Anton Bauer

CREB regulates hepatic gluconeogenesis through the coactivator PGC-1.

When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia* is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.

Mice with an Increased Glucocorticoid Receptor Gene Dosage Show Enhanced Resistance to Stress and Endotoxic Shock

ABSTRACT Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

The thyroid hormone receptor functions as a ligand-operated developmental switch between proliferation and differentiation of erythroid progenitors.

The avian erythroblastosis virus (AEV) oncoprotein v‐ErbA represents a mutated, oncogenic thyroid hormone receptor α (c‐ErbA/ TRα). v‐ErbA cooperates with the stem cell factor‐activated, endogenous receptor tyrosine kinase c‐Kit to induce self‐renewal and to arrest differentiation of primary avian erythroblasts, the AEV transformation target cells. In this cooperation, v‐ErbA substitutes for endogenous steroid hormone receptor function required for sustained proliferation of non‐transformed erythroid progenitors. In this paper, we propose a novel concept of how v‐ErbA transforms erythroblasts. Using culture media strictly depleted from thyroid hormone (T3) and retinoids, the ligands for c‐ErbA/TRα and its co‐receptor RXR, we show that overexpressed, unliganded c‐ErbA/ TRα closely resembles v‐ErbA in its activity on primary erythroblasts. In cooperation with ligand‐activated c‐Kit, c‐ErbA/ TRα causes steroid‐independent, long‐term proliferation and tightly blocks differentiation. Activation of c‐ErbA/ TRα by physiological T3 levels causes the loss of self‐renewal capacity and induces synchronous, terminal differentiation under otherwise identical conditions. This T3‐induced switch in erythroid progenitor development is correlated with a decrease of c‐ErbA‐associated histone deacetylase activity. Our results suggest that the crucial role of the mutations activating v‐erbA as an oncogene is to ‘freeze’ c‐ErbA/ TRα in its non‐liganded, repressive conformation and to facilitate its overexpression.

Targeting of N-CoR and histone deacetylase 3 by the oncoprotein v-erbA yields a chromatin infrastructure-dependent transcriptional repression pathway.

Transcriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. We used Xenopus oocytes to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v‐ErbA, on partly and fully chromatinized TR‐responsive templates in vivo. Repression by v‐ErbA was not as efficient as that mediated by TR, was significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v‐ErbA required mature chromatin to effect repression. We find that both v‐ErbA and TR can recruit the corepressor N‐CoR, but, in contrast to existing models, show a concomitant enrichment for HDAC3 that occurs without an association with Sin3, HDAC1/RPD3, Mi‐2 or HDAC5. We propose a requirement for chromatin infrastructure in N‐CoR/HDAC3‐effected repression and suggest that the inability of v‐ErbA to silence on partly chromatinized templates may stem from its impaired capacity to interfere with basal transcriptional machinery function. In support of this notion, we find v‐ErbA to be less competent than TR for binding to TFIIB in vitro and in vivo.

Molecular Genetic Analysis of Glucocorticoid Signaling Using the Cre/loxP System

Abstract Glucocorticoids (GC) are involved in a plethora of physiological processes that range from the regulation of the stress response and the control of the immune system to modulation of behavior. Most GC effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas in most cases activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interaction with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. Analysis of these mice demonstrated that thymocyte apoptosis and stress erythropoiesis require the DNA binding-dependent function of GR, whereas lung development and the anti-inflammatory activity of GR are mediated by protein-protein interaction. Furthermore, to study the role of GC in the brain, mice were generated specifically lacking GR function in the nervous system. Using these mice we demonstrated that GR is essential for the regulation of the HPA-axis and the stress response, as well as for the control of emotional behavior. Taken together, gene targeting using the Cre/loxP system proved to be highly valuable for the analysis of both molecular mechanism and tissue-specific functions of the GR.

Molecular genetic dissection of glucocorticoid receptor function in vivo

Zusammenfassung Glukokortikoide sind an der Regulation zahlreicher physiologischer Prozesse beteiligt. Die meisten ihrer Wirkungen werden vom Glukokortikoidrezeptor (GR) durch Aktivierung und Repression von Genexpression vermittelt. Während die Aktivierung eine Bindung des Rezeptors and DNA erfordert, erfolgt die Repression in den meisten Fällen durch Protein-Protein-Wechselwirkung mit anderen Transkriptionsfaktoren. Zur Analyse des molekularen Mechanismus des GR wurden mittels “gene targeting” Mäuse mit einer Punktmutation in einer der Dimerisierungsdomänen des Rezeptors erzeugt, welche den Verlust der DNA-Bindung zur Folge hatte. Diese GRdim-Mäuse überleben bis ins erwachsene Alter und erlauben sie die Analyse der Wirkungsweise des GR in physiologischen Prozessen. Insbesondere stellt sich heraus, dass die Stresserythropoese die DNA-bindungsabhängige Funktion des GR benötigt, während die antitumorigene Wirkung des GR in der Haut durch Protein-Protein-Wechselwirkung mit dem Transkriptionsfaktor AP-1 vermittelt wird. Des Weiteren zeigt sich, dass die immunsuppressive und anti-inflammatorische Wirkung des GR weitgehend unabhängig von dessen DNA-Bindung ist. Dies deutet daraufhin, dass GRdim-Mäuse in der Zukunft möglicherweise zur Identifizierung steroidaler anti-inflammatorischer Substanzen mit geringeren Nebenwirkungen verwendet werden können.Summary Glucocorticoids are involved in numerous physiological processes. Most of their effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interactions with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting, carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. These GRdim mice survive to adulthood and thereby allowed analysis of the mechanism used by GR in the control of physiological processes. Specifically, stress erythropoiesis was found to require the DNA binding-dependent function of GR whereas the antitumor-promoting activity of GR in skin is mediated by interaction with the transcription factor AP-1. Furthermore, the immunosuppressive and anti-inflammatory activity of glucocorticoids is largely independent of GR DNA-binding, suggesting that GRdim mice might be useful in the future for the search of steroidal anti-inflammatory drugs with reduced side-effects.

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor.

Abstract Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFα progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor α and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFα progenitors and also do no longer respond to transforming growth factor α and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.

correction: CREB regulates hepatic gluconeogenesis through the coactivator PGC-1

This corrects the article DOI: 35093131

Mechanism of transformation by v-ErbA : Substitution for steroid hormone receptor function in self renewal induction

V-ErbA, a mutated thyroid hormone receptor (TR) α cooperates with tyrosine kinase oncoproteins to induce fatal erythroleukemia in chicks. In vitro, v-ErbA employs a similar cooperation to induce sustained proliferation and arrest differentiation of committed erythroid progenitors. V-ErbA has been proposed to function as a dominant-negative c-ErbA/TRα, since it lacks an AF-2 transactivation domain and cannot be activated by hormone but retains the capacity to bind corepressors. However, v-ErbA fails to heterodimerize with the coreceptor RXR, exhibits an altered DNA binding specificity and fails to suppress the action of coexpressed TRα/c-ErbA in erythroblasts. In this paper, we identify a novel mechanism by which v-ErbA contributes to leukemogenesis. Recently, the glucocorticoid receptor (GR) was identified as a key regulator of proliferation and differentiation in normal erythroid progenitors. For this, the GR required to cooperate with endogenous receptor tyrosine kinases (c-Kit) and with the estrogen receptor (ER). Here, we demonstrate that v-ErbA can substitute for the ligand-activated GR and ER, inducing proliferation and arresting differentiation in the presence of specific GR and ER antagonists. Like the GR, v-ErbA required to cooperate with c-Kit for both proliferation induction and differentiation arrest, being devoid of biological activity in the absence of an active c-Kit. In self-renewing erythroblasts, v-ErbA not only repressed known v-ErbA target genes but also maintained high expression of c-myb. These biological activities of v-ErbA depended on distinct mutations in the DNA-binding domain. Additionally, v-ErbA acted as a partial, weak repressor of c-ErbA/TRα function in normal erythroblasts. It could be converted into a truly dominant-negative receptor by restoring its ability to heterodimerize with RXR.

Erratum: CREB regulates hepatic gluconeogenesis through the coactivator PGC-1 (Nature (2001) 413 (179-183))

This corrects the article DOI: 35095083Nature, 413, 323–327 (2001). In this Letter, the last name of Themis R. Kyriakides was misspelled as ‘Kyriakidis’.