9HWHULQDU\ 6FLHQFH Expression of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in human placenta Phil-Ok Koh 1 , Chung-Kil Won 1 , Hae-Sook Noh 2 , Gye
Trang 19HWHULQDU\ 6FLHQFH
Expression of pituitary adenylate cyclase activating polypeptide and
its type I receptor mRNAs in human placenta
Phil-Ok Koh 1
, Chung-Kil Won 1
, Hae-Sook Noh 2
, Gyeong-Jae Cho 2
, Wan-Sung Choi 2,
*
1Department of Anatomy, College of Veterinary Medicine and Institute of Animal Medicine, Gyeongsang National University, Jinju 660-701, Korea
2
Department of Anatomy and Neurobiology, College of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Korea
Pituitary adenylate cyclase activating polypeptide
(PACAP) was first isolated from ovine hypothalamus and
was known to stimulate the release of growth factor in
various cells Recently, we reported the cellular
localization of PACAP and its type I (PAC 1 ) receptor in rat
placenta during pregnancy Placenta is a critical organ
that synthesizes several growth factors and angiogenic
factors for the fetal development and its own growth.
However, there is little information regarding the cellular
localization of PACAP and its receptor in human placenta
at various gestations The aim of the present study was to
define the expression and distribution of PACAP and
were expressed in stroma cells of stem villi and terminal
villi At the early stage, on 7 and 14 weeks, PACAP and
cells surrounding the blood vessels within stem villi These
genes were strongly expressed in stroma cells of stem villi
and terminal villi on 24 and 38 weeks The expression of
these genes was increased as gestation advanced, and
localized in the same areas Localization of PACAP and
play an important role, as an autoregulator or
findings strongly suggest that PACAP may have a critical
role in physiological function of the placenta for
gestational maintenance and fetal growth.
Key words: PACAP, receptor, placenta, human
Introduction
The placenta is an essential organ for the fetal development and the maintenance of pregnancy It is known that placenta synthesis the growth hormone [21] and several growth factors, such as basic fibroblast growth factor and insulin like growth factor [6,29] Also, placenta produces placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) [6,29], which are critical factors for the placental growth and fetal development As an important regulator of angiogenesis, VEGF contributes to the development and growth of the endothelium during the tissue growth [5,32] Also, another member of the VEGF family, PlGF, promotes endothelial cell proliferation in vitro [16] The previous study showed pituitary adenylate cyclase activating polypeptide (PACAP) stimulates the release of VEGF and acts as a trophic factor in various cells [7,8,17,31, 33] PACAP has considerable homology with vasoactive interstinal peptide (VIP) and growth hormone releasing hormone [17,31,33] Recently, it was reported that PACAP and PACAP receptor are present in both the human and rat placenta at term [23] Also, even more recently, we reported the cellular localization of PACAP and PACAP type I (PAC1) receptor in the rat placenta during pregnancy [14] Therefore, the existence of PACAP in placenta suggests that PACAP affects placental function
PACAP was originally isolated from ovine hypothalamus and was known to stimulate the production of cAMP in anterior pituitary cells [18] PACAP exists in two biologically active forms, PACAP 38 and PACAP 27, sharing the same N-terminal 27 amino acids [19] PACAP binds to three type Ireceptors Among these receptors, PAC1 receptor has high affinity with PACAP 38 and PACAP 27, very low affinity with VIP [12,28] But, VIP1 and VIP2 receptors have approximately equal high affinity for PACAP 38, PACAP
27, and VIP [11,12,28] PACAP and its receptor have been found in the central nervous system and its peripheral tissues, including the hypothalamus, pituitary gland, adrenal
*Corresponding author
Tel: 82-55-751-8716; Fax: 82-55-759-0779
E-mail: choiws@nongae.gsnu.ac.kr
Trang 2PACAP in the hypothalamus, pituitary, and gonads suggests
its roles in the reproductive system However, the existence,
localization of PACAP and PAC1 receptor genes in human
placenta at various gestations has been unknown Thus, the
present study was performed to determine the distribution of
PACAP and PAC1 receptor mRNAs in human placenta
Materials and Methods
Tissue preparation
Human placental tissue from legal abortions aged between
6-7 weeks post menstruation (pm) were collected from
normal pregnancies by curettages Second trimester placenta
from 14-24 weeks of gestation were obtained from induced
abortion of healthy pregnancies and term placenta (38-41
weeks pm) by caesarian section or normal delivery For in
situ hybridization studies, tissues were fixed with 4%
paraformaldehyde in 0.1M phosphate buffered saline (PBS)
and cryoprotected with 20% sucrose phosphate buffer for 24
hr Placental sections were cut in a cryomicrotome at a
thickness of 15µm, mounted on the Probe-on slides (Fisher
Scientific, USA), and stored at −70o
C Slides from each placenta were stained with hematoxylin and eosin for
general morphological observation
In situ Hybridization
All solutions were made with sterile water and glassware was
autoclaved to prevent contamination by RNase In situ
hybridization histochemistry was carried out, as described by
Angerer et al [1] Briefly, the slides were dried, washed with
0.1 M PBS, and treated proteinase K, TE buffer, and an
acetylation solution Sections were covered with prehybridization
buffer containing 50% deionized formamide and incubated at
37o
C for 1 hr After removal of the prehybridization buffer, the
slides were covered with the mixture containing the
prehybridization buffer, 50µg/ml yeast tRNA, 10 mM
dithiothreitol, and 35
S-labeled PACAP cRNA probe or PAC1
receptor cRNA probe [13] The slides were then covered with
cover glasses and incubated at 60o
C for 24 hr 35
S-UTP-labeled
probes were prepared using in vitro transcription kit (Promega,
USA) Antisense and sense cRNA probes were purified with a
Sephadex G-50 nick column (Pharmacia Biotech, Sweden) and
eluted with SET buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris,
and 10 mM DTT) Tissue slides were posthybridized in a
posthybridization buffer Following a wash in 4× SSC for 30
min, the sections were then treated with ribonuclease A (50µg/
ml) at 37o
C for 10 min, washed twice in 2× SSC and 1 × SSC,
transferred to a wash buffer containing 0.1× SSC at 65o
C for
30 mins, and dehydrated in alcohol solutions with ascending
concentrations Slides were exposed to β-max hyperfilm
(Amersham, Sweden) for 4 days in light-tight cassettes at −
70o
C, and were dipped into NTB2 emulsion (1 : 1 dilution,
Eastman Kodak, USA), exposed at 4 for 2 weeks, developed in
Kodak D19 developer (1 : 1 dilution, Eastman Kodak, USA) at
15o
C, and counterstained with hematoxylin The slides were observed under a dark and a bright field microscope, and photographed
Results
The present study showed the expression and distribution
of PACAP and PAC1 receptor mRNAs in the human placenta
at various gestations In situ hybridization revealed the
expression of PACAP and PAC1 receptor mRNAs in stem villi and terminal villi Positive cells of PACAP mRNA were detected in stroma cells surrounding the blood vessels within stem villi on 7 weeks (Figs 1A & 2A) PACAP mRNA was expressed in stroma cells of stem villi on 14 week (Figs 1B & 2B) Furthermore, positive signals of PACAP mRNA were strongly observed in the stroma cells
of stem villi and terminal villi on 21 and 38 weeks (Figs 1C, 1D, 2C & 2D) Signals for PACAP mRNA in these cells were gradually increased as gestation advanced However, PACAP mRNA was very weakly expressed in cytotrophoblast cells and syncytiotrophoblast cells There were no detectable signals in negative control with a sense probe (Fig 1E) The hybridization of adjacent sections with PACAP and PAC1 receptor cRNA probes showed that PACAP and PAC1
receptor mRNAs were expressed in the same areas Positive signals of PAC1 receptor mRNA were detected in stroma cells of stem villi on 7 and 14 weeks (Figs 3A, 3B, 4A & 4B) PAC1 receptor mRNA was strongly expressed in stroma cells of stem villi and terminal villi on 21 and 38 weeks
Fig 1 Dark-field photomicrographs of PACAP mRNA expression
in the human placenta from 7 (A), 14 (B), 24 (C), and 38 (D)
weeks gestation by in situ hybridization A and B: Positive signals
were moderately observed in stroma cells of stem villi C and D: Positive cells were strongly detected in stroma cells of stem villi and terminal villi E: No positive signals were detected in negative control with a sense probe Bar = 200µm
Trang 3(Figs 3C, 3D, 4C & 4D) As similar like to the expression
pattern of PACAP, positive signals for PAC1 receptor in these
cells became strong as gestation advanced But, PAC1
receptor mRNA was very weakly expressed in cytotrophoblast
cells and syncytiotrophoblast cells No positive signals of
PAC1 receptor mRNA was detected in negative control with
a sense probe (Fig 3E)
Discussion
In the present study, we showed the cellular localization of PACAP and PAC1 receptor mRNAs in human placenta on 7,
14, 24, and 38 weeks Recently, we reported evidence that PACAP and PAC1 receptor mRNAs were expressed in decidual cells, chorionic vessels, and stroma cells of
chorionic villi in the rat placenta [14] Also, Scaldaferri et al.
[23] demonstrated the presence of PACAP and PAC1
receptor in both the human and the rat placenta at term, using RT-PCR and immunohistochemistry techniques They showed the expression of PACAP at term placenta, but they did not offer any information as to the distribution of PACAP and PAC1 receptor in human placenta at various
gestations, during pregnancy In this study, we utilized in situ
hybridization to determine the existence of PACAP and PAC1 receptor mRNAs in human placenta Our data showed the expression of PACAP and PAC1 receptor mRNAs in stroma cells of stem villi and terminal villi As gestation advanced, the expression of PACAP and PAC1 receptor mRNAs was increased in these cells
It is known that placenta produces several growth factors such as insulin like growth factor and basic fibroblast growth factor Especially, VEGF and placenta growth factor (PlGF), which are essential factor for the placental growth and fetal development [5,32] VEGF was expressed in stroma cells within villi in human placenta [26] Localization of VEGF
in stroma cells demonstrates that VEGF play an important role in the physiological growth and function of the vascular system in the villous stroma Furthermore, PACAP that acts
as a growth factor in various cells, stimulates VEGF release
Fig 2 Bright-field photomicrographs of PACAP mRNA
expression in the human placenta from 7 (A), 14 (B), 24 (C), and
38 (D) weeks gestation by in situ hybridization A and B: PACAP
mRNA was strongly expressed in stroma cells of stem villi C
and D: Positive signals were strongly detected in stroma cells of
stem villi Arrows indicate the positive cells Bar = 20µm
placenta from 7 (A), 14 (B), 24 (C), and 38 (D) weeks gestation
by in situ hybridization A and B: Positive signals were
moderately observed in stroma cells of stem villi C and D:
Positive cells were strongly detected in stroma cells of stem villi
and terminal villi E: No positive signals were detected in
negative control with a sense probe Bar = 200µm
Fig 4 Localization of PAC1 receptor mRNAs expression in the human placenta from 7 (A), 14 (B), 24 (C), and 38 (D) weeks
gestation by in situ hybridization A and B: PAC1 receptor mRNA was strongly expressed in stroma cells of stem villi C and D: Positive signals were strongly detected in stroma cells of stem villi Arrows indicate the positive cells Bar = 20µm
Trang 4endothelial cells by promoting cell proliferation and
permeability In this study, PACAP was strongly expressed
in stroma cells of stem villi and terminal villi PACAP
mRNA was localized in the stroma cell surrounding the
blood vessel on 14 and 24 weeks Also, PACAP mRNA was
expressed in the whole stroma cells on 38 weeks
Furthermore, PACAP 38 immunostaining was detected in
stroma cells of stem and terminal placental villi [23] Both
PACAP and VEGF were expressed in stroma cells of
terminal villi Thus, our data suggest that PACAP stimulates
the release of VEGF and promotes the growth of placenta
In the previous studies, VIP/PACAP neuropeptide family
regulates the blood flow and hormone secretion in human
placenta [4,9,30] Steenstrup et al [30] reported that PACAP
were expressed in the uteroplacental unit, where it causes a
concentration dependent relaxation on stem villous and
umbilical cord arteries These results suggest that PACAP
mediates the placental growth and fetal development during
the pregnancy period
In the previous studies, many researchers demonstrated
that PACAP acts an autocrine and/or paracrine regulator in
various tissues, including ovarian granulose cells, testicular
Leydig cells, and placental tissue [15,20,22,24] Also, in our
results, PACAP and PAC1 receptor mRNAs were expressed
in the same areas Although our data did not elucidate the
physiological role and action mechanism of PACAP in
human placenta, the localization of PACAP and its PAC1
receptor in the same areas strongly suggest that PACAP may
act as an autoregulator or pararegulator via its PAC1 receptor
in stem villi and terminal villi during pregnancy In
conclusion, our findings suggest that PACAP may have an
important role in physiological function of the placenta for
gestational maintenance and fetal growth
Acknowledgments
This work was supported by the Brain Korea 21 Project,
the Ministry of Education of Republic of Korea, and was
partly supported by the grant from the Korea Science and
Engineering Foundation (KOSEF R04-2003-000-10062-0)
References
1 Angerer LM, Cox KH, Angerer RC Demonstration of
tissue-specific gene expression by in situ hybridization.
Methods Enzymol 1987, 152, 649-661.
2 Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K,
Coy DH, Kitada C Tissue distribution of PACAP as
determined by RIA: highly abundant in the rat brain and
testes Endocrinology 1991, 129, 2787-2789.
3 Arimura A Pituitary adenylate cyclase activating
polypeptide (PACAP): discovery and current status of
research Regul Pept 1992, 37, 287-303.
4 Besson J, Malassine A, Ferre F Autoradiographic
sites in the human term placenta Relationship with activation
of adenylate cyclase Regul Pept 1987, 19, 197-207.
5 Clark DE, Smith SK, He Y, Day KA, Licence DR, Corps
AN, Lammoglia R, Charnock-Jones DS A vascular
endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation
Biol Reprod 1998, 59, 1540-1548.
6 Correia-da-Silva G, Bell SC, Pringle JH, Teixeira N.
Expression of mRNA encoding insulin-like growth factors I and II by uterine tissues and placenta during pregnancy in the
rat Mol Reprod Dev 1999, 53, 294-305.
7 Gloddek J, Pagotto U, Paez Pereda M, Arzt E, Stalla GK,
Renner U Pituitary adenylate cyclase-activating polypeptide,
interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate
cells J Endocrinol 1999, 160, 483-490.
8 Gonzalez BJ, Basille M, Vaudry D, Fournier A, Vaudry
H Pituitary adenylate cyclase-activating polypeptide promotes
cell survival and neurite outgrowth in rat cerebellar
neuroblasts Neuroscience 1997, 78, 419-430.
9 Graf AH, Hutter W, Hacker GW, Steiner H, Anderson V,
Staudach A, Dietze O Localization and distribution of
vasoactive neuropeptides in the human placenta Placenta
1996, 17, 413-421.
10 Hashimoto H, Ishihara T, Shigemoto R, Mori K, Nagata
S Molecular cloning and tissue distribution of a receptor for
pituitary adenylate cyclase-activating polypeptide Neuron
1993, 11, 333-342.
11 Inagaki N, Yoshida H, Mizuta M, Mizuno N, Fujii Y,
Gonoi T, Miyazaki J, Seino S Cloning and functional
characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in
insulin-secreting cells Proc Natl Acad Sci USA 1994, 29,
2679-2683
12 Ishihara T, Shigemoto R, Mori K, Takahashi K, Nagata S.
Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide Neuron 1992,
8, 811-819.
13 Koh PO, Kwak SD, Kim HJ, Roh G, Kim JH, Kang SS,
Choi WS, Cho GJ Expression patterns of pituitary
adenylate cyclase activating polypeptide and its type I receptor mRNAs in the rat placenta Mol Reprod Dev 2003,
64, 27-31.
14 Koh PO, Kwak SD, Kang SS, Cho GJ, Chun SY, Kwon
HB, Choi WS Expression of pituitary adenylate cyclase
activating polypeptide (PACAP) and PACAP type I A receptor mRNAs in granulosa cells of preovulatory follicles
of the rat ovary Mol Reprod Dev 2000, 55, 379-386.
15 Kotani E, Usuki S, Kubo T Rat corpus luteum expresses
both PACAP and PACAP type IA receptor mRNAs Peptides
1997, 18, 1453-1455.
16 Maglione D, Guerriero V, Viglietto G, Delli-Bovi P,
Persico MG Isolation of a human placenta cDNA coding for
a protein related to the vascular permeability factor Proc Natl
Acad Sci USA 1991, 88, 9267-9271.
17 Matsumoto H, Koyama C, Sawada T, Koike K, Hirota K,
Miyake A, Arimura A, Inoue K Pituitary
Trang 5folliculo-stellate-like cell line (TtT/GF) responds to novel hypophysiotropic
peptide (pituitary adenylate cyclase-activating peptide),
showing increasedadenosine 3',5'-monophosphate and
interleukin-6 secretion and cell proliferation Endocrinology
1993, 133, 2150-2155.
18 Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A,
Jiang L, Culler MD,Coy DH Isolation of a novel 38
residue hypothalamic peptide which stimulates adenylate
cyclase in pituitary cells Biochem Biophys Res Commun
1989, 164, 567-574.
19 Miyata A, Jiang L, Dahl RR, Kitada C, Kubo K, Fujino
M, Minamino N, Arimura A Isolation of a neuropeptide
corresponding to the N-terminal 27 residues of the pituitary
adenylate cyclase activating polypeptide with 38 residues
(PACAP38) Biochem Biophys Res Commun 1990, 170,
643-648
20 Moretti C, Mencacci C, Frajese GV, Cerilli M, Frajese G
Growth hormone-releasing hormone and pituitary adenylate
cyclase-activating polypeptide in the reproductive system
Trends Endocrinol Metab 2002, 13, 428-435.
21 Ogilvie S, Buhi WC, Olson JA, Shiverick KT.
Identification of a novel family of growth hormone-related
proteins secreted by rat placenta Endocrinology 1990, 126,
3271-3273
22 Scaldaferri L, Arora K, Lee SH, Catt KJ, Moretti C.
Expression of PACAP and its type-I receptor isoforms in the
rat ovary Mol Cell Endocrinol 1996, 25, 227-232.
23 Scaldaferri ML, Modesti A, Palumbo C, Ulisse S, Fabbri
A, Piccione E, Frajese G, Moretti C Pituitary adenylate
cyclase-activating polypeptide (PACAP) and
PACAP-receptor type 1 expression in rat and human placenta
Endocrinology 2000, 141, 1158-1167.
24 Sherwood NM, Krueckl SL, McRory JE The origin and
function of the pituitary adenylate cyclase-activating
polypeptide (PACAP)/glucagon superfamily Endocr Rev
2000 21, 619-670.
25 Shioda S, Legradi G, Leung WC, Nakajo S, Nakaya K,
Arimura A Localization of pituitary adenylate
cyclase-activating polypeptide and its messenger ribonucleic acid in
the rat testis by light and electron microscopic
immunocytochemistry and in situ hybridization Endocrinology
1994, 135, 818-825.
26 Shiraishi S, Nakagawa K, Kinukawa N, Nakano H,
Sueishi K Immunohistochemical localization of vascular
endothelial growth factor in the human placenta Placenta
1996, 17, 111-121.
27 Shivers BD, Gorcs TJ, Gottschall PE, Arimura A Two
high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions
Endocrinology 1991, 128, 3055-3065.
28 Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert
J, Seeburg PH,Journot L Differential signal transduction
by five splice variants of the PACAP receptor Nature 1993,
365, 170-175.
29 Srivastava RK, Gu Y, Ayloo S, Zilberstein M, Gibori G.
Developmental expression and regulation of basic fibroblast growth factor and vascular endothelial growth factor in rat decidua and in a decidual cell line J Mol Endocrinol 1998,
21, 355-362.
30 Steenstrup BR, Jorgensen JC, Alm P, Hannibal J, Junge
J, Fahrenkrug J, Ottesen B Pituitary adenylate cyclase
activating polypeptide (PACAP): occurrence and vasodilatory
effect in the human uteroplacental unit Regul Pept 1996, 22,
197-204
31 Tischler AS, Riseberg JC, Gray R Mitogenic and
antimitogenic effects of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult rat chromaffin cell cultures
Neurosci Lett 1995, 21, 135-138.
32 Vuorela P, Hatva E, Lymboussaki A, Kaipainen A,
Joukov V, Persico MG, Alitalo K, Halmesmaki E.
Expression of vascular endothelial growth factor and placenta growth factor in human placenta Biol Reprod 1997,
56, 489-494.
33 Wolf N, Krieglstein K Phenotypic development of neonatal
rat chromaffin cells in response to adrenal growth factors and glucocorticoids: focus on pituitary adenylate cyclase activating
polypeptide Neurosci Lett 1995, 24, 207-210.