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Program in Neuroscience, Howard Hughes Medical Institute, Harvard Medical School (S.C.W.), and the Division of Endocrinology, Children’s Hospital, Harvard Medical School (S.C.W., J.A.M.), Boston, Massachusetts 02115; and The Jackson Laboratory (L.L.P.), Bar Harbor, Maine 04609
Address all correspondence and requests for reprints to: Dr. J. A. Majzoub, Division of Endocrinology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115. E-mail: majzoub{at}a1.tch.harvard.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CRH has been implicated as a mediator of many aspects of the stress response, including the endocrine, autonomic, behavioral, and immune responses to stress (3). In addition, the CRH system has been implicated in the pathophysiology of diseases such as depression and anorexia nervosa (4). However, recent evidence has brought into question the relative roles of CRH, urocortin, or potentially other unknown CRH-like molecules in various aspects of the stress response. For example, CRH-deficient [knockout (KO)] mice have normal stress-induced behavioral responses that are attenuated by the administration of CRH antagonists (5), suggesting the possibility that urocortin or another CRH-related molecule, rather than CRH, mediates these behaviors. In support of this possibility, infusion of urocortin into the brain reduces appetite (6) and causes an increase in anxiety-like behavior (7).
However, we have previously shown by in situ hybridization that urocortin expression within the brain is confined to the region of the Edinger-Westphal nucleus (EW), including the nucleus of Darkschewitsch, in mice (5), both regions of unknown function. Early studies suggested that both nuclei were primarily occulomotor nuclei, with the EW classically thought to control pupilloconstriction (8), and the nucleus of Darkschewitsch considered the main center responsible for the inhibitory regulation of vestibularly induced eye movements (9). However, more recent tracing studies have indicated a more complicated role for each of these nuclei. The nucleus of Darkschewitsch has been shown to receive input from the cerebellum, motor cortex, and spinal cord and to have efferent projections to the inferior olivary nucleus and motor cortex, thereby suggesting a role in the coordination of limb movement (10, 11). The role that the nucleus of Darkschewitsch may be playing in the stress response is entirely unclear. However, the EW has been shown to receive input from the spinal cord and the hypothalamus, and although no ascending projections from the EW have been reported, the EW does have descending projections to various nuclei expressing the CRH type I receptor (12), such as to the olivary nucleus, parabrachial nucleus, trigeminal brain stem nuclear complex, facial nucleus, reticular nucleus, and lamina I and V of the spinal cord (8, 13, 14). Therefore, the EW may play a role in autonomic regulation.
If urocortin plays a role in the behavioral response to stress, it would be through an entirely novel pathway, most likely involving descending projections from the EW. At this point little is known about the regulation of the urocortin gene. To begin to elucidate the physiological role of urocortin, we have examined the basal levels and the effect of restraint stress on urocortin messenger RNA (mRNA) expression in the EW (which hereafter we mean to include the nucleus of Darkschewitsch) of wild-type (WT) mice. As it is possible that the regulation of the urocortin gene is affected by CRH or glucocorticoid deficiency, we have also examined the basal levels and the effect of restraint stress on urocortin mRNA of CRH-deficient (KO) mice (15). As the urocortin gene is similar to the CRH gene, and the CRH gene is both positively and negatively regulated by glucocorticoids depending on the cell type (16), we have further examined the regulation of the urocortin gene in the EW by glucocorticoids, both basally in WT and KO mice and in response to stress in WT mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Digestion of the cloned PCR product with AvaII yielded a
222-bp fragment corresponding to the 3'-end of the cloned PCR product.
This AvaII fragment was randomly labeled with
[32P]deoxy-CTP and used to screen a 129SV mouse
genomic library in the 
FIX II vector (Stratagene, La
Jolla, CA). Approximately 280,000 phage plaques grown in
Escherichia coli strain LE392 on NCZYM plates for 9 h
at 37 C were transferred to BA85 0.45-µm pore size nitrocellulose
membranes (Schleicher & Schuell, Inc., Keene, NH) in
duplicate. After denaturation (1 min in 0.5 N
NaOH and 1.5 M NaCl) and neutralization (5 min in
0.5 M Tris, pH 7.4, and 1.5
M NaCl), the membranes were baked for 2 h at
80 C in a vacuum oven. Membranes were prehybridized for 3 h at 65
C in 5 x SSC (1 x = 0.15 M NaCl and
0.015 M sodium citrate), 5 x Denhardt’s
solution (1 x = 0.02% polyvinylpyrrolidine, 0.02% BSA, and
0.02% Ficoll, type 400), 0.5% SDS, and 200 µg/ml sonicated salmon
sperm DNA, after which 500,000 cpm/ml probe were added, and
hybridization was carried out at 65 C for 16 h. Membranes were
washed in 2 x SSC-0.1% SDS (one rinse followed by two 30-min
washes at 65 C) and in 1 x SSC-0.1% SDS (three 30-min washes at
65 C). A positive plaque was identified by overnight exposure to
Kodak XAR 5 film (Eastman Kodak Co.,
Rochester, NY) at -80 C. This positive plaque was plated to
homogeneity and purified for further studies.
Southern blot analysis
To construct a restriction map of the cloned phage, 3.5 µg of
the phage were cut with various restriction enzymes and subjected to
agarose gel electrophoresis and transfer to a nylon membrane
(GeneScreen, NEN Life Science Products, Boston, MA)
following standard protocols (19). Blots were probed with the randomly
labeled 222-bp AvaII fragment previously described.
Hybridization and wash steps were peformed as described above.
DNA sequence analysis
Smaller fragments of the phage DNA containing urocortin were
cloned into Bluescript SK (Stratagene, La Jolla, CA). The
entire urocortin gene was sequenced using overlapping primers to
different regions of the gene, beginning with the sequence already
determined from the 812-bp PCR product. Sequencing was carried out
using a Taq polymerase dideoxy-termination method with an
automated sequencer, as described above, using various primers.
Chromosomal mapping of mouse UCN
The gene encoding urocortin (Ucn) was mapped by
Southern blot analysis using 94 progeny from The Jackson Laboratory BSS interspecific backcross [(C57BL/6JEi x
SPRET/Ei)F1 x SPRET/Ei] (20). An
EcoRI restriction fragment length polymorphism (C57BL/6JEi,
17.5 kb; SPRET/Ei, 13.5 kb) was used to follow the segregation of
Ucn alleles. The 222-bp AvaII fragment of the
Ucn PCR product was used as a hybridization probe.
Hybridization and posthybridization washes were performed at 65 C, as
previously described (21).
Northern blot analysis
Various tissues (hypothalamus, midbrain, cerebral cortex,
cerebellum, pituitary, aorta, heart, kidney, pancreas, adrenal, liver,
lung, thymus, epididymal fat pad, diaphragm, intestine, testis, ovary,
and spleen) of adult male WT mice were collected, and total RNA was
isolated using Tri-Reagent (Sigma, St. Louis, MO). Tissue
was kept on dry ice until homogenized in 1 ml of Tri-Reagent. The
homogenizer was cleaned between samples with 1 M NaOH,
followed by three rinses in diethylpyrocarbonate-treated water, and a
final rinse in Tri-Reagent. RNA (10 µg) was separated on a 1.4%
formaldehyde agarose gel and transferred to GeneScreen (NEN Life Science Products, Boston, MA) following standard protocols (22).
A complementary RNA (cRNA) urocortin riboprobe was made by subcloning
the AvaII fragment described above into pBluescript II
SK± phagemid (Stratagene, La Jolla,
CA), digesting with HindIII, and labeling with
[
-32P]UTP (NEN Life Science Products) and T3 polymerase as previously described (23).
Hybridization was carried out at 65 C for 16 h with
106 cpm riboprobe/ml. The filter was washed
(three times for 20 min in 0.1 x SSC-10% SDS) and exposed to
Kodak XAR 5 film at -80 C for 1 and 8 days.
Animals for in situ studies
Breeding stock for WT and CRH KO mice used in these studies were
derived from heterozygote matings (mixed 129SVJ/C57BL6 background). All
CRH KO mice were adult male offspring of homozygous KO male X
heterozygote female matings, thereby eliminating the need for prenatal
glucocorticoid treatment of the mother (15). All animals were housed
under a 12-h light, 12-h dark cycle (lights on at 0700 h) with
free access to food and water.
For restraint experiments, animals were individually restrained for 3 h in ventilated 50-ml polypropylene tubes (24), which allowed minimal movement except that required for respiration. Animals were rapidly decapitated immediately after the cessation of the restraint. For corticosterone replacement, 6 male KO and 11 male WT mice were anesthetized by ip administration of 2.5% avertin, 0.01 ml/g BW, and a 40-mg 50% corticosterone/50% cholesterol pellet was implanted sc in each mouse using previously described techniques (25). Surgery was performed on sham-operated WT controls (n = 7) just as for the implantation of the pellet, but no pellet was placed in the sc opening. Plasma corticosterone was measured using a commercial RIA kit (ICN Pharmaceuticals, Inc., Costa Mesa, CA) using previously described modifications (26). All animal experiments were approved by the Children’s Hospital animal care and use committee.
In situ hybridization of mRNA
After rapid decapitation of mice between 1100–1200 h, brains
(n = 3–6/genotype/experimental group) were quickly frozen in
isopentane cooled to -30 C on dry ice and stored at -80 C until they
were sectioned. Fifteen-micron cryostat (from Reichert-Jung, Deerfield,
IL, for all except those for the WT/corticosterone study, which were
sectioned on a cryostat by Bright Instrument Co. Ltd., Huntingdon, UK)
sections were thaw-mounted on SuperFrost Plus slides (Fisher Scientific, Pittsburgh, PA). After air-drying at room
temperature, sections were stored at -20 C with desiccant. Fixation
and hybridization were carried out as previously described (5).
Briefly, the same urocortin cRNA probe described above for Northern
analysis was used for in situ hybridization studies. This
probe is complementary to nucleotides 660–882 (17) of the 3'-coding
and untranslated region of urocortin mRNA. The probe has 42% identity
with the corresponding region of CRH mRNA (maximum length of identity,
six nucleotides). We have previously shown that CRH mRNA does not
cross-hybridize to this probe (5). The radiolabeled urocortin cRNA
probe was synthesized using the enzyme T3 and
[-33P]UTP (SA, 1000–3000 Ci/mmol; 10
mCi/mL; New England Nuclear, Boston, MA). Once generated, probes
(3 x 107 cpm/ml) were stored at -20 C in
hybridization solution [50% deionized formamide, 10% dextran
sulfate, 0.5 M NaCl, 1 x Denhardt’s, 10
mM Tris (pH 8), 1 mM EDTA
(pH 8), 500 µg/ml yeast transfer RNA, and 10 mM
dithiothreitol] until use.
After fixation as previously described (5), tissue sections from all experimental groups to be compared were hybridized together in the same experiment. For hybridization, slides were placed in an air-tight chamber on top of filter paper saturated with 50% formamide-0.5 M NaCl and hybridized for a minimum of 16 h at 60 C. Posthybridization wash steps were carried out as follows. Coverslips were soaked off in 2 x SSC at room temperature for 15 min, followed by another 15 min wash in 2 x SSC at room temperature. Ribonuclease digestion was carried out for 30 min at 37 C in a solution containing 20 µg/ml ribonuclease A (Roche Molecular Biochemicals, Indianapolis, IN), 0.5 M NaCl, 10 mM Tris (pH 8), and 1 mM EDTA (pH 8). Next, the slides were washed in 2 x SSC for 10 min at room temperature, followed by three 10-min washes in 0.5 x SSC at room temperature. After washing for 30 min in 0.5 x SSC at 65 C, slides were sequentially treated at room temperature with 0.5 x SSC for 2 min, 50% ethanol-0.3 M ammonium acetate for 1 min, 70% ethanol-0.3 M ammonium acetate for 1 min, 95% ethanol-0.15 M ammonium acetate for 1 min, and finally twice with 100% ethanol for 1 min each. Slides were allowed to air-dry and then were exposed to x-ray film (Hyperfilm ßmax, Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 and 3 days. Quantitation of the signal was accomplished using the public domain NIH image program (developed at the NIH and available on the internet at http://rsb.info.nih.gov/nih-image/) by analysis of scanned images of the autoradiography film. The mean signal in the EW of the top two expressing sections minus a background reading of each section was determined for each animal. Significance was determined by Student’s t test for unpaired measures with appropriate Bonferroni corrections.
After exposure to x-ray film, slides were dipped in NBT-2 emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 in distilled water. The slides were developed after 18–28 days of exposure [4 min in D19 (Eastman Kodak Co.), 1:1 in distilled water, 15 sec in distilled water, and 5 min in Kodak fixer] and counterstained with 0.01% toluidine blue (Fisher Scientific). Comparisons were only made between slides that had been hybridized, emulsion-coated, and exposed in the same batch.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FIX II vector resulted in one positive phage. Restriction
enzyme analysis of this clone revealed a 15.5-kb insert, with
approximately 11 kb of sequences 5' to the urocortin gene and 3.2 kb of
3'-flanking sequence (Fig. 1A
). The
entire urocortin gene was sequenced including over 400 bp 5' to the
transcription start site. Our sequence confirms that reported by Zhao
et al. (17).
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). The gene order and map
distances (given as percent recombination ±
SE) are shown in Fig. 1B. Based on the mouse
consensus map (27), Ucn is approximately 18 centimorgans
(cM) distal to the centromere, a region conserved with human chromosome
2p22-p23. No obvious potential candidate mouse mutations map to this
region of chromosome 5. This assignment is consistent with the known
mapping of the human gene encoding urocortin (UCN) to human
chromosome 2 (28).
Northern blot analysis
Urocortin mRNA is detectable in WT mice by Northern blot analysis
only in the midbrain. No detectable expression was observed in other
brain regions, including the hypothalamus, cortex, and cerebellum, nor
was detectable expression observed in other tissues, including the
pituitary, aorta, heart, kidney, pancreas, adrenal, liver, lung,
thymus, epididymal fat pad, diaphragm, intestine, ovary, or spleen
(Fig. 2A). Although a band in the
vicinity of that of urocortin mRNA was detected in testis, longer
exposures confirmed that this band was significantly larger than
urocortin mRNA. Conceivably, this band could represent an alternatively
spliced form of urocortin. Additionally, the RNA of testis was
significantly degraded (Fig. 2B). Northern blot analysis of midbrain
RNA suggests a primary transcript size of approximately 900 bp.
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).
The signal shown in subsequent higher power darkfield images (
Figs. 4–6) can be seen as black grains in the
center of the brightfield image in Fig. 3. We first examined the
effects of 3 h of restraint stress on urocortin expression in the
EW of WT mice. Compared with the basal state (Fig. 4, A and E),
urocortin mRNA expression within the EW of WT mice is significantly
up-regulated after 3 h of restraint (Fig. 4, B and E; n =
6/experimental group). We next examined the effects of glucocorticoid
supplementation on the basal and stress-induced levels of urocortin
expression in WT mice (Fig. 5).
Corticosterone supplementation with 50% corticosterone pellets for
5 days in WT animals (n = 5) resulted in plasma corticosterone
levels of 360 ± 49 ng/ml. However, compared with sham-operated
controls (Fig. 5, A and E; plasma corticosterone, 18 ± 6
ng/ml; n = 3), corticosterone treatment did not affect basal
urocortin expression (Fig. 5, B and E). Furthermore, although the
expected rise in urocortin was seen after 3 h of restraint in
sham-operated controls (Fig. 5. C and E; P = 0.027,
sham restraint vs. sham basal; plasma corticosterone,
435 ± 22 ng/ml; n = 4), no rise in urocortin expression was
seen in corticosterone-treated WT mice after 3 h of restraint
(Fig. 5, D and E; P = 0.019 sham restraint
vs. corticosterone restraint; plasma corticosterone,
389 ± 29 ng/ml; n = 6).
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, A and E), urocortin mRNA
expression is up-regulated approximately 3-fold in the EW of KO mice
(Fig. 4, C and E; n = 6/genotype). This up-regulation of urocortin
mRNA is not due to the chronically low glucocorticoid levels of KO mice
(15), as corticosterone supplementation with 50% corticosterone
pellets for 5 days in CRHKO mice, although achieving mean plasma
corticosterone levels of 232 ± 24 ng/ml (range, 168–339 ng/ml),
did not affect the level of urocortin expression in the EW of
KO mice (Fig. 6; n = 6/experimental
group). We further sought to determine whether urocortin
expression in CRHKO mice was regulated by stress (Fig. 4). Unlike the
situation in WT mice (Fig. 4, A and B, E), 3 h of restraint had no
effect on urocortin mRNA expression in the EW of KO mice
(Fig. 4, C and D, E; n = 6/experimental group). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that in situ hybridization analysis of urocortin mRNA within brain detects expression only in the midbrain region of the Edinger-Westphal nucleus, including the nucleus of Darkschewitsch, in both WT and KO mice (5). Northern blot analysis of sites within and outside the central nervous system again restricts urocortin mRNA expression to the midbrain. Interestingly, using nested RT-PCR we have detected urocortin mRNA expression in every tissue examined within and outside of the brain (including cerebral cortex, cerebellum, and most peripheral tissues; Weninger, S. C., unpublished observations). This discrepancy is most likely explained by the extreme sensitivity of nested RT-PCR and the likely expression of low levels of urocortin in vascular smooth muscle cells of blood vessels (29). Of note, we also detected an alternatively spliced, slightly smaller product of the urocortin gene by nested RT-PCR in pituitary, kidney, liver, spleen, and diaphragm. Sequencing of the alternatively spliced complementary DNA product reveals that the intron is slightly larger than in midbrain (13 nucleotides at the 5'-end and 7 nucleotides at the 3'-end) and that the splicing of the intron does not follow the GT/AG rule (30) (Weninger, S. C., unpublished observations). As the alternative splicing occurs upstream of the AUG, and the alternatively spliced product is only detectable by nested RT-PCR, the significance is unclear. Of note, Zhao et al. also described alternative splicing of urocortin mRNA within the same region, although the product they reported does obey the GT/AG rule (17).
The factors regulating urocortin in the EW nucleus are unclear. The identification of a cAMP response element (CRE) and putative RE-1 element suggest that this gene may be positively regulated by CRE-binding protein and negatively by RE-1-silencing transcription factor (REST) (17). In vitro analysis has suggested that the CRE is functionally significant (17). We wondered whether urocortin, like CRH in the paraventricular nucleus of the hypothalamus (PVH), is negatively regulated by glucocorticoids (31). We therefore studied CRHKO mice, which are also profoundly glucocorticoid deficient (15). Although our finding of increased urocortin mRNA expression in the EW of CRHKO mice vs. WT mice is consistent with negative regulation by glucocorticoids, increasing plasma corticosterone to levels of greater than 160 ng/ml for 5 days by sc implantation of 50% corticosterone pellets did not suppress these elevated levels of urocortin mRNA in CRHKO mice. We chose this dose of corticosterone based on a previous study which showed that CRH in the PVH is decreased to just detectable levels only with corticosterone levels above 130 ng/ml (31). Although CRH expression is classically thought to be inhibited by glucocorticoids, CRH expression has been found to be up-regulated by glucocorticoids in the central nucleus of the amygdala and the bed nucleus of the stria terminalis (16, 32). However, glucocorticoid treatment of WT animals with sc implantation of 50% corticosterone pellets for 5 days did not affect basal mRNA urocortin levels. Therefore, in contrast to CRH, glucocorticoids appear to neither stimulate nor inhibit basal urocortin expression.
To determine whether urocortin in the EW is regulated by stress, we further characterized modulation of urocortin mRNA expression in the EW after 3 h of restraint in both WT and KO mice. Although compared with the basal state, a significant increase was seen in WT mice after restraint, no increase was detectable in KO mice after restraint. Conceivably, the high basal urocortin mRNA levels in KO mice may preclude a further rise with restraint stress. Although chronic corticosterone treatment did not affect basal levels of urocortin expression in WT mice, this same regimen of corticosterone treatment with 50% corticosterone pellets for 5 days blocked the rise in urocortin expression seen after 3 h of restraint. In this respect, glucocorticoids act similarly to block stress-induced increases in the expression of both CRH mRNA in the PVH and urocortin mRNA in the EW. However, it is possible that the mechanism of action is different in these two cases, as the effects of glucocorticoids on basal mRNA levels of CRH in the PVH and urocortin in the EW are different. Whether the differences in regulation by glucocorticoids are cell specific or gene specific remains to be determined.
CRH KO mice have normal behavioral responses to stress, which are attenuated by CRH antagonists (5). Although it is possible that up-regulated urocortin in the EW of CRHKO mice could compensate for the lack of CRH in other brain regions, it seems highly unlikely that this increased urocortin expression could precisely compensate for the lack of CRH in other brain regions to perfectly replicate complex stress behaviors. Therefore, as no other central differences between WT and KO mice have been observed, and the normal physiological role of urocortin in the EW is unknown, the consequences of up-regulation of urocortin in the EW chronically in CRHKO mice are unclear.
The functional importance of the EW nucleus is largely unknown. There are afferent projections from the hypothalamus, suggesting that the EW nucleus may play a role in some aspects of the stress response (33). Projections from the EW have been examined in both the cat (8, 14) and the rat (13). No evidence for ascending projections has been found, but a peripheral projection to the ciliary ganglion and central descending projections to the olivary nucleus, parabrachial nucleus, trigeminal brain stem nuclear complex, facial nucleus, reticular nucleus, and laminae I and V of the spinal cord have been described. The EW was traditionally thought to primarily regulate pupilloconstriction (8). However, the above tracing studies reveal that the EW may play a much more complex role. The projections described above suggest a possible role in autonomic regulation. A role in pain regulation is also possible, considering the existence of projections to somatosensory relay nuclei as well as laminae I and V of the dorsal horn of the spinal cord, areas that respond to nociceptive stimuli.
All of the descending pathways of the EW project to regions known to express the CRH type I receptor (12). As urocortin can bind to and activate this receptor, it is conceivable that urocortin may act at any or all of these projection sites. The projections of urocortin-specific neurons in the EW have not yet been determined. It has been suggested, based upon CRH receptor localization, that CRH may exert some of its effect on autonomic function via receptors in the parabrachial or medullary reticular formation (34). As the EW projects to both of these regions, it is possible that urocortin from the EW, rather than CRH, is responsible for the autonomic effects of stress previously ascribed to CRH. Autonomic effects of centrally infused CRH have been reported in a variety of species, including rats, dogs, sheep, and monkeys. These include stimulation of plasma catecholamine secretion and cardiovascular parameters such as heart rate and mean arterial pressure (35). As stated above, we have found that chronic glucocorticoid treatment blocked the stress-induced rise in urocortin. Previously, it had been shown that chronic infusion of cortisol attenuates the rise in plasma catecholamines after immobilization stress in rats (36). It is possible that if urocortin is playing a role in stress-induced activation of the sympathetic nervous system, this attenuation in plasma catecholamines after chronic glucocorticoid treatment results from an inhibition of the stress-induced rise of urocortin expression in the EW. Further studies of specific afferent projections to and efferent projections from urocortin neurons of the EW as well as the examination of mice with targeted deletion of the urocortin gene will help to further define the regulation and physiological role of urocortin gene expression.
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Acknowledgments |
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Footnotes |
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Received June 15, 1999.
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 M. L.G. Correia, D. A. Morgan, J. L. Mitchell, W. I. Sivitz, A. L. Mark, and W. G. Haynes Role of Corticotrophin-Releasing Factor in Effects of Leptin on Sympathetic Nerve Activity and Arterial Pressure Hypertension, September 1, 2001; 38(3): 384 - 388. [Abstract] [Full Text] [PDF]
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S. C. Coste, K. A. Heldwein, S. L. Stevens, E. Tobar-Dupres, and M. P. Stenzel-Poore IL-1{alpha} and TNF{alpha} Down-Regulate CRH Receptor-2 mRNA Expression in the Mouse Heart Endocrinology, August 1, 2001; 142(8): 3537 - 3545. [Abstract] [Full Text] [PDF]
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