help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1570
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, A.
Right arrow Articles by Vale, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, A.
Right arrow Articles by Vale, W.
Endocrinology Vol. 145, No. 5 2445-2457
Copyright © 2004 by The Endocrine Society

Urocortin II Gene Is Highly Expressed in Mouse Skin and Skeletal Muscle Tissues: Localization, Basal Expression in Corticotropin-Releasing Factor Receptor (CRFR) 1- and CRFR2-Null Mice, and Regulation by Glucocorticoids

Alon Chen, Amy Blount, Joan Vaughan, Bhawanjit Brar and Wylie Vale

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie Vale, Ph.D, Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptides encoded by the Urocortin (Ucn) II gene, also known as stresscopin-related peptide, were recently identified as new members of the corticotropin-releasing factor (CRF) family. Ucn II is a specific ligand for the type 2 CRF receptor (CRFR). We have demonstrated the peripheral distribution of mouse Ucn (mUcn) II transcripts by using specific mUcn II ribonuclease protection assays, RT-PCR, Southern hybridization, and DNA sequencing. Although Ucn II mRNA is widely expressed in a variety of peripheral tissues, we found it to be most highly expressed in the skin and skeletal muscle tissues. Using a specific RIA for mUcn II, we detected Ucn II-like immunoreactivity (ir) in acid extracts of mouse brain, muscle, and skin. Immunohistochemical studies revealed Ucn II-like ir in both skin epidermis and adnexal structures and in the skeletal muscle myocytes. Ucn II mRNA and ir were also observed in neonatal skeletal muscle cultures in which Ucn II was localized to the myotube. We found a significant increase in Ucn II mRNA levels in the skin, but not in skeletal muscle, of both CRFR1- and CRFR2-null mice compared with their wild-type littermates. We showed that administration of dexamethasone to mice resulted in a decrease of Ucn II mRNA levels in the back skin region 12 h after ip injections. Removal of the adrenal gland significantly increased the levels of Ucn II mRNA in the skin, and the levels were reduced back to normal levels after corticosterone replacement. Further examination of the distribution and regulation of CRFR2 and its specific ligand Ucn II in the skin and skeletal muscle tissues may reveal the manner by which the CRFR2 pathway is involved in the physiological responses to stress in these tissues and in other pathophysiologies of the skin and muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEPTIDES ENCODED BY the Urocortin (Ucn) II gene, also known as the stresscopin-related gene, were recently identified as members of the corticotropin-releasing factor (CRF) family (1, 2). CRF, originally isolated from hypothalamus (3), plays an important and well-established role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis and in other endocrine, autonomic, and behavioral responses to stress (4). In addition to CRF and Ucn II peptides, the mammalian CRF-peptide family contains Ucn (5) and Ucn III, also known as stresscopin (2, 6). Human Ucn II and mouse Ucn (mUcn) II predicted mature 38-amino acid peptides are 76% identical, are more related to Ucn III than to Ucn, and are associated with high biological activity (6, 7, 8). The effects of CRF-related peptides are mediated through activation of two known receptors, CRF receptor (CRFR) 1 (9), and CRFR2 (10, 11, 12, 13). Although mice deficient for CRFR1 display decreased anxiety-like behavior and have an impaired stress response due to low plasma ACTH and corticosterone concentrations (14, 15), the CRFR2-null mice exhibit increased anxiety-like behaviors and an accelerated HPA response to stress (16, 17, 18).

Receptor binding studies have demonstrated that human Ucn II and mUcn II bind to and activate with high affinity and selectivity the CRFR2 compared with CRFR1 (1, 6). In addition, Ucn II exhibits high potency to stimulate intracellular cAMP accumulation in cells stably transfected with CRFR2 but not with CRFR1 (6). The distribution of CRFR1 and CRFR2 in the central nervous system (19, 20, 21) and periphery (10, 11, 12, 13) are distinct and imply diverse physiological functions. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including the paraventricular and arcuate nuclei in the hypothalamus and locus ceruleus in the brainstem (1). PCR analysis using a panel of human tissue cDNAs showed that Ucn II transcript could be detected in most tissues analyzed, with higher expression levels in brain, heart, lung, muscle, stomach, adrenal, and peripheral blood cells (2). The CRF gene is widely expressed in mammalian peripheral tissues, at levels lower than in the hypothalamus. CRF gene expression has been detected in placenta, endometrium, uterus, ovary, testes, spleen, immune system, gastrointestinal tract, adrenal gland, thyroid, and human skin (7, 8, 22, 23). Expression of the Ucn gene has also been documented in peripheral tissues such as pituitary, placenta, uterus, testes, immune system, gastrointestinal tract, adrenal gland, pancreas, heart, and skin (24).

The skin is the largest body organ and functions to maintain internal homeostasis by serving as a barrier between the external environment and the internal milieu (25). The skin is continuously exposed to a variety of stressors represented by solar radiation and biological and chemical insults, which require localized and specific mechanisms for dealing with the immediacy of these interactions (25). Biochemical and molecular studies have demonstrated CRF, Ucn, CRFRs, and proopiomelanocortin (POMC) peptides in human and mouse skin (22, 23, 24, 25, 26, 27, 28, 29). CRF and Ucn have been demonstrated to play an important role in a variety of physiological processes in the skin that lead to the hypothesis of an equivalent HPA axis present in the skin that can be activated in response to stress (22, 23, 24, 25, 26, 27, 28, 29).

In contrast to the extensive literature regarding skin and the CRF system, the skeletal muscle, although expressing high levels of CRFR2 (11), is lacking any anatomical or functional data.

Glucocorticoid receptors are widely expressed in all skin compartments, including the epidermal and follicular keratinocytes, epithelial cells of eccrine and apocrine glands, sebocytes, melanocytes, immune cells of the epidermis and dermis, dermal fibroblasts, and smooth muscle (23). Glucocorticoids are potent drugs used in the treatment of inflammatory skin disease (23), and activation of glucocorticoid receptors regulates specific functions in the corresponding cells, for example, the inhibitory effect of glucocorticoids on hair growth (30).

In the present study, we examine the expression of Ucn II in mouse peripheral tissues. The Ucn II peptide expression and localization in the skin and skeletal muscle tissues is presented. The basal expression of Ucn II in skin and skeletal muscle of CRFR1- and CRFR2-null mice is determined, and skin and skeletal muscle Ucn II regulation in the whole animal after dexamethasone administration and adrenalectomy (ADX) is presented.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA preparation and quantitative RT-PCR
Total RNA was extracted from different mouse tissues, from the skin and skeletal muscle of CRFR1- and CRFR2-null mice and their wild-type littermates, and from primary cultures of neonatal skeletal muscle, using the TRI Reagent isolation reagent (Molecular Research Center, Inc., Cincinnati, OH) based on the acid guanidinium thiocyanate-phenol-chloroform extraction method, according to the manufacturer’s recommendations. To avoid false-positive results caused by DNA contamination, we performed a deoxyribonuclease (DNase) treatment for 30 min at 37 C using the RQ1 ribonuclease (RNase)-free DNase (catalog no. M6101, Promega Corp., Madison, WI). We used quantitative RT-PCR to amplify the levels of endogenous Ucn II that may be present in the different mouse tissues studied. The expression of the mRNA of ribosomal protein S16 (31) served as internal control. The PCR conditions were as follows: the cDNA equivalent of 200 ng of total RNA was amplified by PCR for 35 cycles at an annealing temperature of 60 C, the final MgCl2 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
Total RNA was extracted from different mouse tissues using the TRI Reagent RNA isolation reagent (Molecular Research Center, Cincinnati, OH). mUcn II and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Anbion Inc., Austin, TX) mRNA levels were measured simultaneously by RNase protection, using mouse GAPDH as an internal loading control. A 631-nucleotide Ucn II antisense riboprobe specific to the mUcn II mRNA was synthesized by using T3 RNA polymerase. A 376-nucleotide antisense riboprobe specific to mouse GAPDH mRNA was synthesized by using T3 RNA polymerase. All riboprobes were synthesized in the presence of [{alpha}-32P]uridine 5'-triphosphate (UTP) [3000 Ci/mmol (1 Ci = 37 GBq)] and either 20 µM UTP for Ucn II or 200 µM UTP for GAPDH, as described (32). The fragment sizes protected by Ucn II and GAPDH riboprobes are 592 and 316 nucleotides, respectively.

RNase protection analysis was carried out as described (32). RNA samples (40 µg of peripheral tissues or 20 µg of brainstem/cerebellum tissues) were hybridized in 24 µl of deionized formamide plus 6 µl of hybridization buffer containing 6 x 105 cpm of Ucn II and 4 x 104 cpm GAPDH antisense riboprobes. After heating to 85 C for 5 min, the samples were hybridized at 42 C for 15 h and subsequently digested by RNase (100 µg/ml RNase A and 0.5 U/ml RNase T1) at 24 C for 60 min. The samples were resolved on 4% polyacrylamide 7 M urea gels. Image analysis was performed by using the PhosphorImager system (Molecular Dynamics, Inc., Jersey City, NJ) and the ImageQuant TL 4.0 software package (Amersham Biosciences, Piscataway, NJ).

Southern analysis
The PCR products were transferred onto a nylon membrane (Hybond-N, Amersham Biosciences, Pitcataway, NJ) by overnight capillary blotting in 20x sodium chloride/sodium citrate buffer (SSC) solution, and the nylon was baked in a vacuum oven at 80 C for 2 h. Overnight hybridizations were performed sequentially on the same membrane, using Super Hyb Kit (Molecular Research Center) in the presence of a [32P]-labeled probe specific to the mUcn II or S16 cDNA. Hybridizations were performed at 60 C for mUcn II probe. The corresponding bands could be seen after exposure of the membranes to PhosphorImager plates (445 SI, Molecular Dynamics). Gels were also exposed to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) for 2–16 h at –80 C and were developed in a CURIX 60 processor (AGFA, Köln, Germany).

Oligonucleotide primers
Sense and antisense primers were selected, when possible, to be located on different exons to avoid false-positive results caused by DNA contamination. The following specific Ucn II and S-16 oligonucleotide primers were used in the PCRs: 1) Ucn II, 5'-GGCCGCCGCTGAGACTGAA-3' and 5'-GGCCTGTGGACCTTAGATGGACTT-3' corresponding to nucleotides 329–347 (sense) and 707–730 (antisense), respectively (GenBank accession no. AF33151). The predicted size of the band is 402 bp; and 2) S-16, 5'-TGCGGTGTGGAGCTCGTGCTTGT-3' and 5'-GCTACCAGGCCTTTGAGATGGA-3' corresponding to nucleotides 369–391 (sense) and 1968–1990 (antisense), respectively (GenBank accession no. M11408). The predicted size of the band is 309 bp.

DNA sequencing
The appropriate cDNA fragments of mUcn II obtained from Ucn II-expressing tissues were extracted from gels using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) and subcloned into pGEM-T vector by using the pGEM-T Easy Vector System I (Promega Corp.). Nucleotide sequencing of the specific PCR bands was performed by automated direct DNA sequencing according to the manufacturer’s recommendations (model 377, PE Applied Biosystems, PerkinElmer Corp., Foster City, CA).

RIA
Ucn II antisera was raised in a rabbit immunized with TyrGly-mUcn II conjugated to human {alpha}-globulins via bisdiazotized benzidine using a protocol previously described for inhibin subunits (33). The analog DTyrGly(Ala21)-mUcn II was radiolabeled with Na[125I] using the chloramine-T method and purified by HPLC (33) for use as tracer in the RIA. The procedure for Ucn II RIA was similar to that previously described in detail for inhibin subunits (33). Briefly, rabbit anti-mUcn II (PBL Rabbit no. 6555, 09/12/01 bleed) was used at 1/300,000 final dilution, and mUcn II was used as standard. Murine tissues were acid-extracted and partially purified using octadecyl silica cartridges as described (34). Lyophilized samples for testing were resuspended in assay buffer, the pH was checked and adjusted if necessary, and samples were tested at three- to seven-dose levels. Free tracer was separated from tracer bound to antibody with the addition of sheep antirabbit {gamma}-globulins and 10% (weight/volume) polyethylene glycol. The EC50 and minimum detectable dose for mUcn II are 12–15 pg and 1 pg per tube, respectively.

Immunohistochemical procedure
For the immunohistochemical studies, we used samples of thigh skeletal muscle and back skin taken from C57B6 mice. The tissues were fixed in 10% natural buffered formalin, embedded in paraffin, and cut into 4-µm-thick sections that were used for immunohistochemical studies using specific antibodies for Ucn II. The immunohistochemical procedure was performed using the VECTASTAIN Elite ABC system (catalog no. PK-6101, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s recommendations. To determine the specificity of the signals, we preabsorbed aliquots of the antibody with an excess (10–100 µg) of Ucn II for 24 h. Additional control sections were incubated without the first antibody or with an irrelevant antibody. The sections were also stained with hematoxylin and eosin to demonstrate the morphological structure of the tissues.

Fluorescence immunocytochemical analysis
Primary culture cells obtained from the skeletal muscle of 1- to 2-d-old mice were analyzed by fluorescence immunocytochemistry using Ucn II-specific antibody. The cells were plated on round glass coverslips (13 mm) coated with gelatin in 24-well culture plates. Two days later, the cells were fixed by the addition of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4, 30 min), washed (three times for 5 min each) with PB, and permeabilized for 3 min with 0.5% Triton X-100. After washing three times, the cells were incubated for 2 h at room temperature in a blocking medium (PBS containing 10% normal donkey serum, 2% BSA, 1% glycine, and 0.5% Triton X-100) to saturate nonspecific binding sites for IgG. The primary antibodies were added for 12–15 h at 4 C, and the cells were washed (5 min, three times) with 0.1 M PB. The cells were then incubated for 2 h at room temperature with Cy2-conjugated AffiniPure donkey antirabbit (green fluorescence, Jackson ImmunoResearch Laboratories, West Grove, PA). The cells were washed with PB and coverslipped with fluorescence mounting medium. To determine the specificity of the signals, we have included several control groups in which the antibodies were preabsorbed with excess (2–100 µg) Ucn II for 24 h. Additional control sections were incubated without the first antibody or with normal rabbit serum.

Skeletal myocytes culture
Skeletal myocytes were isolated from the limbs of neonatal C57BL/6 mice that were less than 2 d old. Myocytes were cultured as described previously (35) with the following modifications. After collagenase digestion, the cells were preplated in medium consisting of DMEM [1000 µg glucose/liter, 1 mmol/liter L-glutamine, 100 U/ml penicillin/streptomycin (all from Life Technologies, Inc., Rockville, MD)] supplemented with 15% (vol/vol) fetal bovine serum on 10-cm tissue culture dishes. Preplating of the cell suspension for 30 min allows contaminating fibroblasts to attach, and the myocytes remain free in the culture media. Subsequent to this incubation, the myocyte cell suspension was transferred onto 12-well (1 cm) gelatin-coated plates or to 8-well chamber slides. After 24 h in culture, the media was replaced with media containing reduced fetal bovine serum at 1% (vol/vol) for an additional 48 h before paraformaldehyde fixation.

RNA competitor construction
Homologous RNA competitive internal standards that shared the same primer binding sites (but contained a shortened internal sequence with respect to the endogenous target RNA for mUcn II) were prepared as follows (Fig. 1AGo). The sense primer was primer "A" (5'-GGCCGCCGCTGAGACTGAA-3'), which is a conventional PCR primer and corresponds to nucleotides 329–347 (GenBank accession no. AF33151) (Fig. 1AGo). The antisense primer was 48 nucleotides in length in which 24 nucleotides at the 3' end correspond to primer "B" and 24 nucleotides at the 5' end (123 nucleotides upstream from primer B) correspond to nucleotides 707–730 (primer "C"). Primers A and BC (5'-GGCCTGTGGACCTTAGATGGACTTAACATACATGCTAGAGTCCAAGAT-3'), corresponding to nucleotides 560–583 and 707–730, were introduced to the PCR, and the 279-bp product, which is 123 bp smaller than the product resulting from PCR with primers A and C, was subcloned into pGEM-T vector (Promega) and sequenced. The pGEM-T 279-bp mUcn II vector was linearized by NcoI restriction digestion and transcribed into cRNA template by SP6 RNA Polymerase (Riboprobe System-SP6, catalog no. M6101, Promega). The DNA template after transcription was removed, and the cRNA product was quantified and used as internal standard in RT-PCR for mUcn II gene expression.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 1. The design of mUcn II competitor internal control construct for competitive RT-PCR, and the levels of Ucn II mRNA expressed in the mouse skin and skeletal muscle as determined by competitive RT-PCR. A, Schematic demonstration of the mUcn II gene and the location of the oligonucleotide primers to generate endogenous and competitive fragments. The isolation of the mUcn II competitor internal control fragment was performed using primers A and BC for the PCR. The 279-bp fragment was isolated and subcloned, and a cRNA was transcribed as described in Material and Methods. B and C, After RT reactions containing 2-fold serial dilutions of mUcn II 279-bp internal standard (competitor mUcn II), in addition to the skin (B) or skeletal muscle (C) sample RNA, PCRs were performed using oligonucleotide primers A and C. The PCR products (402 bp for endogenous mUcn II and 279 bp for mUcn II internal standard) were separated on 2.2% agarose gel (B and C, gels), visualized with ethidium bromide, photographed, and quantified using ImageQuant software program 1.2. The ratio of internal standard to endogenous area was plotted as a function of the competitor concentration added to each PCR (B and C, graphs). The concentration of mUcn II mRNA at which the ratio of the internal standard mUcn II and endogenous mUcn II area was equal to 1 (i.e. the equivalence point; B and C, graphs) was determined.

 
Competitive RT-PCR
Ucn II mRNA levels in the mouse skin and skeletal muscle after ADX or dexamethasone administration were quantified by a competitive RT-PCR assay. After DNase treatment, a constant amount of RNA (100 ng) was added to a reverse transcription (RT) mixture (SuperScript II RNase H-Reverse Transcriptase, Invitrogen, Carlsbad, CA) containing 2-fold serial dilutions of mUcn II 279-bp internal standard (7.8–2000 fg). To test for possible pseudogene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. Upon completion of the RT, 25% of the RT mixture was added to separate PCR mixtures containing 100 pmol of specific mUcn II oligonucleotide primers A and C, 100 µM of deoxynucleotide triphosphate, 3 mM MgCl2, 1 x Taq buffer, and 2 U Taq DNA polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd., London, UK) in a total volume of 50 µl. The PCRs were amplified for 35 cycles using a programmable thermal controller (PTC-200, MJ Research Inc., Watertown, MA). Each cycle included denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and primer extension at 72 C for 1 min. PCR products (402 bp for endogenous mUcn II and 279 bp for mUcn II internal standard) (Fig. 1Go, B and C) were separated on 2.2% agarose gel, visualized with ethidium bromide, photographed, and quantified using ImageQuant software program (version 1.2, Molecular Dynamics). The ratio of internal standard to endogenous area was plotted as a function of the competitor concentration added to each PCR. The concentration of mUcn II mRNA at which the ratio of the internal standard mUcn II and endogenous mUcn II area was equal to 1, i.e. the equivalence point, was determined.

Animals
Adult C57BL/6 male mice (27–30 g) were used in all experiments. Animals were housed three per cage in a room with controlled temperature and a fixed lighting schedule (lights on from 0600 h to 1800 h). Food and water were given ad libitum. All experimental protocols were approved by the animal committee of the Salk Institute for Biological Studies.

ADX, corticosterone replacement, and dexamethasone administration
ADX was performed through a dorsal incision under isoflurane anesthesia. Sham operation was performed by manipulating the animal in the same manner but without removing the adrenal gland. One group of ADX mice received a replacement of corticosterone (catalog no. C-2502, Sigma Chemical, St. Louis, MO) in the drinking water at final concentration of 25 µg/ml immediately after ADX surgery. All mice were killed 2 wk after ADX surgery, and the skin and skeletal muscles were isolated and subjected to RNA isolation. Blood collected from control, ADX, and corticosterone replacement mice was measured for plasma corticosterone using the ImmuChem Double Antibody Corticosterone 125I RIA Kit (ICN Biomedicals, Inc., Costa Mesa, CA).

Dexamethasone (75 mg/kg, 150 mg/kg, 300 mg/kg) or saline was injected ip into mice. The mice were killed 2 or 12 h after injection, and the skin and skeletal muscles were isolated and used for RNA isolation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue distribution of Ucn II mRNA in mouse peripheral tissues
RNase protection assays were performed to determine the tissue distribution of mUcn II mRNA. Total RNA from peripheral tissues and from the brainstem and cerebellum regions of the central nervous system were hybridized with a 631-nucleotide antisense RNA probe that spanned the mature peptide region and gave a protected fragment of 592 nucleotides. Ucn II was found to be highly expressed in the skin and skeletal muscle (Fig. 2AGo). Peripheral tissues expressing lower levels of Ucn II mRNA include the lung, adrenal, testes, ovary, brown fat, thymus, and spleen (Fig. 2AGo). The expression of mUcn II mRNA in the gastrointestinal tract is restricted mainly to the stomach, with very low levels of expression in the duodenum, jejunum, ileum, and colon regions (Fig. 2AGo). RNA obtained from the brainstem and cerebellum regions served as a positive control (Fig. 2AGo).



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 2. Expression of Ucn II gene in mouse brain and peripheral tissues. A, Representative image of RNase protection assay of Ucn II mRNA. Total RNA isolated from each tissue was hybridized with the mUcn II and GAPDH antisense probes. The protected fragments were resolved on a 4% polyacrylamide 7 M urea gel. BS/Cer, Brainstem and cerebellum. B, Representative image of electrophoretic analysis of the quantitative RT-PCR products of mUcn II and the ribosomal protein S16 (upper panels) in the mouse tissues. Southern blot hybridization of amplified mUcn II cDNA fragment (lower panels) was also performed. To avoid false-positive results caused by DNA contamination, each PCR was performed both on cDNA and RNA obtained from the same sample. BS/Cer, Brainstem and cerebellum; Hypot, hypothalamus; OB, olfactory bulb; Sk. Mus, skeletal muscle; B. fat, brown fat; W. fat, white fat; mUcn II Hyb, hybridization. C, The radioactive bands were quantified by PhosphorImager, and the normalized values (relative to the control S16 expression) are presented as relative densitometry units.

 
To reveal tissues expressing lower amounts of Ucn II mRNA and to confirm the pattern of expression detected by RNase protection assay, we used RT-PCR followed by Southern hybridization reactions specific for Ucn II (Fig. 2Go, B and C). Total RNA preparations derived from tissue encompassing several regions of the central nervous system (brainstem/cerebellum, hypothalamus, and olfactory bulb) and peripheral tissues were reverse-transcribed to generate cDNA pools. The cDNA products were used as templates for quantitative RT-PCR analysis, followed by Southern hybridization, using specific primers and probes for mUcn II and for the ribosomal protein S16, which served as internal control. To avoid false-positive results caused by DNA contamination, each PCR was performed both on cDNA and RNA obtained from the same sample. Predicted mUcn II cDNA fragments from skin and skeletal muscle were isolated from agarose gels, subcloned into pGEM-T vector, and the nucleotide sequences were determined. The sequences were compared with those in the GenBank database and were found to be identical to the known sequence of mUcn II. The RT-PCR and Southern hybridization demonstrate that the tissue distribution pattern of mUcn II expression is similar to that observed by RNase protection assay (Fig. 2AGo). Due to increased sensitivity of RT-PCR, strong levels of expression were demonstrated (in addition to the brain, skin, and skeletal muscle) in the adrenal, lung, stomach, ovary, spleen, thymus, and brown fat (Fig. 2Go, B and C). Low levels could also been detected in the heart, kidney, and white fat (Fig. 2Go, B and C). The S16 ribosomal protein (Fig. 2BGo, lower panel) served as internal control and was expressed equally in all cDNA preparations.

Ucn II immunoreactivity (ir) in murine skin and skeletal muscle
Using antiserum that we raised in rabbit against mouse TyrGly-Ucn II, we developed an RIA that displays the requisite sensitivity for the measurement of Ucn II-like ir in biological fluids and tissues. The mUcn II RIA has a detection limit of 0.7 to 1 pg/tube and ED50 values in the range of 12–15 pg/tube. As shown in Fig. 3AGo, the RIA is highly specific for mUcn II and, additionally, selective for the murine homolog of this peptide because human Ucn II displays dramatically lower ability to displace the tracer in this RIA. Human Ucn II did not show a competition curve parallel to that of mUcn II in the RIA, nor was full displacement of tracer achieved (Fig. 3AGo). Closely related peptide family members including rat CRF, Ucn, and Ucn III fail to displace the tracer with cross-reactivities less than 0.01% (Fig. 3AGo).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3. Presence of Ucn II-like ir peptide in mouse skin and skeletal muscle tissue extracts. A, Displacement of [125I]D-Tyr-Gly(Ala21)-mUcn II binding to rabbit anti-mUcn II by mUcn II. Human Ucn II shows dramatically lower ability to displace the tracer in RIA, indicating selectivity for the murine homolog. Closely related family peptides including rat Ucn, rat CRF, human Ucn III, or mUcn III showed little or no cross-reactivity (0.01%). B/Bo, Bound to maximum bound ratio; rCRF, rat CRF; rUcn, rat Ucn; hUcn, human Ucn. B, Ucn II-like ir in tissue extracts obtained from skin, skeletal muscle, and brain. Ucn II-like ir was measured in acid-extracted and partially purified tissue isolated from mouse brain, muscle, and skin. Tissue extracts displaced [125I]-labeled D-TyrGly(Ala21)-mUcn II binding to rabbit anti-mUcn II in a dose-dependent manner.

 
Ucn II-like ir was measured in acid-extracted and partially purified tissue isolated from total mouse brain, muscle, and skin (Fig. 3BGo). All tissue extracts tested displaced [125I]-radiolabeled D-TyrGly(Ala21)-mUcn II binding to rabbit anti-mUcn II in a dose-dependent manner (Fig. 3BGo).

Immunohistochemical studies were carried out on paraffin sections obtained from mouse dorsal skin and thigh skeletal muscle tissues using affinity purified antibodies specific for mUcn II (Fig. 4Go, C–F, and Fig. 5Go, C and D) and revealed strong Ucn II ir staining in both tissues. Parallel sections were stained with hematoxylin and eosin (Fig. 4Go, A and B, and Fig. 5AGo, a and b), and sections with Ucn II ir were counterstained with methyl green to demonstrate the morphology of the tissue. The mUcn II ir was observed in both the epidermis and dermis regions of the skin, including blood vessel walls and adnexal structures: hair follicle, sweat, and sebaceous glands (Fig. 4Go, C–F). In the skeletal muscle sections, ir of different intensity could be observed in skeletal muscle myocytes (Fig. 5CGo, a and b). Skin and skeletal muscle sections that were reacted with either buffer alone or normal rabbit sera followed by secondary antibodies did not result in any staining. In addition, preabsorption of mUcn II antibody with excess of synthetic mUcn II abolished the immunoreactive staining (Fig. 4Go, G and H, and Fig. 5AGo, e and f). Further support for the expression and translation of Ucn II mRNA in skeletal muscle comes from RT-PCR and fluorescence immunocytochemical analysis for Ucn II in cultures of neonatal mouse skeletal myocytes (Fig. 5Go, B and C). Total RNA preparations derived from skeletal muscle cultures, from mouse skeletal muscle, and from brain tissues that served as positive control were reverse-transcribed to generate cDNA. The cDNA products were used as templates for the PCR by using specific primers for mUcn II and the ribosomal protein S16 that served as an internal control. The RT-PCR results demonstrate that Ucn II is expressed in skeletal muscle cultures in addition to its expression in the skeletal muscle and brain tissues (Fig. 5BGo, upper panel). The ribosomal protein S16 that served as internal control was expressed in all cDNA preparations (Fig. 5BGo, lower panel). Figure 5CGo, a and b, demonstrates Ucn II-like ir in skeletal myocytes. The Ucn II antibody was reacted with goat antirabbit Cy2 antibody (green fluorescence, Fig. 5CGo, a and b). Culture cells that were reacted with either buffer alone or normal rabbit sera followed by secondary antibodies did not result in any staining (Fig. 5CGo, panel c). In addition, preabsorption of the mUcn II antibody with an excess of synthetic mUcn II abolished the immunoreactive staining (Fig. 5CGo, panel d).



View larger version (93K):
[in this window]
[in a new window]
  		FIG. 4. Expression of Ucn II in mouse dorsal skin. Paraffin-embedded sections were obtained from mouse dorsal skin stained with hematoxylin/eosin (A and B) or incubated with Ucn II-specific antibodies (C–F) revealed Ucn II-like ir in the epidermis, blood vessel walls, and adnexal structures (hair follicle, sweat, and sebaceous glands). Control sections incubated with normal rabbit serum (G) or Ucn II antibodies preabsorbed with Ucn II peptide (H) did not result in any staining. Selected ir signals are indicated by black arrows. Epidermis borders are indicated by gray arrows. Dermis borders are indicated by gray lines. Epi, Epidermis; SwG, sweat glands; SebG, sebaceous glands; BV, blood vessel; Hf, hair follicle. Scale bar, 200 µM for A, C, E, G, and H; 100 µM for B, D, and F.

 


View larger version (39K):
[in this window]
[in a new window]
  		FIG. 5. Expression of Ucn II in mouse skeletal muscle tissue and cells in culture. A, Paraffin-embedded sections obtained from mouse skeletal muscle stained with hematoxylin/eosin (a and b) or incubated with Ucn II-specific antibodies (c and d) revealed Ucn II-like ir in the muscle myocyte. Control sections incubated with normal rabbit serum (e) or Ucn II antibodies preabsorbed with Ucn II peptide (f) did not result in any staining. Scale bars, 200 µM for a, c, e, and f; 100 µM for b and d. B, Representative image of electrophoretic analysis of the semiquantitative RT-PCR products of Ucn II (upper panel) and the ribosomal protein S16 (lower panel) in skeletal muscle tissue and cells in cultere. The brainstem served as positive control. Sk. muscle, Skeletal muscle; R.T., reverse transcription. C, Fluorescence immunostaining of the skeletal muscle culture cells reveals Ucn II-like ir in cultured myotubes indicated by green fluorescence. Scale bars, 100 µM for a, c, and d; 50 µM for b.

 
Basal expression of mUcn II gene in CRFR1- and CRFR2-null mice
To permit reliable quantitation of mUcn II mRNA by competitive PCR, it was necessary to generate a mUcn II cRNA bearing a short internal deletion for use as an internal standard to determine the levels of mUcn II expression in CRFR1- and CRFR2-null mice (Fig. 1Go). We measured the skin (Fig. 6Go) and skeletal muscle (Fig. 7Go) Ucn II mRNA levels in CRFR1- and CRFR2-null mice compared with levels in their wild-type littermates. A significant increase in Ucn II mRNA levels was observed in the skin of CRFR1-null (Fig. 6Go, A, B, and E) and CRFR2-null (Fig. 6Go, C, D, and F) mice compared with their wild-type littermates (Fig. 6Go). We did not find any significant differences in Ucn II mRNA levels in the skeletal muscle tissues of CRFR1-null (Fig. 7Go, A, B, and E) or CRFR2-null (Fig. 7Go, C, D, and F) mice compared with their wild-type littermates (Fig. 7Go).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 6. Competitive RT-PCR assay for skin mUcn II mRNA in CRFR1- and CRFR2-null mice compared with their wild-type littermates. Total RNA was extracted from back skin of CRFR1-null (B) and CRFR2-null (D) mice and their wild-type littermates (A and C). After DNase treatment, the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group included at least four animals. The data shown are representative of four identical experiments. This figure demonstrates a significant increase in expression of skin mUcn II mRNA in CRFR1- and CRFR2-null mice compared with their wild-type littermates (E and F, respectively). *, P < 0.05 vs. wild-type mice.

 


View larger version (37K):
[in this window]
[in a new window]
  		FIG. 7. Competitive RT-PCR assay for skeletal muscle mUcn II mRNA in CRFR1- and CRFR2-null mice compared with their wild-type littermates. Total RNA was extracted from thigh skeletal muscle of CRFR1- (B) and CRFR2-null (D) mice and their wild-type littermates (A and C). After DNase treatment, the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group included at least four animals. The data shown are representative of four identical experiments. This figure demonstrates no significant differences in expression of skeletal muscle Ucn II mRNA in CRFR1- and CRFR2-null mice compared with their wild-type littermates (E and F, respectively).

 
Regulation of Ucn II expression in mouse skin by dexamethasone administration and ADX
To determine whether the increase of mUcn II mRNA in the skin of the CRFR1-null mice is glucocorticoid related, we examined the effect of dexamethasone injections on mUcn II mRNA levels in skin. Dexamethasone injections (150 and 300 µg/kg) significantly decreased the expression of mUcn II in the skin of animals killed 12 h after injection of the steroid (Fig. 8Go, B and C). No change in the level of mUcn II mRNA was observed in animals killed 2 h after dexamethasone injection (Fig. 8Go, A and C).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 8. Competitive RT-PCR assay for skin mUcn II mRNA after dexamethasone injection. A–C, Saline or dexamethasone (75 µg/kg, 150 µg/kg, or 300 µg/kg) were injected ip into C57BL/6 mice. The mice were killed 2 h (A) or 12 h (B) after injection, and a piece of the dorsal skin was isolated. Total RNA was extracted, followed by DNase treatment, and the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group included at least three animals. The data shown are representative of three identical experiments (C). This figure demonstrates a significant decrease in skin mUcn II mRNA 12 h after dexamethasone injection. *, P < 0.05 vs. saline injections.

 
Two weeks after ADX, with or without corticosterone replacement, mUcn II mRNA levels in the skin (Fig. 9Go, A–D) and skeletal muscle (Fig. 9Go, E–H) were determined using competitive RT-PCR. Levels of mUcn II mRNA in the skin (Fig. 9Go, A, B, and D) of ADX mice increased by approximately 300% compared with control mice. The effect was reversed by corticosterone replacement (Fig. 9Go, C and D). In contrast to the skin, neither ADX nor corticosterone replacement affected mUcn II mRNA levels in skeletal muscle (Fig. 9Go, E–H).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 9. Competitive RT-PCR assay for skin (A–D) and skeletal muscle (E–H) mUcn II mRNA levels after ADX or ADX with corticosterone (Cort) replacement. A piece of the dorsal skin and thigh skeletal muscle were isolated 2 wk after ADX or ADX plus corticosterone treatment of C57BL/6 mice. Total RNA was extracted, followed by DNase treatment, and the mUcn II mRNA levels were determined using competitive RT-PCR as described in Materials and Methods. Each experimental group included at least three animals. The data shown are representative of three identical experiments (D and H). This figure demonstrates a significant increase of skin but not skeletal muscle mUcn II mRNA expression levels after ADX. The skin mUcn II mRNA level returned to normal levels after corticosterone administration. *, P < 0.05 vs. control mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated, using specific mUcn II RNase protection assays, RT-PCR, Southern hybridization, and DNA sequencing, the peripheral distribution of mUcn II transcripts. Although mUcn II mRNA is widely expressed in a variety of peripheral tissues, such as adrenal, lung, gastrointestinal tract, and the immune and reproductive systems, we found it to be most highly expressed in the skin and skeletal muscle. The tissue patterns of mUcn II expression observed by the RNase protection assays and RT-PCR followed by Southern hybridization methods were similar. Human Ucn II transcript was detected in most tissues analyzed by RT-PCR, with higher expression levels in brain, heart, lung, muscle, stomach, adrenal, and peripheral blood cells (2). The human Ucn II transcript pattern of expression is correlated with the pattern we report here for the mouse tissues, both regarding the diversity of the tissues and the intensity of expression. It is important to note that in the same study, no human Ucn II expression was detected in the human skin, and the relative amount of Ucn II in the human heart seems to be higher compared with that of the mouse heart (2).

Two other members of the CRF family of peptides, CRF and Ucn, have been reported to have a wide expression in mammalian peripheral tissues. CRF gene expression has been detected, albeit at levels lower than in the hypothalamus, in placenta, endometrium, uterus, ovary, testes, spleen, immune system, gastrointestinal tract, adrenal gland, thyroid, and human skin (7, 8). Expression of the Ucn gene has also been documented in peripheral tissues such as pituitary, placenta, uterus, testes, immune system, gastrointestinal tract, adrenal gland, pancreas, heart, and skin (22, 23, 24). In contrast to Ucn mRNA and peptide, which are expressed both in human and mouse skin, the CRF mRNA and peptide are expressed in human skin, whereas mouse skin contains only CRF peptide, and CRF gene expression is below detectability by RT-PCR (23). An increasing number of reports indicate that peripheral CRF family peptides play important roles in diverse physiological functions. The CRF peptide is a recognized proinflammatory agent (36). CRF, locally produced by the embryonic trophoblast and maternal decidua, promotes implantation and maintenance of early pregnancy, primarily by killing activated T cells (37). CRF, but not Ucn II or Ucn III, up-regulates nitric oxide synthase and modulates the guanylate cyclase activity in cultured pregnant human myometrial cells (38). Ucn can inhibit trauma-induced edema (39), induce placental ACTH and prostaglandins release (40), and protect cardiac myocytes from hypoxia-induced cell death (41). Ucn and Ucn II have recently been reported to increase IL-6 output via a cAMP-dependent pathway in A7r5 aortic smooth muscle cells (42). We (43) recently reported that Ucn III is expressed in pancreatic ß-cells and stimulates insulin and glucagon secretion.

The relatively high levels of Ucn II mRNA in mouse skin and skeletal muscle led us to investigate mUcn II peptide levels and distribution in these tissues. Using a RIA highly specific for mUcn II, we detected Ucn II-like ir in acid extracts of partially purified tissues isolated from mouse brain, muscle, and skin. Immunohistochemical studies carried out on paraffin sections obtained from mouse skin and skeletal muscle revealed Ucn II-like ir in both skin epidermis and dermis regions including blood vessel walls, the adnexal structures (hair follicle, sweat, and sebaceous glands) and skeletal muscle myocytes. Ucn II mRNA and ir were also observed in neonatal skeletal myocytes, in which Ucn II was localized to the myotubes. CRF ir in the skin was detected in the pilosebaceous unit of the hair follicle, the keratinocytes of the basal epidermis, the outer root sheath, the matrix region of the developing hair follicles, and the nerve bundles (24). The Ucn antigen was localized to keratinocytes in epidermis and the outer and inner root sheaths of hair follicles, epithelium of sebaceous and eccrine glands, erector pili muscle, cutaneous blood vessel walls, cutaneous nerves, and dermal mononuclear cells (27). Further studies will be required to clarify the precise localization of Ucn II in epidermis and dermis compartments.

The skin is exposed to multiple physical, chemical, and biological stressors and should be able to respond immediately to reestablish tissue homoeostasis (25). Skin is known to be a target organ for CRF, Ucn, and POMC peptides. Biochemical and molecular studies have demonstrated CRF, Ucn, CRFRs, and POMC peptides in human and mouse skin, as well as in cultured keratinocytes and melanocytes (22, 23, 24, 25, 26, 27, 28, 29). Slominski et al. (22, 26) proposed that an equivalent of the HPA axis composed of the CRF-CRFR-POMC loop is conserved in the skin and may be activated as a response to stress. The intracellular signaling pathway activated through skin CRFRs involves the production of cAMP and subsequent activation of protein kinase A (23). The involvement of calcium-activated pathways via voltage-activated calcium ion channels has also been demonstrated (45). Furthermore, cAMP production after CRF stimulation has been associated with inhibition of HaCaT keratinocytes proliferation and differentiation of melanoma cells (23). Several recent reports indicate that CRF and Ucn can play important roles in physiological processes in the skin. CRF (46) and Ucn (47) were found to induce mast-cell degranulation. CRF can induce an increase in the synthesis of sebaceous lipids and up-regulate mRNA levels of 3ß-hydroxysteroid dehydrogenase isomerase in human sebocytes (48). Roloff et al. (49) also demonstrate the presence of hair cycle-dependent expression of CRF and CRFRs.

To examine whether the Ucn II signaling system in the skin and skeletal muscle is operative and under regulatory control, we examined the Ucn II transcript levels in CRFR1- and CRFR2-null mice and compared them with their wild-type littermates. We found a significant increase in Ucn II mRNA levels in the skin of both CRFR1- and CRFR2-null mice compared with their wild-type littermates. We did not find any differences in Ucn II mRNA levels in the skeletal muscle tissue of CRFR1- and CRFR2-null mice compared with their wild-type littermates. The CRFR1-null mice displayed low plasma corticosterone concentrations that resulted from marked agenesis of the zona fasciculate region of the adrenal gland due to insufficient ACTH production during the neonatal period (14, 15). To examine whether the increase in the Ucn II transcript in the skin is due to the low circulating corticosterone levels, we studied the Ucn II mRNA levels after dexamethasone administration and ADX in mice.

We showed that mUcn II gene transcription is inhibited by glucocorticoid administration and stimulated by removal of glucocorticoids by ADX. Administration of dexamethasone to mice resulted in a decrease of mUcn II levels in the back skin region 12 h after ip injections. Removal of the adrenal gland significantly increased the levels of Ucn II mRNA in the skin, and the levels were reduced back to normal levels after corticosterone replacement. No effect of ADX could be observed on the level of Ucn II mRNA in the skeletal muscle. Dexamethasone at a concentration of 10 µmol/liter has been reported to inhibit CRF peptide content in squamous cell carcinoma and melanoma cells (62 and 68% reduction, respectively), but had no significant effect on the CRF mRNA levels (50), suggesting posttranscriptional regulation of CRF expression.

The potent inhibitory effect of adrenal glucocorticoids on CRF biosynthesis and release from the hypothalamus has been well documented (51, 52, 53, 54, 55, 56, 57, 58, 59, 60). Acute administration of dexamethasone has been found to inhibit significantly CRF mRNA accumulation in the paraventricular nucleus (PVN) after stress (57). Dexamethasone has also been found to reduce CRF mRNA levels in a human primary liver carcinoma cell line, NPLC, which endogenously expresses CRF (57, 58), and in mouse anterior pituitary tumor cells, AtT-20, which were stably transfected with the human CRF gene (59). ADX induced a significant increase in CRF mRNA levels in whole hypothalamus, specifically in the PVN, and these levels were normalized after dexamethasone replacement (59). It has been reported that ADX is accompanied by significant elevations in CRF peptide levels in the hypothalamus and that CRF heteronuclear RNA in the PVN is augmented after ADX and inhibited after dexamethasone treatment (60). In contrast to the hypothalamus, no effects of ADX or dexamethasone replacement were detected in the olfactory bulb, midbrain, cerebral cortex, or brainstem, indicating regional differences in the regulation of CRF mRNA by glucocorticoids (60). We (61) have very recently reported that mUcn II gene expression is up-regulated by dexamethasone in a dose- and time-dependent fashion both in vitro, using a Ucn II-expressing cell line, and in vivo, using dexamethasone injections and ADX. The differential regulation of the mUcn II gene by glucocorticoids in the skin and in the hypothalamus and brainstem regions of the brain could be explained by different cellular mechanisms activated by glucocorticoids in these different cell types. Binding of glucocorticoids to different binding sites in the mUcn II promoter, the involvement of different cofactors, transcription factors, and signaling molecules in these tissues can cause inhibition or activation of the mUcn II gene. Similar effects were observed for the CRF gene; in contrast to the negative effects of glucocorticoids on hypothalamic CRF, corticosterone delivered to the amygdala increases CRF mRNA in the central amygdaloid nucleus (62). In addition, CRF from amnion, chorion, and decidual placental cells is positively regulated by cortisol and dexamethasone (63). We have also recently reported increases in thymic Ucn mRNA after corticosterone or ACTH injection, in the absence of immune stimulation, indicating that the increase in Ucn mRNA by endotoxin is induced by HPA axis activation (24).

The increase in the skin Ucn II transcript in the CRFR2-null mice could be attributed to a feedback mechanism in which the absence of the receptor will lead to increased ligand concentration due to the absence of normal negative feedback. Further studies will be required to establish this putative model.

The expression of high levels of Ucn II by the skeletal muscle in addition to the known high expression of CRFR2 by this tissue may imply on autocrine/paracrine system in the skeletal muscle that remains to be elucidated. Expression of Ucn II in the skin, a selective ligand for CRFR2, is opening new insights into the possible functions of CRFR2 signaling in the skin, which can act as a parallel balancing system to CRFR1 signaling.

Further examinination of the distribution and regulation of CRFR2 and its specific ligand Ucn II in the skin and skeletal muscle tissues may reveal the manner by which the CRFR2 pathway is involved in the physiological responses to stress in these tissues and in other pathophysiologies of the skin and muscle.


   Acknowledgments
 
We thank Y. Haas for corticosterone RIAs and S. Guerra for assistance in the preparation of this manuscript.


   Footnotes
 
This work was supported by National Institute of Diabetes & Digestive & Kidney Diseases Program Project Grant DK 26741, The Robert J. and Helen C. Kleberg Foundation, The Adler Foundation, and The Foundation for Research (to W.V.). W.V. is a Senior Foundation for Research Investigator.

Abbreviations: ADX, Adrenalectomy; CRF, corticotropin-releasing factor; CRFR, CRF receptor; DNase, deoxyribonuclease; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPA, hypothalamus-pituitary-adrenal; ir, immunoreactivity; mUcn, mouse Ucn; PB, phosphate buffer; POMC, proopiomelanocortin; PVN, paraventricular nucleus; RNase, ribonuclease; RT, reverse transcription; Ucn, Urocortin; UTP, uridine 5'-triphosphate.

Received November 19, 2003.

Accepted for publication January 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, Hogenesch JB, Gulyas J, Rivier J, Vale WW, Sawchenko PE 2001 Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 98:2843–2848[Abstract/Free Full Text]
  2. Hsu SY, Hsueh AJ 2001 Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat Med 7:605–611[CrossRef][Medline]
  3. Vale W, Spiess J, Rivier C, Rivier J 1981 Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and ß-endorphin. Science 213:1394–1397[Free Full Text]
  4. Koob GF, Heinrichs SC 1999 A role for corticotropin releasing factor and urocortin in behavioral responses to stressors. Brain Res 848:141–152[CrossRef][Medline]
  5. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Sawchenko PE, Vale W 1995 Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378:287–292[CrossRef][Medline]
  6. Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Reyes TM, Gulyas J, Fischer W, Bilezikjian L, Rivier J, Sawchenko PE, Vale WW 2001 Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 98:7570–7575[Abstract/Free Full Text]
  7. Perrin MH, Vale WW 1999 Corticotropin releasing factor receptors and their ligand family. Ann NY Acad Sci 885:312–328[Medline]
  8. Dautzenberg FM, Hauger RL 2002 The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol Sci 23:71–77[CrossRef][Medline]
  9. Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 90:8967–8971[Abstract/Free Full Text]
  10. Perrin M, Donaldson C, Chen R, Blount A, Berggren T, Bilezikjian L, Sawchenko P, Vale W 1995 Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc Natl Acad Sci USA 92:2969–2973[Abstract/Free Full Text]
  11. Kishimoto T, Pearse 2nd RV, Lin CR, Rosenfeld MG 1995 A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle. Proc Natl Acad Sci USA 92:1108–1112[Abstract/Free Full Text]
  12. Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdorf T 1995 Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc Natl Acad Sci USA 92:836–840[Abstract/Free Full Text]
  13. Stenzel P, Kesterson R, Yeung W, Cone RD, Rittenberg MB, Stenzel-Poore MP 1995 Identification of a novel murine receptor for corticotropin-releasing hormone expressed in the heart. Mol Endocrinol 9:637–645[Abstract]
  14. Smith GW, Aubry JM, Dellu F, Contarino A, Bilezikjian LM, Gold LH, Chen R, Marchuk Y, Hauser C, Bentley CA, Sawchenko PE, Koob GF, Vale W, Lee KF 1998 Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 20:1093–1102[CrossRef][Medline]
  15. Timpl P, Spanagel R, Sillaber I, Kresse A, Reul JM, Stalla GK, Blanquet V, Steckler T, Holsboer F, Wurst W 1998 Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nat Genet 19:162–166[CrossRef][Medline]
  16. Bale TL, Contarino A, Smith GW, Chan R, Gold LH, Sawchenko PE, Koob GF, Vale WW, Lee KF 2000 Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat Genet 24:410–414[CrossRef][Medline]
  17. Kishimoto T, Radulovic J, Radulovic M, Lin CR, Schrick C, Hooshmand F, Hermanson O, Rosenfeld MG, Spiess J 2000 Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. Nat Genet 24:415–419[CrossRef][Medline]
  18. Coste SC, Kesterson RA, Heldwein KA, Stevens SL, Heard AD, Hollis JH, Murray SE, Hill JK, Pantely GA, Hohimer AR, Hatton DC, Phillips TJ, Finn DA, Low MJ, Rittenberg MB, Stenzel P, Stenzel-Poore MP 2000 Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nat Genet 24:403–409[CrossRef][Medline]
  19. Potter E, Sutton S, Donaldson C, Chen R, Perrin M, Lewis K, Sawchenko PE, Vale W 1994 Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. Proc Natl Acad Sci USA 91:8777–8781[Abstract/Free Full Text]
  20. Chalmers DT, Lovenberg TW, De Souza EB 1995 Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression. J Neurosci 15:6340–6350
  21. Van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C, Prins GS, Perrin M, Vale W, Sawchenko PE 2000 Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. J Comp Neurol 428:191–212[CrossRef][Medline]
  22. Slominski A, Wortsman J, Luger T, Paus R, Solomon S 2000 Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 80:979–1020[Abstract/Free Full Text]
  23. Slominski A, Wortsman J, Pisarchik A, Zbytek B, Linton EA, Mazurkiewicz JE, Wei ET 2001 Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors. FASEB J 15:1678–1693[Abstract/Free Full Text]
  24. Kageyama K, Bradbury MJ, Zhao L, Blount AL, Vale WW 1999 Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology 140:5651–5658[Abstract/Free Full Text]
  25. Slominski A, Wortsman J 2000 Neuroendocrinology of the skin. Endocr Rev 21:457–487[Abstract/Free Full Text]
  26. Slominski AT, Botchkarev V, Choudhry M, Fazal N, Fechner K, Furkert J, Krause E, Roloff B, Sayeed M, Wei E, Zbytek B, Zipper J, Wortsman J, Paus R 1999 Cutaneous expression of CRH and CRH-R. Is there a "skin stress response system?" Ann NY Acad Sci 885:287–311[Medline]
  27. Slominski A, Roloff B, Curry J, Dahiya M, Szczesniewski A, Wortsman J 2000 The skin produces urocortin. J Clin Endocrinol Metab 85:815–823[Abstract/Free Full Text]
  28. Pisarchik A, Slominski AT 2001 Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression. FASEB J 15:2754–2756[Free Full Text]
  29. Kono M, Nagata H, Umemura S, Kawana S, Osamura RY 2001 In situ expression of corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) genes in human skin. FASEB J 15:2297–2299[Free Full Text]
  30. Stenn KS, Paus R, Dutton T, Sarba B 1993 Glucocorticoid effect on hair growth initiation: a reconsideration. Skin Pharmacol 6:125–134[Medline]
  31. Wagner M, Perry RP 1985 Characterization of the multigene family encoding the mouse S16 ribosomal protein: strategy for distinguishing an expressed gene from its processed pseudogene counterparts by an analysis of total genomic DNA. Mol Cell Biol 5:3560–3576[Abstract/Free Full Text]
  32. Bilezikjian LM, Corrigan AZ, Blount AL, Vale WW 1996 Pituitary follistatin and inhibin subunit messenger ribonucleic acid levels are differentially regulated by local and hormonal factors. Endocrinology 137:4277–4284[Abstract]
  33. Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Voglmayr JK, Bardin CW, Rivier C, Vale W 1989 Detection and purification of inhibin using antisera generated against synthetic peptide fragments. Methods Enzymol 168:588–617[Medline]
  34. Vaughan JM, Fischer WH, Hoeger C, Rivier J, Vale W 1989 Characterization of melanin-concentrating hormone from rat hypothalamus. Endocrinology 125:1660–1665[Abstract]
  35. Stephanou A, Brar B, Heads R, Knight RD, Marber MS, Pennica D, Latchman DS 1998 Cardiotrophin-1 induces heat shock protein accumulation in cultured cardiac cells and protects them from stressful stimuli. J Mol Cell Cardiol 30:849–855[CrossRef][Medline]
  36. Chrousos GP 1995 The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 332:1351–1362[Free Full Text]
  37. Makrigiannakis A, Zoumakis E, Kalantaridou S, Coutifaris C, Margioris AN, Coukos G, Rice KC, Gravanis A, Chrousos GP 2001 Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance. Nat Immunol 2:1018–1024[CrossRef][Medline]
  38. Aggelidou E, Hillhouse EW, Grammatopoulos DK 2002 Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells. Proc Natl Acad Sci USA 99:3300–3305[Abstract/Free Full Text]
  39. Turnbull AV, Vale W, Rivier C 1996 Urocortin, a corticotropin-releasing factor-related mammalian peptide, inhibits edema due to thermal injury in rats. Eur J Pharmacol 303:213–216[CrossRef][Medline]
  40. Petraglia F, Florio P, Benedetto C, Marozio L, Di Blasio AM, Ticconi C, Piccione E, Luisi S, Genazzani AR, Vale W 1999 Urocortin stimulates placental adrenocorticotropin and prostaglandin release and myometrial contractility in vitro. J Clin Endocrinol Metab 84:1420–1423[Abstract/Free Full Text]
  41. Okosi A, Brar BK, Chan M, D’Souza L, Smith E, Stephanou A, Latchman DS, Chowdrey HS, Knight RA 1998 Expression and protective effects of urocortin in cardiac myocytes. Neuropeptides 32:167–171[CrossRef][Medline]
  42. Kageyama K, Suda T 2003 Urocortin-related peptides increase interleukin-6 output via cyclic adenosine 5'-monophosphate-dependent pathways in A7r5 aortic smooth muscle cells. Endocrinology 144:2234–2241[Abstract/Free Full Text]
  43. Li C, Chen P, Vaughan J, Blount A, Chen A, Jamieson PM, Rivier J, Smith MS, Vale W 2003 Urocortin III is expressed in pancreatic ß-cells and stimulates insulin and glucagon secretion. Endocrinology 144:3216–3224[Abstract/Free Full Text]
  44. Deleted in proof
  45. Wiesner B, Roloff B, Fechner K, Slominski A 2003 Intracellular calcium measurements of single human skin cells after stimulation with corticotropin-releasing factor and urocortin using confocal laser scanning microscopy. J Cell Sci 116:1261–1268[Abstract/Free Full Text]
  46. Theoharides TC, Singh LK, Boucher W, Pang X, Letourneau R, Webster E, Chrousos G 1998 Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects. Endocrinology 139:403–413[Abstract/Free Full Text]
  47. Singh LK, Boucher W, Pang X, Letourneau R, Seretakis D, Green M, Theoharides TC 1999 Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotropin-releasing hormone receptors. J Pharmacol Exp Ther 288:1349–1356[Abstract/Free Full Text]
  48. Zouboulis CC, Seltmann H, Hiroi N, Chen W, Young M, Oeff M, Scherbaum WA, Orfanos CE, McCann SM, Bornstein SR 2002 Corticotropin-releasing hormone: an autocrine hormone that promotes lipogenesis in human sebocytes. Proc Natl Acad Sci USA 99:7148–7153[Abstract/Free Full Text]
  49. Roloff B, Fechner K, Slominski A, Furkert J, Botchkarev VA, Bulfone-Paus S, Zipper J, Krause E, Paus R 1998 Hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors in murine skin. FASEB J 12:287–297[Abstract/Free Full Text]
  50. Slominski A, Ermak G, Mazurkiewicz JE, Baker J, Wortsman J 1998 Characterization of corticotropin-releasing hormone (CRH) in human skin. J Clin Endocrinol Metab 83:1020–1024[Abstract/Free Full Text]
  51. Jingami H, Matsukura S, Numa S, Imura H 1985 Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/ß-lipotropin precursor mRNA in the pituitary in rats. Endocrinology 117:1314–1320[Abstract]
  52. Young 3rd WS, Mezey E, Siegel RE 1986 Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198–203[CrossRef][Medline]
  53. Kovacs KJ, Mezey E 1987 Dexamethasone inhibits corticotropin-releasing factor gene expression in the rat paraventricular nucleus. Neuroendocrinology 46:365–368[Medline]
  54. Beyer HS, Matta SG, Sharp BM 1988 Regulation of the messenger ribonucleic acid for corticotropin-releasing factor in the paraventricular nucleus and other brain sites of the rat. Endocrinology 123:2117–2123[Abstract]
  55. Fink G, Robinson IC, Tannahill LA 1988 Effects of adrenalectomy and glucocorticoids on the peptides CRF-41, AVP and oxytocin in rat hypophysial portal blood. J Physiol 401:329–345[Abstract/Free Full Text]
  56. Swanson LW, Simmons DM 1989 Differential steroid hormone and neural influences on peptide mRNA levels in CRH cells of the paraventricular nucleus: a hybridization histochemical study in the rat. J Comp Neurol 285:413–435[CrossRef][Medline]
  57. Harbuz MS, Nicholson SA, Gillham B, Lightman SL 1990 Stress responsiveness of hypothalamic corticotrophin-releasing factor and pituitary pro-opiomelanocortin mRNAs following high-dose glucocorticoid treatment and withdrawal in the rat. J Endocrinol 127:407–415[Abstract/Free Full Text]
  58. Owens MJ, Nemeroff CB 1991 Physiology and pharmacology of corticotropin-releasing factor. Pharmacol Rev 43:425–473[Medline]
  59. Imaki T, Nahan JL, Rivier C, Sawchenko PE, Vale W 1991 Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress. J Neurosci 11:585–599[Abstract]
  60. Imaki T, Xiao-Quan W, Shibasaki T, Yamada K, Harada S, Chikada N, Naruse M, Demura H 1995 Stress-induced activation of neuronal activity and corticotropin-releasing factor gene expression in the paraventricular nucleus is modulated by glucocorticoids in rats. J Clin Invest 96:231–238
  61. Chen A, Vaughan J, Vale WW 2003 Glucocorticoids regulate the expression of the mouse urocortin II gene: a putative connection between the corticotropin-releasing factor receptor pathways. Mol Endocrinol 17:1622–1639[Abstract/Free Full Text]
  62. Shepard JD, Barron KW, Myers DA 2000 Corticosterone delivery to the amygdala increases corticotropin-releasing factor mRNA in the central amygdaloid nucleus and anxiety-like behavior. Brain Res 861:288–295