Expression and regulation of the rat prostaglandin E2 receptor type 4 (EP4) in pregnant cervical tissue

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Abstract

Objective

Prostaglandins play an important role in the regulation of parturition and cervical ripening. Prostaglandin E2 (PGE2)–induced tissue remodeling is mediated through activation of prostaglandin E2 receptor type 4 (EP4). EP4 is known to regulate matrix metalloproteinase secretion and expression and therefore may play a role in cervical ripening. We hypothesized that EP4 expression is regulated in the rat cervix to coincide with cervical ripening. In addition, we analyzed transcriptional regulation of the rat EP4 gene.

Study design

EP4 expression was evaluated in the cervix in timed pregnancy Sprague-Dawley rats by real-time reverse transcriptase polymerase chain reaction and Western blot. The genomic structure of the rat EP4 gene was determined by sequencing rat genomic clones obtained by plaque hybridization. Northern blots were performed to identify the number of different transcripts. The transcriptional start site was identified on the basis of sequence comparison to the human and mouse EP4 gene. The minimal promoter was identified by using reporter constructs containing portions of the 5′ flanking region. Reporter activity was evaluated in vitro. Identified regulatory regions were mutated to determine their role in transcription.

Results

Cervical EP4 expression (messenger RNA and protein) peaks on the day of parturition. The rat EP4 gene is structurally similar to other prostaglandin receptors as well as the human EP4 gene and contains three exons separated by two introns. The coding regions are located in the second and third exons separated in the sixth transmembrane spanning region. Northern blot identified a single EP4 transcript with an estimated size of 4 kb, indicating a single transcription initiation site. The first 80 bases of the 5′ flanking region were required for constitutive expression of EP4. Expression was lost after mutation of the GC/Sp1 binding site located at position –78 to –66 downstream from the transcription initiation site. Three Sp1 binding sites were identified and appear to cooperate to enhance EP4 transcription.

Conclusion

EP4 expression is regulated in the cervix and peaks on the day of parturition. The genomic structure of the human and rat EP4 gene is conserved between these two species. A GC rich/Sp1 binding site located within the first 80 bases of the transcription start site is important in transcription initiation of the EP4 gene.

Section snippets

Tissue and RNA extraction

For these studies, cervical tissue was surgically obtained from timed-pregnant Sprague-Dawley rats under pentobarbital sodium anesthesia (100 mg/kg) by use of a protocol approved by the Institutional Animal Care and Utilization Committee at the University of Chicago. Specifically, animals were killed in estrus and on gestational days 12, 16, 20, 21, and 22 (day of parturition), and day 0 post partum. All tissue was obtained before midday. Labor begins in the evening of day 22 with delivery

Results

Real-time RT-PCR measurements of cervical EP4 mRNA demonstrated increasing levels with advancing gestation (Fig 1). This increase was determined to be significant (ANOVA, P = .028). EP4 mRNA levels were relatively constant from estrus to day 21 of gestation. EP4 mRNA expression increased significantly on day 22 and postpartum day 0 over levels seen on day 12 and estrus (multiple comparisons, S-N-K method). EP4 mRNA expression doubles on day 22 compared with estrus levels.

EP4 Western blots of rat

Comment

Cervical ripening involves the remodeling of the extracellular matrix. Biochemical studies of the cervix have identified changes in the composition of the extracellular matrix with increases in collagenase activity.3., 20. Anatomic studies have demonstrated fragmentation of collagen fibrils and disruption of collagen bundles in the ripened cervix1., 20., 33. and in cervical tissue treated with PGE2.1., 3., 20. These changes were found to be similar in both the human and the rat in anatomic

References (48)

  • W. Rath et al.

    Biochemical changes in human cervical connective tissue after intracervical application of prostaglandin E2

    Prostaglandins

    (1993)
  • A. Calder et al.

    Prostaglandins and the unfavourable cervix

    Lancet

    (1973)
  • L.M. Wahl et al.

    Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs

    Cell Immunol

    (1987)
  • E. Segi et al.

    Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice

    Biochem Biophys Res Commun

    (1998)
  • E.K. Chien et al.

    Identification of gestationally regulated genes in rat myometrium by use of messenger ribonucleic acid differential display

    Am J Obstet Gynecol

    (1997)
  • S.M. Foord et al.

    The structure of the prostaglandin EP4 receptor gene and related pseudogenes

    Genomics

    (1996)
  • T. Arakawa et al.

    Prostanoid receptors of murine NIH 3T3 and RAW 264.7 cells: structure and expression of the murine prostaglandin EP4 receptor gene

    J Biol Chem

    (1996)
  • J.R. Owiny et al.

    Effect of intravaginal application of prostaglandin E2 gel on the mechanical properties of the ovine cervix uteri at term

    Am J Obstet Gynecol

    (1990)
  • C. Miyaura et al.

    Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

    J Biol Chem

    (2000)
  • J.P. Hapgood et al.

    Regulation of gene expression by GC-rich DNA cis-elements

    Cell Biol Internat

    (2001)
  • L.B. Augustin et al.

    Genomic structure of the mouse delta opoid receptor gene

    Biochem Biophys Res Commun

    (1995)
  • S. Safe

    Transcriptional acitivation of genes by 17 beta-estradiol through estrogen receptor-Sp1 interactions

    Vitam Horm

    (2001)
  • A.A. Calder

    Prostaglandins and biological control of cervical function

    Aust N Z J Obstet Gynaecol

    (1994)
  • M. Winkler et al.

    Changes in the cervical extracellular matrix during pregnancy and parturition

    J Perinat Med

    (1999)
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    Supported by University of Chicago Summer Research Stipend, NIH Grant for Basic Research, Society for Gynecological Investigation Summer Medical Student Grant, and the National Institute of Child Health and Human Development (grant No. HD01232-01).

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