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M Zenke

Publications and source records attributed to M Zenke.

At least 37 records · Page 2Linked to original sources
PubMed1998

Retinoid X receptor and c-cerbA/thyroid hormone receptor regulate erythroid cell growth and differentiation.

Nuclear receptors are important regulators of erythroid cell development. Here we investigated the impact of retinoid X receptor (RXR), retinoic acid receptor (RAR), and of the c-erbA/thyroid hormone (T3) receptor (c-erbA/TR) on growth and differentiation of erythroid cells using an in vitro culture system of stem cell factor-dependent erythroid progenitors. RXR, RAR, and c-erbA/TR-specific ligands were found to induce erythroid-specific gene expression and to accelerate erythroid differentiation in culture, with T3 being most effective. Furthermore, while ligand-activated c-erbA/TR accelerated differentiation, unliganded c-erbA/TR effectively blocked differentiation and supported sustained progenitor growth in culture. Thus, c-erbA/TR appears to act as a binary switch affecting erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated c-erbA/TR induces differentiation. Additionally, to determine the impact of RXR for erythroid cell development, dominant interfering mutant RXRs, lacking the transcriptional activator functions AF-1 and AF-2, or AF-2 only, or the entire DNA-binding domain, were introduced into erythroid progenitor cells via recombinant retrovirus vectors and analyzed for RXR-specific effects. It was found that expression of wild-type RXR and of the RXR mutants devoid of AF-1 and/or AF-2 supported a transient outgrowth of erythroid cells. In marked contrast, expression of the dominant interfering deltaDNA-binding domain RXR, containing a deletion of the entire DNA-binding domain, was incompatible with erythroid cell growth in vitro, suggesting a pivotal role of RXR for erythroid cell development.

Animals↗
PubMed1997

The Myb leucine zipper is essential for leukemogenicity of the v-Myb protein.

The AMV v-Myb oncoprotein causes oncogenic transformation of myelomonocytic cells in vivo and in vitro. Its transforming capacity is strictly dependent upon the N-terminal DNA binding domain, the central transactivation region, and on the C-terminal domain containing a putative leucine zipper motif. Here we show that the v-MybL3,4A mutant, in which Leu325 and Leu332 of the leucine zipper have been replaced by alanines, failed to induce leukemia in virus infected chicken. This demonstrates that the leucine zipper domain is indispensable for v-myb induced leukemogenesis in vivo. v-MybL3,4A was, however, still able to transform myelomonocytic cells from chicken bone marrow in vitro. Yet, while v-mybL3,4A transformed cells were impaired in growth at 37 degrees C, they failed to grow at 42 degrees C, the physiological body temperature of avian species. This might explain the loss of v-MybL3,4A leukemogenic potential in vivo. We also demonstrate that the v-Myb leucine zipper domain interacts in vitro with two host cell proteins, p26 and p28. This interaction is compromised in v-MybL3,4A indicating that binding of v-Myb to p26 and p28 might be important for the leukemogenic potential of v-Myb.

Amino Acid Sequence↗
PubMed1997

[Usefulness of directional coronary atherectomy as a bail-out device for acute closure after coronary angioplasty].

The usefulness of directional coronary atherectomy (DCA) as a bail-out device for acute closure (reclosure) after percutaneous transluminal coronary angioplasty (PTCA) was evaluated. PTCA was performed in 1,023 patients (182 with acute myocardial infarction) between January 1993 and January 1994 in our hospital. Thirty-one patients (11 with acute myocardial infarction) suffered acute closure (reclosure) after PTCA. In six patients (five with acute myocardial infarction), DCA was performed as a rescue treatment for acute closure (reclosure). In three of these patients, angioscopy was performed before DCA, which demonstrated intimal tear and some thrombi although coronary angiography showed no evidence of thrombus. Bail-out DCA was successful in all six patients without complications. DCA is useful as a bail-out device for acute closure (reclosure) after PTCA when the thrombus is not massive.

Angioplasty, Balloon, Coronary↗
PubMed1996

Avian stem cell factor (SCF): production and characterization of the recombinant His-tagged SCF of chicken and its neutralizing antibody.

Recombinant chicken stem cell factor (SCF) was produced in bacteria as a histidine-tagged protein (His delta SCF) and purified by affinity chromatography on an Ni(2+)-NTA agarose column. His delta SCF is devoid of signal peptide and transmembrane domains and therefore mimics a soluble form of SCF. We demonstrate that the factor is biologically fully active in supporting sustained proliferation of SCF-dependent red blood progenitor cells of chicken in vitro. Polyclonal SCF-specific antibodies were raised in rabbits and shown to display potent SCF-neutralizing activity. Thus, recombinant chicken His delta SCF together with the SCF-specific antibody provide valuable tools for studying the role of SCF and c-kit receptor in development, growth and differentiation of avian haematopoietic cells.

Amino Acid Sequence↗
PubMed1996

Regulation and function of transcription factor GATA-1 during red blood cell differentiation.

The tissue-specific transcription factor GATA-1 is a key regulator of red blood cell differentiation. One seemingly contradictory aspect of GATA-1 function is that, while it is abundant in erythroid progenitor cells prior to the onset of overt differentiation, it does not significantly activate known GATA-1 target genes in those cells. To investigate the mechanisms underlying GATA-1 function during the transition from early to late erythropoiesis, we have examined its expression and activity in normal avian erythroid progenitor cells before and after induction of differentiation. In these primary progenitor cells, GATA-1 protein was predominantly located in the cytoplasm, while induction of differentiation caused its rapid relocalization to the nucleus, suggesting that nuclear translocation constitutes an important regulatory step in GATA-1 activation. As an alternative way of addressing the same question, we also ectopically expressed a GATA-1/estrogen receptor fusion protein (GATA-1/ER) in red blood cell progenitors, where nuclear translocation of, and transcriptional activation by, this hybrid factor are conditionally controlled by estrogen. We found that hormone-activated GATA-1/ER protein accelerated red blood cell differentiation, and concomitantly suppressed cell proliferation. These phenotypic effects were accompanied by a simultaneous suppression of c-myb and GATA-2 transcription, two genes thought to be involved in the proliferative capacity of hematopoietic progenitor cells. Thus, GATA-1 appears to promote differentiation in committed erythroid progenitor cells both by inducing differentiation-specific genes and by simultaneously suppressing genes involved in cell proliferation.

Animals↗
PubMed1995

Generation of cell lines from embryonic quail retina capable of mature neuronal differentiation.

The avian embryonic retina is widely used as a model system for cellular and molecular studies on central nervous system neurons. We aimed at the generation of cell lines from the early embryonic quail retina by retroviral oncogene transduction. For this, we made use of the retina organ culture system which exhibits both proliferation, necessary for stable oncogene transduction, and initial neuronal differentiation, a prerequisite for the generation of cell lines with mature neuronal properties. The oncogene myc was chosen as it is both proliferation-inducing and differentiation-compatible. A chimeric gene, mycER, containing v-myc and the hormone-binding domain of the estrogen receptor, was used for transduction in order to allow for hormone regulation of myc activity. Transduced organ-cultured cells from temporal and nasal retina were passaged into sparse single cell cultures. From these, colonies of rapidly dividing cells were isolated and the progeny expanded as cell lines. The lines contained cells with features of neuroepithelial cells, showing vimentin and A2B5. They also contained spontaneously differentiated neuronal cells showing neurofilament L and N-CAM180. A subpopulation of the neuronal cells exhibited the morphological characteristics of retinal ganglion cells, i.e., large pear-shaped somata each emitting one long process with a distinct growth cone. In addition, they showed the marker profile of retinal ganglion cells, i.e., expression of Thy-1, G4, DM-GRASP, Nr-CAM, neurofilament H, and tau. Neuronal differentiation could be induced by the addition of db cAMP and retinoic acid. The mature neuronal features of the lines open new possibilities to study properties of retinal neurons, including ganglion cells, in a defined and manipulable experimental system.

Animals↗
PubMed1995

Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-RelER.

A conditional v-Rel estrogen receptor fusion protein, v-RelER, causes estrogen-dependent but otherwise unaltered v-rel-specific transformation of chicken bone marrow cells. Here, we demonstrate that such v-relER-transformed cells exhibit B lymphoid determinants in line with earlier studies on v-rel-transformed cells. However, following inactivation of v-RelER oncoprotein activity by administration of an estrogen antagonist, cells differentiate into antigen-presenting dendritic cells as judged by several morphological and functional criteria. Additionally, under yet different culture conditions, v-relER cells differentiate into cells resembling polymorphonuclear neutrophils. Our studies therefore suggest that the conditional v-RelER, and probably also the authentic v-Rel, transform a common progenitor for neutrophils and dendritic cells.

Animals↗
PubMed1994

Structure and expression of the chicken retinoblastoma gene.

The molecular cloning and initial characterization of the complementary DNA encoding the chicken homologue of the human retinoblastoma susceptibility gene product Rb are described. Chicken Rb (chRb) was found to be highly homologous to human, mouse, and Xenopus Rb in primary amino acid sequence, underlining its conserved function in cell cycle control in higher eukaryotes. The highest level of homology was found within the Rb "pocket domains" and at the Rb COOH terminus, whereas the extreme NH2 terminus of chRb is divergent. We also show that transcription of chRb initiates at multiple start sites within a DNA segment which represents a CpG island. ChRb is transcribed as a 4.7-kilobase (kb) mRNA species in a variety of normal chicken tissues and oncogene-transformed hematopoietic cells. However, in v-rel-transformed cell lines, a variant 3.1-kb Rb-specific mRNA was detected in addition to the 4.7-kb transcript. ChRb is expressed as a 104 kilodalton protein and is therefore slightly smaller than the mammalian Rb proteins.

Amino Acid Sequence↗
PubMed1993

Unliganded T3R, but not its oncogenic variant, v-erbA, suppresses RAR-dependent transactivation by titrating out RXR.

V-erbA is thought to be an antagonist of thyroid hormone receptor (T3R) function. Here we show that unliganded T3R, but not v-erbA, suppresses retinoic acid (RA)-dependent induction of the RAR-beta 2 promoter by competing for the common dimerization partner, the retinoid X receptor (RXR). Firstly, T3R suppression can be alleviated by co-transfection of RXR. Secondly, T3R, but not v-erbA, competes with RAR for RXR and causes the dissociation of a preformed RAR/RXR-RARE ternary complex in vitro. A single point mutation located in the dimerization interface of v-erbA (Pro349 to Ser) abolishes the transdominant phenotype when introduced at the respective position in T3R. The hypertransforming v-erbA variant r12, in which this mutation is reversed (Ser349 to Pro) suppresses RA-induced differentiation in chicken erythroid progenitors, while v-erbA does not. Our data thus suggest that unliganded T3R and v-erbA act as dominant suppressors through mechanistically distinct pathways.

Base Sequence↗
PubMed1993

Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner.

The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.

Amino Acid Sequence↗
PubMed1993

A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor).

The thyroid hormone (T3) receptor type alpha, the c-ErbA alpha proto-oncoprotein, stimulates transcription of T3-dependent promoters, interferes with AP-1 activity, and induces erythroid differentiation in a ligand-dependent manner. The v-ErbA oncoprotein does not bind hormone and has lost all of these activities. Using c-ErbA/v-ErbA chimeras, we found that a deletion of 9 amino acids, conserved among many members of the nuclear receptor superfamily, which are located at the extreme carboxy terminus of c-ErbA alpha is responsible for loss of both transactivation and transcriptional interference activities. Single, double, and triple amino acid substitutions within this region completely abolished T3-dependent transcriptional activation, interference with AP-1 activity, and decreased T3 binding by c-ErbA alpha. However, the lower T3 binding by these mutants does not fully account for the loss of transactivation and transcriptional interference, since a c-ErbA/v-ErbA chimera which was similarly reduced in T3 binding activity has retained both of these functions. Deletion of homologous residues in the retinoic acid receptor alpha (RAR alpha) resulted in a similar loss of transactivation and transcriptional interference activities. The ability of c-ErbA alpha to induce differentiation of transformed erythroblasts is also impaired by all of the mutations introduced into the conserved carboxy-terminal sequence. We conclude that this 9-amino-acid conserved region is essential for normal biological function of c-ErbA alpha and RAR alpha and possibly other T3 and RA receptors.

Amino Acid Sequence↗
PubMed1992

Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression.

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes.

Animals↗
PubMed1992

Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.

Animals↗
PubMed1992

Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action.

The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis. Here we demonstrate that the endogenous thyroid hormone receptor alpha (c-erbA/TR-alpha) and the closely related retinoic acid receptor alpha (RAR-alpha) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the RAR-alpha is essential for the RA effect. Likewise, enhanced expression of exogenous c-erbA/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors.

Bone Marrow Cells↗
PubMed1991

v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII.

The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein.

Base Sequence↗
PubMed1990

v-erbA oncogene activation entails the loss of hormone-dependent regulator activity of c-erbA.

The v-erbA oncogene, one of the two oncogenes of the avian erythroblastosis virus, efficiently blocks erythroid differentiation and suppresses erythrocyte-specific gene transcription. Here we show that the overexpressed thyroid hormone receptor c-erbA effectively modulates erythroid differentiation and erythrocyte-specific gene expression in a T3-dependent fashion, when introduced into erythroid cells via a retrovirus. In contrast, the endogenous thyroid hormone receptor does not detectably affect erythroid differentiation. The analysis of a series of chimeric v-/c-erbA proteins suggests that the v-erbA oncoprotein has lost one type of thyroid hormone receptor function (regulating erythrocyte gene transcription in response to T3), but constitutively displays another function: it represses transcription in the absence of T3. The region responsible for the loss of hormone-dependent regulator activity of v-erbA has been mapped to the very C-terminus of c-erbA, encompassing a cluster of highly conserved amino acid residues with the potential to form an amphipathic alpha-helix.

Alpharetrovirus↗