- Email: [email protected]
A new mRNA splice variant coding for the human EP3-I receptor isoform
ARTICLE IN PRESS
Prostaglandins, Leukotrienes and Essential Fatty Acids 77 (2007) 195–201 www.elsevier.com/locate/plefa
A new mRNA splice variant coding for the human EP3-I receptor isoform L. Kotelevetsa,b, N. Foudia,c, L. Louedeca,c, A. Couvelardd, E. Chastrea,b, X. Norela,c, a
CHU X-Bichat, Paris F-75018, France INSERM U773, Paris F-75018, France c INSERM U698, Paris F-75018, France d Department of Pathology, Beaujon Hospital, Clichy F-92110, France b
Received 13 June 2007; received in revised form 9 September 2007; accepted 15 September 2007
Abstract The cellular localization of prostaglandin E2 receptors (EP) and their corresponding transcripts were investigated in human gastric and vascular tissues. A strong staining of the EP3 receptor on the gastric glands, mucous cells, media of the mammary and pulmonary arteries was observed by immunohistochemistry. We identified a new mRNA splice variant of the EP3 gene in human gastric fundic mucosa, mammary artery and pulmonary vessels. This EP3-Ic transcript contains exons 1, 2, 3, 5 and 6 of the EP3 gene and should be translated in the EP3-I isoform. In addition, the EP3-Ib, EP3-II, EP3-III, EP3-IV and EP3-e mRNAs were detected in these tissues. r 2007 Elsevier Ltd. All rights reserved. Keywords: EP3 receptor isoforms; Human vascular preparations; Human gastric fundic mucosa; EP1; EP2; EP4; Prostaglandin
1. Introduction Prostanoid synthesis is dependent on the metabolism of arachidonic acid by the initial and rate-limiting enzymatic activity of cyclooxygenase (COX). The different prostaglandins (PG) and thromboxane produced also depend on the presence of other specific enzymes, such as prostaglandin E synthase (PGES) which is responsible for the synthesis of PGE2. The inducible COX-2 and PGES-1 isoforms are up-regulated in some patho-physiological situations: in human gastric ulcer tissues [1] and carotid atherosclerosis plaques [2]. Abbreviations: COX, cyclooxygenase; PG, prostaglandin; PGES, prostaglandin E synthase; IMA, internal mammary artery; PA, pulmonary artery; PV, pulmonary vein; GFM, gastric fundic mucosa; Epith, gastric fundic epithelium. Corresponding author. INSERM U698, CHU X. Bichat, secteur Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France. Tel.: +33 1 40 25 75 29; fax: +33 1 40 25 86 02. E-mail address: [email protected] (X. Norel). 0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.09.005
PGE2 mediates its effects via four receptor subtypes (EP1, EP2, EP3, EP4) and these receptors are found in many human tissues. In humans, these subtypes are involved in the control of physiological events, such as, the vascular smooth muscle tone [3–5], angiogenesis [6], inflammation of the vascular wall [7] and the gastric mucosa secretions [8]. Among the EP receptor subtypes, the expression of the EP3 is more complex since different isoforms derive from alternative splicing of the gene. In human tissues, nine mRNAs and eight isoforms (EP3-I, EP3-II, EP3-III, EP3-IV, EP3-V, EP3-VI, EP3-e, EP3-f) have been described [9–11]. These EP3 isoforms differ in their C-terminus and their different signal transduction pathways [9]. The activation of the EP3-I, EP3-II, EP3III, EP3-IV, EP3-e and EP3-f isoforms have been shown to inhibit cAMP production, while stimulation of EP3-I, EP3-II and EP3-III could also increase IP3/[Ca2+]i [9,10]. The expression patterns of the EP3 isoforms are poorly documented and their different physiological roles remain unknown.
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The aim of the present study was to characterize the EP3 isoforms present in the human gastric fundic mucosa (GFM), internal mammary artery (IMA), pulmonary artery (PA) and vein (PV). In addition, the cellular localization of the EP3 receptor subtype in these tissues as well as a comparison with the localization of the other EP receptor subtype in the GFM was also performed by immunohistochemistry.
ratus. RNAs were isolated using ultracentrifugation on CsCl. The identification of the EP receptor (32 cycles) and GAPDH transcripts were performed by RT-PCR and confirmed by Southern blot analysis: hybridization with P32-end-labeled internal probes of RT-PCR products was performed by autoradiography (see Table 1 for the primers and probes used). The autoradiographic bands were quantified as optical density band area by Scion Image software and normalized with the GAPDH mRNA product.
2. Materials and methods 2.4. DNA sequencing 2.1. Isolation of human tissue All research programs involving the use of human tissue were approved by the medical ethics committee at Beaujon and Bichat hospitals. The human gastric preparations, the IMA and the pulmonary vessels were obtained from patients (12 males, 63703 years old and 1 female, 56 years old) who had undergone surgery for coronary bypass, stomach or lung cancer. Each surgical sample was examined by a pathologist and the remaining macroscopically healthy tissue was provided for experimentation. The GFM as well as the pulmonary vessels were dissected from these samples. The fundic epithelial cells were obtained after incubation of the gastric mucosa at 4 1C in a solution containing 0.24 M NaCl, 2.5 mM EDTA for 1 h at 4 1C followed by gentle shakings. All the preparations were snap frozen and stored at 80 1C. 2.2. Immunohistochemistry Transverse slices (5 mm) of human GFM, IMA, PA and PV were obtained from paraffin embedded preparations. The sections were submitted to high temperature (80 1C) antigen unmasking technique using Vector’s solution (H-3300). Cayman (Cay: anti-EP1, -EP2, -EP3), LifeSpam (LS: anti-EP3), Santa-Cruz (SC: anti-EP1) or Alpha Diagnostic International (ADI: antiEP2, -EP3, -EP4) polyclonal antibodies were used as primary antibodies (1/100) raised against human EP receptors. The anti-EP1 SC antibody was raised in goat while all the others were rabbit primary antibodies. Biotinylated anti-rabbit or anti-goat was the secondary antibody, and peroxydase Vectastains Elite ABC kits were used for detection followed by haematoxylin treatment for cell nuclei staining. In addition, control slices were incubated without primary antibody, with a rabbit non-immune antibody (Dako Cytomation), or with the Cayman anti-EP3 primary antibody and its respective blocking peptide. 2.3. RT-PCR and Southern blot analysis The vascular and gastric preparations were disrupted in guanidinium isothyocyanate, using a polytron appa-
The RT-PCR product of about 500 bp in Fig. 3a was extracted from agarose gel and purified with a Nucleospin Extract II kit (Macherey-Nagel); this RT-PCR product was subjected to direct sequencing (GENOME express; Meylan, France).
3. Results Immunohistochemistry, performed on gastric preparations using at least two different polyclonal antibodies for each EP receptor subtype provided similar results (Fig. 1). A low to moderate staining of the gastric glands was obtained with the EP1 antibodies. The presence of the EP2, EP3 and EP4 receptor subtypes was identified on the gastric glands. The EP3 subtype was also strongly detected on the mucous cells covering the gastric pits. This staining was localized either on the basolateral (Cay, ADI) or the lateral/apical (LS) side of the cells depending of the antibody used. The EP2 receptor subtype was also observed in the lamina propria (Fig. 1). RT-PCR experiments revealed the presence of the four transcripts corresponding to EP1, Table 1 Primers and probes used in RT-PCR and Southern blot for human EP3 receptor splice variants and GAPDH Primer/P32-probe
Exon-S/AS
Sequence (50 –30 )
EP3 primers P1 P2 P3 P6 P10
Exon1-S Exon2-AS Exon3-AS Exon6-AS Exon10-AS
cgagacggccattcagctta agagcagctggagacagcat aggtggagctggatgcatag gctgctcacgagtacctccat gagtcatggagcttccagtgatg
Exon1-S Exon5-S Exon10-AS
cgagacggccattcagctta atgagaaaaagaagactcagagagcaa atcctggcagaaaggcaggt
S AS
atcaccatcttccaggagcg cctgcttcaccaccttcttg
EP3 P32-probes
GAPDH Primer Primer
S, sense; AS, anti-sense.
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Fig. 1. Paraffin section of human gastric fundic mucosa, immunohistochemistry using anti-EP1, -EP2, -EP3 or -EP4 antibodies 1/100 (Cay, cayman; SC, santa-cruz; ADI, alpha diagnostic intl.; LS, lifespam). Staining is observed on the surface mucous epithelial cells (black arrow), on the lamina propria (arrow head) and on the gastric glands (white arrow). The nuclei are stained in blue by haematoxylin. Representative results derived from four different experiments and 40 paraffin sections analysed.
EP2, EP3 and EP4 receptor subtypes in the human gastric mucosa (data not shown). In addition, the EP3 receptor staining was observed in the human IMA, PA and to a lesser extend in PV (Fig. 2). This staining was mainly observed within the media on the smooth muscle cells and not on the endothelium (Fig. 2). A similar staining was observed with the three different EP3 antibodies in human IMA (data not shown). Since the EP3 receptor subtype proved to be highly expressed in gastric and vascular preparations, we further investigated the pattern of expression of the EP3 isoform mRNA. The results presented in Fig. 3 revealed that the transcripts encoding EP3-Ib, EP3-II, EP3-III, EP3-IV and EP3-e are expressed in the human GFM, IMA and pulmonary vessels. In addition, the absence of 500, 479 or 575 bp autoradiographic band within the RT-PCR products obtained using the combination of primers P1 and P10, excludes, respectively, the presence of the EP3-V, EP3-VI and EP3-f isoforms in these human tissues. An unexpected cDNA (500 bp) was identified (Fig. 3a); this mRNA was expressed in all the tissues and tentatively designed EP3Ic. Sequencing of 463 bp of this cDNA revealed the presence of exons 1, 2a, 3, 5 and 6 of the EP3 gene
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Fig. 2. Paraffin section of human mammary artery (IMA), pulmonary artery (PA) and pulmonary vein (PV). Representative results derived from nine different individuals, seven different experiments, 46 paraffin sections analysed. Control indicates immunohistochemistry performed without primary antibody (IMA) or with a non-immune antibody (PA, PV). Immunohistochemistry was performed using anti-EP3 antibodies (IMA: LifeSpam; PA and PV: Cayman). Staining was detected in the smooth muscle layer (black arrow) while the endothelium was not stained (arrow head).
(Tables 2 and 3). The quantity of different EP3 isoform transcripts normalized for GAPDH expression revealed that the isoforms EP3-Ib, EP3-Ic, EP3-III, EP3-IV and EP3-e were more prominent in human PA as compared to PV (Fig. 4).
4. Discussion Our results demonstrate the expression of a new mRNA splice variant (EP3-Ic) coding for the EP3 receptor in human gastric and vascular tissues. This transcript would be translated into EP3-I isoform similarly to the EP3-Ia and EP3-Ib mRNA transcripts, where only the first three exons are translated. The expected length of the complete EP3-Ic transcript with exons 1, 2a, 3, 5, and 6 would be 2055 bp. There are now, three mRNA transcripts coding for the EP3-I isoform and a suitable approach to quantify selectively these three transcripts by RT-PCR together, will be to use a (sense) primer localized in exon (1 or 2a) and the (anti-sense) primer in exon 3. The biological significance of the different 30 -UTR region of EP3-I isoforms RNA remains elusive. Nevertheless, these regions are the target of microRNA that could control both RNA stability and translation. In addition, the present report suggests that the EP3-I and EP3-II mRNAs are the most expressed isoforms in each tissue. PGE2 has been largely implicated in gastric cytoprotection by the control of the gastric acid, the mucus or
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GFM
Epith IMA IMA PA
PV 463 bp (EP3-Ic) 380 bp (EP3-e)
600 bp 500 bp
400 bp
300 bp
432 bp (EP3-Ib) 340 bp (EP3-IV)
600 bp 500 bp 432 bp (EP3-Ib)
400 bp
340 bp (EP3-IV)
300 bp
313 bp (EP3-III)
270 bp (EP3-II)
283 bp (EP3-I)
566 bp (GAPDH)
Fig. 3. Southern hybridation of RT-PCR products showing the relative levels of expression of the EP3 isoforms and GAPDH in different human tissues (GFM: gastric fundic mucosa; Epith: gastric fundic epithelium; IMA: internal mammary artery; PA: pulmonary artery; PV: pulmonary vein). The size markers (2-log ladder, New England Biolabs) are shown on the left (b, c). The exposure time was 45 min without intensifying screen (d, e), 3 h with screen (a, b, f), 2 h 30 min with screen (c: left) and 15 h with screen (c: right). The primers and probes used are indicated beneath each panel and described in Table 1. (a) Primers P1 and P6 and P32-probe in exon 10; (b) primers P1 and P10 and P32-probe in exon 5; (c) primers P1 and P10 and P32-probe in exon 10; (d) primers P1 and P2 and P32-probe in exon 1; (e) primers P1 and P3 and P32-probe in exon 1 and (f) RT-PCR products correlating to GAPDH.
the HCO secretions. In the dog gastric fundic 3 epithelium, it has been shown that the inhibition of acid secretion is mediated via the EP3 receptor [12]. The present report determines more specifically the EP3 isoform mRNAs which are expressed in the human GFM and epithelial cells. In another study with samples of human stomach, the expression of EP3-Ia, EP3-Ib, EP3III, EP3-IV, EP3-V and EP3-VI mRNAs had not been detected while only the EP3-II mRNA was weakly expressed [9,13]. This discrepancy with our results might be attributed to the origin of the biological samples (autopsy versus surgical samples) and/or the use of different area of the stomach.
Takafuji et al. [8] reported the EP2, EP3, EP4 but not the EP1 receptor subtypes using immunohistochemistry, in normal human gastric mucosa and more specifically, on gastric epithelia lining gastric pits. These results are partially in accordance with the results obtained in the present report using human GFM (Fig. 1) where EP2 and EP3 receptor subtypes are dominant in the upper gastric mucosa. However, in the present report, the EP4 receptor was only detected in the gastric glands, while the EP2 receptor was found in both the glands and lamina propria (Fig. 1). This difference may be explained by the use of a different stomach region in the study of Takafuji et al. [8]. In the rodent stomach,
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Table 2 The different exons found in mRNA transcripts generated by alternative splicing of the human EP3 gene
EP3-Ia EP3-Ib EP3-Ic EP3-II EP3-III EP3-IV EP3-V EP3-VI EP3-e EP3-f
1 1 1 1 1 1 1 1 1 1
2a 2a 2a 2b 2a 2a 2a 2a 2a 2a
3 3 3
4 5 5
10 6
5 5 5 5
7 7
9 9
10 10 10 10
9
10
6 8
Consensus polyadenylation signals are located at the end of exons 2, 4, 6 and 10. The termination codons are positioned at the end of the translated region of exons 2b, 3, 6, 10 and at the beginning of exon 5.
1.2
Table 3 Nucleotide sequence of the purified 463 bp cDNA of the human EP3-Ic isoform detected in Fig. 3a
Exon 5
94020 A TGA GAA AAA GAA GAC TCA GAG AGC AA 94046
Exon 6
94750 T TAA TCT GCA GTT TGC AGA ACT CTC AGA TAC AGA GGG CAA CTG CAC ATT GTG GGC AAG TAC AAA CCT ATC GTG TGC TGA ACA GAG AAG AAA TGG AGG TAC TCG TGA GCA GCA 94861
This nucleotide sequence is registered under Genbank/BankIt ID number: EF534325. The numeration starts from the first nucleotide of the first exon determined in the EP3 gene sequence (NT_032977.8). At the beginning of exon 5, a termination codon (bold text) is found.
the EP receptor subtypes responsible for the control of the mucus and HCO 3 secretions are the EP4 and EP1, respectively [14–17]. In contrast, the human stomach is markedly different since the mucous cells covering the gastric pits preferentially exhibited EP3 but neither the EP4 or EP1 receptors (present report; [8]). Finally,
0.0 EP3-e
73421 ATC AGG TAC CAC ACA AAC AAC TAT GCA TCC AGC TCC ACC TCC TTA CCC TGC CAG TGT TCC TCA ACC TTG ATG TGG AGC GAC CAT TTG GAA AG 73512
0.2
EP3-IV
Exon 3
0.4
EP3-III
35325 ATA ATG ATG TTG AAA ATG ATC TTC AAT CAG ACA TCA GTT GAG CAC TGC AAG ACA CAC ACG GAG AAG CAG AAA GAA TGC AAC TTC TTC TTA ATA GCT GTT CGC CTG GCT TCA CTG AAC CAG ATC TTG GAT CCT TGG GTT TAC CTG CTG TTA AGA AAG ATC CTT CTT CGA AAG TTT TGC CAG 35504
EP3-II
Exon 2a
0.6
EP3-Ic
1077 T CAG CTT ATG GGG ATC ATG TGC GTG CTG TCG GTC TGC TGG TCT CCG CTC CTG 1128
0.8
EP3-Ib
Exon 1
1.0
EP3-I
cDNA sequence 50 –30
EP3 / GAPDH
EP3-Ic exons
Pulmonary artery Pulmonary vein
Fig. 4. EP3 isoform mRNAs detected in human pulmonary vessels derived from one individual. Southern hybridation of RT-PCR products of the EP3 isoforms were normalized with the GAPDH products (arbitrary units). The exposure time was 45 min without screen (EP3-I; EP3-II), with screen 2 h 30 min (EP3-III) and 3 h (EP3-Ib; EP3-Ic; EP3-IV; EP3-e; GAPDH).
different cellular localization of the staining was obtained with the LS antibody (lateral/apical) in comparison with the other anti-EP3 antibodies (basolateral). This discrepancy could be due to the different epitopes recognized; Cay and ADI antibodies recognize extracellular epitopes while LS antibody recognizes an intracellular epitope. Another explanation could be the different sub-cellular localization of the different EP3 isoforms: Cay and ADI antibodies recognize epitopes common to all isoforms while LS antibody was raised against an EP3-f epitope which is found in part in EP3-IV, EP3-V and EP3-e.
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In the vascular wall, there is increasing evidence for different roles of the EP3 receptor subtype. M&B28767, a selective agonist for EP3 receptors, stimulates the migration of the human vascular smooth muscle cells derived from the IMA [18]. Neovascularization induced by PGE2 via the EP3 receptor have been shown in rodents [19,20]. Activation of the EP3 receptor is responsible for the contraction of human PA [3] and IMA [21]. These results are in accordance with the present report where in human IMA and PA, the EP3 receptor mRNAs were detected and the protein localized in the vascular smooth muscle by immunohistochemistry (Figs. 2–4). However, the EP3 isoforms which are involved in the physiological events described above remains to be determined. The absence of the EP3-V, EP3-VI or EP3-f receptor in the present report excludes their involvement in the vasoconstriction of human IMA or PA. Previous reports have shown that in human pulmonary vessels the contractions induced by PGE2 involve the EP3 receptor only in the artery and not in the vein [3,4]. The results obtained by immunohistochemistry (Fig. 2) provide the biochemical basis for these pharmacological reports since they suggest a stronger expression of the EP3 receptor subtype in human PA in comparison with PV. Furthermore, the data presented (Fig. 4) indicate that most of the EP3 isoform mRNAs were markedly expressed in the human PA when compared with the PV. In all the preparations, the EP3-Ia mRNA was not investigated. However, in the IMA and the PV low EP3-Ib, EP3-Ic mRNA expression (Fig. 3a and b) and the greater signal obtained for the total EP3-I (Fig. 3e) suggest the presence of the EP3-Ia mRNA. Finally, the different EP3 isoform mRNAs found in IMA and pulmonary vessels are similar to those reported in smooth muscle cells derived from human umbilical artery (EP3-Ib, EP3-III, EP3-IV and no EP3-II, EP3-VI) [22] and aorta (EP3-Ib, EP3-II, EP3-III, EP3IV) [9,13,23]. In conclusion, our results suggest that there are at least 10 mRNAs splice variants coding for the eight different EP3 isoforms. The different cellular localizations of the EP3 isoforms and the EP subtypes may be associated with different physiological roles. Finally, the cellular distribution and expressions of the EP receptors are dependent on the tissue area and the species studied.
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Prostaglandins, Leukotrienes and Essential Fatty Acids 77 (2007) 195–201 www.elsevier.com/locate/plefa
A new mRNA splice variant coding for the human EP3-I receptor isoform L. Kotelevetsa,b, N. Foudia,c, L. Louedeca,c, A. Couvelardd, E. Chastrea,b, X. Norela,c, a
CHU X-Bichat, Paris F-75018, France INSERM U773, Paris F-75018, France c INSERM U698, Paris F-75018, France d Department of Pathology, Beaujon Hospital, Clichy F-92110, France b
Received 13 June 2007; received in revised form 9 September 2007; accepted 15 September 2007
Abstract The cellular localization of prostaglandin E2 receptors (EP) and their corresponding transcripts were investigated in human gastric and vascular tissues. A strong staining of the EP3 receptor on the gastric glands, mucous cells, media of the mammary and pulmonary arteries was observed by immunohistochemistry. We identified a new mRNA splice variant of the EP3 gene in human gastric fundic mucosa, mammary artery and pulmonary vessels. This EP3-Ic transcript contains exons 1, 2, 3, 5 and 6 of the EP3 gene and should be translated in the EP3-I isoform. In addition, the EP3-Ib, EP3-II, EP3-III, EP3-IV and EP3-e mRNAs were detected in these tissues. r 2007 Elsevier Ltd. All rights reserved. Keywords: EP3 receptor isoforms; Human vascular preparations; Human gastric fundic mucosa; EP1; EP2; EP4; Prostaglandin
1. Introduction Prostanoid synthesis is dependent on the metabolism of arachidonic acid by the initial and rate-limiting enzymatic activity of cyclooxygenase (COX). The different prostaglandins (PG) and thromboxane produced also depend on the presence of other specific enzymes, such as prostaglandin E synthase (PGES) which is responsible for the synthesis of PGE2. The inducible COX-2 and PGES-1 isoforms are up-regulated in some patho-physiological situations: in human gastric ulcer tissues [1] and carotid atherosclerosis plaques [2]. Abbreviations: COX, cyclooxygenase; PG, prostaglandin; PGES, prostaglandin E synthase; IMA, internal mammary artery; PA, pulmonary artery; PV, pulmonary vein; GFM, gastric fundic mucosa; Epith, gastric fundic epithelium. Corresponding author. INSERM U698, CHU X. Bichat, secteur Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France. Tel.: +33 1 40 25 75 29; fax: +33 1 40 25 86 02. E-mail address: [email protected] (X. Norel). 0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.09.005
PGE2 mediates its effects via four receptor subtypes (EP1, EP2, EP3, EP4) and these receptors are found in many human tissues. In humans, these subtypes are involved in the control of physiological events, such as, the vascular smooth muscle tone [3–5], angiogenesis [6], inflammation of the vascular wall [7] and the gastric mucosa secretions [8]. Among the EP receptor subtypes, the expression of the EP3 is more complex since different isoforms derive from alternative splicing of the gene. In human tissues, nine mRNAs and eight isoforms (EP3-I, EP3-II, EP3-III, EP3-IV, EP3-V, EP3-VI, EP3-e, EP3-f) have been described [9–11]. These EP3 isoforms differ in their C-terminus and their different signal transduction pathways [9]. The activation of the EP3-I, EP3-II, EP3III, EP3-IV, EP3-e and EP3-f isoforms have been shown to inhibit cAMP production, while stimulation of EP3-I, EP3-II and EP3-III could also increase IP3/[Ca2+]i [9,10]. The expression patterns of the EP3 isoforms are poorly documented and their different physiological roles remain unknown.
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The aim of the present study was to characterize the EP3 isoforms present in the human gastric fundic mucosa (GFM), internal mammary artery (IMA), pulmonary artery (PA) and vein (PV). In addition, the cellular localization of the EP3 receptor subtype in these tissues as well as a comparison with the localization of the other EP receptor subtype in the GFM was also performed by immunohistochemistry.
ratus. RNAs were isolated using ultracentrifugation on CsCl. The identification of the EP receptor (32 cycles) and GAPDH transcripts were performed by RT-PCR and confirmed by Southern blot analysis: hybridization with P32-end-labeled internal probes of RT-PCR products was performed by autoradiography (see Table 1 for the primers and probes used). The autoradiographic bands were quantified as optical density band area by Scion Image software and normalized with the GAPDH mRNA product.
2. Materials and methods 2.4. DNA sequencing 2.1. Isolation of human tissue All research programs involving the use of human tissue were approved by the medical ethics committee at Beaujon and Bichat hospitals. The human gastric preparations, the IMA and the pulmonary vessels were obtained from patients (12 males, 63703 years old and 1 female, 56 years old) who had undergone surgery for coronary bypass, stomach or lung cancer. Each surgical sample was examined by a pathologist and the remaining macroscopically healthy tissue was provided for experimentation. The GFM as well as the pulmonary vessels were dissected from these samples. The fundic epithelial cells were obtained after incubation of the gastric mucosa at 4 1C in a solution containing 0.24 M NaCl, 2.5 mM EDTA for 1 h at 4 1C followed by gentle shakings. All the preparations were snap frozen and stored at 80 1C. 2.2. Immunohistochemistry Transverse slices (5 mm) of human GFM, IMA, PA and PV were obtained from paraffin embedded preparations. The sections were submitted to high temperature (80 1C) antigen unmasking technique using Vector’s solution (H-3300). Cayman (Cay: anti-EP1, -EP2, -EP3), LifeSpam (LS: anti-EP3), Santa-Cruz (SC: anti-EP1) or Alpha Diagnostic International (ADI: antiEP2, -EP3, -EP4) polyclonal antibodies were used as primary antibodies (1/100) raised against human EP receptors. The anti-EP1 SC antibody was raised in goat while all the others were rabbit primary antibodies. Biotinylated anti-rabbit or anti-goat was the secondary antibody, and peroxydase Vectastains Elite ABC kits were used for detection followed by haematoxylin treatment for cell nuclei staining. In addition, control slices were incubated without primary antibody, with a rabbit non-immune antibody (Dako Cytomation), or with the Cayman anti-EP3 primary antibody and its respective blocking peptide. 2.3. RT-PCR and Southern blot analysis The vascular and gastric preparations were disrupted in guanidinium isothyocyanate, using a polytron appa-
The RT-PCR product of about 500 bp in Fig. 3a was extracted from agarose gel and purified with a Nucleospin Extract II kit (Macherey-Nagel); this RT-PCR product was subjected to direct sequencing (GENOME express; Meylan, France).
3. Results Immunohistochemistry, performed on gastric preparations using at least two different polyclonal antibodies for each EP receptor subtype provided similar results (Fig. 1). A low to moderate staining of the gastric glands was obtained with the EP1 antibodies. The presence of the EP2, EP3 and EP4 receptor subtypes was identified on the gastric glands. The EP3 subtype was also strongly detected on the mucous cells covering the gastric pits. This staining was localized either on the basolateral (Cay, ADI) or the lateral/apical (LS) side of the cells depending of the antibody used. The EP2 receptor subtype was also observed in the lamina propria (Fig. 1). RT-PCR experiments revealed the presence of the four transcripts corresponding to EP1, Table 1 Primers and probes used in RT-PCR and Southern blot for human EP3 receptor splice variants and GAPDH Primer/P32-probe
Exon-S/AS
Sequence (50 –30 )
EP3 primers P1 P2 P3 P6 P10
Exon1-S Exon2-AS Exon3-AS Exon6-AS Exon10-AS
cgagacggccattcagctta agagcagctggagacagcat aggtggagctggatgcatag gctgctcacgagtacctccat gagtcatggagcttccagtgatg
Exon1-S Exon5-S Exon10-AS
cgagacggccattcagctta atgagaaaaagaagactcagagagcaa atcctggcagaaaggcaggt
S AS
atcaccatcttccaggagcg cctgcttcaccaccttcttg
EP3 P32-probes
GAPDH Primer Primer
S, sense; AS, anti-sense.
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Fig. 1. Paraffin section of human gastric fundic mucosa, immunohistochemistry using anti-EP1, -EP2, -EP3 or -EP4 antibodies 1/100 (Cay, cayman; SC, santa-cruz; ADI, alpha diagnostic intl.; LS, lifespam). Staining is observed on the surface mucous epithelial cells (black arrow), on the lamina propria (arrow head) and on the gastric glands (white arrow). The nuclei are stained in blue by haematoxylin. Representative results derived from four different experiments and 40 paraffin sections analysed.
EP2, EP3 and EP4 receptor subtypes in the human gastric mucosa (data not shown). In addition, the EP3 receptor staining was observed in the human IMA, PA and to a lesser extend in PV (Fig. 2). This staining was mainly observed within the media on the smooth muscle cells and not on the endothelium (Fig. 2). A similar staining was observed with the three different EP3 antibodies in human IMA (data not shown). Since the EP3 receptor subtype proved to be highly expressed in gastric and vascular preparations, we further investigated the pattern of expression of the EP3 isoform mRNA. The results presented in Fig. 3 revealed that the transcripts encoding EP3-Ib, EP3-II, EP3-III, EP3-IV and EP3-e are expressed in the human GFM, IMA and pulmonary vessels. In addition, the absence of 500, 479 or 575 bp autoradiographic band within the RT-PCR products obtained using the combination of primers P1 and P10, excludes, respectively, the presence of the EP3-V, EP3-VI and EP3-f isoforms in these human tissues. An unexpected cDNA (500 bp) was identified (Fig. 3a); this mRNA was expressed in all the tissues and tentatively designed EP3Ic. Sequencing of 463 bp of this cDNA revealed the presence of exons 1, 2a, 3, 5 and 6 of the EP3 gene
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Fig. 2. Paraffin section of human mammary artery (IMA), pulmonary artery (PA) and pulmonary vein (PV). Representative results derived from nine different individuals, seven different experiments, 46 paraffin sections analysed. Control indicates immunohistochemistry performed without primary antibody (IMA) or with a non-immune antibody (PA, PV). Immunohistochemistry was performed using anti-EP3 antibodies (IMA: LifeSpam; PA and PV: Cayman). Staining was detected in the smooth muscle layer (black arrow) while the endothelium was not stained (arrow head).
(Tables 2 and 3). The quantity of different EP3 isoform transcripts normalized for GAPDH expression revealed that the isoforms EP3-Ib, EP3-Ic, EP3-III, EP3-IV and EP3-e were more prominent in human PA as compared to PV (Fig. 4).
4. Discussion Our results demonstrate the expression of a new mRNA splice variant (EP3-Ic) coding for the EP3 receptor in human gastric and vascular tissues. This transcript would be translated into EP3-I isoform similarly to the EP3-Ia and EP3-Ib mRNA transcripts, where only the first three exons are translated. The expected length of the complete EP3-Ic transcript with exons 1, 2a, 3, 5, and 6 would be 2055 bp. There are now, three mRNA transcripts coding for the EP3-I isoform and a suitable approach to quantify selectively these three transcripts by RT-PCR together, will be to use a (sense) primer localized in exon (1 or 2a) and the (anti-sense) primer in exon 3. The biological significance of the different 30 -UTR region of EP3-I isoforms RNA remains elusive. Nevertheless, these regions are the target of microRNA that could control both RNA stability and translation. In addition, the present report suggests that the EP3-I and EP3-II mRNAs are the most expressed isoforms in each tissue. PGE2 has been largely implicated in gastric cytoprotection by the control of the gastric acid, the mucus or
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GFM
Epith IMA IMA PA
PV 463 bp (EP3-Ic) 380 bp (EP3-e)
600 bp 500 bp
400 bp
300 bp
432 bp (EP3-Ib) 340 bp (EP3-IV)
600 bp 500 bp 432 bp (EP3-Ib)
400 bp
340 bp (EP3-IV)
300 bp
313 bp (EP3-III)
270 bp (EP3-II)
283 bp (EP3-I)
566 bp (GAPDH)
Fig. 3. Southern hybridation of RT-PCR products showing the relative levels of expression of the EP3 isoforms and GAPDH in different human tissues (GFM: gastric fundic mucosa; Epith: gastric fundic epithelium; IMA: internal mammary artery; PA: pulmonary artery; PV: pulmonary vein). The size markers (2-log ladder, New England Biolabs) are shown on the left (b, c). The exposure time was 45 min without intensifying screen (d, e), 3 h with screen (a, b, f), 2 h 30 min with screen (c: left) and 15 h with screen (c: right). The primers and probes used are indicated beneath each panel and described in Table 1. (a) Primers P1 and P6 and P32-probe in exon 10; (b) primers P1 and P10 and P32-probe in exon 5; (c) primers P1 and P10 and P32-probe in exon 10; (d) primers P1 and P2 and P32-probe in exon 1; (e) primers P1 and P3 and P32-probe in exon 1 and (f) RT-PCR products correlating to GAPDH.
the HCO secretions. In the dog gastric fundic 3 epithelium, it has been shown that the inhibition of acid secretion is mediated via the EP3 receptor [12]. The present report determines more specifically the EP3 isoform mRNAs which are expressed in the human GFM and epithelial cells. In another study with samples of human stomach, the expression of EP3-Ia, EP3-Ib, EP3III, EP3-IV, EP3-V and EP3-VI mRNAs had not been detected while only the EP3-II mRNA was weakly expressed [9,13]. This discrepancy with our results might be attributed to the origin of the biological samples (autopsy versus surgical samples) and/or the use of different area of the stomach.
Takafuji et al. [8] reported the EP2, EP3, EP4 but not the EP1 receptor subtypes using immunohistochemistry, in normal human gastric mucosa and more specifically, on gastric epithelia lining gastric pits. These results are partially in accordance with the results obtained in the present report using human GFM (Fig. 1) where EP2 and EP3 receptor subtypes are dominant in the upper gastric mucosa. However, in the present report, the EP4 receptor was only detected in the gastric glands, while the EP2 receptor was found in both the glands and lamina propria (Fig. 1). This difference may be explained by the use of a different stomach region in the study of Takafuji et al. [8]. In the rodent stomach,
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Table 2 The different exons found in mRNA transcripts generated by alternative splicing of the human EP3 gene
EP3-Ia EP3-Ib EP3-Ic EP3-II EP3-III EP3-IV EP3-V EP3-VI EP3-e EP3-f
1 1 1 1 1 1 1 1 1 1
2a 2a 2a 2b 2a 2a 2a 2a 2a 2a
3 3 3
4 5 5
10 6
5 5 5 5
7 7
9 9
10 10 10 10
9
10
6 8
Consensus polyadenylation signals are located at the end of exons 2, 4, 6 and 10. The termination codons are positioned at the end of the translated region of exons 2b, 3, 6, 10 and at the beginning of exon 5.
1.2
Table 3 Nucleotide sequence of the purified 463 bp cDNA of the human EP3-Ic isoform detected in Fig. 3a
Exon 5
94020 A TGA GAA AAA GAA GAC TCA GAG AGC AA 94046
Exon 6
94750 T TAA TCT GCA GTT TGC AGA ACT CTC AGA TAC AGA GGG CAA CTG CAC ATT GTG GGC AAG TAC AAA CCT ATC GTG TGC TGA ACA GAG AAG AAA TGG AGG TAC TCG TGA GCA GCA 94861
This nucleotide sequence is registered under Genbank/BankIt ID number: EF534325. The numeration starts from the first nucleotide of the first exon determined in the EP3 gene sequence (NT_032977.8). At the beginning of exon 5, a termination codon (bold text) is found.
the EP receptor subtypes responsible for the control of the mucus and HCO 3 secretions are the EP4 and EP1, respectively [14–17]. In contrast, the human stomach is markedly different since the mucous cells covering the gastric pits preferentially exhibited EP3 but neither the EP4 or EP1 receptors (present report; [8]). Finally,
0.0 EP3-e
73421 ATC AGG TAC CAC ACA AAC AAC TAT GCA TCC AGC TCC ACC TCC TTA CCC TGC CAG TGT TCC TCA ACC TTG ATG TGG AGC GAC CAT TTG GAA AG 73512
0.2
EP3-IV
Exon 3
0.4
EP3-III
35325 ATA ATG ATG TTG AAA ATG ATC TTC AAT CAG ACA TCA GTT GAG CAC TGC AAG ACA CAC ACG GAG AAG CAG AAA GAA TGC AAC TTC TTC TTA ATA GCT GTT CGC CTG GCT TCA CTG AAC CAG ATC TTG GAT CCT TGG GTT TAC CTG CTG TTA AGA AAG ATC CTT CTT CGA AAG TTT TGC CAG 35504
EP3-II
Exon 2a
0.6
EP3-Ic
1077 T CAG CTT ATG GGG ATC ATG TGC GTG CTG TCG GTC TGC TGG TCT CCG CTC CTG 1128
0.8
EP3-Ib
Exon 1
1.0
EP3-I
cDNA sequence 50 –30
EP3 / GAPDH
EP3-Ic exons
Pulmonary artery Pulmonary vein
Fig. 4. EP3 isoform mRNAs detected in human pulmonary vessels derived from one individual. Southern hybridation of RT-PCR products of the EP3 isoforms were normalized with the GAPDH products (arbitrary units). The exposure time was 45 min without screen (EP3-I; EP3-II), with screen 2 h 30 min (EP3-III) and 3 h (EP3-Ib; EP3-Ic; EP3-IV; EP3-e; GAPDH).
different cellular localization of the staining was obtained with the LS antibody (lateral/apical) in comparison with the other anti-EP3 antibodies (basolateral). This discrepancy could be due to the different epitopes recognized; Cay and ADI antibodies recognize extracellular epitopes while LS antibody recognizes an intracellular epitope. Another explanation could be the different sub-cellular localization of the different EP3 isoforms: Cay and ADI antibodies recognize epitopes common to all isoforms while LS antibody was raised against an EP3-f epitope which is found in part in EP3-IV, EP3-V and EP3-e.
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In the vascular wall, there is increasing evidence for different roles of the EP3 receptor subtype. M&B28767, a selective agonist for EP3 receptors, stimulates the migration of the human vascular smooth muscle cells derived from the IMA [18]. Neovascularization induced by PGE2 via the EP3 receptor have been shown in rodents [19,20]. Activation of the EP3 receptor is responsible for the contraction of human PA [3] and IMA [21]. These results are in accordance with the present report where in human IMA and PA, the EP3 receptor mRNAs were detected and the protein localized in the vascular smooth muscle by immunohistochemistry (Figs. 2–4). However, the EP3 isoforms which are involved in the physiological events described above remains to be determined. The absence of the EP3-V, EP3-VI or EP3-f receptor in the present report excludes their involvement in the vasoconstriction of human IMA or PA. Previous reports have shown that in human pulmonary vessels the contractions induced by PGE2 involve the EP3 receptor only in the artery and not in the vein [3,4]. The results obtained by immunohistochemistry (Fig. 2) provide the biochemical basis for these pharmacological reports since they suggest a stronger expression of the EP3 receptor subtype in human PA in comparison with PV. Furthermore, the data presented (Fig. 4) indicate that most of the EP3 isoform mRNAs were markedly expressed in the human PA when compared with the PV. In all the preparations, the EP3-Ia mRNA was not investigated. However, in the IMA and the PV low EP3-Ib, EP3-Ic mRNA expression (Fig. 3a and b) and the greater signal obtained for the total EP3-I (Fig. 3e) suggest the presence of the EP3-Ia mRNA. Finally, the different EP3 isoform mRNAs found in IMA and pulmonary vessels are similar to those reported in smooth muscle cells derived from human umbilical artery (EP3-Ib, EP3-III, EP3-IV and no EP3-II, EP3-VI) [22] and aorta (EP3-Ib, EP3-II, EP3-III, EP3IV) [9,13,23]. In conclusion, our results suggest that there are at least 10 mRNAs splice variants coding for the eight different EP3 isoforms. The different cellular localizations of the EP3 isoforms and the EP subtypes may be associated with different physiological roles. Finally, the cellular distribution and expressions of the EP receptors are dependent on the tissue area and the species studied.
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