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W Schmid

Publications and source records attributed to W Schmid.

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

DNA binding of the glucocorticoid receptor is not essential for survival.

Transcriptional regulation by the glucocorticoid receptor (GR) is essential for survival. Since the GR can influence transcription both through DNA-binding-dependent and -independent mechanisms, we attempted to assess their relative importance in vivo. In order to separate these modes of action, we introduced the point mutation A458T into the GR by gene targeting using the Cre/loxP system. This mutation impairs dimerization and therefore GRE-dependent transactivation while functions that require cross-talk with other transcription factors, such as transrepression of AP-1-driven genes, remain intact. In contrast to GR-/- mice, these mutants termed GRdim are viable, revealing the in vivo relevance of DNA-binding-independent activities of the GR.

Amino Acid Sequence↗
PubMed1998

Distribution of Na,K-ATPase is normal in the inner ear of a mouse with a null mutation of the glucocorticoid receptor.

This study was performed in order to test the hypothesis that the glucocorticoid hormone stimulates the formation of Na,K-ATPase in the inner ear of the mouse. An immunohistochemical study with respect to the presence and distribution of glucocorticoid receptors (GR) and Na,K-ATPase in the vestibular and cochlear regions of the inner ear was performed on a C57BL mouse with a null mutation of the glucocorticoid receptor (GR mutant mouse). The wild type C57BL mouse and the CBA mouse served as normal controls. As expected, the homozygous GR mutant mouse showed no specific staining for GR in the inner ear. The heterozygous GR mutant mouse showed faint staining of GR in the spiral limbus, the spiral ganglion, the organ of Corti and the utricle. This staining was markedly less than in the wild type C57BL mouse. Antibody labelling of Na,K-ATPase in the inner ear showed no significant difference between the homozygous and the heterozygous GR mutant mouse as compared to the control wild type C57BL mouse or the CBA mouse. Although earlier studies have shown a positive correlation between levels of glucocorticoid hormone in serum and the concentration of Na,K-ATPase in the inner ear, the hypothesis that glucocorticoid hormones alone stimulate the formation of Na,K-ATPase in the inner ear could not be confirmed by this study. Thus other regulating substances must be considered.

Adrenal Cortex Hormones↗
PubMed1998

Analysis of glucocorticoid signalling by gene targeting.

Glucocorticoids are involved in the regulation of numerous physiological processes. The majority of these effects are thought to be mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. In most cases activation requires binding of a receptor-dimer to DNA while repression is mediated by protein-protein-interaction of GR-monomers with other transcription factors. To analyse the molecular mechanisms that underlie glucocorticoid effects, mouse mutations in the GR gene were generated and analysed. In order to address the role of glucocorticoid receptor signalling during development and in physiology, the gene was disrupted by gene targeting. Most of the mice homozygous for the mutation die shortly after birth due to severe lung atelectasis. Additional defects were found in the adrenals, liver, brain, bone marrow and thymus as well as in the feedback-regulation of the HPA-axis. To approach the question which functions of the GR are regulated by DNA-binding and which by protein-protein-interaction, a point mutation was introduced into the dimerization domain of the GR which is located in the DNA-binding domain. By homologous recombination in ES-cells using the Cre/loxP-system, mice carrying this mutation were generated [GR(dim) mice]. The mice are fully viable although they show impaired inducibility of gluconeogenetic enzymes in liver, defects in longterm renewal of erythroid progenitors and increased expression of POMC and ACTH in the pituitary. However neither in the lung nor the adrenals were any histological abnormalities found. In conclusion GR(dim)-mice represent a valuable tool to further analyse mechanisms of physiological effects of the GR.

Animals↗
PubMed1997

Ginkgo thrives.

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Cognition Disorders↗
PubMed1997

CREB is activated by UVC through a p38/HOG-1-dependent protein kinase.

Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)-p62TCF which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position -60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2-p62TCF pathway.

Activating Transcription Factor 1↗
PubMed1997

Regulation of hippocampal 5-HT1A receptor mRNA and binding in transgenic mice with a targeted disruption of the glucocorticoid receptor.

Corticosterone is known to suppress levels of 5-HTA(1A) receptor mRNA in rat hippocampus. We describe hippocampal 5-HT(1A) receptor mRNA regulation in mice that have a targeted disruption of the glucocorticoid receptor gene. 5-HT(1A) receptor mRNA levels as well as binding of [3H]8-OH-DPAT, were measured in the hippocampus of heterozygous and homozygous GR-deficient mice and in wild-type control mice. The effect of adrenalectomy in wild-type mice and heterozygous knockouts was also studied. We hypothesized that if the glucocorticoid receptor is important as a mediator of the suppressive effect of corticosterone, this would be revealed by changed (enhanced) expression of 5-HT(1A) receptor mRNA in mice with a genetically changed glucocorticoid receptor status. It was found that 5-HT(1A) receptor mRNA levels and 5-HT(1A) receptor binding were not different in GR-deficient mice. The 5-HT(1A) receptor mRNA levels were responsive to corticosterone, as adrenalectomy led to increased levels of hippocampal 5-HT(1A) receptor mRNA both in wild-type as in heterozygous knockout mice. These increases were paralleled by small but statistically significant changes in [3H]8-OH-DPAT binding. These results support a suppressive control of B over 5-HT(1A) receptor expression in the hippocampus of the mouse, which is predominantly mediated via the mineralocorticoid receptor. The data indicates that no interaction between the two corticosteroid receptors is required for this effect of corticosterone, and that mineralocorticoid receptor-mediated suppression of gene expression can take place in the complete absence of glucocorticoid receptor.

Adrenalectomy↗
PubMed1997

Expression of activating transcription factor-2, serum response factor and cAMP/Ca response element binding protein in the adult rat brain following generalized seizures, nerve fibre lesion and ultraviolet irradiation.

The expression of the constitutive transcription factors activating transcription factor-2 (ATF-2), serum response factor (SRF) and cAMP/Ca response element binding factor (CREB), and the phosphorylation of SRF and CREB were studied in the untreated adult rat nervous system and following seizure activities and neurodegenerative stimuli. In the untreated rat, intense nuclear SRF immunoreactivity was present in the vast majority of neurons in the forebrain, cortex, striatum, amygdala and hippocampus, and in some scattered neurons in the medulla and spinal cord. In contrast, SRF immunoreactivity was absent in the midline areas of the forebrain, e.g., the globus pallidum and septum, and in the hypothalamus, thalamus, mesencephalon and motoneurons. Nuclear ATF-2 was expressed at high levels in apparently all neurons, but not glial cells, throughout the neuraxis except for those neuronal populations which exhibit a high basal level of c-Jun, i.e. dentate gyrus and the motoneurons of cranial and somatosensory neurons. CREB immunoreactivity was present at a rather uniform intensity in all neuronal and glial cells throughout the neuraxis. Two hours, but not 5 h or 24 h, following systemic application of kainic acid, an increase in SRF was detectable by western blot analysis in hippocampal and cortical homogenates whereas the expression of ATF-2 and CREB did not change. Phosphorylation of CREB at serine 133 and of SRF at serine 103 were studied with specific antisera. In untreated rats, intense phosphoCREB and phosphoSRF immunoreactivities labelled many glial cells and/or neurons with the highest levels in the dentate gyrus, the entorhinal cortex and the retrosplenial cortex. Following kainate-induced seizures, phosphoSRF-IR but not phosphoCREB-IR transiently increased between 0.5 h and 2 h. Following transection of peripheral or central nerve fibres such as optic nerve, medial forebrain bundle, vagal and facial nerve fibres, ATF-2 rapidly decreased in the axotomized neurons during that period when c-Jun was rapidly expressed. SRF remained unchanged and CREB disappeared in some axotomized subpopulations. Similar to axotomy, c-Jun increased and ATF-2 decreased in cultured adult dorsal root ganglion neurons following ultraviolet irradiation. The distribution of SRF and ATF-2 suggests that their putative target genes c-fos, junB, krox-24 and c-jun can be independently regulated from SRF and ATF-2. The suppression of ATF-2 and the expression of c-Jun following axotomy and ultraviolet irradiation might be part of a novel neuronal stress response in the brain that strongly resembles the stress response characterized in non-neuronal cells.

Activating Transcription Factor 2↗
PubMed1997

Spatial learning deficits in mice with a targeted glucocorticoid receptor gene disruption.

Previous studies in rats using the Morris water maze suggested that the processing of spatial information is modulated by corticosteroid hormones through mineralocorticoid and glucocorticoid receptors in the hippocampus. Mineralocorticoid receptors appear to be involved in the modulation of explorative behaviour, while additional activation of glucocorticoid receptors facilitates the storage of information. In the present study we used the water maze task to examine spatial learning and memory in mice homozygous and heterozygous for a targeted disruption of the glucocorticoid receptor gene. Compared with wild-type controls, homozygous and heterozygous mice were impaired in the processing of spatial but not visual information. Homozygous mutants performed variably during training, without specific platform-directed search strategies. The spatial learning disability was partly compensated for by increased motor activity. The deficits were indicative of a dysfunction of glucocorticoid receptors as well as of mineralocorticoid receptors. Although the heterozygous mice performed similarly to wild-type mice with respect to latency to find the platform, their strategy was more similar to that of the homozygous mice. Glucocorticoid receptor-related long-term spatial memory was impaired. The increased behavioural reactivity of the heterozygous mice in the open field points to a more prominent mineralocorticoid receptor-mediated function. The findings indicate that (i) the glucocorticoid receptor is of critical importance for the control of spatial behavioural functions, and (ii) mineralocorticoid receptor-mediated effects on this behaviour require interaction with functional glucocorticoid receptors. Until the development of site-specific, inducible glucocorticoid receptor mutants, glucocorticoid receptor-knockout mice present the only animal model for the study of corticosteroid-mediated effects in the complete absence of a functional receptor.

Animals↗
PubMed1996

Hippocampal cell responses in mice with a targeted glucocorticoid receptor gene disruption.

Previous studies in rats have shown that cellular properties of hippocampal CA1 neurons are under coordinative control of mineralocorticoid and glucocorticoid receptors (MRs and GRs, respectively). In the present study, we examined electrical properties under conditions of exclusive MR occupation, by using mice with a genetic defect in GRs obtained by homologous recombination techniques. It appeared that in the animals homozygous for the genetic defect, the properties studied, i.e., the voltage-gated Ca currents and responses to serotonin and the cholinergic analog carbachol, resembled the effects observed in adrenalectomized mice, i.e., when no steroid receptors are activated. This may point to the necessity of functional GRs for the development of MR-induced actions. Ca current amplitude and transmitter responses in the heterozygous animals, which combine a reduced amount of GRs in the hippocampus with relatively high circulating levels of corticosterone, were large compared with those in the wild-type controls; this resembles the responses that were observed previously in rats subjected to a very high dose of corticosterone. These findings exemplify the use of GR knockout mice for the study of cellular properties in the brain. Further substantiation of the observations, however, awaits the development of site-specific, inducible GR knockouts.

Animals↗
PubMed1996

Targeting of the CREB gene leads to up-regulation of a novel CREB mRNA isoform.

To define the role of cAMP signaling in gene control, we have generated mice with a mutation in the cAMP response element binding protein (CREB) gene. Mice carrying this mutation are viable but show an impairment in memory consolidation. In further analysis of these mice, we have found an up-regulation of a CREB isoform that has not been described previously . The new isoform, termed CREB beta, has nearly the same transactivation potential as the other CREB isoforms and is expressed ubiquitously. The up-regulation appears to be due to an increase in alternative splicing or mRNA stability, but not to an increase in transcriptional rate. Due to the relatively low levels of expression in all tissues, the role of this isoform is likely to be minor in the wild-type mouse. However, its dramatic up-regulation in the mutant mouse, together with the specific deficiencies recently observed in these mice, suggest that it has a very specific role in compensating for CREB alpha and delta in some, but not all, areas where CREB function has been implicated. Together with the up-regulation of the cAMP response element modulator protein (CREM) mRNA and protein levels demonstrated previously in CREB mutant mice, we suggest that the up-regulation of CREB beta may also contribute to compensation within the CREB/ATF family of transcription factors, when CREB delta and CREB alpha are absent.

Alternative Splicing↗
PubMed1996

Psychiatric symptoms and CAG expansion in Huntington's disease.

The mutation responsible for Huntington's disease (HD) is an elongated CAG repeat in the coding region of the IT15 gene. A PCR-based test with high sensitivity and accuracy is now available to identify asymptomatic gene carriers and patients. An inverse correlation between CAG copy number and age at disease onset has been found in a large number of affected individuals. The influence of the CAG repeat expansion on other phenotypic manifestations, especially specific psychiatric symptoms has not been studied intensively. In order to elucidate this situation we investigated the relation between CAG copy number and distinct psychiatric phenotypes found in 79 HD-patients. None of the four differentiated categories (personality change, psychosis, depression, and nonspecific alterations) showed significant differences in respect to size of the CAG expansion. In addition, no influence of individual sex on psychiatric presentation could be found. On the other hand in patients with personality changes maternal transmission was significantly more frequent compared with all other groups. Therefore we suggest that clinical severity of psychiatric features in HD is not directly dependent on the size of the dynamic mutation involved. The complex pathogenetic mechanisms leading to psychiatric alterations are still unknown and thus genotyping does not provide information about expected psychiatric symptoms in HD gene carriers.

Adult↗
PubMed1996

Somatic stability of the expanded CAG trinucleotide repeat in X-linked spinal and bulbar muscular atrophy.

Expansion of trinucleotide repeats has now been associated with eight inherited diseases: X-linked spinal and bulbar muscular atrophy, two fragile X syndromes, myotonic dystrophy, Huntington's disease, spinocerebellar ataxia type I, dentatorubral pallidoluysian atrophy and Machado-Joseph disease. It has been shown that these expanded DNA repeats are unstable in number when transmitted from parents to offspring ("meiotic instability"), while somatic variation in repeat number has also been found in the fragile X syndrome and myotonic dystrophy. Moderate meiotic instability has been demonstrated in X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease). In order to determine if the expanded CAG repeat in SBMA also shows somatic instability, we compared different tissues from two patients with SBMA. We then examined the in vitro stability of the CAG repeat expansion by analyzing fibroblast cell cultures. Length comparison of expanded CAG repeats from all these materials clearly demonstrates that the CAG trinucleotide repeat in SBMA does not exhibit somatic variation.

Cells, Cultured↗
PubMed1996

Molecular genetic analysis of glucocorticoid and mineralocorticoid signaling in development and physiological processes.

To understand the role of glucocorticoid and mineralocorticoid signalling during development and in whole animal physiology, we have disrupted the mouse glucocorticoid and mineralocorticoid receptor gene by gene targeting. Most of the mice with a disrupted glucocorticoid receptor gene die within the first hours after birth due to severe lung atelectasis. Perinatal induction of gluconeogenic enzymes in the liver is impaired. Regulation of the glucocorticoid synthesis via the hypothalamic-pituitary-adrenal axis is perturbed, leading to increased plasma levels of corticosterone and adrenocorticotrophic hormone. Activation of the hypothalamic-pituitary-adrenal axis results in extensive hypertrophy and hyperplasia of the cortical zones of the adrenal and induction of genes involved in steroid biosynthesis. The adrenal medulla is disorganized and severely reduced in size; no cells capable of adrenaline synthesis can be detected. Mineralocorticoid receptor deficient mice die mainly at day 9/10 after birth. Weightloss precedes death of homozygous mutant mice and is correlated with an increase in the haematocrit. As a consequence of this mutation, plasma levels of renin and aldosterone are high elevated.

Adrenal Medulla↗
PubMed1996

Analysis of glucocorticoid and mineralocorticoid signalling by gene targeting.

To get a better understanding of the role of glucocorticoid and mineralocorticoid signalling during development and in whole animal physiology, we have disrupted the mouse glucocorticoid and mineralocorticoid receptor gene by gene targeting. Most of the mice with a disrupted glucocorticoid receptor gene die within the first hours after birth due to severe lung atelectasis. Perinatal induction of gluconeogenic enzymes in the liver is impaired. Feed back control of the glucocorticoid synthesis via the hypothalamic-pituitary-adrenal axis is perturbed leading to increased plasma levels of corticosterone and adrenocorticotrophic hormone. Increased activity of the hypothalamic-pituitary-adrenal axis results in extensive hypertrophy and hyperplasia of the cortical zones of the adrenal and induction of genes involved in steroid biosynthesis. The adrenal medulla is disorganized and severely reduced in size; cells capable of adrenaline synthesis are missing. Mineralocorticoid receptor deficient mice die around day 10 after birth. Weight loss precedes death of homozygous mutant mice and is correlated with an increase in the haematocrit. As a consequence of this mutation plasma levels of renin, angiotensin II, and aldosterone are highly elevated.

Animals↗
PubMed1995

Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation.

The role of the glucocorticoid receptor (GR) in glucocorticoid physiology and during development was investigated by generation of GR-deficient mice by gene targeting. GR -/- mice die within a few hours after birth because of respiratory failure. The lungs at birth are severely atelectatic, and development is impaired from day 15.5 p.c. Newborn livers have a reduced capacity to activate genes for key gluconeogenic enzymes. Feedback regulation via the hypothalamic-pituitary-adrenal axis is severely impaired resulting in elevated levels of plasma adrenocorticotrophic hormone (15-fold) and plasma corticosterone (2.5-fold). Accordingly, adrenal glands are enlarged because of hypertrophy of the cortex, resulting in increased expression of key cortical steroid biosynthetic enzymes, such as side-chain cleavage enzyme, steroid 11 beta-hydroxylase, and aldosterone synthase. Adrenal glands lack a central medulla and synthesize no adrenaline. They contain no adrenergic chromaffin cells and only scattered noradrenergic chromaffin cells even when analyzed from the earliest stages of medulla development. These results suggest that the adrenal medulla may be formed from two different cell populations: adrenergic-specific cells that require glucocorticoids for proliferation and/or survival, and a smaller noradrenergic population that differentiates normally in the absence of glucocorticoid signaling.

Adrenal Cortex↗