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HIGHLIGHT European Journal of Endocrinology (1998) 139 479–480 ISSN 0804-4643 Glucocorticoid-stimulated gene expression knocked out by knock-in mutation Jens P Berg Hormone Laboratory, Aker University Hospital, Trondheimsveien 235, N-0514 Oslo, Norway Glucocorticoid receptors (GRs) are ligand-dependent the D loop of the receptor protein (9, 10). This loop transcription modulators. They belong to a superfamily consists of ﬁve amino acids critical for receptor of nuclear hormone receptors including receptors for dimerization. Changing the amino acid alanine in steroid and thyroid hormones, retinoids, vitamin D position 458 in the mouse GR to threonine (A458T) and peroxisomal activators among others (1). The abolished the formation of receptor dimers and activa- receptor can be divided into three functional domains: tion of gene transcription via GREs, whereas effects on (1) an N-terminal activation domain (AF1 or t1); (2) transcriptional repression were almost unaffected. a central DNA-binding domain; and (3) a ligand- The consequences of this dimerization defect have now dependent activation domain (AF2 or t2), which is a been studied invivo. Reichardt et al. (11) (reviewed by Karin part of the ligand-binding domain in the C-terminus. (12)) created a strain of mice with alanine 458 replaced by The GR is essential for survival. ‘Knock-out’ of the threonine inthe GRprotein.In contrast withGR knock-out receptor in mice resulted in impaired lung development, mice, there were no signs of impaired lung development and the newborn mice died shortly after birth because and both sexes were fertile. An unexpected almost of respiratory failure (2). Several other abnormalities Mendelian ratio of the three genotypes with 21% homo- associated with a loss of glucocorticoid stimulation zygous mutants (denoted GRdim/dim) was found among were observed in these mice including a reduced level the offspring in contrast with the lethal effect of of gluconeogenic enzymes in the liver, increased serum homozygous GR knock-out in mice. levels of corticotrophin-releasing hormone (CRH), As expected a series of experiments demonstrated that adrenocorticotrophic hormone (ACTH) and cortico- the mutation speciﬁcally affected DNA-binding-depen- sterone, hyperplasia of the adrenal cortex, loss of dent transcriptional activation by the GR. Immortalized glucocorticoid-dependent thymocyte apoptosis, and embryonic ﬁbroblasts from GRdim/dim and control mice impaired proliferation of erythroid progenitor cells. were incubated in the absence or presence of dexa- Activation of GRs may stimulate or repress gene methasone and two reporter genes with different transcription (3). The glucocorticoid-induced transcrip- GREs. Only minimal activation of the reporter tion of target genes is mediated by glucocorticoid- genes was observed in GRdim/dim cells in contrast with responsive elements (GREs), which are palindromic the activation seen in control cells. Dexamethasone- sequences in the regulatory region of these genes. A induced transactivation of gluconeogenic enzymes in GRE is a binding site for the activated GR, and the liver was also lost in the A458T mutants, indicating transactivation depends on the dimerization of two the need for dimerization and GRE binding for these GRs on the GRE. There are probably three ways in effects. However, the protein–protein interactions which GRs can repress gene transcription. The pro- were preserved, as the expression of collagenase 3 and opiomelanocortin (POMC) gene is negatively regulated gelatinase B, which is transcriptionally activated by by a direct interaction of GR with its promoter (4). phorbol esters via an AP-1 element, was almost Secondly, it may interact with the DNA-binding domain completely abolished by dexamethasone in control but together with another transcription factor, and, ﬁnally, also in GRdim/dim cells. glucocorticoid-induced repression of gene transcription Glucocorticoid effects have typically been demon- can be independent of direct binding of GR to DNA. As strated in the autoregulatory circuit consisting of the examples, the activated receptor has been shown to hypothalamus, pituitary and adrenal glands. Negative interfere with signalling via the cAMP-responsive feedback via the GR has been shown on both the transcription factor (CREB) or transcription factors expression and secretion of CRH and ACTH. In GRdim/dim using AP-1 elements by protein–protein interactions mice, CRH immunoreactivity in the hypothalamic (5–7). Furthermore, some of the immunosuppressive median eminence was unaltered in contrast with the effects of glucocorticoids have been associated with a increase observed in the GR knock-out mice. However, repression of the activity of the transcription factor the expression of POMC and ACTH in the anterior NF-kB by a direct interaction between GRs and NF-kB, pituitary was increased in mice with the A458T GR even though this regulation may be even more complex mutation. This indicated that GR uses different mecha- (8). In cell culture, the consequences of a defect in the nisms to regulate CRH and POMC/ACTH. The lack of dimerization of GRs have been studied by introducing a CRH up-regulation in GRdim/dim mice indicated that GR point mutation in the region of the GR gene coding for repressed CRH transcription without binding to DNA, 1998 Society of the European Journal of Endocrinology 480 Highlight EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 139 whereas repression of POMC expression seemed to and limited side effects on glucose, lipid and calcium require GR–DNA binding activity. The increase in metabolism. ACTH immunoreactivity in the anterior pituitary of GRdim/dim mice did not result in increased serum levels of ACTH, which indicated that ACTH secretion was References regulated by a mechanism independent of DNA binding. 1 Meier CA. Regulation of gene expression by nuclear hormone However, serum levels of corticosterone, the major receptors. Journal of Receptor and Signal Transduction Research glucocorticoid in mice, increased. The adrenal medullas of 1997 17 319–335. GR knock-out mice were disorganized, and the expression 2 Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, of phenylethanolamine-N-methyltransferase (PNMT), an Aguzzi A et al. Targeted disruption of the glucocorticoid receptor enzyme involved in adrenaline synthesis, was impaired. gene blocks adrenergic chromafﬁn cell development and severely retards lung maturation. Genes and Development 1995 9 1608– Although the PNMT gene has a classical GRE in its 1621. promoter, the expression of the gene and the development 3 Beato M, Herrlich P & Schutz G. Steroid hormone receptors: many ¨ of the adrenal medulla were unaltered in GRdim/dim mice. actors in search of a plot. Cell 1995 83 851–857. Reichardt et al. (11) speculate that basal expression of 4 Drouin J, Sun YL, Chamberland M, Gauthier Y, De Lean A, ´ Nemer M & Schmidt TJ. Novel glucocorticoid receptor complex the gene is independent of the DNA-binding activity of with DNA element of the hormone-repressed POMC gene. EMBO the GR. Additional experiments are needed to exclude the Journal 1993 12 145–156. possibility that GR A458T mutants transactivate target 5 Akerblom IE, Slater EP, Beato M, Baxter JD & Mellon PL. Negative genes by binding to other not yet deﬁned responsive regulation by glucocorticoids through interference with a cAMP elements on DNA. It will be particularly interesting to responsive enhancer. Science 1988 241 350–353. 6 Jonat C, Rahmsdorf HJ, Park K-K, Cato AC, Gebel S, Ponta H & know how PNMT expression and the production and Herrlich P. Antitumor promotion and antiinﬂammation: down- secretion of CRH, ACTH and corticosterone change modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. during acute stress. Cell 1990 62 1189–1204. The mouse model developed by Reichardt et al. (11) 7 Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J & Karin M. Transcriptional interference between c-Jun may become a tool that can be used to indicate and the glucocorticoid receptor: mutual inhibition of DNA mechanisms by which GRs induce their effects, i.e. binding due to direct protein–protein interaction. Cell 1990 62 whether they are mediated by activation or repression of 1205–1215. gene expression. In mice, T-lymphocyte development 8 Auphan N, DiDonato JA, Rosette C, Helmberg A & Karin M. and differentiation are controlled by glucocorticoid- Immunosuppression by glucocorticoids: inhibition of NF-kB activity through induction of IkB synthesis. Science 1995 270 induced apoptosis, but there has been controversy about 286–290. the mechanism. T-cells from GRdim/dim mice were found ˚ 9 Dahlman-Wright K, Wright A, Gustafsson JA & Carlstedt-Duke J. to be refractive to apoptosis induced by dexamethasone, Interaction of the glucocorticoid receptor DNA-binding domain which indicated a mechanism requiring binding of GR with DNA as a dimer is mediated by a short segment of ﬁve amino acids. Journal of Biological Chemistry 1991 266 3107–3112. to GRE and transcriptional activation. 10 Heck S, Kullmann M, Gast A, Ponta H, Rahmsdorf HJ, Herrlich P GRdim/dim mice survive in the absence of glucocorti- & Cato AC. A distinct modulating domain in glucocorticoid coid-activated GR binding to GRE and the subsequent receptor monomers in the repression of activity of transcription direct stimulation of gene transcription under standard factor AP-1. EMBO Journal 1994 13 4087–4095. laboratory conditions. The most important effects of 11 Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R et al. DNA binding of the glucocorticoid receptor is not glucocorticoids and GRs seem to be via cross-talk with essential for survival. Cell 1998 93 531–541. other transcription factors. This opens a pharmaco- 12 Karin M. New twists in gene regulation by glucocorticoid logical window of opportunity. New GR ligands that receptor: is DNA binding dispensable? Cell 1998 93 487–490. activate the mechanisms used for transcriptional ´ 13 Vayssiere BM, Dupont S, Choquart A, Petit F, Garcia T, Marchandeau C et al. Synthetic glucocorticoids that dissociate repression more than those for activation have already transactivation and AP-1 transrepression exhibit antiinﬂamma- been tested (13). They bring hope of ﬁnding glucocorti- tory activity in vivo. Molecular Endocrinology 1997 11 1245– coid analogues with potent anti-inﬂammatory properties 1255.
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