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Biomedical subjects

T Wirth

Publications and source records attributed to T Wirth.

At least 19 recordsLinked to original sources
PubMed1999

Regulation of the human interleukin-5 promoter by Ets transcription factors. Ets1 and Ets2, but not Elf-1, cooperate with GATA3 and HTLV-I Tax1.

Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.

Base Sequence↗
PubMed1999

NF-kappaB is activated and promotes cell death in focal cerebral ischemia.

The transcription factor NF-kappaB is a regulator of cell death or survival. To investigate the role of NF-kappaB in neuronal cell death, we studied its activation in a rodent model of stroke. In the ischemic hemisphere, NF-kappaB was activated, as determined by increased expression of an NF-kappaB-driven reporter transgene, nuclear translocation of NF-kappaB in neurons and enhanced DNA binding of NF-kappaB subunits RelA and p50. In p50 knockout mice, ischemic damage was significantly reduced. This indicates a cell death-promoting role of NF-kappaB in focal ischemia. NF-kappaB may provide a new pharmacological target in neurologic disease.

Animals↗
PubMed1998

No change of brain extracellular catecholamine levels after acute catechol-O-methyltransferase inhibition: a microdialysis study in anaesthetized rats.

Catechol-O-methyltransferase inhibitors have been newly introduced as adjunct drugs to the levodopa/dopa decarboxylase inhibitor therapy in Parkinson's disease. When given alone, catechol-O-methyltransferase inhibitors seem to affect behaviour. We wanted to determine whether the concentrations of free amine would be increased by catechol-O-methyltransferase inhibition with tolcapone and underpin the positive behavioural effects. To this end, dopamine and noradrenaline levels were analyzed in the microdialysis perfusion fluid collected from several brain regions in chloral hydrate anaesthetized rats. We also analyzed the turnover rate of catecholamines in the brain after single doses of tolcapone and entacapone using the alpha-methyl-p-tyrosine method. On their own, tolcapone (at 10 or 30 mg/kg) did not elevate dopamine or noradrenaline levels in any brain region studied although the formation of catechol-O-methyltransferase-dependent metabolites was strongly reduced. Neither tolcapone nor entacapone (at 30 mg/kg) affected the turnover rate of catecholamines. It seems that catechol-O-methyltransferase inhibitors do not alter behaviour by elevating extracellular levels of free catecholamines levels but other explanations are needed.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1998

Inhibition of nuclear factor kappaB activation attenuates apoptosis resistance in lymphoid cells.

Death-inducing ligands (DILs) such as tumor necrosis factor alpha (TNFalpha) or the cytotoxic drug doxorubicin have been shown to activate a nuclear factor kappaB (NFkappaB)-dependent program that may rescue cells from apoptosis induction. We demonstrate here that TRAIL (TNF-related apoptosis-inducing ligand), a recently identified DIL, also activates NFkappaB in lymphoid cell lines in a kinetic similar to TNFalpha. NFkappaB activity is independent from FADD, caspases, and apoptosis induction. To study the influence of NFkappaB activity on apoptosis mediated by TRAIL, CD95, TNFalpha, or doxorubicin, NFkappaB activation was inhibited using the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal or transient overexpression of mutant IkappaBalpha. Sensitivity for induction of apoptosis was markedly increased by these treatments in apoptosis sensitive cell lines. Moreover, both in cell lines and in primary leukemia cells that are resistant towards induction of apoptosis by DILs and doxorubicin, antagonization of NFkappaB activity partially restored apoptosis sensitivity. These data suggest that inhibition of NFkappaB activation may provide a molecular approach to increase apoptosis sensitivity in anticancer treatment.

Antibiotics, Antineoplastic↗
PubMed1998

The mutant plasmacytoma cell line S107 allows the identification of distinct pathways leading to NF-kappaB activation.

Studies on the mechanisms of inducible and constitutive activity of NF-kappaB transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-kappaB activity. Our analysis shows that these cells bear a specific defect that interferes with NF-kappaB induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-kappaB in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-kappaB. Interestingly, both the potent physiological inducer of NF-kappaB TNFalpha as well as endoplasmic reticulum overload can induce NF-kappaB via a PDTC sensitive pathway. In all cases, DNA-binding NF-kappaB complexes are comprised predominantly of p50-RelA heterodimers, and NF-kappaB activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-kappaB-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-kappaB induction. These routes utilized by tumor necrosis factor alpha and endoplasmic reticulum overload are still intact in this cell line.

Animals↗
PubMed1998

Induction of nuclear factor-kappa B during primary B cell differentiation.

We have investigated activation of nuclear factor-kappa B (NF-kappa B) in the process of primary B cell differentiation in vitro. In this system, NF-kappa B is strongly induced when B cells develop from the pre-B cell to the immature B cell stage. Unlike the typical NF-kappa B activation in response to exogenous stimuli, induction proceeds with a slow time course. NF-kappa B induction is only observed in B cells that undergo differentiation, not in Rag2-deficient cells. Nuclear DNA binding complexes predominantly comprise p50/RelA heterodimers and, to a lesser extent, c-Rel-containing dimers. The increase in NF-kappa B binding activity is accompanied by a slow and steady decrease in I kappa B beta protein levels. Interestingly, absolute RelA protein levels remain unaffected, whereas RelB and c-Rel synthesis is induced. The reason for preferential nuclear translocation of RelA complexes appears to be selective inhibition by the I kappa B beta protein. I kappa B beta can efficiently inhibit p50/RelA complexes, but has a much reduced ability to interfere with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly, p50/RelB complexes are not at all targeted by I kappa B beta, and coimmunoprecipitation experiments show no evidence for an association of I kappa B beta and RelB in vivo. Consistent with these observations, I kappa B beta cotransfection can inhibit p50/RelA-mediated trans-activation, but barely affects p50/RelB mediated trans-activation.

3T3 Cells↗
PubMed1998

Application of differential cDNA screening techniques to the identification of unique gene expression in tumours and lymphocytes.

Development, differentiation and cell death in all organisms are mediated by tightly regulated programs of differential gene expression. Furthermore, changes in gene expression profiles are responsible for tumour formation and tumour progression, as well as for many other human diseases. Thus, the definition of distinct patterns of gene expression is not only essential for understanding complex biological processes but also leads to the identification of novel targets for therapy of various diseases.

DNA, Complementary↗
PubMed1997

Glutamate-dependent activation of NF-kappaB during mouse cerebellum development.

NF-kappaB and activator protein 1 (AP-1) are dimeric transcription factors involved in transcriptional regulation in many cells, including neurons. We have examined their activity during mouse cerebellum development, a postnatal process starting just after birth and completed by the fourth postnatal (PN) week. The activity of these factors was analyzed by binding of nuclear extracts to a synthetic oligonucleotide representing the kappaB site of human immunodeficiency virus or the AP-1 site of the urokinase promoter. NF-kappaB activity was observed from 7 PN, was restricted to the developing cerebellum, and was not observed in the early postnatal neocortex and hippocampus. On the other hand, AP-1 activity was not found in cerebellum but was present in both neocortex and hippocampus. Moreover, a kappaB-driven transgene was found to be increasingly expressed in the cerebellum from 5 PN to 10 PN but not in the adult. The regulation of NF-kappaB activation in mouse cerebellum was analyzed by intraperitoneal injection of glutamate receptor antagonists to 9 PN mice, which abolished NF-kappaB-binding activity, suggesting an endogenous loop of glutamate receptor activation. Glutamate receptor agonists, on the other hand, induced NF-kappaB nuclear translocation in the cerebellum of 5 PN mice, which is a stage in which NF-kappaB is not yet endogenously activated. This effect was specific for NF-kappaB and not observed for AP-1. In adult mice, NF-kappaB activity was absent in the cerebellum and was not induced by intraperitoneal injection of glutamate receptor agonists. These data show that NF-kappaB is specifically activated during cerebellum development and indicate an important role of glutamate receptors in this process.

2-Amino-5-phosphonovalerate↗
PubMed1997

Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.

BOB.1/OBF.1 is a transcriptional coactivator that is constitutively expressed in B cells and interacts with the Oct1 and Oct2 transcription factors. Upon activation of Jurkat T cells and primary murine thymocytes with phorbol esters and ionomycin, BOB.1/OBF.1 expression and transactivation function were induced. BOB.1/OBF.1 was phosphorylated at Ser184 both in vivo and in vitro, and this modification was required for inducible activation. Mutation of Ser184 also diminished transactivation function in B cells, suggesting that the activating phosphorylation that is inducible in T cells is constitutively present in B cells. Thus, BOB.1/OBF.1 is a transcriptional coactivator that is critically regulated by posttranslational modifications to mediate cell type-specific gene expression.

Amino Acid Sequence↗
PubMed1997

RelB is a key player for both kappa B-dependent transcription and demethylation in B cells.

NF-kappa B was originally identified as a B cell-specific nuclear factor binding to the intronic kappa-light-chain enhancer element. It is found constitutively in the nucleus of mature B and plasma cells. In other cell types including pre-B cells, NF-kappa B is sequestered in the cytoplasm but can be induced by a variety of stimuli. In contrast to essentially all other mature B cells and plasma cell lines, the S107 plasmacytoma cell line lacks both constitutive and inducible kappa B-binding activity. A molecular characterization of the defect in these S107 cells suggests that the primary defect lies in the signal transduction pathway leading to NF-kappa B induction. Ectopic expression of RelB after stable transfection of these cells restores constitutive nuclear kappa B-binding activity. Moreover, kappa B-dependent transcription is also restored. Finally we demonstrate, that in contrast to parental S107 cells, the stable RelB transfectants have also regained the ability to specifically demethylate a transfected immunoglobulin kappa-locus. These data suggest that RelB is critically involved in both B cell-specific transcription and demethylation directed by the intronic kappa-enhancer element.

B-Lymphocytes↗
PubMed1997

Identification of target genes of the lymphoid-specific transcription factor Oct2.

The Oct2 transcription factor is expressed predominantly in B lymphocytes and plays an essential role during the terminal phase of B cell differentiation. The regulatory regions of several genes specifically expressed in B cells contain functional binding sites for Oct2. Nevertheless, none of the genes originally thought to be regulated by Oct2 were affected in their expression in Oct2-deficient B cells. In an attempt to find such elusive Oct2 target genes and to understand the molecular function of Oct2 in B cell development, we isolated cDNAs for Oct2 target genes. So far, we have identified five potential targets for Oct2: the membrane glycoprotein CD36, the cysteine-rich secreted protein 3 (CRISP-3), a mouse homolog of the human monocyte/neutrophil elastase inhibitor (mEI) and two unknown cDNA sequences Nov1 and Nov2. These target genes show quite distinct expression patterns demonstrating that transcription factors in addition to Oct2 are involved in their regulation. Whereas CD36 and mEI were expressed in all hematopoetic cell lines containing Oct2,. CRISP-3 is pre-B cell-specific, Nov1 is plasma B cell-specific and Nov2 is B cell-specifically expressed.

Animals↗
PubMed1996

CD40, but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes, leads to a persistent nuclear accumulation of RelB.

In this study we analyzed the effect of CD40 stimulation on the activity and nuclear appearance of Rel/nuclear factor kappaB (NF-kappaB) factors in primary murine B lymphocytes. We show that triggering of CD40 signaling pathway(s) by CD40 ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice. Analyses of nuclear translocation of individual members of Rel proteins after CD40 induction of primary B cells showed a strong and long-lasting accumulation of RelB and, less pronounced, of c-Rel. LPS stimulation did not give rise to a persistent nuclear accumulation of RelB and c-Rel, whereas nuclear c-Rel, but not RelB, accumulated after B cell receptor stimulation. CD40 induced not only nuclear translocation but also de novo synthesis of RelB RNA and protein. S107 plasmacytoma cells, which express CD40 but are defective for the nuclear appearance of p50/p65-NF-kappaB, do not express RelB after CD40 stimulation. In S107 cells stably transfected with relB genes, stimulation of nuclear RelB translocation by CD40 was observed. These results indicate that stimulation of CD40 signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of RelB. Since LPS and anti-IgM were unable to activate RelB, CD40 appears to trigger a special program of gene expression involved in the proliferation and/or differentiation of B lymphocytes.

Animals↗
PubMed1996

Coordinate suppression of myeloma-specific genes and expression of fibroblast-specific genes in myeloma X fibroblast somatic cell hybrids.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of the differentiation-specific traits of the non-fibroblast parental cell. To explore the genetic basis of this phenomenon, we have studied a series of somatic cell hybrids between mouse myeloma and fibroblasts. All the hybrids were adherent having a fibroblast-like phenotype. Molecular analysis revealed that plasma cell specific genes like the productively rearranged Ig genes, the J chain gene and genes for the cell surface markers CD20 and PC1, were extinguished in the hybrids. In contrast, fibroblast specific genes like fibronectin, alpha 2(I) and III collagens, as well as the receptor for fibroblast growth factor (flg), were expressed. Extinction was not due to chromosomal loss or lack of the relevant genes. To learn about the mechanism(s) of this phenomenon we have looked for the presence of positive and negative transcription factors in our hybrids. Expression of the PU.1 transcription factor, a member of the Ets transcription factor family normally expressed in B cells and macrophages, was lost in the cell hybrids. Interestingly, we found that the B-cell-specific Oct-2 transcription factor was still expressed at somewhat variable levels in several of the hybrid cell lines. In contrast, expression of the recently identified octamer coactivator BOB.1/OBF.1 was extinguished in all cell hybrids. This supports a critical role of this transcriptional coactivator for B-cell-specific gene expression. In addition, the Id and HLH462 genes coding for proteins known to repress bHLH transcription factors by formation of heterodimers, were found to be expressed at increased levels in fibroblasts and in the hybrids, indicating that their increased levels might also contribute to the suppression of myeloma-specific genes. Our results show that in myeloma x fibroblast hybrids, the phenotype of the fibroblast is dominant. It is suggested that fibroblasts contain regulatory "master" genes that are responsible for activation of the fibroblast differentiation pathway and suppress differentiation programs of other cell types.

Animals↗
PubMed1996

A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus.

The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

B-Lymphocytes↗
PubMed1996

CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2.

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.

3T3 Cells↗
PubMed1995

Functional characterization of the murine homolog of the B cell-specific coactivator BOB.1/OBF.1.

B cell-specific transcriptional promoter activity mediated by the octamer motif requires the Oct1 or Oct2 protein and additional B cell-restricted cofactors. One such cofactor, BOB.1/OBF.1, was recently isolated from human B cells. Here, we describe the isolation and detailed characterization of the murine homolog. Full-length cDNAs and genomic clones were isolated, and the gene structure was determined. Comparison of the deduced amino acids shows 88% sequence identity between mouse and human BOB.1/OBF.1. The NH2-terminal 126 amino acids of BOB.1/OBF.1 are both essential and sufficient for interaction with the POU domains of either Oct1 or Oct2. This protein-protein interaction does not require the simultaneous binding of Oct proteins to DNA, and high resolution footprinting of the Oct-DNA interaction reveals that binding of BOB.1/OBF.1 to Oct1 or Oct2 does not alter the interaction with DNA. BOB.1/OBF.1 can efficiently activate octamer-dependent promoters in fibroblasts; however, it fails to stimulate octamer-dependent enhancer activity. Fusion of subdomains of BOB.1/OBF.1 with the GAL4 DNA binding domain reveals that both NH2- and COOH-terminal domains of BOB.1/OBF.1 contribute to full transactivation function, the COOH-terminal domain is more efficient in this transactivation assay. Consistent with the failure of full-length BOB.1/OBF.1 to stimulate octamer-dependent enhancer elements in non B cells, the GAL4 fusions likewise only stimulate from a promoter-proximal position.

Amino Acid Sequence↗