoter involved in regulation by strain. Strain-responsive regions were found between -262 bp and -92 bp and between -92 bp and -41 bp of the promoter. Since these regions are GC-rich and contain response elements for Egr-1 and Sp-1, we examined expression of these transcription factors in response to strain. mRNA for both factors increased over 0.5 to 4 hours of strain, while protein expression for both increased gradually over a 24 hours period. Gel shift assays with a probe specific for Egr-1 demonstrated at least 1 prominent new shifted band after 4 to 12 hours exposure to strain. An Sp-1 probe demonstrated constitutive shifted bands that did not change in response to strain. Thus, GC-rich regions in the proximal 92 bp of the PDGF-A promoter contain mechanical strain-responsive elements that bind Egr-1 and possibly Sp-1.

Animals↗
PubMed1998

Egr-1 and Sp1 interact functionally with the 5-lipoxygenase promoter and its naturally occurring mutants.

5-Lipoxygenase (5-LO), an enzyme essential for the formation of leukotrienes, is functionally modulated by a number of mechanisms, including transcriptional controls. The 5-LO promoter has a unique G+C-rich sequence, located between 176 and 147 base pairs upstream of the ATG translation start site, which contains five tandem Sp1 (a zinc-finger transcription factor) consensus binding sites overlapping five tandem early growth response protein 1 (Egr-1), a zinc-finger transcription factor, consensus binding sites. A family of naturally occurring mutations has been identified that consists of additions or deletions of these binding sites. The role of these overlapping Sp1/Egr-1 sites in the regulation of 5-LO transcription and the effects of these mutations on transcriptional regulatory mechanisms are unknown. We now show that Sp1 and Egr-1 bind specifically to the G+C-rich promoter sequence using in vitro deoxyribonuclease I footprinting. Both Sp1 and Egr-1 activate 5-LO promoter-reporter constructs in a minimally active drosophila SL2 cotransfection system, and the G+C-rich sequence is involved in this process. Moreover, studies comparing mutant promoter function indicate that both Sp1 and Egr-1 trans-activation are proportional to the number of Sp1/Egr-1 consensus binding sites within the G+C-rich sequence. It is possible that basal and inducible 5-LO gene transcriptions are mediated by an interplay of Sp1, Egr-1, and other transcription factors within the G+C-rich promoter region, and the naturally occurring mutations alter transcription by modifying their trans-activation potential.

Animals↗
PubMed1997

Knowledge of perfusion and contractile reserve improves the predictive value of recovery of regional myocardial function postrevascularization: a study using the combination of myocardial contrast echocardiography and dobutamine echocardiography.

BACKGROUND: This study was designed to determine the value of myocardial contrast echocardiography (MCE) and dobutamine echocardiography (DE), alone or in combination, in predicting functional recovery in patients with resting wall motion abnormalities due to CAD. MCE and DE have been independently shown to be useful in detecting myocardial viability in the post-myocardial infarction setting. METHODS AND RESULTS: Thirty-nine patients with significant coronary artery disease and resting wall motion abnormalities underwent DE (2.5 to 20 microg x kg(-1) x min(-1)) and wall motion analysis (16-segment model). MCE was performed with selective intracoronary injections of sonicated meglumine (2 cm3). Myocardial viability was defined as presence of contrast effect by MCE and contractile reserve or an ischemic response by DE. Functional recovery (improvement in wall motion) was assessed after revascularization (percutaneous transluminal coronary angioplasty, n=20; coronary artery bypass surgery, n=19). When the two groups of patients were analyzed, MCE was associated with excellent sensitivities (84%) yet poor specificities (19% to 26%); DE had lower sensitivities (79% to 80%) but also poor specificities (30% to 36%). The combination of both was associated with excellent sensitivities (90% to 93%) and modest specificities (48% to 50%) for predicting functional recovery. A biphasic response with DE was infrequent (14% to 42%) but highly specific of functional recovery (84% to 94%). MCE had an excellent negative predictive value for functional recovery (83%). CONCLUSIONS: The prediction of functional recovery post-revascularization can be enhanced by combining MCE and DE.

Adult↗
PubMed1997

Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-alpha-induced phosphorylation of IkappaB-alpha, resulting in decreased nuclear factor-kappaB and decreased expression of adhesion molecules. The effects on both IkappaB-alpha phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of approximately 10 microM. These agents selectively and irreversibly inhibited the tumor necrosis factor-alpha-inducible phosphorylation of IkappaB-alpha without affecting the constitutive IkappaB-alpha phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130-140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IkappaBalpha phosphorylation exert anti-inflammatory activity in vivo.

Animals↗
PubMed1997

Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions.

Although several adhesion molecules expressed on leukocytes (beta1 and beta2 integrins, platelet endothelial cell adhesion molecule 1 [PECAM-1], and CD47) and on endothelium (intercellular adhesion molecule 1, PECAM-1) have been implicated in leukocyte transendothelial migration, less is known about the role of endothelial lateral junctions during this process. We have shown previously (Read, M.A., A.S. Neish, F.W. Luscinskas, V.J. Palambella, T. Maniatis, and T. Collins. 1995. Immunity. 2:493-506) that inhibitors of the proteasome reduce lymphocyte and neutrophil adhesion and transmigration across TNF-alpha-activated human umbilical vein endothelial cell (EC) monolayers in an in vitro flow model. The current study examined EC lateral junction proteins, principally the vascular endothelial (VE)-cadherin complex and the effects of proteasome inhibitors (MG132 and lactacystin) on lateral junctions during leukocyte adhesion, to gain a better understanding of the role of EC junctions in leukocyte transmigration. Both biochemical and indirect immunofluorescence analyses of the adherens junction zone of EC monolayers revealed that neutrophil adhesion, not transmigration, induced disruption of the VE-cadherin complex and loss of its lateral junction localization. In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil transmigration, was not altered. These findings identify new and interrelated endothelial-dependent mechanisms for leukocyte transmigration that involve alterations in lateral junction structure and a proteasome-dependent event(s).

Acetylcysteine↗
PubMed1997

CREB-binding protein/p300 are transcriptional coactivators of p65.

CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-kappaB (NF-kappaB) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1-CAT reporter constructs. The N- and C-terminal domains of both CBP/p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP/p300 with p65 were demonstrated by glutathione S-transferase fusion protein binding, and coimmunoprecipitation/Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inflammatory genes.

Adenovirus E1A Proteins↗
PubMed1997

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Alleles↗