Neuroendocrine Regulation of the Hypothalamic Pituitary Adrenal Axis by the nurr1/nur77 Subfamily of Nuclear Receptors
Evelyn P. Murphy and
Orla M. Conneely
Department of Cell Biology Baylor College of Medicine
Houston, Texas 77030
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ABSTRACT
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The present study was designed to examine the role
of the nurr1/nur77 subfamily of nuclear receptor transcription factors
in the regulation of the hypothalamic/pituitary/adrenal axis at the
neuroendocrine level. We demonstrate that this nuclear receptor
subfamily can regulate the expression of the CRF and POMC genes by
interacting with a specific cis-acting sequence in their
proximal promoter regions. To examine the physiological significance of
this response, we have focused on the POMC gene. We provide evidence
that nurr1 and nur77 are rapidly induced by CRF in primary pituitary
cells and that this induction is mimicked by forskolin in an anterior
pituitary cell line. Further, we demonstrate that both nurr1- and
forskolin-dependent induction of a POMC-chloramphenicol
acetyltransferase reporter gene are inhibited by mutation of the
nurr1-binding site within the POMC promoter and that this site alone
can confer cAMP responsiveness to a heterologous promoter. Finally, we
provide evidence that the nurr1/nur77 response sequence is pivotal to
both nurr1/nur77-dependent positive regulation and glucocorticoid
receptor-dependent negative regulation of the POMC gene. These data
strongly support the conclusion that the nurr1/nur77 subfamily plays an
important coordinate neuroendocrine-regulatory role at all levels of
the hypothalamic/pituitary/adrenal axis.
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INTRODUCTION
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Nuclear receptors comprise a superfamily of structurally related
transcription factors that control a variety of developmental,
physiological, and behavioral processes (1, 2, 3). The family includes
receptors for lipophilic hormones and vitamins as well as a majority of
orphan members whose physiological function is poorly understood (4).
Nurr1 (also called rnr-1 and NOT) (5, 6) is an orphan member of the
superfamily (7) that is expressed predominantly in the central nervous
system (7, 8). The protein exhibits a close structural relationship to
the orphan receptors nur77 (also called NGFIB/N10/NAK) (9, 10, 11, 12) and
NOR-1 (also called MINOR/TEC) (13, 14, 15, 16). All three proteins are members
of a nuclear receptor subgroup (hereafter referred to as the nurr1
subfamily) that bind as monomers to the cis-acting sequence,
AAAGGTCA, to regulate gene expression without a requirement for ligand
binding (14, 17, 18, 19). Nurr1 and nur77 have also been implicated in the
regulation of retinoid-signaling pathways by heterodimerizing with the
9-cis retinoic acid receptor, RXR, and binding to the
AAAGGTCA motif when arranged as two directly repeated elements (20).
Thus, the nurr1 subfamily has the capacity to regulate overlapping gene
networks if expressed in the same cells.
Transcripts for the nurr1 subfamily are constitutively expressed in a
differentially restricted but partially overlapping temporal and
spatial pattern (7, 8). Whereas nurr1 expression appears to be
restricted to brain tissue in the developing and adult mouse, the
constitutive expression of nur77 and NOR-1 is observed in some
peripheral tissues in addition to brain (7, 14). Nur77 mRNA is present
in several tissues including testis, ovary, and muscle (7) whereas low
NOR-1 expression is detected in the thymus, kidney, and spleen (14).
Further, unlike most nuclear receptors, these proteins are products of
immediate early genes whose expression can be differentially induced in
response to a variety of extracellular stimuli including growth factors
(9, 10, 21), neurotransmitters (22, 23), and polypeptide hormones (24, 25).
Several lines of evidence indicate that the members of the nurr1
subfamily may play an important role in the coordinate neuroendocrine
regulation of the activity of the hypothalamic/pituitary/adrenal (HPA)
axis. This axis is regulated at the level of the hypothalamus by CRF,
which is synthesized in the hypothalamic paraventricular nucleus (PVN).
In response to stressful stimuli, CRF is released from the PVN and
transported to the anterior pituitary causing an increase in synthesis
of POMC. POMC is a precursor molecule of several neuropeptides
including ACTH, which is released from the pituitary and regulates the
synthesis of glucocorticoids from the adrenal cortex. To maintain
homeostasis, glucocorticoids inhibit CRF and POMC synthesis and
secretion at the level of the hypothalamus and anterior pituitary. It
has previously been shown that while nurr1 is constitutively expressed
in the PVN (8), nur77 mRNA is rapidly induced in this region by stress
(26) and interleukin-1ß (27), both important regulators of
hypothalamic CRF. Also, central administration of CRF to conscious rats
significantly increases the expression of nur77 within the PVN (28). We
have shown that nurr1 and nur77 are both expressed in the anterior
pituitary, the site of POMC synthesis (8). Further, nur77 and nurr1
transcripts are strongly induced by stress in the adrenal cortex (25).
The induction of nur77 in this region has been implicated in the
transcriptional induction of the steroidogenic enzyme
steroid-21
-hydroxylase (24), a rate-limiting enzyme in
glucocorticoid synthesis. However, recent reports reveal that nur77
null mutant mice display no abnormal functions of the HPA axis (29).
The absence of detectable phenotypic changes in the HPA axis has been
proposed to reflect a functional redundancy by nurr1 because levels of
this mRNA, after HPA axis stimulation, are compensatorily increased in
the adrenal gland of nur77 null mutant mice (29). Finally, our
laboratory recently identified specific DNA-binding sites for nurr1 and
nur77 in the proximal promoter region of the CRF and POMC genes that
may mediate nurr1 subfamily-dependent regulation of these genes in the
hypothalamus and pituitary, respectively (19).
The aim of this study was to examine the neuroendocrine regulation of
the HPA axis by the nurr1 subfamily. We report here that, as predicted
by our previous DNA-binding studies (19), nurr1 and nur77 interact
specifically with the CRF (-352/-332) and POMC (-70/-47) promoter
elements in electrophoretic mobility shift assays (EMSA).
Cotransfection experiments in pituitary-derived cells show that nurr1
can increase the transcriptional activity of both promoters. Further,
mutational analysis of the nurr1 consensus site within the POMC
promoter results in loss of nurr1-stimulated expression. CRF functions
through the secondary messenger cAMP to potently stimulate POMC gene
transcription within pituitary cells. By increasing cAMP levels within
a pituitary cell line, we observe a rapid and robust increase of nurr1
and nur77 mRNAs, suggesting CRF induction of POMC synthesis may be
mediated through these transcription factors. Finally, examination of
the nurr1 consensus sequence in the POMC promoter reveals that the
element overlaps with a well characterized negative glucocorticoid
receptor response element (nGRE). We provide evidence to indicate that
glucocorticoid repression of the POMC gene may be mediated, at least in
part, by glucocorticoid receptor (GR)-dependent inhibition of
activation of the POMC gene by nurr1 subfamily members. Our results
strongly support the conclusion that the nurr1 subfamily of nuclear
receptors plays a coordinate role in neuroendocrine regulation of the
activity of the HPA axis.
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RESULTS
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Specific Binding of nurr1 and nur77 to Proximal Promoter Fragments
of the CRF and POMC Genes
In a previous study, we identified three
cis-acting sequences, GAAGGTCA, AAAGGTCG, and GAAGGTCG (19),
in addition to the previously characterized AAAGGTCA (NBRE) site (18),
that bind specifically to both nurr1 and nur77. Examination of Genbank
for sequences containing one of these sequences, GAAGGTCA, revealed
several genes of neuronal and neuroendocrine origin whose proximal
promoters contain this cis-acting sequence (19). Two
identified genes, ovine CRF (oCRF) (30) and rat POMC (rPOMC) (31), were
of particular interest since previous analysis of the spatial
expression of nurr1 and nur77 within the central nervous system
indicated that nurr1 and nur77 are either expressed or induced by
HPA-activating signals in the hypothalamic and pituitary structures
that express the CRF and POMC genes (8, 26). The GAAGGTCA sequence of
the POMC is highly conserved across the rat, human, and mouse species
(31, 32, 33), suggesting this sequence has important regulatory
functions.
To test whether nurr1 and nur77 interact directly with the POMC
-70/-47 and CRF -352/-332 regions, we prepared 32P-
labeled oligonucleotides containing these regions and used EMSA to
examine their binding to nurr1 and nur77 translated in vitro
in the reticulolysate system. The results of these assays are shown in
Fig. 1
. Incubation with nurr1 resulted in a retarded
radiolabeled complex that was observed when either the POMC (panel A,
lane 2) or CRF (panel C, lane 2) promoter fragments were used. These
complexes were nurr1 dependent and were not observed in the absence of
nurr1 in the reticulolysate (lane 1, both panels). Furthermore, complex
formation on both promoters was specifically inhibited by increasing
concentrations of unlabeled homologous oligonucleotide (lanes 35) but
not by a heterologous oligonucleotide (lane 6), indicating that binding
to these DNA fragments was specific and competitive. Finally, mutation
of the GAAGGTCA motif to GAACATCA or
GTACGTCA within these sequences resulted in
loss of ability to competitively inhibit nurr1-dependent binding,
indicating that this sequence is essential for the nurr1 interaction
(lanes 79). Similar results were obtained when nur77 was used in EMSA
instead of nurr1 (panels B and D).

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Figure 1. EMSA of nurr1 and nur77 Binding to the rPOMC and
oCRF Promoters
Nurr1 (panels A and C) and nur77 (panels B and D) were transcribed and
translated in vitro and incubated with
32P-labeled rPOMC (panels A and B) and oCRF (panels C
and D) oligonucleotides (lane 2). For competition analysis 1050x
molar excess of homologous oligonucleotide (lanes 35), 50x molar
excess of heterologous oligonucleotide (lane 6), and 1050x molar
excess of mutant oligonucleotide (lanes 79) were used.
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Nurr1 Enhances the Transcriptional Activity of the CRF and POMC
Promoter Regions
To determine whether nurr1 and nur77 were capable of
regulating the expression of the CRF and POMC promoters, we generated
target gene constructs containing the proximal promoter regions of both
genes and used these in cotransfection experiments with nurr1 or nur77
expression constructs. In these experiments, we constructed target
vectors in the promoterless pBL3CAT plasmid (34) using a
-483/+1 promoter fragment of the rPOMC gene (31), a -483/+81 fragment
of the human POMC promoter (32), and a -372/+11 fragment of the oCRF
gene (30). Transcriptional regulation of these target constructs by
nurr1 and nur77 was then measured by transfection in the anterior
pituitary corticotropic cell line, AtT20/D. As shown in Fig. 2A
, cotransfection of these target genes with nurr1
results in a 7- to 12-fold stimulation of the rat, human POMC, and oCRF
promoters over that observed when the target genes are cotransfected
with the parent expression vector lacking nurr1 (p91023B). To confirm
that the nurr1-dependent induction transcription is due to the presence
of the GAAGGTCA motif within the promoter, we introduced point
mutations within this element in the rPOMC promoter to
GAACATCA, which we have shown to result in loss of nurr1
binding in vitro (Fig. 1
), and we examined the regulation of
this promoter construct by nurr1. As shown in Fig. 2B
, nurr1 induction
of the POMC promoter is lost when the mutated promoter construct (rPOMC
MT1) is used, indicating that a functional GAAGGTCA motif is essential
for transcriptional induction of the POMC promoter by nurr1. When nurr1
was replaced by nur77 in these assays, similar results were observed
(data not shown), indicating that both subfamily members can regulate
the expression of the CRF and POMC promoters and may function
redundantly to do so in a physiological context if coexpressed.

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Figure 2. Activation of Transcription from the rPOMC and oCRF
Promoters by nurr1
AtT20/D cells were transfected with 500 ng -483/+1 rPOMC, -483/+81
hPOMC, or -372/+11 CRF reporter plasmids (panel A) and 500 ng -483/+1
rPOMC or -483/+1 rPOMC MT1 reporter plasmids (panel B) together with
either p91023B or p91023B-nurr1 expression vector. The results shown
are representative of four individual experiments. Each data bar
represents two replicates.
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CRF Rapidly Induces nurr1 and nur77 Expression in Pituitary
Cells
To determine whether the nurr1/nur77 subfamily is likely to
contribute to the regulation of POMC expression by CRF in a
physiological context, we incubated isolated mouse pituitary cells with
CRF (10-8 M) and examined its ability to
induce expression of the nurr1 and nur77 transcripts. As indicated in
Fig. 3
, both nurr1 and nur77 transcripts are rapidly
induced by CRF within 15 min of treatment and are maximal at
approximately 30 min. Further, this induction slightly precedes the
previously reported time course of POMC transcription by CRF, which is
maximal by 3060 min (35, 36).

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Figure 3. CRF Treatment of Isolated Pituitary Cells Rapidly
Increases nurr1 and nur77 mRNA Levels
After treatment with 10-8 M CRF for the
indicated times, total RNA was extracted, and Northern blots were
performed as described in Materials and Methods. The
same filter was hybridized with a cDNA probe for nurr1 and nur77 and
also with cyclophilin to control for RNA loading and transfer.
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Forskolin Induces Expression of the p-483/+1 rPOMC Reporter
Construct
While AtT20/D cells are a suitable model to examine the
regulation of POMC in corticotropic cells, the induction of POMC by CRF
in these cells is very weak and variable and most likely due to loss of
membrane CRF receptors in the transformed cell line. However, since
CRF-dependent regulation of POMC expression is known to be mediated by
the secondary messenger cAMP pathway (37, 38), CRF-dependent POMC
induction can be mimicked by incubation of cells with forskolin to
activate this pathway. Direct activation of adenylate cyclase with
forskolin induced both nurr1 and nur77 mRNAs rapidly (Fig. 4
). The time course of this induction correlates with
the time course of CRF induction of nurr1 and nur77 in the isolated
pituitary cells. Further, we show that forskolin can significantly
stimulate the expression of the rPOMC-chloramphenicol acetyltransferase
(CAT) reporter gene when transfected into AtT20/D cells. Most
importantly, however, the induction of POMC-CAT by forskolin is reduced
when the nurr1 binding site is destroyed by point mutation of the
GAAGGTCA motif (rPOMC MT1), and the basal activity of the promoter is
also decreased. Finally, when the GAAGGTCA alone is placed in front of
a heterologous promoter [thymidine kinase (tk) (34)], this enhancer
element is sufficient to mediate induction of this promoter by
forskolin (Fig. 4
). The results confirm that the nurr1-binding site
plays an important role in the induction of this promoter by cAMP
pathways and also contributes to the basal activity of the POMC
promoter in pituitary cells.

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Figure 4. Forskolin Mimics CRF Induction of nurr1 and nur77
mRNA and Induces rPOMC-CAT in AtT20/D16 Cells
Panel A, AtT20/D cells were treated with 25 µM forskolin
for 01.5 h. Nurr1 and nur77 mRNA levels were measured using 20 µg
total RNA. Each membrane was probed with a glyceraldehyde-3-phosphate
dehydrogenase cDNA fragment to control for transfer and loading. Panel
B, AtT20/D cells were transfected with -483/+1 rPOMC, -483/+1 rPOMC
MT1, or M2G (GAAGGTCA)2-tk reporter plasmids and treated
with 25 µM forskolin for 10 h. These results are
representative of four individual experiments. Each data bar represents
two replicates.
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The nurr1-Binding Site Mediates Both cAMP-Dependent Up-Regulation
and Dexamethasone-Dependent Down-Regulation of the POMC Promoter
Upon identification of the nurr1 enhancer element, we noted that
its location overlapped with a previously characterized nGRE that
mediates GR-regulated repression of the POMC promoter (39) (Fig. 5A
). Mutation of the GAAGGTCA motif to
GAACATCA has been shown to convert the nGRE into a positive
enhancer element that mediates induction of the enhancer element by GR
(40) when placed upstream of a heterologous basal promoter. In
preliminary studies to test the hypothesis that the nurr1-binding site
is pivotal to both positive regulation by nurr1 and negative feedback
by GR, we confirm that forskolin regulation of the POMC promoter is
down-regulated by pretreatment with dexamethasone (36) and that
rPOMC-MT1, while not responding to forskolin or nurr1, is up-regulated
by dexamethasone (Fig. 5B
). These data predicted that GR may inhibit
expression of POMC in the pituitary, at least in part, by either
directly or indirectly inhibiting binding of the nurr1 subfamily to the
GAAGGTCA motif. To directly test this predication, we carried out EMSA
on nuclear extracts to examine the DNA-binding properties of nurr1 in
forskolin- and dexamethasone-treated cells. The results are shown in
Fig. 5C
. In unstimulated AtT20/D cells, two proteins, presumably nurr1
and nur77, bound to the rPOMC -70/-47 probe (lane 1). Stimulation of
the cells with 25 µM forskolin for 1 h resulted in
significant increased binding of both proteins to DNA (lane 4). Binding
was inhibited by 25x molar excess of homologous oligonucleotide (lanes
2 and 5) but not by the oligonucleotide containing a mutation of the
nurr1-binding site rPOMC -70/-47MT1 (lanes 3 and 6). The larger
protein complex was selectively blocked by nurr1-specific antiserum
(lane 8) (8). These results confirm that the cAMP-dependent increases
in nurr1 mRNA in these cells correlate with an increase in specific
binding of nurr1 protein to the POMC promoter sequence. Further, as
predicted by our transactivation results, pretreatment with
dexamethasone inhibited binding of both proteins to DNA and also
diminished the basal DNA-binding activity (lane 10). These results
confirm that the nurr1-binding site can play a pivotal role in both
basal and cAMP-mediated up-regulation of the POMC promoter and its
down-regulation by GRs.

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Figure 5. The nurr1-Binding Site on the POMC Promoter Is Also
Required for Glucocorticoid Feedback Inhibition of the POMC Promoter
Panel A, Nucleotide sequence of rPOMC promoter (-70/-23). Positions
of the nGRE (underlined) and nurr1 response element
(nurr1 RE, italics) are indicated. Panel B, AtT20/D
cells transfected with -483/+1 rPOMC or -483/+1 rPOMC MT1 and
cultured in the presence of forskolin (25 µM),
dexamethasone (10-8 M), or dexamethasone and
forskolin (pretreated with dexamethasone and followed by the addition
of forskolin). Panel C, Nuclear extracts from AtT20/D cells, untreated
(lanes 13) or treated with 25 µM forskolin (lanes
410) for 1 h, were prepared and used in EMSA with
32P-labeled rPOMC -70/-47 oligonucleotide. For
competition analysis 25x molar excess of homologous oligonucleotide
(lanes 2 and 5) and mutant oligonucleotide rPOMC -70/-47 MT1 (lanes 3
and 6) was used. Nurr1-specific antiserum was included in the binding
reaction (lane 8). AtT20/D cells were pretreated with 10-8
M dexamethasone for 2 h before the addition of 25
µM forskolin (lane 10).
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DISCUSSION
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We have provided substantial evidence to support the conclusion
that members of the nurr1 subfamily of nuclear receptors play an
important role in the coordinate neuroendocrine regulation of the
activity of the HPA axis as well as its negative feedback inhibition by
glucocorticoids. Using in vitro DNA binding and cell-based
transactivation assays in an anterior pituitary cell line, we have
demonstrated that nurr1 and nur77 can bind and regulate the expression
of the CRF and POMC promoters. To examine the physiological
significance of this transcriptional regulation, we focused on the POMC
promoter for several reasons. First, the proximal binding site for the
nurr1 subfamily is positionally conserved across species in this
promoter and is contained in a region important for both positive and
negative regulation of the POMC gene (31, 40). Second, the POMC
promoter fragment used in these studies contains all of the sequences
(-323/-34) that have been shown to be necessary and sufficient for
the correct spatiotemporal and hormone-inducible expression of a
ß-galactosidase reporter gene in the anterior pituitary when
expressed in transgenic animals (41). Third, the availability of a well
characterized anterior pituitary-derived cell line (AtT20/D) (31),
which produces endogenous POMC and responds to cAMP activation by
induction of POMC expression in a manner that mimics induction by CRF,
facilitates examination of the regulation of POMC by the nurr1
subfamily and the promoter elements responsible for this regulation in
a physiologically relevant context.
As predicted by the species conservation of the nurr1-binding site, we
confirmed that transactivation of the POMC gene by nurr1 is conserved
between rat and human species. Further, we showed that mutation of the
nurr1-binding site within the POMC promoter results in loss of ability
to bind nurr1 and inhibits nurr1-dependent activation of this promoter,
confirming that the GAAGGTCA sequence motif is essential for
nurr1-dependent induction of POMC expression. To determine whether
nurr1 and/or nur77 are likely to mediate CRF- dependent induction of
POMC expression in the anterior pituitary, we confirmed that the
expression of both nurr1 and nur77 is rapidly induced by stimulation of
isolated mouse pituitary cells with CRF. Further, we demonstrated that
this induction can be mimicked in AtT20/D cells by activation of
cAMP-dependent pathways by forskolin. Forskolin stimulation results in
increased binding of endogenous nurr1 to DNA and a functional POMC-CAT
transactivation response. These data indicate that cAMP may regulate
the POMC promoter, at least in part, by increasing expression of the
nurr1 subfamily. However, nur77 has also been shown to be
phosphorylated by cAMP (25), and cAMP has also been shown to alter the
transcriptional activity of nurr1 and nur77 (42). Thus, both covalent
modification of existing pools of nur proteins and de novo
synthesis are likely to contribute to cAMP-dependent induction of the
POMC promoter.
The observation that forskolin induction of the rPOMC-CAT target gene
is diminished by mutation of the nurr1-binding site illustrates the
importance of this cis-acting sequence in mediating
cAMP-dependent induction of POMC expression in the anterior pituitary
cell line. While CRF is known to induce expression of POMC through a
cAMP-dependent pathway, previous studies have not uncovered a
recognizable cis-acting cAMP response element that may
mediate this response (43). The location of cAMP-responsive sequences
in the POMC promoter has therefore been controversial. Previous studies
have indicated that sequences located upstream (-236/-133) of the
nurr1-binding site are responsive to CRF when placed upstream of a
heterologous promoter and may contribute to hormonal regulation of the
endogenous gene (43). While our data support a major role for the
nurr1-binding site located at -60/-70 in mediating cAMP responses in
the context of the endogenous POMC promoter, the lack of complete
inhibition of POMC induction by mutation of this sequence indicates
that additional sequences outside of this region may also contribute to
cAMP-mediated induction of expression of this gene.
The nurr1-binding site overlaps with a previously identified nGRE that
has been shown to be important for GR-mediated repression of the POMC
gene and is also important for basal expression of this promoter (31, 39, 40, 44). Consistent with the reported contribution of this region
to basal promoter activity (44), we observed that mutation of the
nurr1-binding site also results in decreased basal promoter activity.
Two critical nucleotides within the nGRE are critical for both GR
repression (40) and nurr1 transactivation. Mutation of these
nucleotides, as we have done in our study (POMC-MT1), converted the
nGRE into a positive GR response element when placed in front of a
heterologous promoter (40). We have demonstrated that although this
mutated sequence no longer responds to nurr1 and demonstrates
diminished response to forskolin, the mutant target gene is induced by
dexamethasone. These data support the conclusion that the nurr1-binding
site plays an important role in negative regulation of the POMC gene by
glucocorticoids as well as nurr1-mediated basal and cAMP-inducible
expression of POMC. Further, we show that GR-mediated inhibition of the
POMC gene is accompanied by an inhibition of nurr1 subfamily-dependent
DNA binding to the GAAGGTCA response element, demonstrating functional
antagonism between these two nuclear receptors. Thus, the nurr1
subfamily may play a pivotal role in regulation of neuroendocrine
homeostasis at the pituitary level.
The data we have provided in the present study, together with the
demonstrated expression (8) and induction (26, 27, 28) of nurr1 subfamily
members in the hypothalamic PVN, and the demonstration by others (24)
that nur77 can mediate the regulation of expression of the
steroidogenic enzyme, steroid-21
-hydroxylase by the POMC processing
product, ACTH, indicate that members of the nurr1 subfamily may be
important coordinators of the activity of the HPA axis at all levels.
Despite these observations, however, recent analysis of HPA activity in
homozygous nur77 null mutant mice has detected no disturbance in this
neuroendocrine pathway (29). In fact, the only significant difference
between wild type and homozygous animals observed in this pathway was a
compensatory increase in the induction of nurr1 by stress in nur77 null
mutant animals that was not observed in the wild type mice (29). This
observation highlights the capacity for redundancy of function between
nurr1 subfamily members. Our analysis of the comparative developmental
expression of nurr1 and nur77 has indicated that nurr1 is selectively
expressed during embryonic development, particularly in the
diencephalic regions that give rise to the hypothalamus at a time that
coincides with the developmental organization of the HPA axis, whereas
nur77 is not expressed until the postnatal stage (O. Saucedo-Cardenas
and O. Conneely, manuscript in preparation). Given the ability of nurr1
to substitute functionally for nur77, it is not surprising that nur77
null mutant mice do not show any detectable aberrant phenotypes in the
HPA because nur77 expression does not begin until the postnatal stage
of development at a time when nurr1 is already expressed. Thus, nurr1
may play a selective role in the developmental organization and
activity of the HPA axis that is not substituted by nur77. Finally, the
ontogeny and impact, if any, of the third subfamily member, NOR-1, on
this pathway remain to be established. With the use of gene-targeting
strategies, null mutation of these genes in mice should provide
valuable insights into the selective and collective functions of these
proteins in vivo, including their essential role, if any, in
the neuroendocrine development and activity of the HPA axis.
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MATERIALS AND METHODS
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Plasmid Construction
The nurr1 and nur77 cDNAs were cloned into the plasmid
pT7ß-6 Sal (45) at the NcoI site of the
ß-Globin linker and the SalI site of the polylinker. This
generated pT7ß-nurr1 and pT7ß-nur77, which
drives the expression of these cDNAs under the control of the
T7 promoter in vitro. For expression in tissue
culture cells, cDNAs were ligated to the EcoRI site of
p91023ß and expressed under the control of the adenoviral major late
promoter (46). The reporter plasmid p-372CRF-CAT was generated by PCR
using, as the template, a 5-kb BglII/HindIII
fragment isolated from the previously identified
CRF-1 genomic
clone (30). p-483POMC-CAT reporters were also generated by PCR using
rat and human genomic DNA. The PCR products were subcloned into
pBL3CAT, which lacks the minimal tk promoter (34). All PCR
products were sequenced by the dideoxy method (47). The M2G
(GAAGGTCA)2 tk-CAT reporter plasmid was made by ligating
double-stranded oligonucleotides, containing two inverted copies of the
response element oligonucleotide separated by 10 nucleotides, into the
BamHI site of pBL2CAT, upstream of the minimal
tk promoter (34).
In Vitro Transcription and Translation
In vitro transcription and translation was
accomplished with the TNT kit (Promega, Madison, WI) with the addition
of RNAsin (Promega). Nurr1 and nur77 were transcribed with
T7 RNA polymerase from pT7ß-6 recombinant
plasmid (45), a derivative of pGEM 2 in which the ß-globin insert of
pSP6 Hß 166 6 was inserted at the initiation codon to
create the sequence CCATGCCTCGACCATGG (48). The translation was carried
out in the presence of [35S]methionine and run on an
8.5% denaturing gel or cold methionine for use in the mobility shift
assay, according to the manufacturers directions; 12.5 µl of a
translation mixture was used in each gel shift-binding reaction.
EMSA
EMSA were performed with in vitro translated proteins
in a rabbit reticulocyte lysate system (TNT, Promega) or AtT20/D
nuclear extracts. Proteins were mixed with 100,000 cpm of
Klenow-labeled probes in the reaction buffer, 20 mM HEPES,
pH 7.9, 5 mM MgCl2, 20% glycerol, 100
mM KCl, 0.2 mM EDTA, 8% Ficoll, 600
mM KCl, 500 ng/µl poly(deoxyinosinic-deoxycytidylic)acid,
and 50 mM dithiothreitol (DTT). The reaction was incubated
for 20 min at room temperature and then electrophoresed through a 5.5%
nondenaturing polyacrylamide gel in 0.5x Tris-Borate-EDTA (TBE)
electrophoresis buffer. Nurr1-specific antiserum (8) was incubated with
nuclear extract for 15 min before the addition of probe. For
competition studies, the reaction was performed as described with the
indicated concentrations of unlabeled probe. The sequences of the
oligonucleotides studied are listed as follows: rPOMC -70/-47
5'-GATCT-70CAGGAAGGTCACGTCCAAGGCTCA-47
rPOMC -70/-47MT1
5'-GATCT-70CAGGAACATCACGTCCAAGGCTCA-47
oCRF -352/-332
5'-GA-352TCTTTCTGACCTTCCCTTTA-332 oCRF
-352/-332MT1
5'-GA-352TCTTTCTGACGTACCCTTTA-332
Preparation of Nuclear Extracts
AtT20/D16V-F2 cells untreated or treated with 25
µM forskolin were washed with cold PBS, resuspended, and
incubated for 5 min in 0.25 ml buffer A (10 mM HEPES-KOH pH
7.9, 1.5 mM MgCL2, 10 mM KCL, 0.5
mM DTT, 0.1 mM EGTA, 0.5 mM
phenylmethylsulfonylfluroide, and 2 µg each of the protease
inhibitors antipain, pepstatin A, and aprotinin per ml). Then, 1.25
µl of 10% Nonidet P-40 were added, and the cells were incubated for
2 min on ice. The cells were centrifuged at low speed (1,7000 rpm), and
the supernatant was removed (cytosolic fraction). To the pellet, 0.125
ml of buffer B (0.4 M NaCL, 10 mM HEPES-KOH, pH
7.9, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5
mM DTT, 5% glycerol, and 0.5 mM
phenylmethylsulfonylfluoride) was added. The mixture was vortexed at 4
C and left on ice for 5 min. The extracts were then centrifuged, and
the supernatant was dialyzed against 50 volumes of buffer C (20
mM HEPES-KOH, pH 7.9, 75 mM NaCl, 0.1
mM EDTA, 0.5 mM DTT, 20% glycerol, and 0.5
mM phenylmethylsulfonylfluoride) for 4 h at 4 C with
one change of buffer C. After 4 h, the materials that precipitated
during dialysis were removed by centrifugation, and the supernatant was
aliquoted, flash frozen in liquid N2, and stored at
-80°C until further use. The protein concentration was estimated
with the Bradford protein assay kit (Bio-Rad, Richmond, CA). One
microgram of protein was used in the EMSA.
Cell Culture and Transfection
AtT20/D16V-F2 were grown in DMEM supplemented with 10% FBS,
penicillin at 100 µg/ml, and streptomycin at 100 µg/ml in a
humidified atmosphere of 5% CO2 and 95% air. Twenty four
hours before transfection, 2 x 105 cells were plated
in 3-cm dishes in DMEM supplemented with 10% FBS and were allowed to
attach. The cells were then washed with Hanks Balanced Salt Solution
(HBSS) lacking calcium and magnesium and incubated in DMEM supplemented
with 10% horse serum for AtT20/D cells. Cells treated with forskolin
(25 µM) or dexamethasone (10-8
M) were grown in DMEM supplemented with 10% stripped
serum. DNA (50100 ng p91023-nurr1/nur77; 0.51 µg p-372CRF CAT and
p-483POMC CAT; 200 ng M2G-tk CAT) in a volume of 250 µl
HEPES-buffered saline was added to 1 x 1010 d1312
adenovirus particles (49) in a volume of 333 µl HEPES-buffered saline
and incubated at room temperature for 30 min. Poly-L-lysine
was added (the amount required was based on the size of the DNA used)
and incubated at room temperature for 30 min. The DNA-modified
virus-poly-L-lysine was added to the cells and incubated
for 2 h at 37 C. The virus-containing medium was removed, and 3 ml
of specific medium were added to the cells. The cells were incubated at
37 C for 24 h before harvesting. Pituitaries from adult mice
(BALB/c) were rapidly isolated intact. Four whole pituitaries were
pooled per sample and collected in DMEM containing 10% stripped serum
and equilibrated in a 95% air-5% CO2 mixture.
Northern Blot
Total RNA from cultured cells was isolated at specific times
after treatment. RNA was quantitated by UV absorption, and 20 µg of
total RNA were electrophoresed on a standard Northern gel and
transferred to nylon membrane (50). Nurr1 and nur77 cDNA probes spanned
the amino-terminal region to avoid cross-hybridization. All membranes
were probed under high stringency conditions.
CAT Assay
Each plate of cells was washed once with PBS without calcium and
magnesium, scraped into 1 ml TEN buffer (40 mM Tris, 1
mM EDTA, 150 mM NaCl, pH 8.0) and collected by
centrifugation at 13,000 rpm for 30 sec. Cells were resuspended in 250
mM Tris-HCl, pH 7.5, and lysed by four freeze/thaw cycles.
Protein concentrations were determined by the micro-plate Bradford
assay (51). CAT activity was determined by incubating 510 µg
protein with 0.2 µCi [3H]chloramphenicol (20
µCi/µmol) and 250 µM butryl-Coenzyme A in 100 µl
250 mM Tris-HCl, pH 7.5, for 3 h at 37 C. Acylated
chloramphenicol was extracted using a mixture of 200 µl 2:1
2,6,10,14-tetramethylpentadecane and Xylenes and counted in a
scintillation counter (52). The background of the CAT activity ranges
from 300500 cpm and has been subtracted from the assay. Therefore,
1000 cpm represents a low but significant level of basal activity.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Aileen Ward for technical
assistance. We are very grateful to Dr. J. P. Coghlan (Howard Florey
Institute of Experimental Physiology and Medicine, Parkville, Victoria
3052, Australia) for providing the oCRF genomic clone, Dr J. P. Lydon
for providing pituitaries and the tissue culture facility for providing
AtT20/D cells. We also thank Laura Berkin for secretarial
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Orla M. Conneely, Ph.D., Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston,Texas 77030.
Received for publication June 18, 1996.
Revision received October 2, 1996.
Accepted for publication October 22, 1996.
 |
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