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Endocrinology, doi:10.1210/en.2004-0194
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Endocrinology Vol. 145, No. 8 3971-3983
Copyright © 2004 by The Endocrine Society

Promoter Analysis of Human Corticotropin-Releasing Factor (CRF) Type 1 Receptor and Regulation by CRF and Urocortin

Kelly L. Parham, Sevasti Zervou, Emmanouil Karteris, Rob D. Catalano, Robert W. Old and Edward W. Hillhouse

Department of Biological Sciences (K.L.P., S.Z., E.K., R.D.C., R.W.O., E.W.H.), University of Warwick, Coventry CV4 7AL, United Kingdom; The Leeds Institute of Genetics (E.W.H.), Therapeutics and Health, The Medical School, University of Leeds, Leeds LS2 9NL, United Kingdom; and Reproductive Molecular Research Group (R.D.C.), Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom

Address all correspondence and requests for reprints to: Edward W. Hillhouse, Office of the Dean, Worsley Building, The Medical School, The University of Leeds, Leeds LS2 9NL, United Kingdom. E-mail: e.w.hillhouse{at}leeds.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report the full genomic organization of the human gene for the corticotropin-releasing factor (CRF) receptor type 1 (CRFR1), with complete mapping of exons 1–14. The 5' flanking region (2.4 kb) of the gene encoding for human CRFR1 was isolated, sequenced, and characterized. Two major transcriptional start sites were determined at –265 and –238, relative to the ATG start site (+1). Transient expression of constructs containing sequentially deleted 5'-flanking sequences of CRFR1 fused to luciferase, revealed the minimal promoter sequence 370 bp in size, as shown by assays in neuroblastoma (SH-5YSY), teratocarcinoma (NT2), and adenocarcinoma (MCF 7) cell lines. CRF and UCN markedly increased promoter activity during transient CRFR1 expression studies. Similarly, CRF and UCN up-regulate the endogenous CRFR1 at the mRNA level in NT2 and MCF 7 cells. To dissect further the mechanisms involved, we have used primary myometrial cells transfected with the CRFR1 promoter. CRF and UCN increased the promoter activity, an effect blocked by protein kinase (PK)A and PKC inhibitors. Both CRF and UCN cause a positive feedback effect in primary cultures of human pregnant myometrial cells, by increasing mRNA expression of CRFR1. This effect appears to be dependent on activation of both PKA and PKC by CRF, whereas UCN's effect was mediated solely via PKC activation. Collectively, our data suggest that the CRFR1 gene is under the influence of both CRF and UCN, acting via distinct signaling pathways to create a positive feedback loop and regulate further the transcription of the receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORTICOTROPIN-RELEASING FACTOR (CRF; a 41-amino-acid peptide) is responsible for triggering the secretion of ACTH from the anterior pituitary (1, 2). CRF has also been implicated in immune and cardiovascular responses. It regulates energy balance as an anorectic agent, stimulates thermogenesis, and elicits anxiogenic effects (3, 4, 5). Moreover, CRF exerts profound effects in the female reproductive system by promoting blastocyst implantation, regulating placental vascular tone, and myometrial activity (6, 7).

CRF belongs to a family of structurally related peptides that include sauvagine (amphibians; Ref.8), urotensin I (fish; Ref.9), and the urocortins (UCNs) in mammals (10, 11, 12, 13). CRF and related peptides act by activating the two subtypes of CRF receptors (CRFRs) (3), CRFR1 (14, 15, 16, 17, 18) and CRFR2 (19, 20), which are encoded by different genes. These receptors belong to the family of class II G protein-coupled receptors, which include receptors for vasopressin, calcitonin, and PTH (21).

CRF and its homologues (UcnI, urotensin, and sauvagine) bind with high affinities to the mammalian CRFR1 in a nonselective manner, whereas the CRFR2 has higher affinity for the UCNs than CRF (7).

CRFR1 is a 415-amino acid protein. Four isoforms of human CRFR1 were initially described: CRFR1ß (containing all known 14 exons), CRFR1{alpha} (lacking exon 6), CRFR1c (lacking exons 3 and 6), and CRFR1d (lacking exons 6 and 13). In addition, Pisarchik and Slominski (22) identified four new mRNA transcripts, which may encode new receptor isoforms e, f, g, and h. All have exon 6 spliced out from the final transcript. This work also describes how the translation products of CRFR alternative splicing may influence CRF actions in human tissues. More recent work describes how CRFR1{alpha} is the most prevalent isoform in human skin compared with mouse skin, where CRFR2 is widely expressed and may have a role in hair growth (23). In general, CRFR1{alpha} appears to be the most prevalent isoform in all tissues studied in rodents and humans, and it appears to be abundantly expressed in the brain and pituitary but also in the periphery in tissues such as the adrenals, placenta, endometrium, and myometrium (15, 16, 22, 23, 24, 25).

Targeted disruption of CRFR in mice has provided insight into the importance of the different receptor subtypes (21). These studies have implicated CRFR1 in mediating normal responses to stress (26), whereas CRFR2 appears to play important roles in fine-tuning stress responses (27). CRFR1 mediates anxiogenic actions that are opposed by the anxiolytic properties of CRFR2 (28). The CRFR2 gene expresses three anatomically distinct splice variants (2ß, 2{gamma}, and 2{alpha}) that are produced by 5' alternative splicing mechanisms (19, 29, 30, 31). Their expression is more confined to the periphery in tissues such as the heart, lungs, genitourinary tract, and skeletal muscle. The differential expression of these receptors points toward distinct tissue-specific functions, mediated by CRF and its related peptides.

Our group has recently published how CRFR2 is regulated by multiple promoters, giving a better insight into the processing of the gene (31). Currently we present sequencing analysis of the 5' region and the genomic structure of the human CRFR1 gene. We also describe mechanisms regulating its transcription and therefore propose a positive feedback mechanism on the CRFR1 gene transcription, mediated by CRF and UCN in human cell lines and primary cell culture systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of genomic clones by genomic library screening
A human chromosome 17 specific cosmid (LA1780-B1) library was obtained from the HGMP Resource Centre, Cambridge, UK. To isolate the promoter, an oligonucleotide probe was designed to encompass exon 1 of the gene for use in screening of a genomic library. Our library screening experiments and cosmid studies for determining the promoter sequence revealed the position of exons 1 and 2 on the human crfr1 genomic sequence.

For library screening, two 100-bp probes were designed, covering a region of 171 bp of exon 1 (Table 1Go). The probes were end-labeled with {gamma}-32 phosphorous using T4 polynucleotide kinase. The membrane was hybridized with the radiolabeled probes. Clones that provided the strongest signals were ordered from the HGMP Resource Centre for subsequent analysis. Upstream sequencing was performed, and we eventually sequenced a 2.4-kb continuous region.


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  		TABLE 1. Oligonucleotide sequences for RT-PCR

 
Determination of the transcription start site
5' Rapid amplification of cDNA ends (RACE) was performed by using the Marathon Ready human hippocampus cDNA Amplification Kit (BD Biosciences Clontech, Oxford, UK), according to manufacturers’ instructions. Primary PCR was carried out using a gene-specific primer (CRFR-E) and the anchor primer (AP)1. Secondary PCR was carried out using a nested gene-specific primer (CRFR-D) and AP2, with 1 µl primary PCR product as a template. PCR products were analyzed on a 2% agarose gel, excised, and subcloned into pGEM T Easy (Promega, Southampton, UK) for sequencing.

Construction of luciferase-expression vectors
Flanking sequences 5' to the transcriptional start site were PCR-amplified from cosmid LA1780-B1 as a template, cloned into the pGL3-Basic mammalian expression vector (Promega), and sequence-verified. For cloning purposes, primers used for PCR had a 5' NheI site and a 3' KpnI site, in the sense and the antisense primer, respectively. Constructs were designed using sense (5') primers located at positions –2453 (for reasons of brevity, called the 2.4 kb-promoter fragment), –1990 (2.0-kb promoter), –1563 (1.5-kb promoter), –1002 (1.0 kb-promoter), –764 (0.75-kb promoter), –566 (0.5-kb promoter), –374 (0.37-kb promoter), and –289 (0.29-kb promoter) relative to ATG (+1). The antisense primer was located at –8 relative to ATG.

Cell culture, transient transfection, and reporter gene analysis
NTera/D1 cells (NT2, human neuronal-like teratocarcinoma cells; Stratagene Europe, Amsterdam, The Netherlands), MCF 7 (human breast cancer; European Collection of Animal Cell Cultures (ECACC), Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK), and SH-5YSY cells (human neuroblastoma; ECACC) were seeded in six-well plates and cultured according to the ECACC recommendations, until they were approximately 60–70% confluent. Primary cultures of myometrial cells were used at passages 2–4 and maintained as described before (32). Cell cultures were maintained in phenol red-free media containing charcoal-stripped fetal calf serum (FCS), for 24 h before transfection. The cells were transfected with 4 µg plasmid DNA, using Exgen 500 (MBI Fermentas, Helena Biosciences Europe, Sunderland, UK) in 0.5 ml phenol red-free culture media, containing charcoal-stripped FCS, and were incubated for 3 h at 37 C. Cells were treated with either of the following: dexamethasone (500 nM; Sigma-Aldrich, Dorset, UK), all-trans retinoic acid (1 µM; Sigma-Aldrich), human CRF (1–100 nM; Peninsula Laboratories Europe Ltd., St. Helens, UK), human UCN (10–100 nM; Peninsula Laboratories), for 16–24 h. Myometrial cells were pretreated with either bisindolylmaleimide or H-89 (both from Calbiochem, Merck Biosciences LTD, Nottingham, UK; used at 100 nM each) for 30 min before incubation with human CRF or human UCN, as shown before (33, 34).

All plasmid constructs were cotransfected with 0.5 µg of the pRL-TK vector (Promega), to determine the transfection efficiency. Media were replaced with 2 ml fresh culture medium (containing various factors for stimulation of promoter activity). The cells were grown until 90% confluent. Cell extracts were prepared up to 24 h after transfection and assayed using a dual-luciferase reporter assay system (Promega). Luciferase activity was measured for 10 sec using a Luminoscan RS Luminometer (Labsystems, Helsinki, Finland).

RNA isolation, cDNA synthesis, and PCR
NT2 and MCF 7 cells were maintained in phenol red-free culture media containing charcoal-stripped FCS, for 24 h before treatments. Cells were treated with either of the following: dexamethasone (500 nM), all-trans retinoic acid (1 µM), human CRF (1–100 nM), human (1–100 nM), for 1, 4, or 16 h. Myometrial cells were pretreated with either bisindolylmaleimide or H-89 (used at 100 nM each) for 30 min before incubation with CRF or UCN.

Total RNA was extracted by using ULTRASPEC (Biotecx Laboratories, Houston, TX), according to manufacturer’s instructions. RNA concentration was determined by spectrophotometric analysis and agarose gel electrophoresis. A set concentration of RNA (500 ng) was reverse-transcribed into cDNA, by using 5 IU/µl RNase H reverse transcriptase (Superscript II; Invitrogen, Paisley, UK). PCR amplification was carried out with 8 µl cDNA, using Taq polymerase (Invitrogen) and oligonucleotide primers (Invitrogen; Table 1Go) for human glyceraldehyde-6-phosphate dehydrogenase (GAPDH; Ref.35) and human CRFR1, respectively. For quantitation purposes, the number of cycles was optimized for each of the two genes assessed, and per cDNA template, to confirm that all reactions were at the linear range of amplification. The PCR would be expected to yield a product, at 290 bp for CRFR1 and 485 bp for GAPDH. Both products for CRFR1 and GAPDH were cloned by ligation into pGEM T Easy plasmid vector (Promega) and transformation of Escherichia coli cells. Bacterial DNA was lysed using the QIAPrep Miniprep Kit (Qiagen, Crawley, West Sussex, UK). Consequent sequencing of the PCR products confirmed both the specificity of the reactions and the identity of the products by comparing to the already published human sequences for GAPDH (Accession JO4038) and CRFR1 (Accession L23333), respectively.

In silico approach and statistical analysis
Gene structure and exon mapping were determined using the DNA Star SeqMan Software (University of Warwick, UK). The transcriptional start sites were investigated using the prediction programs TSSW and TSSG on www.sanger.ac.uk. Sequence alignments were performed using ClustalW on the BioEdit software program (www.ebi.ac.uk/clustalw). To identify potential transcription factor binding sites, the MatInspector software program on www.genomatrix.de and the AliBaba2 programs on the TRANSFAC database (www.gene-regulation.com), were employed (36). To predict possible CpG islands, the EMBOSS-CpG Plot (www.ebi.ac.uk) and NNPP (37) programs were used.

For luciferase assays, ±SEM values were calculated for each experiment, and data were analyzed using one-way ANOVA. When a significant effect was found (P < 0.05), one-way ANOVA was followed by a post hoc test (Tukey procedure) to locate the difference in the groups.

Analysis of the RT-PCR products was performed as follows. For each of the cell culture treatments corresponding to individual samples, the PCR product for GAPDH and the product for CRFR1 were analyzed on 2% agarose gels. The densities were measured using a scanning densitometer coupled to scanning computer software (ImageQuant; Molecular Dynamics, Amersham Pharmacia, Little Chalfont, UK). For quantitation purposes, data were recorded as the ratio of CRFR1 over GAPDH. Results were evaluated using one-way ANOVA. When a significant effect was found (P < 0.05), post hoc tests (Tukey procedure) were done to locate the difference in the groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Structure of the CRFR1 gene
Sequence analysis of contig AC003662 led to mapping of exons 1–14 and determined the distance between exons 3 and 4 and exons 4 and 5. Restriction digestion of cosmid LA1780-B1 led to the determination of the distance between exons 1 and 2, which was later confirmed from sequence analysis of contig AC106030. The distance between exons 1 and 2 is approximately 23 kb, whereas the distance between exons 3 and 4 is 4.7 kb, and the distance between exons 4 and 5 is 7.8 kb (Fig. 1Go).



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  		FIG. 1. Full genomic diagram of the crfr1 gene. The genomic structure of the crfr1 gene, showing exons 1–14. The positions of exons 1 and 2 were identified on contig AC003662, using the SeqMan software program (DNA Star, University of Warwick, UK). Exon 1 is situated approximately 23 kb upstream of exon 2. This schematic representation of the human CRFR1 gene includes exon 6, which is present in the CRFR1ß, but absent from the CRFR1{alpha}, splice variant.

 
Characterization and in silico analysis of the CRFR1 promoter region
Initial analysis of the promoter sequence showed that the promoter lacks consensus sites for TATA or CCAAT initiator sequences, whereas the region is exceptionally GC-rich (Fig. 2Go). The defined 2.4-kb promoter region is 65% GC rich, whereas the region around the transcriptional start sites (–600 bp to +1 bp) is highly GC rich, at 78%.



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  		FIG. 2. The promoter region and 5'UTR for the crfr1 gene. Identification of promoter sequence and transcriptional start site. Transcription factor binding sites identified within the CRFR1 promoter. The 5' UTR is in bold. ATG (+1) and possible putative transcription factor binding sites are also indicated. The two arrows in bold, at –265 and –238 upstream of ATG, represent the points of the two major transcriptional start sites.

 
Although there is no typical TATA or CCAAT sequence in the promoter region of CRFR1, a cluster of contig Sp1 consensus sites (–1752 to –1705 and –474 to –340, relative to ATG) was found throughout the sequence. Several AP-2{alpha} elements (–1140, –1023, and –557 relative to ATG) were also present. Two CpG islands were predicted within the 5' end of the crfr1 gene, separated by 73 bp, indicating that they are probably an entity representing a 1081-bp CpG island, extending from –942 to +139, relative to ATG.

Putative elements for the ubiquitously expressed brain factors Egr-1/Egr-2 (–900, –800, –319, and –1165 nucleotide positions) were identified in addition to two elements for the retinoid X receptor (RXR sites) at –1493 and –1480, relative to ATG. Other putative elements present are Oct-1 at –1926; GATA 3 and GATA 1 at –2086 and –1321, respectively; C/EBP at –1836; ERE at –1698; GRE at –2046; PRE at –958 nucleotides; YY1 at –2426; and NF-{kappa} at –53.

Characterization of the 5' untranslated region (UTR) for crfr1
The 5' UTR of human CRFR1 was determined in the human hippocampus. The two products observed, at 195 and 160 bp, were excised from the gel, subcloned into pGEM-T Easy (Promega), and sequenced to verify their identity (Fig. 3Go).



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  		FIG. 3. 5' RACE. Results of nested PCRs during 5' RACE experiments, as described in Materials and Methods. Lane 1, marker; lane 2, negative control; lane 3: human hippocampus Marathon Ready cDNA was used as a template. The two bands (indicated by the arrows) at 195 and 160 bp were subcloned into pGEM T Easy, as described in Materials and Methods.

 
Using the human genome database, we confirmed the presence of a cluster of transcriptional start sites, around a 10-bp region. Two major transcriptional start points were identified at –265 and –238, relative to ATG (Fig. 2Go). The 5'UTR region of human CRFR1 is highlighted in Fig. 2Go.

The 5'UTR region approximately 700 bp upstream of ATG shows considerable homology among rat, mouse, and human (highlighted conserved sequence shown on Fig. 4Go). Sequence alignment analysis revealed that the three promoters share 58% homology. The conserved region among the three species includes mainly Sp1 sites, in addition to AP2, Egr2 (Krox-20), and NF{kappa} sequences. The homology shared between the mouse and rat CRFR1 promoter regions is approximately 85%.



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  		FIG. 4. Alignment of 5' UTR for rat, mouse, and human CRFR1. Comparison of genomic sequences 5' to the ATG start codon of rat (NM_030999), mouse (NM_007762) and human (accession number AF488558) 5'UTR sequence of the crfr1 gene, using ClustalW sequence alignment program (http://www.ebi.ac.uk/clustalw). The highlighted regions indicate sequences in the promoter that are conserved in the three species. Nucleotides in bold indicate predicted transcriptional start sites.

 
Determining basal promoter activity and the minimal promoter sequence
To examine promoter activity, the human cell lines NT2 and SH-5YSY, expressing CRFR1 endogenously, were transiently transfected with luciferase constructs containing eight different fragments of the promoter (2.4–0.29 kb), as described in Materials and Methods. Transfection efficiency was monitored by cotransfection with the pRL-TK vector. In both cell lines, maximal activity was displayed by the positive control pGL3-Control vector (Fig. 5Go).



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  		FIG. 5. Functional analysis of CRFR1 promoter in (A) SH-SY5Y and in (B) NT2 (NTera/D1) cells. Luciferase reporter plasmids (in equimolar amounts) containing serially truncated putative CRFR1 promoter fragments (2.4–0.29 kb), positive control (pGL3-SV40) or vector alone (pGL3-Basic), were transiently transfected into NT2 and SH-5YSY cells (Materials and Methods). These data are representative of two independent experiments, each performed in triplicate. Values shown are ±SEM. Data were analyzed by one-way ANOVA, followed by a post hoc test. *, P < 0.001.

 
The highest level of CRFR1 promoter activity was exhibited by the 0.37-kb promoter construct. Reducing the promoter length by 84 bp (0.29 kb promoter construct) caused a significant decrease in activity, by 10- and 3-fold in NT2 and SH-5YSY cells, respectively (P < 0.001). The basal promoter may lie –374 bp upstream of the ATG. Plasmid constructs of the promoter, which extend further upstream from –374 bp, generally showed a gradual decrease in promoter activity. There was a significant decrease in the activity observed between the 0.37-kb promoter construct and the 2.4-kb (full-length) promoter construct, both in NT2 cells and in SH-5YSY cells (P < 0.001). Interestingly, the 2.0-kb promoter construct displayed higher activity than the 2.4-kb promoter construct in both cell types (P < 0.001).

Transient expression analysis and regulation of the CRFR1 promoter region
To investigate CRFR1 promoter activity further, MCF 7 and NT2 cells were transiently transfected with the full-length promoter construct (2.4 kb) and the 2.0-kb construct. The 2.0-kb promoter construct displayed higher basal activity levels compared with the full-length (2.4 kb) in MCF 7 (Fig. 6Go), consistent with the observations in NT2 and SH-5YSY cells (P < 0.001) (see Figs. 5–7GoGoGo). First we examined the effect of CRF (100 nM) and UCN (100 nM), which are both naturally occurring CRFR1 ligands. We also investigated dexamethasone, a synthetic steroid that inhibits hypothalamo-pituitary-adrenal activity; and retinoic acid because the promoter contains consensus, potential RXR sites.



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  		FIG. 6. CRFR1 promoter activity. Luciferase reporter constructs containing the full-length promoter (2.4 kb) and a 2.0-kb region immediately upstream of ATG were transiently transfected into NT2 and MCF 7 cells. Basal activity of the constructs containing 2.4 kb, 2.0 kb, the SV40 promoter (pGL3-SV40; positive control), and promoterless luciferase vector (pGL3-Basic). Data were analyzed by one-way ANOVA, followed by a post hoc test. *, P < 0.001.

 


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  		FIG. 7. Transient expression studies of the full-length promoter (2.4 kb) and the 2.0-kb fragment of the promoter in NT2 (A) and MCF 7 (B) cells. The effects of dexamethasone, all-trans retinoic acid, CRF (100 nM), and UCN (100 nM) on the activity of the CRFR1 promoter were assessed 16 h after transient transfection (Materials and Methods). These data are representative of two independent experiments, each performed in triplicate and analyzed using one-way ANOVA, followed by a post hoc test. Values shown are ±SEM. *, P < 0.001.

 
NT2 cells
In NT2 cells, CRF significantly up-regulated CRFR1 promoter activity for the 2.0-kb construct (P = 0.0008), whereas dexamethasone and UCN had no effect (Fig. 7AGo).

Furthermore, the CRF effect was completely abolished by the antagonist {alpha}-helical (used at 1 µM) (data not shown). All-trans retinoic acid caused a decrease in promoter activity when the cells were transiently transfected with the 2.0-kb, but not the 2.4-kb, construct (P = 0.001).

MCF 7 cells
When MCF 7 cells were transfected with the full-length (2.4 kb) and the 2.0-kb constructs, neither dexamethasone, retinoic acid, nor CRF were able to influence promoter activity (Fig. 7BGo).

Regulation of endogenous crfr1 gene in cell lines
To investigate our initial observations further, we examined the effects of dexamethasone, retinoic acid, CRF, and UCN in cell lines, using semiquantitative RT-PCR. NT2 and MCF 7 cells were assessed for this purpose, because both cell lines express endogenously CRFR1 (Fig. 8AGo).



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  		FIG. 8. Regulation of endogenous crfr1 in cell lines. A, Products from RT-PCR experiments were analyzed on 2% agarose gels and quantitated using a scanning densitometry system. The expected CRFR1 product had a size of 290 bp. To confirm that the PCR were at the linear range of amplification, we showed that increasing amounts of cDNA input resulted in accordingly increasing amounts of PCR product. CRFR1 messenger RNA levels were monitored in NT2 (B) and MCF 7 (C) cells after incubation with dexamethasone (Dex.), all-trans retinoic acid, CRF, UCN. Semiquantitation was performed by comparing CRFR1 mRNA to GAPDH levels by using a scanning densitometer. These data are representative of two independent experiments, each performed in triplicate. Values shown are ±SEM. Levels of significance were determined using one-way ANOVA, followed by a post hoc test. Values shown are ±SEM. No supplement (NS) was set at 100. *, P < 0.001.

 
NT2 cells
The cell line was used to monitor mRNA levels after 4-h treatments with dexamethasone, retinoic acid, CRF, UCN. From these experiments, it was evident that CRF acts as a potent regulator of CRFR1 mRNA, because it dramatically induced expression levels by almost 40-fold (P < 0.001). Similarly, UCN increased mRNA by 30-fold (P < 0.001). Dexamethasone did not have any effect, whereas retinoic acid decreased the mRNA expression, although the effect did not reach significance levels (P = 0.195) (Fig. 8BGo).

MCF 7 cells
We also studied the human adenocarcinoma cell line MCF 7. Interestingly, in this cell line, CRF (but not UCN) induced CRFR1 mRNA expression by 4-fold (P < 0.001) (Fig. 8CGo). Dexamethasone up-regulated mRNA levels by 10-fold (P < 0.001), and retinoic acid had no effect on CRFR1 mRNA expression (Fig. 8CGo).

For both NT2 and MCF7 cells, similar effects for CRF, UCN, and retinoic acid were demonstrated for 4 h and 1 h of treatment (data not shown).

Transient expression analysis and regulation of the CRFR1 promoter region in human pregnant myometrial cells by CRF and UCN
In a manner similarly to our transient expression experiments in cell lines, we employed a primary human cell culture system to further study the positive feedback effects mediated by CRF and UCN. Human pregnant myometrial smooth muscle cells were used at passages 2–4.

The activity levels of the 2.4-kb promoter were lower than the levels of the 2.0-kb promoter region, consistent with our previous experiments in NT2, SH-5YSY, and MCF-7 cells (Figs. 5–7GoGoGo).

Treatment (16 h) with CRF or UCN (optimized at 100 nM) resulted in up-regulation of the CRF-R1 promoter, with UCN being more potent than CRF (P < 0.001) (Fig. 9AGo).



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  		FIG. 9. Transient expression analysis of the CRFR1 promoter region in human pregnant myometrial cells. Primary cultures of human myometrial cells were transfected with the 2.0- and 2.4-kb promoter region of human CRFR1. A, The effects of dexamethasone, all-trans retinoic acid, CRF, and UCN on the activity of the CRFR1 promoter were assessed 16 h after transient transfection (Materials and Methods). B, CRF and UCN were supplemented in the presence/absence of PKC inhibitor bisindolylmaleimide (PKCi; 100 nM) or PKA inhibitor H-89 (PKAi; 100 nM) as reported in Materials and Methods. Untr, Nontransfected control culture; Vector, the luciferase reporter vector pGL3-Basic. These data are representative of two independent experiments, each performed in triplicate. Values shown are ±SEM. For CRF vs. CRF+PKCi, P = 0.089; and for CRF vs. CRF+PKAi, P = 0.0763 (one-way ANOVA, followed by a post hoc test). *, P < 0.001.

 
We then used specific inhibitors to dissect the mechanistics of these effects. Use of Bisindolylmaleimide (PKC inhibitor) or H-89 (PKA inhibitor) partially inhibited the CRF effect and more potently reduced the UCN effect (P < 0.001) (Fig. 9BGo). There was no apparent difference in the effect of either CRF or UCN in myometrial cells pretreated with Pertussis toxin (Gi/Go inhibitor-data not shown). The specificity of the CRF and UCN effects was also assessed using the antagonist {alpha}-helical (data not shown), which was able to totally inhibit the action of both peptides. PKC and PKA inhibitors alone did not alter CRFR1 promoter activity.

Transcriptional regulation of the CRFR1 gene in human pregnant myometrial cells by CRF and UCN
In addition to examining the regulation of CRFR1 promoter region in primary cultures of myometrial cells, we assessed the effects of dexamethasone, retinoic acid, CRF, and UCN on the endogenous expression of crfr1 (Fig. 10Go).



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  		FIG. 10. Transcriptional regulation of the CRFR1 gene in myometrial cells. A, CRFR1 messenger RNA levels were monitored in human myometrial cells, after incubation with Dex., all-trans retinoic acid, CRF, UCN. Semiquantitation was performed as described before. Values shown are ±SEM. Statistical significance was determined using one-way ANOVA, followed by Tukey post hoc analysis. Data are representative of two independent experiments, each performed in triplicate. NS was set at 100. *, P < 0.001. B, CRF and UCN were supplemented in the presence or absence of PKC inhibitor bisindolylmaleimide (PKCi, 100 nM) or PKA inhibitor H-89 (PKAi; 100 nM). These data are representative of two independent experiments, each performed in triplicate and analyzed using one-way ANOVA, followed by a post hoc test. Values shown are ±SEM. For CRF vs. CRF+PKCi, P = 0.065. *, P < 0.001.

 
Both CRF and UCN caused an increase of CRFR1 mRNA expression (P < 0.001) over 4 h of treatment with 100 nM of either of the two agonists (Fig. 10AGo). The CRF effect was completely abolished by the PKA inhibitor and partially reversed by the PKC inhibitor (P < 0.001). On the contrary, the induction of CRFR1 mRNA by UCN was significantly inhibited by the PKC, but not the PKA, inhibitor (P < 0.001). PKC and PKA inhibitors alone did not alter CRFR1 promoter activity (Fig. 10BGo).

Finally, dose-response studies in primary cultures of myometrial cells have revealed a positive correlation between the concentration of CRF or UCN (1, 10, and 100 nM) and the levels of expression for endogenous CRFR1 (Fig. 11Go; P = 0.0008 after one-way ANOVA, followed by Tukey post hoc test).



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  		FIG. 11. Dose-response experiment on transcriptional regulation of CRFR1 in myometrial cells. A range of increasing concentrations for CRF or UCN (1, 10, or 100 nM) resulted in accordingly increasing levels of mRNA for CRFR1. Levels of significance were determined using one-way ANOVA, followed by a post hoc test. Values shown are ±SEM. NS was set at 100. *, P < 0.001.

 
The data presented on Figs. 5–11GoGoGoGoGoGoGo on cell lines and myometrial cells are representative of two independent experiments, each performed in triplicate. There was no apparent difference between the two experiments in the response at either the promoter activity or mRNA levels of CRFR1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we present our findings on the structure of human CRFR1 gene, including characterization of the 5' UTR, sequence analysis of its promoter region, and signaling mechanisms involved in the regulation of CRFR1 by CRF and UCN both at promoter and mRNA level. Using the human genome database, we revealed that CRFR1 spreads over a 50.3-kb region (exons 1–14). Our data complements previous work by Sakai et al. (18), who partially elucidated the CRFR1 gene sequence.

In addition to exploring the 5' structural organization of the human CRFR1 gene, we performed a sequence analysis of the promoter region. Interestingly, the promoter lacks a TATA box and TATA-associated factor binding sites. It is well known that Sp1 sites can drive transcription from TATA-less promoters (38). It is possible that Sp1 might participate in initiating transcription of CRFR1, due to the lack of a TATA box, because there are a high number of potential Sp1 elements clustered around the transcriptional start sites. The rat CRFR1 also displays no canonical TATA box, and it is an Sp1- and AP2-rich region (39). Similarly, we have also shown that the 5'-flanking region of the CRFR2ß, 2{gamma}, and 2{alpha} lack a functional TATA box (31). Other members of the same family of G protein-coupled receptors are also found to lack TATA boxes in the promoter region of the gene but do have a high number of potential Sp1 sites in its place, including the calcitonin receptor gene (40), glucagon-like peptide receptor-1 (41), vasointestinal peptide 1 receptor (42). Identification of the rat and mouse CRFR1 promoters has allowed recognition of sites that are likely to have an important role in controlling transcription because they have been conserved through evolution. The vast majority of the conserved sites among rat, mouse, and human are Sp1 elements, which are important in TATA-less transcription (43). The conserved Egr 2 (Krox-20) transcription factors in this region are particularly important because Egr factors are members of the zinc finger DNA binding transcription factors, participate in neuronal development, and are expressed in the adult pituitary, hippocampal, and hypothalamic neurons, all of which express CRFR1 (30, 44, 45). The absence of a TATA-box in the promoter region dictated a strategy of systematic deletion of the promoter regions to localize and identify the minimal promoter region necessary for basal transcription. The shortest DNA fragment with significant promoter activity encompassed the region from –374 to –9, relative to ATG (0.37-kb promoter). Sequence analysis of the 0.37-kb region (minimal promoter) for potential transcription factor binding sites predicts that Sp1 factors almost solely bind to this region.

Interestingly, the 2.0-kb region displays higher basal activity compared with the 2.4-kb full-length promoter region, as seen consistently in SH-5YSY, NT2, and MCF 7 cells.

This points toward potential repressive elements being present between –1990 and –2452, relative to ATG, that could determine the low basal levels of transient expression for 2.4 kb compared with the 2.0-kb promoter sequence. The 400-bp region contains repressor elements such as the YY1 (yin-yang 1) element at –2426 relative to ATG. YY1 is a highly conserved DNA-binding zinc finger transcription factor, which has been shown to act as an activator, repressor, or initiator of transcription (46, 47). The YY1 site may interact with other binding sites on the 2.4-kb promoter region and result in lower basal activity levels.

CRF has been previously shown to up-regulate the activity of the proopiomelanocortin and macrophage migration-inhibitory factor promoters (48, 49). It is attractive to speculate that CRF drives transcription of CRFR1, via promoter-initiated events. In contrast to NT2 cells, in MCF 7 cells there were no apparent CRF-mediated fluctuations in promoter activity when the latter were transfected with the 2.4- or 2.0-kb CRFR1 promoter. Furthermore, dexamethasone was unable to induce a change in CRFR1 promoter activity. Retinoic acid slightly down-regulated activity of both 2.4- and 2.0-kb regions, suggesting that the putative target element(s) for all-trans retinoic acid lies in the 2.0-kb region upstream of ATG.

Our investigation on the regulation of endogenous CRFR1 in human cell line systems has clearly shown a positive feedback on CRFR1, mediated by CRF and UCN. This might represent an important physiological system for amplifying stress responses. In NT2 cells, CRF and UCN up-regulate CRFR1 mRNA levels by 40-fold and 30-fold, respectively. Similarly, in MCF 7, CRF increases CRFR1 expression by 4-fold, and UCN by 1-fold. It was evident that although dexamethasone did not influence endogenous CRFR1 expression in NT2 cells, it acts as a potent mediator in MCF 7 cells, causing a 10-fold increase of mRNA. A plausible explanation for this differential action of glucocorticoids displayed by MCF 7 cells may be the high endogenous expression of steroid and glucocorticoid receptors in the particular cell type (50, 51, 52). MCF 7 cells are known to be glucocorticoid-responsive, and glucocorticoids were shown to stabilize the mRNA for insulin receptor in MCF 7 cells (53).

Our experiments showed differences between the effects of CRF and UCN in NT2 cells. Although CRF causes an increase in promoter activity for CRFR1, UCN has no effect.

A possible explanation might be far more potent coupling to Gq shown for UCN, when compared with CRF, in HEK293 cells stably expressing CRFR1 (54). This may be due to structural differences between the two peptides (55). The time-points of the treatments for luciferase assays (16–24 h) were different from those of the treatments for the RT-PCR studies in NT2 cells (4 h). As a result, the differences observed in NT2 cells raise the possibility that the two ligands can induce different conformations in the same receptor with different signaling consequences.

Several studies have reported positive effects mediated by CRF on CRFR1s. The hypothalamic paraventricular nucleus (PVN) is often a site of CRF-induced up-regulation of CRFRs. An example is the autoregulation of CRF biosynthesis in PVN through up-regulation of CRFR1, showing the mediation of positive effects on the amygdaloid CRF system (56). In rats, CRFR1 mRNA in the PVN is increased after stress, and this response is attenuated by central CRF blockade.

The attenuation of stress-induced CRFR1 mRNA responses suggests direct or indirect positive feedback effects of CRFR ligands on CRF hormone expression (57). This suggests that elevated levels of central CRF may trigger CRFR1 transcription selectively in the PVN and there is a positive feedback of CRF on its own receptor, acting as a functional adaptation of the hypothalamic-pituitary-adrenal axis in response to stress (58). Similarly, in the rat pituitary, CRF had a positive effect on CRFR1 mRNA levels, whereas dexamethasone had the opposite action in vivo (59). CRF has been shown to exert negative effects on CRFR1, such as the mRNA decrease of the receptor by CRF, in rat anterior pituitary cells (60) and a CRF-mediated down-regulation of CRFR1 binding efficiency in frontal rat cortex (61).

Dexamethasone has been shown to act as a negative regulator of CRFR1 mRNA expression in rodent anterior pituitary and PVN (62, 63, 64, 65, 66). A positive feedback of dexamethasone has been reported on the CRF gene in the human placenta (67).

CRF has also been implicated in the events related to endometrial implantation and pregnancy (68, 69). Uterine smooth muscle cells express endogenously CRF, UCN, and multiple CRFRs, the functionality of which is well documented (70, 71, 72). CRF has a central role in coordinating the smooth transition from a state of myometrial relaxation to one of contraction during human pregnancy (54, 73). We have therefore used this extensively studied system (i.e. primary myometrial cell cultures) to dissect the mechanism of the action of CRF and UCN.

Our findings demonstrate that UCN is more potent than CRF in inducing promoter activity and this phenomenon appears to be mediated by PKC- and PKA-dependent mechanisms, because inhibitors for both signaling cascades were able to inhibit the response of CRF and UCN. It appears that both peptides induced mRNA levels equipotently. The CRF effect was completely abolished by the PKA inhibitor and partially reversed by the PKC inhibitor, thus suggesting that CRF mediates its actions primarily via the activation of the adenylyl cyclase/cAMP/PKA pathway. Interestingly, the effect of UCN was abolished only by the PKC, but not the PKA, inhibitor. These data suggest that there may be a distinct UCN effect on the CRFR1 gene mediated exclusively via activation of a PKC-dependant pathway. This is in agreement with previous studies showing that UCN is far more potent than CRF in inducing coupling of myometrial CRFR1 to Gq (54). The induction of CRFR1 by UCN and CRF appears to be CRFR-specific, because use of the antagonist {alpha}-helical abolished their effects (data not shown).

However, the use of inhibitors of PKA and PKC may not be sufficient to define signal transduction cascade(s) activated by CRF or UCN ligands. The cells expressing the same receptor isoform may have surprisingly different coupling to second messengers for CRF and UCN (74).

Furthermore, the fact that both peptides induce a similar effect on crfr1 gene expression, points toward CRFR1-mediated effects, because both peptides have equal affinities for the CRFR1, and also primary myometrial cells tend to have a dramatic down-regulation of their CRFR2 in vitro, whereas the CRFR1 levels remain constant (75). Studies in our laboratory have shown that CRFRs are highly promiscuous and can couple to multiple G proteins upon activation of CRF and UCN. In our system, their effect appears to be PKA- and/or PKC-specific, because inhibition of Gi and Go by Pertussis toxin had no effect either on the promoter or the gene activity (data not shown).

Collectively, our data suggest a distinctive role for CRF and UCN in the control of CRFR1 gene expression. It is attractive to speculate that CRF and UCN might be implicated in potential positive feedback mechanisms acting at the transcriptional level to further modulate myometrial contractility by activating distinct signaling cascades.


   Acknowledgments
 
The authors thank Dr. Alison Jackson for her contribution in establishing the primary myometrial cell cultures.


   Footnotes
 
This work was supported by The Wellcome Trust, The City of Coventry General Charities, and the Diabetes Medical Research Fund UK (to E.W.H.).

K.L.P. and S.Z. contributed equally to this work and should both be considered first authors.

Data deposition: sequences reported in this paper have been deposited in the GenBank Database (accession no. AF488558).

Clone ID: LA1780-B1 HGMP transfer agreement documents are available.

Abbreviations: AP, Anchor primer; CRF, corticotropin-releasing factor; CRFR, CRF receptor; ECACC, European Collection of Animal Cell Cultures; FCS, fetal calf serum; GAPDH, glyceraldehyde-6-phosphate dehydrogenase; PK, protein kinase; PVN, paraventricular nucleus; UCN, urocortin; RACE, rapid amplification of cDNA ends; UTR, untranslated region.

Received February 13, 2004.

Accepted for publication May 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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