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T A Libermann

Publications and source records attributed to T A Libermann.

At least 37 records · Page 2Linked to original sources
PubMed1999

Bradykinin induces interleukin-6 expression in astrocytes through activation of nuclear factor-kappaB.

Bradykinin, a mediator of inflammation, is produced in the brain during trauma and stroke. It is thought to open the blood-brain barrier, although the mechanism is unclear. We have investigated, therefore, the effect of bradykinin on the expression of interleukin-6 (IL-6), a putative modulator of the blood-brain barrier, in astrocytes. IL-6 gene transcription was evaluated by transient transfection of the human IL-6 promoter linked to the luciferase gene. In murine astrocytes, bradykinin stimulated IL-6 secretion and gene transcription. The effect of bradykinin was blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases, and by bisindolylmaleimide I, an inhibitor of protein kinase C, suggesting the involvement of these protein kinases. Mutations in the multiple response element and the binding site for nuclear factor-kappaB (NF-kappaB), but not in other known elements of the IL-6 promoter, interfered with induction of IL-6 transcription. The involvement of NF-kappaB was supported further by the finding that overexpression of nmIkappaB alpha, a stable inhibitor of NF-kappaB, inhibited the induction of IL-6 by bradykinin. Bradykinin activated NF-kappaB in primary astrocytes as shown by increased DNA binding of NF-kappaB. These data demonstrate that bradykinin stimulates IL-6 expression through activation of NF-kappaB, which may explain several inflammatory effects of bradykinin.

Animals↗
PubMed1998

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Amino Acid Sequence↗
PubMed1998

Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response.

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a beta-galactosidase-expressing virus (Ad-betagal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented beta-galactosidase expression from Ad-betagal, whereas the expression of the FasL transgene was unaffected. When Ad-betagal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and beta-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.

Adenoviridae Infections↗
PubMed1998

Tissue-restricted expression of the cardiac alpha-myosin heavy chain gene is controlled by a downstream repressor element containing a palindrome of two ets-binding sites.

The expression of the alpha-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in alpha-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac alpha-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the alpha-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the alpha-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the alpha-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the alpha-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the alpha-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the alpha-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the alpha-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

Animals↗
PubMed1997

Parathyroid hormone (1-34)-mediated interleukin-6 induction.

Parathyroid hormone (PTH) functions in part by regulating osteoblast cytokine expression. We recently demonstrated that PTH induced a rapid and transient increase in interleukin-6 (IL-6) mRNA expression in rat bones in vivo. To determine the molecular basis of this effect, we analyzed the human IL-6 promoter fused (-1,179 to +9) with the chloramphenicol acetyltransferase (CAT) reporter gene in stable transfections into human osteoblast-like osteosarcoma SaOS-2 cells. We compared the effects of PTH on IL-6 expression with adenylate cyclase activator forskolin, PKC activator phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, interleukin-1 alpha (IL-1 alpha), prostaglandin E-2 (PGE-2), RS-66271 (a parathyroid hormone-related peptide analog), and platelet-derived growth factor-BB (PDGF-BB). Analyses of cell clones showed that IL-6 promoter expression was extremely low in the unstimulated state. Exposure to PTH (0.001-100 nM) for 12 h stimulated CAT expression in a dose-dependent manner (200-500% of control). Treatment with IL-1 alpha was more potent than PTH in inducing transcription of the IL-6 promoter (900-1,000%). Activation of the cAMP-PKA pathway by treatment with forskolin induced a comparable level of induction with PTH. Together, the effects of PTH and forskolin were additive. RS-66271, previously shown to have PTH-like effects, induced a comparable level of IL-6 promoter expression. When examined together, PTH+RS-66271 effects were comparable to PTH effects alone. Exposure to PGE-2, PMA, PDGF-BB, or A23187 for 12 h did not significantly alter IL-6 promoter expression. These results demonstrate PTH, forskolin, the PTHrP analog RS-66271, and IL-1 alpha stimulate IL-6 expression by stimulating gene transcription. The response to forskolin suggests that the messenger system mediated by PKA is sufficient to induce IL-6 expression.

Becaplermin↗
PubMed1997

Ex vivo adenovirus-mediated gene delivery leads to long-term expression in pancreatic islet transplants.

BACKGROUND: Replication-deficient adenovirus, one of the most efficient vectors in gene therapy, has been limited by transient transgene expression due to its episomal location and loss during cell division, as well as a host immune response against viral proteins. METHODS: Murine pancreatic islets were infected ex vivo with ad5-cytomegalovirus (CMV)-beta-galactosidase and transplanted into diabetic recipients with normalization of glucose metabolism. RESULTS: High levels of beta-galactosidase activity were detectable histologically for at least 20 weeks after transplant, and beta-galactosidase and viral mRNA were also present that long. Sera from transplanted animals did not significantly inhibit ad5-CMV-interleukin-2-Ig infection of HeLa cells in vitro, whereas sera from intravenously delivered ad5-CMV-beta-galactosidase drastically diminished HeLa cell infection, suggesting the presence of reduced levels of antibodies in transplanted animals as compared with intravenously infected animals. Immunofluorescent staining of islet isografts infected with ad5-CMV-beta-galactosidase revealed the presence of CD8+ and CD4+ T lymphocytes at all time points, however, no islet destruction was seen. CONCLUSIONS: Treatment of islet isografts ex vivo with ad5-CMV-beta-galactosidase results in prolonged transgene expression, possibly due to an attenuated immune response against adenovirus.

Adenoviridae↗
PubMed1997

Adenovirus-mediated expression of Fas ligand induces hepatic apoptosis after Systemic administration and apoptosis of ex vivo-infected pancreatic islet allografts and isografts.

Fas ligand (FasL) mediates apoptosis of Fas-bearing cells and is expressed on a limited number of tissues, predominantly activated T lymphocytes. We describe the construction and biological activity of a replication-deficient type-5 adenovirus encoding murine FasL under the control of the cytomegalovirus (CMV) promoter (adCMV-FasL). In vitro, Jurkat cells undergo apoptosis when co-incubated with adCMV-FasL-infected COS cells. Systemic administration of adCMV-FasL to Wistar rats or DBA/2J mice results in widespread hepatic apoptosis and death in a dose-dependent manner within 72 hr, an effect not seen in lpr mice, or animals administered equivalent doses of adCMV-beta gal. Murine pancreatic islets also undergo apoptosis when infected ex vivo with adCMV-FasL, resulting in uniform primary nonfunction when transplanted into syngeneic or allogeneic diabetic recipients. These results indicate that adCMV-FasL is a potentially useful tool to study Fas/FasL biology.

Adenoviridae↗
PubMed1997

Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family.

We report here the isolation of a novel, highly tissue-restricted member of the ets transcription factor/oncogene family, ESE-1 (for epithelium-specific Ets), which has features distinct from those of any other ets-related factor. ESE-1 contains two putative DNA binding domains: an ETS domain, which is unique in that the 5' half shows relatively weak homology to known ets factors, and an A/T hook domain, found in HMG proteins and various other nuclear factors. In contrast to any known ets factors, ESE-1 is expressed exclusively in epithelial cells. ESE-1 expression is induced during terminal differentiation of the epidermis and in a primary human keratinocyte differentiation system. The keratinocyte terminal differentiation marker gene, SPRR2A, is a putative target for ESE-1, since SPRR2A expression during keratinocyte differentiation correlates with induction of ESE-1 expression, and ESE-1 binds with high affinity to and transactivates the ets binding site in the SPRR2A promoter. ESE-1 also binds to and transactivates the enhancer of the Endo A gene, a potential target for ESE-1 in simple epithelia. Due to the important role that other ets factors play in cellular differentiation, ESE-1 is expected to be a critical regulator of epithelial cell differentiation.

Alternative Splicing↗
PubMed1996

ELF-1 interacts with and transactivates the IgH enhancer pi site.

We previously identified a B-cell-specific regulatory element in the immunoglobulin heavy chain (IgH) enhancer, pi, with striking similarity to binding sites for ets-related transcription factors. Whereas the ability of ets-related factors to bind to and transactivate the pi site has been substantiated, the identification of the particular member of the ets family responsible for B-cell-specific regulation of the pi site has remained controversial. We have used antibodies specific for individual members of the ets family to evaluate which ets-related factor in B-cell nuclear extracts interacts with the IgH pi site. We present strong evidence that ELF-1 is highly expressed in B-cells and is one of two major factors specifically interacting with the murine IgH enhancer pi site in B-cell nuclear extracts. Binding of ELF-1 correlates with activity of the pi site, since mutations abolishing function of pi also inhibit binding of ELF-1. Furthermore, we demonstrate that ELF-1 can transactivate the IgH enhancer in HeLa cells, suggesting a role for ELF-1 in B-cell-specific IgH gene expression.

Animals↗
PubMed1996

Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B.

Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) not only localizes MM cells in the marrow microenvironment, but also triggers interleukin-6 (IL-6) secretion by BMSCs and related MM cell proliferation. In the present study, we characterized the regulation of IL-6 gene expression in BMSCs during MM cell adhesion. Adhesion of ARH-77, HS-Sultan, IM-9, and U266 MM cell lines to BMSCs and BMSC lines (LP 101 and AA 101) triggered 5-through 15-fold and 2-through 4-fold increases in IL-6 secretion, respectively. IL-6 mRNA transcripts were undetectable by Northern blotting in IM-9 MM cells or LP 101 BMSCs cultured alone; however, adherence of IM-9 cells to LP 101 cells induced a transient increase in IL-6 transcripts at 6 hours, followed by peak IL-6 secretion at 24 hours. To confirm increased IL-6 transcription and characterize its regulation, LP101 BMSCs were transiently transfected with full length and deletion fragments of the IL-6 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection of LP101 BMSCs with plasmid containing an intact NF-kappa B site showed a 6.8 +/- 0.4-fold increase in CAT activity triggered by IM-9 MM cell adhesion (n = 3, P < .05). Transfection of LP 101 cells with plasmid containing a single base pair deletion from the NF-kapp B binding motif abolished the MM adhesion-induced increase in CAT activity, whereas transfection with plasmid containing three copies of synthetic NF-kappa B sequence resulted in an 8.1 +/- 0.7-fold increase in CAT activity related to MM adhesion (n = 3, P < .05). These data suggest that the NF-kappa B site is one of the essential regulatory elements for MM cell adhesion-induced IL-6 transcription in BMSCs. Electrophoretic mobility shift assays confirmed the involvement of NF-kappa B activation in regulating MM adhesion-induced IL-6 transcription in BMSCs. Further characterization of the upstream events in the signalling cascade regulating IL-6 may not only delineate mechanisms of IL-6 regulation during paracrine MM cell growth, but also provide new therapeutic strategies based on interruption of IL-6 mediated tumor cell growth.

Base Sequence↗
PubMed1996

Characterization of NERF, a novel transcription factor related to the Ets factor ELF-1.

We have cloned the gene for a novel Ets-related transcription factor, new Ets-related factor (NERF), from human spleen, fetal liver, and brain. Comparison of the deduced amino acid sequence of NERF with those of other members of the Ets family reveals that the level of homology to ELF-1, which is involved in the regulation of several T- and B-cell-specific genes, is highest. Homologies are clustered in the putative DNA binding domain in the middle of the protein, a basic domain just upstream of this domain, and several shorter stretches of homology towards the amino terminus. The presence of two predominant NERF transcripts in various fetal and adult human tissues is due to at least three alternative splice products, NERF-1a, NERF-1b, and NERF-2, which differ in their amino termini and their expression in different tissues. Only NERF-2 and ELF-1, and not NERF-1a and NERF-1b, function as transcriptional activators of the lyn and blk gene promoters, although all isoforms of NERF bind with affinities similar to those of ELF-1 to a variety of Ets binding sites in, among others, the blk, lck, lyn, mb-1, and immunoglobulin H genes and are expressed at similar levels. Since NERF and ELF-1 are coexpressed in B and T cells, both might be involved in the regulation of the same genes.

Adult↗
PubMed1994

ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development.

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

3T3 Cells↗
PubMed1994

Multiple regulatory elements in the interleukin-6 gene mediate induction by prostaglandins, cyclic AMP, and lipopolysaccharide.

Induction of interleukin-6 (IL-6) gene expression is mediated by numerous agents involving all major signal transduction pathways. We have compared the effects of prostaglandins and their second messenger cyclic AMP (cAMP) with the effect of lipopolysaccharide (LPS) on IL-6 gene expression. We demonstrate that secretion of IL-6 is induced by cAMP in murine monocytic PU5-1.8 cells, even though to a lesser extent than by LPS. Nevertheless, cAMP and prostaglandins of the E series in the presence of theophylline induce transcription of the IL-6 promoter more strongly than LPS, suggesting distinctive effects of cAMP and LPS on posttranscriptional events. Mutations within four regulatory elements, namely, the multiple response element (MRE), AP-1, NF-IL6, and NF-kappa B sites, significantly reduce, but do not completely abrogate, inducibility by cAMP and prostaglandin E1, whereas alterations of four additional sites have no effects. LPS-induced promoter activity, however, is almost completely abolished by mutations in the NF-kappa B site, suggesting that a single regulatory element is crucial for inducibility by LPS. Stimulation by cAMP is correlated with the binding of inducible factors to the AP-1, NF-IL6, and NF-kappa B elements, whereas factors binding to the MRE are constitutively expressed. Recombinant cAMP response element-binding protein binds to the MRE, indicating a potential role for this factor in the cAMP response. Our results suggest that cAMP and prostaglandins act through multiple, partially redundant regulatory elements to induce IL-6 expression in monocytic cells. Nuclear events that overlap partially with the LPS response but also exhibit distinctive features are involved.

Alprostadil↗
PubMed1994

Coexpression of interleukin-1 beta and interleukin-6 in human brain tumors.

Deregulation of the expression of cytokine genes appears to play a role in the development of gliomas. Interleukin-1 beta has been shown to be synthesized in small amounts by astrocytes and can induce the expression of interleukin-6. Interleukin-6 is not expressed by unstimulated astrocytes, but the deregulation of its expression has been implicated in the progression of several tumor types. In this study, tumor specimens from 16 brain tumors and 22 glioma cell lines were studied for the gene expression of both interleukin-1 beta and interleukin-6, and the coexpression of these two cytokines was found in a significant number of these specimens. The expression of these two growth factor genes may play an important role in the growth and development of human gliomas.

Adolescent↗
PubMed1993

Pi, a pre-B-cell-specific enhancer element in the immunoglobulin heavy-chain enhancer.

We have identified a new immunoglobulin heavy-chain enhancer element, designated pi, between the microE2 and microE3 elements. The pi enhancer element is transcriptionally active primarily during early stages of B-cell development but becomes virtually inactive during B-cell maturation at about the stage of immunoglobulin kappa light-chain gene rearrangement. Mutational analysis suggests that the pi element is crucial for immunoglobulin heavy-chain enhancer activity at the pre-B-cell stage but is almost irrelevant for enhancer activity at the mature B-cell or plasma-cell stage. The activity of the pi enhancer element correlates with the presence of an apparently pre-B-cell-specific protein-DNA complex. The similarity of the pi site to recognition sequences for members of the ets gene family suggests that the protein(s) interacting with the pi site most likely are ets-related transcription factors.

3T3 Cells↗
PubMed1992

HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.

Several putative NF-kappa B-binding sites in the ICP0 and Vmw65 herpes simplex virus type-1 (HSV-1) genes have been identified. Oligonucleotides encoding some of these sites bind specifically to purified NF-kappa B protein and an NF-kappa B-like protein in nuclear extracts of phorbol ester- or cycloheximide-induced human embryonic lung (HEL) cells. HSV-1 infection of HEL cells induced a nuclear factor that binds specifically to kappa B sites in the ICP0 and Vmw65 gene regions and comigrates with complexes formed by purified NF-kappa B. The HSV-1-inducible nuclear factor bound to the authentic immunoglobulin (Ig) kappa B site. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing two copies of the Ig kappa B site upstream of the c-fos promoter (kappa B2-CAT) showed activity in HEL cells. HSV-1 infection of kappa B2-CAT-transfected HEL cells, however, induced a dramatic increase in CAT activity; mutation in the NF-kappa B-binding site of kappa B2-CAT abolished the inducibility of CAT gene expression. Our results demonstrate that the HSV-1 ICP0 and Vmw65 gene regions contain binding sites for NF-kappa B, and that HSV-1-inducible proteins bind to NF-kappa B-like sites in the HSV-1 genome.

Base Sequence↗
PubMed1990

Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.

The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.

Amino Acid Sequence↗