Edward K Chien1, Candace Macgregor. 1. Section of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University of Vermont, 89 Beaumont Drive, Given C-262, Burlington, VT 05405, USA. Edward.Chien@uvm.edu
Abstract
OBJECTIVE: Prostaglandins play an important role in the regulation of parturition and cervical ripening. Prostaglandin E(2) (PGE(2))-induced tissue remodeling is mediated through activation of prostaglandin E(2) 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.
OBJECTIVE:Prostaglandins play an important role in the regulation of parturition and cervical ripening. Prostaglandin E(2) (PGE(2))-induced tissue remodeling is mediated through activation of prostaglandin E(2) 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 ratEP4 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 ratEP4 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 mouseEP4 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 ratEP4 gene is structurally similar to other prostaglandin receptors as well as the humanEP4 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 ratEP4 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.
Authors: Christine M O'Brien; Jennifer L Herington; Naoko Brown; Isaac J Pence; Bibhash C Paria; James C Slaughter; Jeff Reese; Anita Mahadevan-Jansen Journal: Sci Rep Date: 2017-07-28 Impact factor: 4.379