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Expression and Regulation of Corticotropin-Releasing
Factor Receptor Type 2

in Developing and Mature
Mouse Skeletal Muscle
Yael Kuperman, Orna Issler, Joan Vaughan, Louise Bilezikjian, Wylie Vale,
and Alon Chen
Department of Neurobiology (Y.K., O.I., A.C.), Weizmann Institute of Science, Rehovot, 76100, Israel;
and Clayton Foundation Laboratories for Peptide Biology (J.V., L.B., W.V.), Salk Institute for Biological
Studies, La Jolla, California
Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM)
tissue where it is suggested to inhibit interactions between insulin signaling pathway components
affecting whole-body glucose homeostasis. However, little is known about factors regulating SM
CRFR2 expression. Here, we demonstrate the exclusive expression of CRFR2, and not CRFR1, in
mature SM tissue using RT-PCR and ribonuclease protection assays and report a differential
expression of CRF receptors during C2C12 myogenic differentiation. Whereas C2C12 myoblasts
exclusively express CRFR1, the C2C12 myotubes solely express CRFR2. Using cAMP luciferase assays
and calcium mobilization measurements, we further demonstrate the functionality of these dif-
ferentially expressed receptors. Using luciferase reporter assays we show a differential activation
of CRFR promoters during myogenic differentiation. Transfections with different fragments of the
5⬘-flanking region of the mCRFR2

gene fused to a luciferase reporter gene show a promoter-
dependent expression of the reporter gene and reveal the importance of the myocyte enhancer
factor 2 consensus sequence located at the 3⬘-proximal region of CRFR2

promoter. Furthermore,
we demonstrate that CRFR2 gene transcription in the mature mouse is stimulated by both high-fat
diet and chronic variable stress conditions. Performing a whole-genome expression microarray
analysis of SM tissues obtained from CRFR2-null mice or wild-type littermates revealed a robust
reduction in retinol-binding protein 4 expression levels, an adipokine whose serum levels are
elevated in insulin-resistant states. In correlation with the SM CRFR2

levels, the SM retinol-
binding protein 4 levels were also elevated in mice subjected to high-fat diet and chronic variable
stress conditions. The current findings further position the SM CRFR2 pathways as a relevant
physiological system that may affect the known reciprocal relationship between psychological
and physiological challenges and the metabolic syndrome. (Molecular Endocrinology 25: 157–169,
2011)
Abdominal obesity and insulin resistance have each been
proposed as the primary factors underlying metabolic
syndrome (1, 2). Skeletal muscle (SM) comprises the largest
insulin-sensitive tissue in humans, and thus, insulin resis-
tance in this organ impacts whole-body glucose homeostasis
(3). Insulin resistance in SM was proposed to promote
atherogenic dyslipidemia by decreasing muscle glycogen
synthesis and elevating hepatic de novo lipid synthesis and
very-low-density lipoprotein production (2).
The corticotropin-releasing factor (CRF)/urocortin
(Ucn) family of peptides and receptors is involved in the
maintenance and adaptive responses necessary for energy
homeostasis (4–11). The CRF/Ucn family of neuropep-
tides signals through the activation of two G protein-
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/me.2010-0308 Received August 2, 2010. Accepted October 14, 2010.
First Published Online November 17, 2010
Abbreviations: Ant, Antalarmin; Ast 2b, Astressin 2B; CHO, Chinese hamster ovary; CRE,
cAMP-responsive element; CRF, corticotropin-releasing factor; CRFR2, corticotropin-re-
leasing factor receptor type 2; CVS, chronic variable stress; DM, differentiation medium;
HFD, high-fat diet; HPRT1, hypoxanthine guanine phosphoribosyl transferase 1; iv, inser-
tion variant; KO, knockout; MEF, myocyte enhancer factor; RBP4, retinol-binding protein
4; RNase, ribonuclease; SM, skeletal muscle; Ucn, urocortin; WT, wild type.
ORIGINAL RESEARCH
Mol Endocrinol, January 2011, 25(1):157–169 mend.endojournals.org 157
coupled receptors, CRF receptor type 1 (CRFR1) (12–14)
and CRF receptor type 2, CRFR2 (15–18). Mouse
CRFR2 has three apparent splice variants, which results
in two putative receptor proteins of 411 and 431 amino
acids (CRFR2
␣
and CRFR2

, respectively) and in a 422-
amino acid insertion-variant (iv) with dominant-negative
activity. In rodents, CRFR2
␣
is predominantly expressed
in the brain (19). The CRFR2

splice variant is expressed
primarily in the SM, the heart, the brain choroid plexus, the
gastrointestinal tract, and the skin (17, 20, 21) whereas
ivCRFR2

is exclusively expressed in the heart (22).
In SM tissue, CRFR2

was suggested to be involved in
different cellular processes. SM CRFR2

activation was
suggested to impede glucose metabolism. CRFR2-null
mice have enhanced glucose tolerance, increased insulin
sensitivity and are protected from high-fat diet-induced
insulin resistance (6). Ucn2, which is highly expressed in
SM tissue (23) and most likely serves as the endogenous
ligand for SM CRFR2

, inhibits the interactions between
insulin-signaling pathway components and insulin-in-
duced glucose uptake in cultured SM cells, and in C2C12
myotubes (8). The Ucn2-null mice exhibit increased insu-
lin sensitivity and are protected from fat-induced insulin
resistance (8). In addition, CRFR2

activation was dem-
onstrated to increase SM mass (24), reduce SM mass loss
in atrophying SM due to denervation or casting, and to
increase nonatrophying SM mass (25).
Given the importance of CRFR2 in regulating the cen-
tral stress response and its beneficial effect on cardiovas-
cular function (26), the regulation of its hypothalamic
and heart expression has been extensively studied (Refs.
27–31 and Refs. 22 and 32–35, respectively). However,
little is known regarding factors regulating SM CRFR2

expression. Here, we demonstrate the differential expres-
sion of CRFR1 and CRFR2 mRNA during C2C12 myo-
genic differentiation. The functional signaling of those
receptors was determined, and promoter analysis studies
demonstrated the importance of muscle-specific tran-
scription factors putative binding sites. Additionally, we
show the in vivo regulation of SM CRFR2

mRNA by
chronic physiological or psychological stressors and its
association with insulin-resistant states.
Results
Differential expression of CRFR1 and CRFR2 during
myogenic differentiation
To verify expression of SM CRFRs, total RNA pre-
pared from SM and brain tissues was reverse transcribed
to generate cDNAs. The cDNA products were used as
templates for specific semiquantitative RT-PCR demon-
strating selective CRFR2 expression in SM tissue whereas
the brain cDNA served as a positive control for CRFR1
and CRFR2 expression (Fig. 1A). The selective expression
of CRFR2, and not CRFR1, in SM tissue was further
verified using ribonuclease (RNase) protection assay (Fig.
1B). The multinucleated SM fibers are formed in succes-
sive distinct steps involving different types of myoblasts
(36). For in vitro investigation of the molecular basis of
SM cell differentiation, C2C12 cells, mouse-derived myo-
blasts that can be propagated as undifferentiated mono-
nuclear cells in serum, serve as a useful experimental
model. On serum withdrawal, muscle-specific genes are
expressed leading to the formation of differentiated
multinucleated myotubes (37, 38). To study the CRFR2
expression profile during myogenic differentiation, RNA
extracted from C2C12 myoblasts at different time points
during myogenic differentiation was reverse transcribed
and used as a template for semiquantitative RT-PCR. The
myogenic determination factors MyoD and myogenin, as
well as the negative regulator of myogenesis, Id2 (39),
were used to monitor the differentiation process. Unex-
pectedly, C2C12 myoblasts were found to exclusively ex-
press CRFR1 whereas C2C12 myotubes were found to
exclusively express CRFR2 (Fig. 1C). The time-depen-
dent differential expression of the two-receptor forms can
be observed during the differentiation process (Fig. 1, C and
D). CRFR2 shows expression kinetics similar to MyoD and
myogenin expression profiles whereas CRFR1 expression
mirrors the expression profile Id2 (Fig. 1, C and D).
C2C12 cells were further used for demonstrating
CRFR1- and CRFR2-selective activation in nondifferenti-
ated (myoblasts) or differentiated (myotubes) state. Recep-
tor functionality was demonstrated by measuring the acti-
vation of the cAMP and calcium pathways using CRF or
Ucn2/3, which are specific ligands for CRFR1 and CRFR2,
respectively (40–42). Nondifferentiated C2C12 cells were
transfected with a luciferase reporter containing a fragment
of the EVX1 gene that contains a cAMP-responsive element
(CRE) site. Luciferase activity was used as a measure of
receptor activation and was determined after4hoftreat-
ment with vehicle, and various doses of CRF, or Ucn3 in
nondifferentiated, or 48-h differentiated, C2C12 cells. The
CRE-luciferase reporter gene was differentially activated in
the myoblasts and myotubes after stimulation with CRF or
Ucn3, respectively (Fig. 2A). In myoblasts, CRF signaling
induced CRE-luciferase activity, which was blocked by the
CRFR1-specific antagonist Antalarmin (Ant), whereas in
the myotubes, Ucn3 signaling induced CRE-luciferase activ-
ity that was blocked by the CRFR2-specific antagonist As-
tressin 2B (Ast 2B).
Additionally, calcium mobilization kinetics were mea-
sured in nondifferentiated (myoblasts) or differentiated
(myotubes) C2C12 cells by CRF or Ucn2 (Fig. 2B). Acti-
158 Kuperman et al. Expression and Regulation of Muscle CRFR2

Mol Endocrinol, January 2011, 25(1):157–169
vation of calcium flux was differentially activated in the
myoblasts and myotubes after stimulation with CRF.
CRF strongly activated calcium flux in myoblasts, but not
in myotubes, (Fig. 2B, a and b). Ucn2 activated calcium
flux to a lesser extent in myoblasts, probably due to its
low affinity for CRFR1, but did not activate calcium flux
in myotubes (Fig. 2B, c and d). To further explore this
phenomenon, the calcium mobilization kinetic studies
were duplicated in Chinese hamster ovary (CHO) cells
stably expressing CRFR1 or CRFR2 treated with CRF or
Ucn2, respectively (Fig. 2C). Only CRF activation of
CHO cells expressing CRFR1, but not Ucn2 activation of
CHO cells expressing CRFR2, promoted calcium mobi-
lization. Demonstrating that CRFR1, but not CRFR2,
activation will promote calcium mobilization supports
the finding of differential expression of the CRFRs during
myogenic differentiation.
Differential activation of mCRFR1 and mCRFR2

promoters during C2C12 myoblast differentiation
To explore the molecular mechanisms mediating the
differential regulation of CRFR1 and CRFR2 during
myogenic differentiation, and to examine whether the dif-
ferential expression is regulated at the promoter level, we
FIG. 1. Expression of mCRFR2 mRNA in mouse SM and differential expression of mCRFR1 and mCRFR2 mRNA during C2C12 cells myogenic
differentiation. A, Representative image of electrophoretic analysis of the semiquantitative RT-PCR products of mCRFR2 (upper panel), mCRFR1
(middle panel), and the ribosomal protein S16 (lower panel) in the mouse SM. Brain samples served as positive controls for both CRFR1 and CRFR2
gene expression. PCR without reverse transcriptase (RT) enzyme (⫺R.T) or without cDNA (⫺cDNA) served as negative controls. B, Representative
image of RNase protection assay of mCRFR1 (right panel) and mCRFR2 (left panel) mRNA. SM total RNA was hybridized with the mCRFR1 (right
panel), mCRFR2 (left panel), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (both panels) antisense probes. Brain and pituitary gland
served as positive controls for CRFR2 and CRFR1 gene expression, respectively. C, Representative image of electrophoretic analysis of the
semiquantitative RT-PCR products of mCRFR1, mCRFR2, muscle differentiation markers, MyoD, myogenin, and Id2 and the ribosomal protein S16
in C2C12 myoblasts cultured in differentiation media (DM). RNA extracted from C2C12 myoblasts cultured in DM (containing 2% horse serum) for
0–6 d were reverse transcribed to generate cDNA, which were used as templates to the PCR using specific primers for mCRFR1, mCRFR2, MyoD,
myogenin, Id2, and the ribosomal protein S16 that served as an internal control. RNA extracted from mouse SM and brain served as positive
controls. PCR without RT enzyme (⫺R.T) or without cDNA (⫺cDNA) served as negative controls. D, The bands were quantified, and the normalized
values (relative to the control S16 expression) are presented as fold increase. Three independent experiments were conducted and showed similar
kinetic of gene expression.
Mol Endocrinol, January 2011, 25(1):157–169 mend.endojournals.org 159
isolated the 5⬘-flanking region of both genes. Subcloning
mCRFR1 or mCRFR2

5⬘-flanking sequences upstream
to a luciferase gene allowed us to study their activity dur-
ing the myogenic differentiation (Fig. 3). C2C12 cells
were transfected with the reporter plasmid DNA, and the
luciferase activity over3dofmyogenic differentiation
was determined. Interestingly, myoblast differentiation
was accompanied by a significant and rapid inhibition of
the mCRFR1 promoter, and time-dependent and robust
activation of the mCRFR2

promoter (Fig. 3, A and B,
respectively). These sequential changes parallel the de-
crease and the increase in mRNA level of CRFR1 and
CRFR2

, respectively (Fig. 3), and confirm that mCRFR2

expression during differentiation is regulated at the tran-
scriptional level.
To further study the involvement of putative muscle-
specific transcription factors in the activation of the
CRFR2

5⬘-flanking region, the 5⬘-flanking region of
FIG. 2. CRFR1 and CRFR2 signaling pathways are differentially activated during the myogenic differentiation. A, Activation of CRE-luciferase
reporter by CRF and Ucn3 peptides in C2C12 myoblasts or myotubes, respectively. C2C12 myoblasts were transfected with CRE-luciferase, and
luciferase activity was measured after treatment (4 h) with vehicle or 1 pM, 0.1 nM,10nM, and 1
MCRF or Ucn3 in nondifferentiated C2C12 cells
(black bars) or 48 h differentiated C2C12 cells (white bars). Assays were normalized to cotransfected

-gal activity. The mean SEM of three
independent experiments is presented as relative activity. The activation of CRE-luciferase in myoblasts by CRF, or in myotubes by Ucn3, was
blocked by the CRFR1-specific antagonist (Ant) or CRFR2-specific antagonist Ast 2B, respectively [*, P⬍0.05 vs. vehicle treatment; **, P⬍0.05
vs. CRF (10 nM) or Ucn3 (10 nM) treatment]. B. Calcium mobilization in nondifferentiated (myoblasts) or differentiated (myotubes) C2C12 cells by
CRF and Ucn2. Nondifferentiated C2C12 cells (a and c) and differentiated C2C12 (b and d) were treated with CRF (a and b) or Ucn2 (c and d),
respectively, and the calcium mobilization kinetic was measured using FlexStation (Molecular Devices Corp.). Bar graphs represent the maximum
values. Interestingly, CRFR1 but not CRFR2 activation promotes calcium mobilization. Activation of calcium flux was differentially activated in the
myoblasts and myotubes after stimulation with CRF. * P⬍0.05 vs. control buffer. C, CHO cells stably expressing the CRFR1 (a) or CRFR2 (b) were
treated with CRF or Ucn2, respectively, and the calcium mobilization kinetic was measured using FlexStation. Bar graphs represent the maximum
values. *, P⬍0.05 vs. control buffer. ND, Not determined; Con., control.
160 Kuperman et al. Expression and Regulation of Muscle CRFR2

Mol Endocrinol, January 2011, 25(1):157–169
CRFR2

(⫺2495 to ⫹23) was analyzed for SM transcrip-
tion factor consensus sequences using the TESS program
(Transcription Element Search System) (Fig. 4A). Six
fragments of the 5⬘-flanking region, with different
lengths and numbers of putative muscle-specific tran-
scription factors consensus sequences, were subcloned
into a luciferase pGL3 basic vector and used for trans-
fecting C2C12 cells (Fig. 4, B and C). The luciferase
activity of each fragment during myogenic differentia-
tion was studied. No basal differences were detected
between the different fragments. When differentiation
medium (DM) was introduced, the promoter activity
increased in a time-dependant manner. The differences
between the promoter fragments could be detected as
early as 24 h in DM, where the full isolated 5⬘-flanking
region was strongly activated and its activity was sig-
nificantly higher compared with the truncated frag-
ments, regardless of their length (Fig. 4C). After 96 h in
DM, all truncated fragments were robustly activated.
However, their activity was significantly lower com-
pared with the full 5⬘-flanking region (Fig. 4C). Al-
though the activity level of the truncated fragments
varied, there was no significant difference between
them. Interestingly, even the shortest fragment, consist-
ing of 168 bp, was strongly activated, indicating the
importance of the proximal site in mediating CRFR2

transcription (Fig. 4C).
Given the strong potency of the short 5⬘-flanking frag-
ment (⫺146 to ⫹23), we examined the importance of the
putative myocyte enhancer factor (MEF)2 consensus se-
quence (located ⫺91 to ⫺82) for this activation. This MEF2
consensus sequence in the minimal 5⬘-flanking region
was mutated and subcloned into pGL3 basic vector.
The MEF2 consensus sequence is
YTWWAAATAR, where Y stands for
T or C, W stands for A or T, and R
stands for A or G (43). The mutation
included C to A and A to C substitutions
(CtatAaataa to AtatCaataa) (Fig. 5A).
The mutated sequence is not recognized
as MEF2 consensus sequence using the
TESS analysis. The WT or mutated frag-
ment were transfected into C2C12 cells,
and luciferase activity during myogenic
differentiation was measured (Fig. 5B).
The mutated fragment was not able to
induce transcription as demonstrated by
a constant low activation of the lucif-
erase gene throughout the differentia-
tion process, indicating the significance
of this MEF2 site for CRFR2

expres-
sion during myogenic differentiation.
Regulation of SM CRFR2

expression by stress and
its correlation to retinol-binding protein 4 (RBP4)
expression level
Given the high expression level of CRFR2

and its
suggested role in modulating insulin sensitivity and glu-
cose uptake, we further studied its regulation in the ma-
ture mouse after exposure to chronic stressors. High-fat
diet (HFD) and chronic-variable stress (CVS) paradigms
were chosen because they represent prolonged physiolog-
ical and psychological stressors, respectively. SM RNA
obtained from mice maintained for 15 wk on HFD or
from mice subjected to CVS protocol, and the respective
controls, was reverse transcribed, and CRFR2

expres-
sion levels were determined using real-time PCR. Interest-
ingly, both stressors triggered a significant elevation in
CRFR2

expression level. HFD induced a 2.3-fold in-
crease (Fig. 6A) whereas CVS induced a 2.0-fold increase
(Fig. 6B) in CRFR2

expression level. The expression of
Ucn2, the local ligand for SM CRFR2

, did not change
significantly under these conditions (data not shown).
CRFR2 signaling was previously demonstrated to in-
hibit insulin signaling in SM (8). To further understand
the molecular mechanisms mediating the effect of Ucn2/
CRFR2 signaling on insulin sensitivity in SM, we com-
pared the gene expression profile of SM obtained from
both CRFR2 knockout (KO) and WT littermates using
gene expression microarray. The microarray analysis
demonstrated a significant reduction of 43.5% in the ex-
pression level of RBP4. RBP4 is an adipokine whose se-
rum levels are increased in insulin-resistant subjects, and
its administration leads to impaired insulin signaling in
muscle (44). To confirm our microarray data, SM was
FIG. 3. Differential activation of mCRFR1 and mCRFR2

promoters during C2C12 myoblast
differentiation. Schematic demonstration of the mCRFR1 (A) and mCRFR2

(B) 5⬘-flanking
region construct fused to the luciferase gene in PGL3 basic vector. C2C12 cells were
transfected with the reporter plasmid DNA, and luciferase activity during the myogenic
differentiation was determined. The luciferase activity was corrected to

-gal values. (Results
are shown as mean ⫾SEM of six independent experiments).
Mol Endocrinol, January 2011, 25(1):157–169 mend.endojournals.org 161
collected from CRFR2-null mice and from their WT litter-
mates, and SM cDNA was used for measuring RBP4 expres-
sion level by real-time PCR. The real-time PCR results were
in agreement with the microarray findings and showed a
significant reduction of 52% in RBP4 expression level (Fig.
7A). Several genes that were found to be up- or down- reg-
ulated in the microarray analysis are listed in Supplemental
Table 1 (published on The Endocrine Society’s Journals
Online web site at http://mend.endojournals.org). These
changes were not further verified using additional quantita-
tive methods. Because CRFR2-KO mice are a developmen-
tal KO model and therefore may represent developmental
compensatory changes, we further examined RBP4 muscle
expression level in conditions that up-regulate CRFR2

ex-
pression, namely HFD and CVS, and found a positive cor-
relation between RBP4 and CRFR2

expression. SM RBP4
FIG. 4. Sequence-, fragmentation-, and differentiation-induced activation of CRFR2

5⬘-flanking region. A, Genomic sequence of mCRFR2

5⬘-
flanking region. 5⬘-Untranslated region is shown in italic letters. The six primers used for promoter fragmentation are underlined, and putative sites
for muscle-specific transcription factors, recognized using TESS (Transcription Element Search System), are indicated. B, Electrophoretic analysis
of pGL3-basic vectors containing truncated mCRFR2

5⬘-flanking region. C, Activity of CRFR2

fragmented promoter during myogenic
differentiation. C2C12 cells were transfected with the reporter plasmid preceded by the different 5⬘-flanking region fragments. Luciferase activity
during the myogenic differentiation in 2% horse serum containing DM was determined. The relative luciferase activity was corrected to

-gal
activity (results are shown as fold increase over the basal activity of each fragment, shown as mean ⫾SEM). AS, Antisense.*, P⬍0.05; **, P⬍
0.001 vs. the full fragment.
162 Kuperman et al. Expression and Regulation of Muscle CRFR2

Mol Endocrinol, January 2011, 25(1):157–169
expression level was significantly elevated in mice subjected
to both HFD and CVS manipulations (Fig. 7, B and C).
Discussion
SM tissue has been demonstrated to express high levels of
CRFR2 transcript (20), which was shown to be associated
with controlling glucose transport into the SM. In the
present study we demonstrated, using specific mCRFR2
RNase protection assays, RT-PCR, and DNA sequencing,
that adult SM tissue expresses CRFR2

, but not the
CRFR1, transcripts. Previous reports showed CRFR2

expression in SM to be localized in neural structures,
blood vessels, myotendinous junctions, and endomysial/
perimysial spaces, but not in myocytes (45).
Here, we showed that C2C12 myoblasts exclusively
express CRFR1, whereas the C2C12 myotubes exclu-
sively express CRFR2

. In the myoblast state, serum in-
duces the expression of Id proteins, transcription factors
that sequester E12 and E47 into complexes unable to bind
DNA (39). Upon serum removal, MyoD family proteins,
MyoD, Mrf4, and myogenin, are activated to promote the
expression of muscle-specific genes with Mef2 family of
transcription factors, which play an important role in this
context (46). This sequential expression pattern is also
demonstrated in the CRFR expression kinetics, where
CRFR1 expression mirrors the expression profile of Id2,
and CRFR2

expression kinetics parallels the MyoD and
myogenin expression profile. Additional examples of dif-
ferential expression of CRFRs were reported in other
types of muscle tissues. In human nonpregnant myome-
trium the CRFR1
␣
and CRFR1

-receptor subtypes were
found, whereas at term R2
␣
and C variant CRFR sub-
types were expressed as well (47). Moreover, we recently
reported differential regulation of mCRFR2

by stress in
heart myocardium. The mRNA levels of mCRFR2

were
down-regulated in hearts of mice that underwent CVS
whereas the mRNA levels of a new splice variant of
CRFR2

, iv-mCRFR2

, were up-regulated (22). Lipo-
polysaccharide was also shown to differentially regulate
CRFR2

expression in the heart and SM. Systemic injec-
tion of lipopolysaccharide up-regulated SM CRFR2
mRNA levels and markedly down-regulated its mRNA
heart levels (48).
The pharmacological properties of CRFR1 and
CRFR2 activation by different ligands of the CRF peptide
family are well established. CRF has relatively lower af-
finity for CRFR2 compared with its affinity for CRFR1,
Ucn1 has equal affinities for both receptors, and Ucn2
and Ucn3 appear to be selective for CRFR2 (40, 42, 49).
Both CRFRs belong to the B1 subfamily of seven-trans-
membrane-domain receptors that signal by coupling to G
proteins (50). CRFR1 and CRFR2 signaling primarily
stimulates the adenylyl cyclase/cAMP pathway via cou-
pling and activation of G
␣
s
proteins and protein kinase A
activation (13, 18, 51). In addition, CRFR1 is coupled to
activation of plasma membrane calcium channels and CRF
signaling and was shown to generate changes
in corticotrope cytosolic free calcium concen-
tration (52). The increase in Ca
2⫹
influx in-
volves voltage-gated channels, namely L- and
P-type channels (53). That CRFR1 coupling
to activation of plasma membrane calcium
channels depends on cell type (54) was dem-
onstrated in melanocytes (55). We assessed
the differential expression of CRFR signaling
in assays based on these similarities and dif-
ferences. CRF and Ucn3 activated CRE-
luciferase reporter gene in myoblasts or myo-
tubes, respectively. The use of selective
antagonists and the subsequent activation of
FIG. 6. Up-regulation of SM CRFR2

after exposure to chronic stressors. CRFR2

mRNA level determined by real-time PCR in SM obtained from mice kept on HFD
compared with control low-fat diet (A) or subjected to CVS (B). CRFR2

expression was
corrected by HPRT1 expression level and normalized to control levels (results are shown
as mean ⫾SEM). *, P⬍0.05 vs. control.
FIG. 5. Activity of the WT and mutated MEF2 site in the minimal
CRFR2

5⬘-flanking region during the myogenic differentiation. C2C12
cells were transfected with the WT or the mutated minimal (⫺146 to
⫹23) 5⬘-flanking region-luciferase vectors and luciferase activity during
myogenic differentiation in 2% HS containing media (DM) was
determined. The relative luciferase activity was corrected to

-gal
activity (results are shown as mean ⫾SEM). Mut, Mutated.
Mol Endocrinol, January 2011, 25(1):157–169 mend.endojournals.org 163
the cAMP pathway further emphasized the ligand specificity
and functionality of the CRFR subtypes. Furthermore, the
ability of CRF to induce calcium mobilization selectively in
myoblasts provides an additional level of support to the
absence of CRFR1 in differentiated myotubes.
Transient transfection of C2C12 myoblasts with con-
structs containing the 5⬘-flanking region of the mCRFR1
or mCRFR2

genes fused to a luciferase reporter showed
differential promoter activity during myogenic differenti-
ation. The CRFR1 promoter activity was negatively reg-
ulated, whereas CRFR2

promoter activity was posi-
tively regulated, during differentiation. This differential
regulation is due to the varying responsiveness of the pro-
moters to myogenic transcription factors. Computer-
aided sequence analysis revealed the presence of putative
muscle-specific transcription factor consensus sequences
in the mCRFR2

5⬘-flanking region. Different fragments
of the 5⬘-flanking region were cloned into a luciferase
vector to identify the crucial area needed for CRFR2

transcription. A robust activation of all the fragmented re-
gions was observed revealing the importance of the 3⬘-prox-
imal region. The importance of this region was verified by
mutating the MEF2 consensus sequence. The mutated frag-
ment was incapable of transcription, as demonstrated by
blunted luciferase activity. This short but powerful minimal
5⬘-flanking region may be further used as a minimal pro-
moter for muscle-specific expression of target genes.
Understanding the regulation of SM CRFR2

may
provide further insight into the physiological functions of
this receptor. Mice lacking either CRFR2 or Ucn2 dem-
onstrate enhanced glucose tolerance, increased insulin
sensitivity, and protection from high fat diet-induced in-
sulin resistance (6, 8). In Ucn2 KO mice, systemic Ucn2
administration before glucose tolerance test or insulin tol-
erance test impaired glucose clearance and reduced insu-
lin sensitivity, respectively (8), showing that this pheno-
type is mediated by peripheral CRFR2. Both obesity and
high stress, hallmarks of a modern lifestyle, are correlated
with insulin resistance (56, 57). We showed that both a
physiological stressor (chronic consumption of a HFD)
and a psychological stressor (CVS)
share the same consequence of ele-
vated SM CRFR2

expression level.
Consequently, the increased SM CRFR2

expression may contribute to the re-
duced insulin sensitivity, which char-
acterizes these conditions. Elucidation
of this phenomenon is essential for bet-
ter management of the metabolic con-
sequences that coincide with both HFD
and chronic psychological stress. Inter-
estingly, a similar CVS protocol medi-
ated a reduction in CRFR2

mRNA lev-
els in the hearts of mice (22); however, these tissue-specific
differences might be attributed to the up-regulation of the
dominant-negative iv-mCRFR2

isoform.
A positive correlation between leptin serum levels and
CRFR2
␣
mRNA levels in the ventromedial hypothala-
mus has been shown (30). Because leptin is produced in
proportion to fat stores (58), and full-length leptin recep-
tor is expressed by SM (59), it is intriguing to hypothesize
that the increased CRFR2

expression under HFD is reg-
ulated by leptin. However, the association with leptin
does not explain the increased CRFR2

expression in
mice subjected to CVS, because CVS was demonstrated to
reduce serum leptin levels (60). Both HFD consumption
and chronic stress lead to elevated glucocorticoids (60–
62), which may regulate CRFR2

expression under these
conditions. HFD and CVS may be considered representa-
tives of modern lifestyle characteristics, which include
high stress load and increased intake of high-fat foods.
The indication that mice lacking Ucn2 exhibited in-
creased insulin sensitivity and better glucose tolerance (8)
implies that Ucn2 is endogenously secreted under hypo-
glycemic and hyperglycemic states. Glucose serves as the
primary fuel molecule in the fight or flight response and is
crucial for the organism’s survival (4, 63). Therefore, the
inhibitory effect of SM CRFR2

on insulin signaling may
function to regulate the stress-induced elevation in blood
glucose levels and to allow availability of glucose to other
tissues. Whereas this function is beneficial under normal
conditions, it may be maladaptive under chronic stress
conditions, under which SM CRFR2

expression is ele-
vated and consequently insulin sensitivity is reduced.
Whole-genome microarray expression data comparing
the expression profile of SM obtained from CRFR2 KO
or WT littermates showed a robust reduction in RBP4
expression, which was further confirmed by real-time
PCR. RBP4 is mainly expressed in liver and adipose tissue
(64). RBP4 serum levels are increased in insulin-resistant
mice and in humans with type 2 diabetes (65), and weight
loss in morbidly obese patients reduces RBP4 serum level
FIG. 7. SM RBP4 and CRFR2

expression levels are positively correlated. RBP4 mRNA level
determined by real-time PCR in SM obtained from CRFR2 KO mice and their WT littermates
(A), mice kept on HFD (B) or mice subjected to CVS (C). RBP4 expression was corrected by
HPRT1 expression level and normalized to WT/control (results are shown as mean ⫾SEM).
*, P⬍0.05 vs. WT/control.
164 Kuperman et al. Expression and Regulation of Muscle CRFR2

Mol Endocrinol, January 2011, 25(1):157–169
(66). Adipose-specific glucose transporter 4 KO mice
demonstrated elevated serum RBP4 levels and secondary
insulin resistance in the muscle and the liver (65), a met-
abolic phenotype that mirrors the observed phenotype of
the CRFR2 and Ucn2 KO mice.
RBP4 and CRFR2 signaling disrupt components of SM
insulin signaling that play a role in the control of glucose
transporter 4 translocation. RBP4 reduces both phospho-
inositide3-kinase activity and insulin-stimulated tyrosine
phosphorylation of insulin receptor substrate-1 at ty-
rosine residue 612, a docking site for the p85 subunit of
phosphoinositide3-kinase (65), whereas Ucn2 signaling
inhibits insulin-induced Akt phosphorylation and reduces
ERK1/2 phosphorylation (8). It was demonstrated that
RBP expressed ectopically in mice muscle can elevate se-
rum RBP levels (65). Here, we demonstrate that HFD and
CVS conditions mediate an increase both in SM CRFR2

and RBP4 expression levels. This dual increased expression
may synergistically act in an autocrine fashion to inhibit
insulin signaling and magnify metabolic complications.
The current findings further position the SM-CRFR2
pathways as a relevant physiological system that may af-
fect the known reciprocal relationship between psycho-
logical and physiological challenges and the metabolic
syndrome. A better understanding of SM CRFR2

path-
way, its physiological roles, and its regulation may pro-
vide benefits in related pathological conditions, such as
obesity and type 2 diabetes.
Materials and Methods
Animals
Mice were housed and handled in a pathogen-free temperature-
controlled (22 C ⫾1) mouse facility on a 12-h light, 12-h dark cycle
(lights on from 1900 h–0700 h), with food and water given ad
libitum, according to institutional guidelines. Adult C57BL/6 male
mice were used in all experiments. CRFR2-null (129⫻C57Bl/6
mixed background) mice were used for microarray and real-time
PCR studies. All experimental protocols were approved by the
Institutional Animal Care and Use Committee of The Weizmann
Institute of Science.
Cell lines
C2C12 myoblasts were grown to 50% confluence in DMEM
(Invitrogen Life Technologies, Carlsbad, CA), containing 10%
FBS supplemented with 100
g/ml of penicillin/streptomycin
(Invitrogen Life Technologies) (normal growth medium). For
differentiation, C2C12 were grown to 90% confluency and
washed with serum-free medium, and their medium was replaced
with DMEM containing 2% horse serum. CHO cells were grown
in normal growth medium as previously described (22).
RNA and cDNA preparation
RNA was extracted from brain, pituitary, gastrocnemius
muscle, or C2C12 cells using Tri-Reagent RNA isolation re-
agent (Molecular Research Center, Cincinnati, OH) according
to the manufacturer’s recommendations. To avoid false-positive
results caused by DNA contamination, a deoxyribonuclease
treatment was performed for 30 min at 37 C using the RQ1 RNase-
free deoxyribonuclease (Promega Corp., Madison, WI). RNA
preparations were reverse transcribed to generate cDNA using
High Capacity cDNA Reverse Transcription Kit (Applied Biosys-
tems, Inc., Foster city, CA). The cDNA products were used as
templates for semiquantitative and quantitative PCR analysis.
Semiquantitative RT-PCR
Semiquantitative RT-PCR was used to amplify the levels of
endogenous mCRFR2 and mCRFR1 present in the mouse SM and
brain. The expression of mCRFR1, mCRFR2 as well as muscle
differentiation markers MyoD, myogenin and Id2 levels were stud-
ied during C2C12 differentiation. The cDNA products were used
as templates for semiquantitative RT-PCR analysis using specific
primers for mCRFR2, mCRFR1, MyoD, myogenin, and Id2 and
the ribosomal protein S16 (for sequences see Table 1).
TABLE 1. Sequence of PCR primers
Gene Primer sequence (5ⴕto 3ⴕ) GenBank accession no.
CRFR1 NM_007762
Sense GGT GTG CCT TTC CCC ATC ATT
Antisense CAA CAT GTA GGT GAT GCC CAG
CRFR2 NM_009953
Sense GGC AAG GAA GT GGT GAT TTG
Antisense GGC GTG GTG GTC CTG CCA GCG
MyoD1 NM_010866
Sense GAG CAA AGT GAA TGA GGC CTT
Antisense CAC TGT AGT AGG CGG TGT CGT
Myogenin NM_031189
Sense TCA GAA GAG GAT GCT CTC TGC
Antisense TCA GAA GAG GAT GCT CTC TGC
Id2 NM_010496
Sense ATG AAA GCC TTC AGT CCG GTG
Antisense TTA GCC ACA GAG TAC TTT GCT
S16 M11408
Sense TGC GGT GTG GAG CTC GTG CTT GT
Antisense GCT ACC AGG CCT TTG AGA TGG A
Mol Endocrinol, January 2011, 25(1):157–169 mend.endojournals.org 165
PCR without reverse transcriptase enzyme (⫺R.T) or with-
out cDNA (⫺cDNA) served as negative control. The expression
of ribosomal protein S16 served as internal control. The PCR
conditions were as follows: cDNA equivalent to 200 ng of total
RNA was amplified by PCR for 35 cycles at an annealing tem-
perature of 62 C. The final MgCl
2
concentration was 3 mM, and
each reaction contained 2.5 U of Taq DNA polymerase (BIO-
X-ACT DNA polymerase; Bioline UK Ltd., London, UK).
RNase protection assay
SM total RNA was hybridized with the mCRFR1, mCRFR2,
and glyceraldehyde-3-phosphate dehydrogenase antisense probes.
Brain and pituitary gland served as positive control for CRFR2 and
CRFR1 gene expression, respectively. RNase protection assay was
performed as previously described (23).
Transient transfections and luciferase assay
C2C12 were used for the CRE activation and for the pro-
moter studies. All transfections were carried out in 12-well
plates using Lipofectamine 2000 Transfection Reagent (Invitro-
gen Life Technologies) according to manufacturer’s instruc-
tions. For CRE-luciferase activation, C2C12 myoblasts were
plated to 90% confluency and transfected with 1.5
gofthe
luciferase reporter containing a fragment of the EVX1 gene,
which contains a potent CRE site (kindly provided by Marc
Montminy, The Salk Institute) and 50 ng

-gal expression plas-
mid. Cells were treated for 4 h with vehicle or 1 pM, 0.1 nM,10
nM, and 1
MCRF or Ucn3 in nondifferentiated C2C12 cells, or
48 h differentiated C2C12 cells with or without the presence of
CRFR1- and CRFR2-selective antagonists (Ant and Ast 2B, re-
spectively). For promoter studies, C2C12 myoblasts were plated
to 90% confluency and transfected with 1.5
g of the luciferase
reporter plasmid or empty pGL3 vector and 50 ng

-gal expres-
sion plasmid. After 24 h the medium was replaced with DM.
The promoter activity was monitored at the basal state and after
the indicated times (24–96 h) in DM. The cells were harvested,
and the luciferase reporter activity was assayed as previously
described (20). Transfections were performed at least three
times (in triplicate) for each construct or treatment tested. To
correct for variations in transfection efficiencies, luciferase ac-
tivities were normalized to

-gal activity. Results were corrected
by the activity of the promoterless pGL3 vector.
Calcium-mobilization assay
Calcium-mobilization kinetics in nondifferentiated or differ-
entiated C2C12 cells or in CHO cells stably transfected with
either mCRFR1 or mCRFR2 after treatment of CRF or Ucn2
(50 nM) were measured using FlexStation (Molecular Devices,
Sunnyvale, CA) as previously described (67).
Construction of luciferase reporter plasmids
The mCRFR1 and mCRFR2

5⬘-flanking region constructs
were cloned by PCR using mouse genomic DNA. The primers
used for the construct were designed to include artificial restric-
tion sites (KpnI and XhoI for mCRFR1; KpnI and NheI for
mCRFR2

). The primer sequences were as follows: for
mCRFR1 sense primer (⫺2685 to 2663): 5⬘-TTG GGT TAC
GTA TGC TGC TCC TT-3⬘and antisense primer (⫹196 to
⫹217): 5⬘-CCT CGG GCT CGC TCT GTC AGC-3⬘. For
mCRFR2

sense primer (⫺2495 to 2473): 5⬘-GGA AAT GCA
GGA AAG CCA AGA CA-3⬘and antisense primer (⫹4to⫹23):
5⬘-CTG CCC GAC CTA CCC ACC AA-3⬘. Fragmentation of
the mCRFR2

5⬘-flanking region was done using the above
mentioned antisense primer along with six sense primers located
at: (⫺1649 to ⫺1630), (⫺1028 to ⫺1009), (⫺955 to ⫺932),
(⫺747 to ⫺726), (⫺512 to ⫺492), (⫺146 to ⫺127). The prim-
ers sequences are indicated in Fig. 4; all primers contained an
artificial KpnI restriction site. For mutating the MEF2 recogni-
tion site located at ⫺91 to ⫺82, the proximal 5⬘-flanking area
was amplified using the above-mentioned antisense primer with
the following sense primer, which contains an endogenous PstI
restriction site: 5⬘-CTGCAGAAGTTGCTGCCCAGAGCCA-
GATATCAATAACCTGG-3⬘. The mutation introduces a
unique EcoRV restriction site (in italics), which was later used
for identifying mutated clones. The PCR products were ana-
lyzed by agarose gel electrophoresis and eluted from the gel.
After digestion by the appropriate restriction enzymes, the DNA
fragments were cloned into the luciferase reporter plasmid
pGL3 (Promega Corp.), and the sequences were verified using
automated direct DNA sequencing.
HFD
Mice were fed ad libitum a high-fat (60% of calories) (n ⫽
13) or low-fat (10% of calories) (n ⫽5) diet (D12492 and
D12450B, respectively; Research Diets, Inc., New Brunswick,
NJ) for 15 wk.
CVS
CVS mice (n ⫽5) were housed in a temperature-controlled
room (22 C ⫾1) and were subjected to the CVS protocol for a
period of 4 wk as previously described (22).
Real-Time PCR
SM cDNA products were used as templates for real-time PCR
analysis. Sense and antisense primers were selected to be located on
different exons to avoid false-positive results caused by DNA con-
tamination. The following specific primers were designed using
Primer Express software (Applied Biosystems, PerkinElmer, Foster
City, CA). For mCRFR2: 5⬘-TACCGAATCGCCCTCATTGT-3⬘
and 5⬘-CCACGCGATGTTTCTCAGAAT-3⬘corresponding to
nucleotides 479-498 and 640-620, respectively (GenBank acces-
sion no. AY445512); for mRBP4: 5⬘-GCTTCCGAGTCAAG-
GAGAACTTC-3⬘and 5⬘-TCCACAGAAAACTCAGC-
GATGA-3⬘corresponding to nucleotides 479-498 and 640-620,
respectively (GenBank accession no. NM_011255). For mouse
hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1),
which served as an internal control: 5⬘-GCAGTACAGC-
CCCAAAATGG-3⬘and 5⬘-GGTCCTTTTCACCAG-
CAAGCT-3⬘corresponding to nucleotides 389-411 and 509-488,
respectively (GenBank accession no. NM_013556). Real-time PCRs
were carried out on a 7500 Real-Time PCR system (Applied Bio-
systems, Inc.), using fluorescent SYBR Green technology (Abgene;
Epsom, Surrey, UK). Reaction protocols had the following format:
15 min at 95 C for enzyme activation, followed by 45 cycles of 15
sec at 94 C and 60 sec at 60 C. The specificity of the amplification
products was checked by melting curve analysis. All reactions con-
tained the same amount of cDNA, 10
l Master Mix, and 250 nM
primers to a final volume of 20
l.
Microarray preparation and data are described under Gene
Expression Omnibus accession number GSE25045. Briefly, to-
tal RNA was extracted from adult skeletal muscle obtained
from four CRFR2 KO mice and four WT littermates, using
Tri-Reagent RNA isolation reagent (Molecular Research Cen-
166 Kuperman et al. Expression and Regulation of Muscle CRFR2

Mol Endocrinol, January 2011, 25(1):157–169
ter, Cincinnati, OH) according to the manufacturer’s protocol.
The RNA was pooled such that each sample consisted of two
muscles from each genotype (a total of four samples). Total
RNA (100
g) was further cleaned using Qiagen RNA purifi-
cation kit (QIAGEN Inc., Valencia, CA), and RNA integrity was
verified using gel electrophoresis and 260/280 ratios. cRNA
synthesis and hybridization to Affymetrix Murine Genome-
U74Av2 array (Affymetrix, Santa Clara, CA) was performed by
the UCSD Biological Services Unit. Data was analyzed using
Affymetrix Microarray Analysis Suite 5.1.
Acknowledgments
We thank Mr. S. Ovadia for his devoted assistance with animal
care.
This research was supported in part by the Clayton Medical
Research Foundation, Inc. A.C. was supported by the following:
Roberto and Renata Ruhman, Brazil; Mark Besen and the Pratt
Foundation, Australia; the Israel Science Foundation; the Leg-
acy Heritage Biomedical Science Partnership D-Cure Fellow-
ship; Nella and Leon Benoziyo Center for Neurosciences; Nella
and Leon Benoziyo Center for Neurological Diseases; Carl and
Micaela Einhorn-Dominic Brain Research Institute; Irwin
Green Alzheimer’s Research Fund; Gerhard and Hannah Ba-
charach (Fort Lee, NJ) and is incumbent of the Philip Harris and
Gerald Ronson Career Development Chair. W.V. was sup-
ported by Award No. 5P01DK026741-30 from the National
Institute of Diabetes and Digestive and Kidney Diseases and is a
Clayton Medical Research Foundation, Inc. Senior Investigator
and the Helen McLoraine Professor of Molecular Neurobiology.
Address all correspondence and requests for reprints to:
Alon Chen, Ph.D., Department of Neurobiology, Weizmann
Institute of Science, Rehovot, Israel 76100. E-mail: alon.
chen@weizmann.ac.il.
Disclosure Summary: W.V. is a cofounder, consultant, equity
holder, and member of the Board of Directors of Neurocrine
Biosciences, Inc., and Acceleron Pharma, Inc. These companies
are developing products that are related to some of the topics
discussed. However, none if these products are as yet on the
market. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the Na-
tional Institute of Diabetes and Digestive and Kidney Diseases
or the National Institutes of Health. Y.K., O.I., J.V., L.B., and
A.C. have nothing to disclose.
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