Glucocorticoid-stimulated gene expression knocked out by knock-in by dfgh4bnmu


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.

To top