General and Comparative Endocrinology 146 (2006) 9–18
www.elsevier.com/locate/ygcen
Widespread tissue distribution and diverse functions
of corticotropin-releasing factor and related peptides
Graham C. Boorse a,1, Robert J. Denver a,b,¤
a
b
Department of Ecology and Evolutionary Biology, The University of Michigan, Ann Arbor, MI 48109-1048, USA
Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109-1048, USA
Received 22 September 2005; revised 18 November 2005; accepted 26 November 2005
Available online 18 January 2006
Abstract
Peptides of the corticotropin-releasing factor (CRF) family are expressed throughout the central nervous system (CNS) and in peripheral tissues where they play diverse roles in physiology, behavior, and development. Current data supports the existence of four paralogous genes in vertebrates that encode CRF, urocortin/urotensin 1, urocortin 2 or urocortin 3. Corticotropin-releasing factor is the major
hypophysiotropin for adrenocorticotropin, and also functions as a thyrotropin-releasing factor in non-mammalian species. In the CNS,
CRF peptides function as neurotransmitters/neuromodulators. Recent work shows that CRF peptides are also expressed at diverse sites
outside of the CNS in mammals, and we found widespread expression of CRF and urocortins, CRF receptors and CRF binding protein
(CRF-BP) genes in the frog Xenopus laevis. The functions of CRF peptides expressed in the periphery in non-mammalian species are
largely unexplored. We recently found that CRF acts as a cytoprotective agent in the X. laevis tadpole tail, and that the CRF-BP can
block CRF action and hasten tail muscle cell death. The expression of the CRF-BP is strongly upregulated in the tadpole tail at metamorphic climax where it may neutralize CRF bioactivity, thus promoting tail resorption. Corticotropin-releasing factor and urocortins are
also known to be cytoprotective in mammalian cells. Thus, CRF peptides may play diverse roles in physiology and development, and
these functions likely arose early in vertebrate evolution.
 2005 Elsevier Inc. All rights reserved.
Keywords: Corticotropin-releasing factor; Urocortin; Urotensin I; Sauvagine; Stress; Amphibian; Xenopus; Metamorphosis; CRF-BP
1. Introduction
Corticotropin-releasing factor (CRF) is the primary
neuroregulator of the vertebrate stress response. This 41amino acid peptide was originally isolated and characterized
from ovine hypothalamus and shown to be the major hypothalamic releasing factor for pituitary adrenocorticotropic
hormone (ACTH; Vale et al., 1981). Physical or emotional
stress increases CRF production in the paraventricular
nucleus of the mammalian hypothalamus, and the release of
the peptide from neurohemal axon terminals in the median
eminence. The peptide binds to speciWc receptors on pituitary
*
Corresponding author. Fax +1 734 647 0884.
E-mail address: [email protected] (R.J. Denver).
1
Present address: Integrated Natural Science, Arizona State University,
Glendale, AZ 85306-2352, USA.
0016-6480/$ - see front matter  2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2005.11.014
corticotropes to increase production and secretion of ACTH,
and ACTH stimulates glucocorticoid biosynthesis by the
adrenal gland (interrenal gland in nonmammalian species).
Glucocorticoids mediate many metabolic changes associated
with the stress response (Sapolsky et al., 2000).
In the mammalian brain, CRF is also expressed in the
limbic system, cortex, and brainstem nuclei associated with
autonomic functions. Corticotropin-releasing factor acts as
a neurotransmitter or a neuromodulator at these sites
where it can induce anxiety-like behaviors, decrease food
intake, enhance learning, increase arousal, alter blood pressure, diminish sexual behavior, and inXuence locomotor
activity (Koob and Heinrichs, 1999; Smagin et al., 2001).
Thus, CRF not only controls the hypothalamo–pituitary–
adrenal (HPA) axis, but also integrates autonomic and
behavioral responses to stress through its actions within the
central nervous system (CNS).
10
G.C. Boorse, R.J. Denver / General and Comparative Endocrinology 146 (2006) 9–18
Neuroanatomical mapping studies showed that CRFimmunoreactivity (CRF-ir) is widely distributed throughout the CNS in non-mammalian species as it is in
mammals, suggesting a conserved role as a neurotransmitter/neuromodulator (Lovejoy and Balment, 1999). In frogs,
CRF-ir is found in the anterior preoptic area and the
median eminence where its primary function is as a hypophysiotropin (Gonzalez and Lederis, 1988; Ogawa et al.,
1995; Olivereau et al., 1987; Verhaert et al., 1984; Yao et al.,
2004). Strong CRF-ir is also found throughout the telencephalon, diencephalon, mesencephalon, rhombencephalon, tectum, cerebellum, and rostral spinal cord in the South
African clawed frog Xenopus laevis (Calle et al., 2005; Yao
et al., 2004). Exposure to a physical stressor activated CRF
neurons in the anterior preoptic area, the amygdala, and
the bed nucleus of the stria terminalis of X. laevis (Yao
et al., 2004), brain regions that are also responsive to physical stress in mammals (Herman et al., 2003). Conserved
behavioral actions of CRF peptides are suggested by the
potent eVects of intracerebroventricular CRF injections on
food intake and locomotion in amphibians (Carr, 2002;
Crespi and Denver, 2004; Crespi et al., 2004; Crespi and
Denver, 2005; Lowry et al., 1996).
The expression of CRF in the hypothalamus and the stimulation of pituitary ACTH secretion is common to all vertebrates that have been studied (Denver, 1999; Fryer et al.,
1983, 1985; Tonon et al., 1986; Tran et al., 1990; Yao et al.,
2004). In non-mammalian species CRF is also known to be a
potent thyrotropin (TSH)-releasing factor (TRF; reviewed
by Denver, 1999; see also De Groef et al. this symposium.)
Thyroid hormone is the primary morphogen controlling
amphibian metamorphosis (Kikuyama et al., 1993; Shi, 2000)
and CRF has been shown to directly stimulate TSH secretion
by the amphibian pituitary gland (Denver, 1988; Okada
et al., 2004) thus elevating thyroid hormone production
(Denver, 1993, 1997a; Gancedo et al., 1992) and accelerating
metamorphosis (Boorse and Denver, 2002; Denver, 1993,
1997a,b; Gancedo et al., 1992; Miranda et al., 2000).
2. Corticotropin-releasing factor receptors and binding
protein
The actions of CRF are mediated by two G protein-coupled receptors, CRF type 1 (CRF1) and CRF type 2 (CRF2;
Hauger et al., 2003, Fig. 1). These receptors are widely
expressed in the CNS and in peripheral tissues (Chalmers
et al., 1996; Chen et al., 1993; Potter et al., 1994; SumanChauhan et al., 1999). The CRF1 and CRF2 couple to Gs in
most cells and ligand binding leads to the activation of
adenylyl cyclase with a resultant increase in intracellular
cAMP. However, coupling to other G proteins has been
reported in some cell types (e.g., to Gq; reviewed by Dautzenberg and Hauger, 2002; Hillhouse et al., 2002). Orthologs of the mammalian CRF1 and CRF2 receptors have
been isolated by molecular cloning from frog (Dautzenberg
et al., 1997) chicken (De Groef et al., 2003; Yu et al., 1996)
and Wsh (Arai et al., 2001; Cardoso et al., 2003; Pohl et al.,
2001); a novel third receptor was isolated from the catWsh
that is expressed in the pituitary and urophysis. While each
CRF receptor gene gives rise to multiple splicing variants
with distinct expression proWles in mammals, splice variants
of CRF receptors have not been identiWed in non-mammalian vertebrates.
The bioactivity of CRF is modulated by a secreted CRFbinding protein (CRF-BP) with aYnity for CRF peptides
that is similar to or greater than the aYnity for the receptors (Seasholtz et al., 2002). The CRF-BP was originally
isolated and characterized from human liver and rat brain
(Potter et al., 1991) and its major function appears to be to
modulate access of CRF and related peptides to CRF
receptors (Seasholtz et al., 2001). Genes for the CRF-BP
have been isolated and characterized in rat, mouse, sheep,
frog (X. laevis) and the honeybee (Behan et al., 1996; Brown
et al., 1996; Cortright et al., 1995; Huising and Flik, 2005;
Potter et al., 1992; Valverde et al., 2001). Also, CRF-BP
protein was detected in brain extracts from sea lamprey,
tilapia, turtle and chicken, thus showing that the CRF-BP is
Fig. 1. Schematic representation of relative binding aYnities and speciWcities of: (A) mammalian and (B) X. laevis corticotropin-releasing factor-like peptides, their receptors (CRF1 and CRF2), and the CRF binding protein (CRF-BP). The question mark indicates that a urocortin 2 gene has not yet been
identiWed in X. laevis. Solid lines with arrows indicate high aYnity, while dashed lines with arrows indicate lower aYnity binding.
G.C. Boorse, R.J. Denver / General and Comparative Endocrinology 146 (2006) 9–18
a phylogenetically ancient protein that has retained both
structural and functional properties (Seasholtz et al., 2002).
Another soluble CRF binding protein that is a splice variant of the type 2a CRF receptor was recently isolated from
mouse brain (Chen et al., 2005a); however, the physiological signiWcance of this splice variant is unknown as is its
existence in nonmammalian species.
3. Urotensins I and urocortins
At the time that CRF was discovered, structurally similar peptides were isolated from the caudal neurosecretory
organ (urophysis) of several Wshes (urotensin I; Lederis
et al., 1982) and from the skin of the frog Phyllomedusa sauvageii (sauvagine; Montecucchi and Henschen, 1981). These
peptides were originally thought to be orthologs of mammalian CRF. However, peptides with even greater similarity to the mammalian CRFs were subsequently discovered
in Wshes and amphibians (Okawara et al., 1988; StenzelPoore et al., 1992). About 15 years passed before a second
CRF-like peptide gene was isolated from rat brain that had
high sequence similarity to urotensin I and sauvagine and
was subsequently named urocortin (now urocortin 1; Donaldson et al., 1996; Vaughan et al., 1995). Both CRF and
urocortin 1 bind to and activate the CRF1 and CRF2 receptors, but CRF has higher aYnity for the CRF1 receptor,
while urocortin 1 has higher aYnity for the CRF2 receptor
(Dautzenberg et al., 1997, 2001).
Two other CRF-like peptides that are selective for the
CRF2 receptor were recently isolated by genomic analysis
and subsequent molecular cloning from human and mouse
(Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al.,
2001). The accepted nomenclature for these peptides is now
urocortin 2 and urocortin 3 (Hauger et al., 2003). Thus, in
mammals there are at least four CRF paralogs. Urocortin 3
genes were also described in two species of puVerWsh (Takifugu rubripes and Tetraodon nigroviridis; Hsu and Hsueh,
2001; Lewis et al., 2001; Reyes et al., 2001).
11
cortin 3 (Boorse et al., 2005a). The deduced X. laevis
urocortin 1 mature peptide is 40 amino acids and shares
70% sequence similarity with mouse/rat urocortin 1 and
63% similarity with Wsh urotensin I (trout), but only 50%
sequence similarity with sauvagine. A full length sauvagine
cDNA sequence isolated from the skin of P. sauvageii was
recently deposited in GenBank (Accession # AY943910).
Sauvagine appears to be a highly divergent urocortin 1 that
may be speciWc to P. sauvageii, and more work is required
to determine the relationship of sauvagine to other vertebrate urocortin genes. The X. laevis urocortin 3 deduced
mature peptide is highly conserved with other vertebrate
urocortins 3, sharing 90 and 75% sequence similarity with
mouse and puVerWsh urocortin 3, respectively. Although we
were unable to identify a urocortin 2 in the frog, we found
urocortin 2 genes by genome analysis in two puVerWsh species and in the chicken (Boorse et al., 2005a).
Currently, the South African clawed frog X. laevis is the
only non-mammalian vertebrate for which CRF, urocortins,
CRF receptors (CRF1 and CRF2) and the CRF-BP have
been cloned and characterized. We synthesized the deduced
mature peptides of X. laevis urocortin 1 and urocortin 3 and
determined their receptor and binding protein pharmacology (Boorse et al., 2005a). We found that the absolute and
rank order aYnities of frog CRF-like peptides for their
receptors and binding protein are largely conserved compared with the known ligand-binder relationships in mammals (see Fig. 1). As with mammals, frog CRF binds to and
activates the frog CRF1 receptor with high aYnity and
potency, that is slightly greater than frog urocortin 1. These
potency relationships are reversed for the CRF2 receptor,
and as in mammals, frog urocortin 3 is a selective CRF2
receptor agonist. The binding aYnities of frog CRF ligands
for the frog CRF-BP are largely similar to those reported
for mammals, except that unlike mammals, the frog urocortin 1 has low aYnity for the CRF-BP. Similar to mammals,
the frog CRF-BP does not bind urocortin 3.
4. Amphibian CRF peptides
5. Tissue distribution of CRF peptides, receptors, and binding
protein
Two CRF genes were isolated from the South African
clawed frog X. laevis by Stenzel-Poore et al. (1992). Both
code for an identical mature peptide of 41 amino acids that
diVers from rat/human CRF in only three positions. Corticotropin-releasing factor genes have also been isolated
from the Western spadefoot toad Spea hammondii (Boorse
and Denver, 2004a), the North American bullfrog Rana
catesbeiana (Ito et al., 2004) and the monkeyfrog Phyllomedusa sauvageii (G.C. Boorse and R.J. Denver, unpublished;
GenBank Accession # AY596828).
Until very recently the only paralog of CRF known in
nonmammalian tetrapods was the amphibian peptide sauvagine (Montecucchi and Henschen, 1981). We isolated and
characterized two urocortins from the South African
clawed frog X. laevis, one orthologous to mammalian urocortin 1/Wsh urotensin I, the other to mammalian/Wsh uro-
In mammals, CRF, urocortin 1, urocortin 3, CRF1,
CRF2, and CRF-BP are expressed in the brain and pituitary (Oki and Sasano, 2004; Suda et al., 2004) and we
observed a similar expression pattern in the frog (Fig. 2A).
To our knowledge, the expression of urocortin 2 has not
been reported in the mammalian pituitary gland.
Many peptide hormones once thought to be restricted to
the brain and pituitary are now known to be widely
expressed in peripheral tissues. Shortly after CRF was isolated, CRF-immunoreactivity (CRF-ir) was detected outside
the brain in the spinal cord, lung, pancreas, gastrointestinal
tract, and adrenal glands (Petrusz et al., 1985, 1983; Suda
et al., 1984). Systemic intravenous delivery of CRF induced
physiological changes in mammals that diVered from intracerebroventricular administration (Lenz et al., 1985; MacCannell et al., 1984), and CRF applied directly to isolated
12
G.C. Boorse, R.J. Denver / General and Comparative Endocrinology 146 (2006) 9–18
Fig. 2. Tissue distribution of mRNAs for corticotropin-releasing factor system components in juvenile Xenopus laevis tissues analyzed by RT-PCR. RNA
isolation and RT-PCR is described by Boorse et al. (2005a) and the PCR primer sequences are given in Table 1. (A) Expression analysis in brain and pituitary. (B) Expression analysis in diverse peripheral tissues. Corticotropin-releasing factor (CRF), urocortin 1 (UCN1), urocortin 3 (UCN3), CRF binding
protein (CRF-BP), CRF receptor 1 (CRF1) and CRF receptor 2 (CRF2). Ribosomal L8 (rpL8) was used as a housekeeping gene to control for RNA quality and loading. Shown are representative ethidium bromide-stained gels of RT-PCR products; RNA was harvested from tissues of three females and similar results were obtained with each. PCR reactions conducted on reverse transcription reactions in which the reverse transcriptase was omitted (minus RT
reactions) produced no bands, thus conWrming the absence of genomic DNA contamination + and ¡ on the Wgure. RT-PCR reactions conducted on RNA
isolated from whole blood or packed blood cells produced no bands, which shows that the positive results that we obtained in other tissues are not due to
contamination from blood cells. However, we cannot rule out the possibility that some blood cell types express CRF system signaling components but
their abundance was below the level of detection of our assay.
heart preparations resulted in increased coronary output and
the rapid release of atrial natriuretic peptide (Grunt et al.,
1993, 1992), suggesting that CRF receptors and ligands could
act directly on tissues such as the heart, perhaps functioning
in either a paracrine or autocrine manner. Subsequently, the
expression of mRNAs for CRF-like peptides and receptors
has been shown in a diversity of peripheral tissues in mammals. CRF peptides are known to be vasoactive (Chen et al.,
2005b; Lederis et al., 1982) and recent data shows that they
play important roles in cardiac cell survival and function
(Bale et al., 2004; Coste et al., 2002; Huang et al., 2004;
Takahashi et al., 2004a). These peptides have also been found
to stimulate proliferation (Emanuel et al., 2000; Ikeda et al.,
2002; Jessop et al., 1997; Mitsuma et al., 2001) and to protect
diverse cell types from environmental insults (i.e., they are
cytoprotective; Brar et al., 2000, 1999; Fox et al., 1993; Pedersen et al., 2002).
In mammals, mRNA for the CRF1 receptor is highly
expressed in brain and anterior pituitary, but low levels of
expression have been found in the testis, ovary, adrenal
gland (Kageyama et al., 1999; Palchaudhuri et al., 1998),
skin (Slominski et al., 2004), GI tract (Chatzaki et al.,
2004a,b) and adipose tissue (Seres et al., 2004). The mRNA
for the CRF2 receptor is widely expressed in peripheral tis-
sues, particularly the heart, GI tract, lung, ovary, skeletal
muscle (Chatzaki et al., 2004a,b; Heldwein et al., 1997;
Kishimoto et al., 1995; Lovenberg et al., 1995; Nozu et al.,
1999; Palchaudhuri et al., 1999), skin (Slominski et al., 2004),
and adipose tissue (Seres et al., 2004). The interactions of
CRF peptides with their receptors in certain peripheral tissues may be modulated by the CRF-BP. While highest
expression of CRF-BP mRNA has been shown in brain
and pituitary gland (Cortright et al., 1995; Potter et al.,
1991) the CRF-BP is also expressed in liver and placenta of
humans (Behan et al., 1995) and in the skin (Slominski
et al., 2004).
The expression of CRF-like peptides in peripheral tissues of mammals overlaps with the tissue distribution of
CRF receptors. In the periphery, CRF mRNA is expressed
in the adrenal gland, testis, placenta, GI tract, spleen, thymus, and skin (Vale et al., 1997). Urocortin 1 mRNA was
found in the GI tract, testis, cardiac myocytes, thymus,
spleen, and kidney (Kageyama et al., 1999), skin (Slominski
et al., 2000, 2001) and adipose tissue (Seres et al., 2004).
Urocortin 2 mRNA has been detected in the heart, adrenal
gland, peripheral blood cells (Hsu and Hsueh, 2001; Reyes
et al., 2001) skin and skeletal muscle (Chen et al., 2004; Slominski et al., 2004). Finally, urocortin 3 mRNA expression
G.C. Boorse, R.J. Denver / General and Comparative Endocrinology 146 (2006) 9–18
Table 1
Sequences of oligonucleotides used for RT-PCR analysis of X. laevis genes
Gene
Oligonucleotide sequence
(forward primer above, reverse primer below)
GenBank
Accession
Number
CRF
5⬘-TCTCCTGCCTGCTCTGTCCAA-3⬘
5⬘-CTTGCCATTTCTAAGACTTCACGG-3⬘
S50096
UCN1
5⬘-GGGTTAAATGGGCTGTTAGGTGATG-3⬘
5⬘-GGCAATCTCTATCATCTGTCTGAG-3⬘
AY596827
UCN3
5⬘-CAGAGAGGTCTTAGAGGAGGCCA-3⬘
5⬘-TGGGCATTGGCTGCTGCTTT-3⬘
AY596826
CRF1
5⬘-GATCTAAATAGCAGGATGCTGTTGGC-3⬘ Y14036
5⬘-GCCGTTCAGGTGACATTCTCTGTAC-3⬘
CRF2
5⬘-GTACCTCCCACATCCCACAGCTTCAC-3⬘
5⬘-GACGCCCAGGTTCCATTCTCAAAGC-3⬘
Y14037
CRF-BP 5⬘-TGACTCCTGCTTCCAGACCT-3⬘
5⬘-TGACCTTGTAATGCTCCCCAC-3⬘
U41858
rpL8
U00920
5⬘-CACAGAAAGGGTGCTGCTAAG-3⬘
5⬘-CAGGATGGGTTTGTCAATACG-3⬘
has been found in the GI tract, skeletal muscle, adrenal
gland, (Hsu and Hsueh, 2001; Lewis et al., 2001), skin
(Saruta et al., 2005) and adipose tissue (Seres et al., 2004).
To our knowledge no studies have investigated the tissue
distribution of CRF signaling components outside of the
CNS/pituitary in non-mammalian species. We used RTPCR to analyze CRF, urocortin 1, urocortin 3, CRF1 and
CRF2 receptor, and CRF-BP mRNAs in juvenile X. laevis
13
peripheral tissues (Table 1). These data are shown in
Fig. 2B (with brain included for comparison), and summarized in Table 2 with comparison to the mammalian distribution pattern. Tissues in which we found at least one of
the ligands and one of the receptors expressed include:
heart, pancreas, kidney, adipose tissue, skin, GI tract
(includes intestine and stomach), lungs and ovary (we did
not analyze the testis). The kidney, lungs and ovary
expressed all components analyzed similar to the brain and
pituitary. The heart and GI tract expressed all genes except
for the CRF1 receptor. These Wndings suggest that CRF
peptides could have paracrine/autocrine functions in each
of these tissues. We used radioimmunoassay on tissue
extracts to verify that the expression of CRF mRNA corresponds to CRF peptide in brain, skin and heart (Fig. 3A;
we were unable to detect the peptide in other tissues). As in
mammals, the distribution and relative level of expression
of the CRF2 receptor was greater than the CRF1 receptor
in frog peripheral tissues. This expression pattern of the
CRF2 receptor corresponds with widespread expression of
urocortin 1 (which has higher aYnity for CRF2 than for
CRF1) and urocortin 3 (which is a selective agonist for the
CRF2; Boorse et al., 2005a).
In several tissues including the spleen, skeletal muscle
and liver we found that the genes for CRF peptides were
expressed, but we could not Wnd evidence for expression of
CRF receptors. This could be due to the limits of detectability of our assay, or these tissues could be a source of
Table 2
Comparison of the tissue distribution of CRF peptides, receptors and binding protein in adult mammals and Xenopus laevis
Tissue
Brain
Pituitary
Heart
Spleen
Skeletal muscle
Pancreas
Kidney
Liver
Adipose
Skin
GI tract
Lung
Ovary
CRF
UCN1
CRFRf
UCN3
CRF-BP
XL
Ma
XL
Mb
XL
Mc
XL
Md
XL
Me
+
+
+
+
¡
+
+
+
+
+
+
+
+
+
+
+
+
?
+
+
+
ND
+
+
+
+
+
+
+
+
+
¡
+
+
+
+g
+
+
+
+
+
+
+
?
ND
+
ND
+
+
+
ND
+
+
+
+
+
+
¡
+
+
+
¡
+
+
+
+
+
?
?
+
+
+
?
+
¡
+
?
+
+
+
+
¡
¡
+
+
¡
+
+
+
+
+
+
+
+
+
+
+
+
ND
+
+
+
+
+
+
+
+
¡
¡
¡
+
+
¡
¡
+
+
+
+
+
¡
¡
ND
ND
+
+
ND
+
ND
¡
+
XL D Xenopus laevis; M D mammal.
+ D detected; ¡ D not detected; ND D not determined; ? D We could not determine from the literature if it had been analyzed, or there are conXicting
reports.
a
Compilation of mRNA and protein expression data from: Asakura et al., 1997; Baigent and Lowry, 2000; Chatzaki et al., 2004a,b; Dotzler et al., 2004;
Muglia et al., 1994; Petrusz et al., 1985, 1983, 1984; Slominski et al., 1996, 2000, 2001; Suda et al., 1984.
b
Compilation of mRNA and protein expression data from: Baigent and Lowry, 2000; Iino et al., 1999, 1997; Kageyama et al., 1999; Lewis et al., 2001;
Oki et al., 1998; Seres et al., 2004; Slominski et al., 2000; Takahashi et al., 2004b; Wong et al., 1996.
c
Compilation of mRNA and protein expression data from: Hsu and Hsueh, 2001; Lewis et al., 2001; Li et al., 2003; Saruta et al., 2005; Seres et al., 2004;
Takahashi et al., 2004b.
d
Compilation of mRNA and protein expression data from: Baigent and Lowry, 2000; Chatzaki et al., 2004a,b; Cortright et al., 1995; Heldwein et al.,
1997; Kishimoto et al., 1995; Lovenberg et al., 1995; Potter et al., 1991; Seres et al., 2004; Slominski et al., 2004, 2000.
e
Compilation of mRNA and protein expression data from: Aguilera et al., 1987; Asakura et al., 1997; Baigent and Lowry, 2000; Chatzaki et al., 2004a,b;
Emanuel et al., 2000; Guzman et al., 2003; Muramatsu et al., 2000; Potter et al., 1994; Reubi et al., 2003; Seres et al., 2004; Slominski et al., 2004, 2000.
f
Includes both CRF1 and CRF2 receptors.
g
Detected in skin by Boorse et al. (2005a) but not in the current study.
14
G.C. Boorse, R.J. Denver / General and Comparative Endocrinology 146 (2006) 9–18
CRF peptides acting in a hemocrine fashion. CRF receptors have been shown to be expressed in mammalian spleen
and skeletal muscle (summarized in Table 2).
We found widespread expression of the CRF-BP mRNA
in frog tissues that included the heart, kidney, GI tract,
lungs and ovary. This is a much broader expression pattern
than that observed in mammals, and it suggests that perhaps in the frog, the CRF-BP plays a role in modulating the
Fig. 3. Detection of CRF and CRF-BP proteins in frog tissue extracts by
radioimmunoassay and crosslinking assay, respectively. (A) To determine
if mRNA expression correlates with protein we used a homologous radioimmunoassay for X. laevis CRF (Boorse and Denver, 2004b) to analyze
CRF-immunoreactivity (CRF-ir) in frog tissue extracts. The graph shows
the relative displacement of [125I]-Xenopus laevis CRF (xCRF) by increasing concentrations of radioinert CRF (closed squares) or dilutions of tissue extracts (closed circles D skin extract; open circles D heart extract;
open diamonds D liver extract). Each tissue was acetic-acid extracted then
analyzed for CRF peptide content by speciWc radioimmunoassay as
described (Boorse and Denver, 2004b). Serial dilutions of extracts from
the skin or heart produced displacement curves that were parallel to the
frog CRF (xCRF) standard. The CRF peptide content of skin was
0.303 § 0.004 pg CRF/mg tissue and heart 0.508 § 0.126 pg CRF/mg tissue
(mean § SEM; n D 3); liver was nondetectable. These values are considerably lower than peptide content in the brain (121.63 § 13.44 pg CRF/mg
tissue; mean § SEM; n D 5) but well within the limits of detection of our
assay (Boorse and Denver, 2004b). We were unable to detect CRF-ir in
other tissues that expressed CRF mRNA (data not shown). (B) We used a
chemical crosslinking assay with [125I]-CRF (Valverde et al., 2001) to
detect CRF-BP in tissue extracts (brain, heart, lung, kidney, intestine,
stomach, and liver was analyzed.) We detected the CRF-BP in brain and
intestine but not other tissues (data not shown). For brain extracts we
used 30 g while for intestine we used 75 g of total protein. SpeciWc binding of crosslinked proteins was shown by the addition of excess (1 M)
radioinert xCRF prior to crosslinking and fractionation by 10% SDS–
PAGE. Data shown are representative of results obtained with tissues isolated from three female frogs.
actions of CRF peptides throughout the body. We were
able to verify that the expression of CRF-BP mRNA corresponds to functional protein in brain and intestine (Fig. 3B)
but not other tissues, perhaps owing to a lower level of
expression in these tissues.
6. Roles for CRF peptides in peripheral tissues of
amphibians: CRF is cytoprotective in X. laevis tadpole tail
The widespread tissue distribution of CRF peptides and
receptors in mammals and the frog suggests the potential
for diverse actions in tissue maintenance and function.
Some of these roles have begun to be studied in mammals
(discussed above) but such functions in non-mammalian
species have not been investigated. We recently discovered
a function for CRF peptides in the X. laevis tadpole tail.
The frog CRF-BP was one of 17 genes found to be strongly
induced by thyroid hormone in a gene expression screen of
premetamorphic X. laevis tadpole tail (Brown et al., 1996).
The CRF-BP is upregulated during spontaneous metamorphosis, but the functional signiWcance of this gene regulation for tadpole tail life history was unknown. We
hypothesized that CRF peptides and receptors are
expressed in tail, and that the CRF-BP would play a modulatory role. Using a series of in vitro and in vivo approaches
we found that CRF functions as a cytoprotective factor in
tadpole tail, and that the upregulation of the CRF-BP at
metamorphic climax serves to neutralize CRF bioactivity
(Boorse et al., 2005b).
Findings in mammalian cells showing cytoprotective
and proliferative eVects of CRF and related peptides (Brar
et al., 2000, 1999; Fox et al., 1993; Radulovic et al., 2003)
and the induction of proliferation in diVerent cell types
(Emanuel et al., 2000; Ikeda et al., 2002) led us to hypothesize that CRF plays a similar role in the tadpole tail. We
also predicted that these actions would be neutralized by
the CRF-BP, especially at metamorphic climax. We Wrst
showed that CRF and CRF receptors are expressed in the
tail (Boorse et al., 2005b). We then found that CRF slows
spontaneous resorption and decreases caspase 3/7 activity
in tail cultures, and increases [3H]-thymidine uptake in the
tail muscle derived cell line XLT-15 (Yaoita and Nakajima,
1997). The actions of CRF on tail or XLT-15 cells were
blocked by coincubation with the speciWc CRF1 receptor
antagonist antalarmin, or by CRF-BP. We also overexpressed CRF-BP using in vivo electroporation-mediated gene
transfer in tail muscle cells and found that this accelerated
cell death. Our Wndings show that the cytoprotective
actions of CRF peptides are evolutionarily conserved and
that CRF action can be neutralized by the CRF-BP.
Given that CRF can play a tissue maintenance role in
the tadpole tail, and since this gene is regulated by stress
(both mammalian and frog CRF genes possess promoter
elements that mediate responses to stress; M. Yao and
R.J. Denver, unpublished results), we reasoned that tail
CRF expression might be inXuenced by environmental
stressors acting directly on tail cells. In support of this
G.C. Boorse, R.J. Denver / General and Comparative Endocrinology 146 (2006) 9–18
hypothesis we found that exposure to environmental
insults such as hypoxia can act directly on the tail to
upregulate CRF and urocortin 1, but to strongly downregulate CRF-BP mRNA expression. Since the tadpole
tail is an essential locomotory organ without which the
animal would be unable to forage or escape predation, we
hypothesize that this stressor-induced gene regulation is
important for maintaining tissue viability in the face of
environmental insults.
7. Conclusions
The signaling components of the CRF system are
widely expressed in peripheral tissues of mammals and
frogs, suggesting that the expression and function of these
peptides outside of the CNS/pituitary arose early in vertebrate evolution. These peptides likely inXuence most if not
all physiological systems, including the nervous, endocrine, vascular, cardiovascular, skeletomuscular and
reproductive systems. However, relatively few studies
have investigated roles for these peptides outside of the
CNS in mammals, and similar (or diVerent) roles have yet
to be studied in nonmammalian species. To our knowledge the only example of a role for a CRF peptide
expressed outside of the CNS in a nonmammalian vertebrate is our Wnding that CRF is cytoprotective in tadpole
tail. Similar cytoprotective actions of CRF in mammalian
cells suggest that this role is evolutionarily conserved.
Acknowledgments
R.J.D. was supported by NSF grant IBN9974672 and
IBN0235401. G.C.B. was supported by a NSF predoctoral
fellowship.
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