Local expression of a POU family transcription factor, Pit-1, in the rat placenta

hholecular and Cellular Endocrinology Molecular and Cellular Endocrinology

118 (1996) 9- 14

Local expression of a POU family transcription the rat placenta

factor, Pit-l, in

Byung J. Leea*, Jin K. Jeong”, Jin H. Kim”, Sung G. Kangb, Myeong 0. Kim”, Wan S. Choi” “Department of Biology, College of Natural Sciences, University of Ulsan, Ulsan 680-749, South Korea bDepartment of Biology, College of Natural Sciences, Inje University, Kimhae, South Korea ‘Department of Anatomy, College of Medicine, Gyeongsang National University, Chinju, South Korea

Received 22 November 1995; accepted 3 January 1996

Abstract A pituitary-specific trans-acting factor, Pit-l regulates transcriptional activity of growth hormone (GH) and prolactin (PRL) genes. Pit-l can bind and activate the promoters of human chorionic somatomammotropin (hCS-A) and placental GH variants (hGH-V) as well. However, expression of Pit-l in the rat placenta has not yet been elucidated. The present study aims to determine whether the Pit-l gene is locally expressed in the rat placenta using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot and Western blot hybridization, in situ hybridization and immunohistochemistry. PCR products were further analyzed by Southern hybridization and DNA sequencing. The estimated size of Pit-l mRNA in placenta was very similar to that in anterior pituitary (AP). PCR products from placenta were exactly the same size with that from AP and confirmed as Pit-l-specific by Southern hybridization. The Pit-l specific sequence was also confirmed by sequencing of partial amplification fragments. Immunoreactive 33 kDa Pit-l was present in the placenta as well as in AP. Pit-l specific mRNA and protein were localized in the trophoblast cells of placenta. These data suggest that Pit-l is locally synthesized in the rat placenta and may be involved in the regulation of GH- and/or PRL-like gene expression in the placenta. Keywords:

Pit-l gene expression; Extra-pituitary

(rat placenta); Placental lactogens

1. Introduction

The placenta of a number of species produces polypeptides that are members of prolactin (PRL) and growth hormone (GH) gene family (Ogren and Talamantes, 1988; Soares et al., 1991). Human placenta synthesizes and secretes several kinds of these members, which are called chorionic somatomammotropin (hCS) or placental lactogen (hPL), while in the rodent placenta, a number of PRL-like proteins (PLPs) and PLs are included among the GH-PRL family members. The strict pituitary-specific expression of the GH and PRL genes is due to their promoter region, which contains binding sites for a transcription factor, Pit-l (Ingraham et al., 1990; Karin et al., 1990). Pit-l is a member of the superfamily of DNA binding proteins containing a homeodomain (Bodner et al., 1988; Ingraham et al., * Corresponding author. 0303.7207,‘96/$15.00 SSDl

1988; Ingraham et al., 1990). Pit-l is closely related to the Ott-1, Ott-2 and Unc-86 proteins, which contain a second highly conserved sequence motif known as the POU-specific domain (Herr et al., 1988). The role of Pit-l in the transcriptional activation of the GH and PRL genes in anterior pituitary (AP), and pituitary cell development is well established (Ingraham et al., 1990; Karin et al., 1990; Li et al., 1990). Pit-l binds 2 and 8 sites in the 5’-flanking regions of GH and PRL genes (Lefevre et al., 1987; Nelson et al., 1988), respectively, while Pit-l can bind and activate the promoters of hCS-A and placental GH variants (hGH-V) in their 150 base pairs (bp) of 5’-flanking DNA (Lemaigre et al., 1989; Nachtigal et al., 1989; Nickel et al., 1991). However, expression of Pit-l in the rat placenta has not been previously reported. Therefore, the present study aims to determine whether the Pit-l gene is locally expressed in the rat placenta using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot and in situ hybridization to detect Pit-l mRNA,

0 1996 Elsevier Science Ireland Ltd. All rights reserved

0303-7207(95)03758-6

B.J. Ler et ul. : hloleculur und Cellular Endocrinology 118 (1996) 9-14

10

and Western blots and immunohistochemistry Pit-l protein.

to detect

2. Materials and methods 2.1. Animals and tissue preparations

Timed pregnancy was generated by housing the Sprague-Dawley female rats (UOU Animal Breeding Center) with males. The presence of a copulatory plug or sperm in the vaginal smear was designated day 0 of pregnancy. Placentas were removed from the uterus on the morning of the pregnant days 12 to 20 at 2-day intervals. Anterior pituitaries from every pregnant female rat were pooled and used as a control tissue. For histochemical studies, pregnant female rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.2) through the left cardiac ventricle. 2.2. Pit-l cDNA and cRNA probes To exclude the possible detection of other POUhomeo family gene products, we reconstructed the Pit-l cDNA fragments. The EcoRI fragment (about 440 bp) of Pit-l cDNA structure (a generous gift from Dr. Richard A. Maurer, Oregon Health Science University, OR), which corresponds to the trans-activation domain of Pit-l and is Pit-l -specific, was inserted into pBluescript II KS (Stratagene, La Jolla, CA). After linearization with PstI, 32P- or 35S-labeled cRNA probes were synthesized using T3 RNA polymerase. The cDNA fragment by PstI and Hind111 was used for the synthesis of cDNA probe with random priming method (Feinberg and Vogelstein, 1983) to detect Southern blots of PCR products as follows. 2.3. RNA extraction and Northern blot hybridization Total RNA from tissues was extracted using the acid guanidium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). RNA samples (20 pg) were electrophoresed on a 1% agarose/2.2 M formaldehyde gel at 100 V for 1.5 h. Hybridization with 32P-labeled Pit-l cRNA probe was carried out at 55°C overnight (Day and Day, 1994a). The blots were rinsed in 2 x SSC (1 x SSC: 0.15 M NaCl and 0.015 M Na citrate) and washed at high stringency with 2 x SSC0.1% SDS, 0.5 x SSC-0.1% SDS, and 0.1 x SSC-0.1% SDS at 60°C to exclude the non-specific signals with other members of POU-homeo family genes. 2.4. RT-PCR, sequencing

Southern blot hybridization nnd DNA

RT-PCR for Pit-l was performed

as previously de-

scribed (Lee et al., 1995). Briefly, RNA (10 pg) was reverse transcribed with Moloney Murine leukemia virus (MMLV) RNaseH ~ reverse transcriptase kit (Promega, Madison, WI). Pit-l specific primers were designed to detect Pit-l transcripts only among various POU-homeo family genes using Mac-Vector program (Eastman Kodak, Rochester, NY). An upstream primer (5’-CACCTCGGCTGATACCTTT-3’) and a downstream primer (5’-GTTTGCTCCCACTTTTTC-3’) correspond to the trans-activation domain and the linker region between POU and homeo domains. The primer set lies on the first and fifth exon of the rat Pit-l cDNA and mouse Pit-l genomic DNA structure (Ingraham et al., 1988; Li et al., 1990). Five microlitres of cDNA products were added to the primer set. Thirty cycles of PCR amplification were performed using Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). To confirm that the PCR bands were Pit-l mRNA specific, resulting products were electrophoresed on a 1.4% agarose gel, transferred to Nytran membrane (pore size: 0.45 pm, Schleicher and Schuell, Keene, NH), and hybridized with Pit-l cDNA probe labeled with an enhanced chemiluminesence (ECL) random priming kit (Amersham, Arlington Heights, IL). Hybridization signals were amplified by an ECL detection system (Amerand autoradiographed onto X-ray film sham) (Hyperfilm-ECL, Amersham). The PCR product was purified with Geneclean II kit (BIO 101, Vista, CA), and partially sequenced with an upstream PCR primer and the sequenase DNA sequencing kit (U.S. Biochemical Corp., Cleveland, OH) according to a previous report (Winship, 1989). 2.5. Western blots Nuclear fractions from placentas and control tissues were prepared as described by Tanaka et al., 1992. Samples were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis on 12% gels and subjected to Western blot hybridization according to the previous report (Day and Day, 1994a) with minor modifications. Briefly, the proteins were transferred to nitrocellulose paper by electroblotting. The membranes were blocked with 1% bovine serum albumin (BSA) in 10 mM PBS, washed in PBS-Tween (O.l%), and incubated with anti-Pit-l rabbit antiserum (Santa Cruz Biotech., Santa Cruz, CA, 1:5000), which was raised against full length human Pit-l and is able to react with rat Pit-l, for 2 h at room temperature. After washing with PBS-Tween, the membrane was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO) followed by addition of BCIP/ NBT phosphatase substrate, consisting of 5-bromo-4chloro-3-indolyl phosphate sodium salt 0.15 mg/ml and p-nitro blue tetrazolium chloride 0.3 mg/ml in 0.1 M carbonate buffer.

B.J. Lee et al. I Molecular and Cellular Endocrinology 118 (1996) 9- 14

2.6. In situ hybridizution and immunohistochemistry In situ hybridization histochemistry was performed basically based on a reported protocol (Petraglia et al., 1992). The fixed placental tissues were coronally sectioned at 10 pm with a cryostat at - 20°C. The tissue sections were hybridized with Y-labeled Pit-l cRNA probe for 1 day at 60°C in a humid chamber. Then, sections were incubated with RNase A to exclude the possibility of mis-matched sequences. After washing and drying, sections were apposed to NTB2 emulsion (Eastman Kodak) for 2 weeks. The signals were observed under a darkfield microscope. Tissue sections were processed for immunofluorescence as previously described (Camps et al., 1991). Tissue sections were incubated at 4°C overnight with the anti-Pit-l rabbit antiserum (1:50). After washing, sections were incubated with the fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, Bar Harbor, ME, 1:50) at room temperature for 2 h. Sections were mounted (Mowisol solution, Hoechst, Somervill, NJ) and visualized under a fluorescence microscopy.

3. Results and discussion Pit-l was originally thought to be synthesized in the pituitary, and regulate pituitary development as well as gene expression of GH and PRL (Ingraham et al., 1988; Ingraham et al., 1990; Karin et al., 1990; Li et al., 1990). However, we report here Pit-l gene product in the rat placenta, based on Northern and Western blot hybridization, PCR analysis and histochemical studies. This finding coincides well with the recent report of Pit-l expression in the human placenta (Bamberger et al., 1995). The level of Pit-l mRNA in the rat placenta was high enough for reasonable detection by the methods used in the present study. It appears then that Pit-l mRNA in the rat placenta is unlike illegitimate transcription products of the gene (Chelly et al., 1989). Northern blot hybridization showed that placentas from day 12 to day 20 of gestation expressed the same Pit-l mRNA with that expressed in AP (Fig. 1). In agreement with previous reports (Ingraham et al., 1988; Day and Day, 1994a), major and minor transcripts of approximately 2.5 kb and 1.2 kb were detected in the rat placenta as well as in AP. The densities of Pit-l transcripts in the placentas from the late pregnancy (16, 18 and 20 days of gestation) were much stronger than those from mid-pregnancy (12 and 14 days of gestation) when the same amount of RNA (20 pg) from tissues was loaded on each lane, and relative mRNA signals were normalized by rehybridization of blots with 32P-labeled cDNA probe for 18s ribosomal RNA. This result indicates changes of the gene expression and physiolog-

L AP

12

16

14

18 20

Fig. I. Northern blot analysis of Pit-l mRNA in the rat placentas, anterior pituitary (AP, as a positive tissue) and lung (L, as a negative tissue). Rat placentas 12-20 were removed from the uterus on pregnant days 12 to 20 at 2-day intervals. RNA samples (20 ,ug) were separated by I’% agarose gel electrophoresis and hybridized with “P-labeled Pit-l cRNA probe (upper panel). Rehybridization signals for 18s ribosomal RNA (18s) were used to certify equal loading of RNA (lower panel). The migration of 28s and 185 ribosomal RNAs is shown.

ical role of Pit-l in the placenta during the progress of pregnancy. Especially in the late pregnancy, Pit-l may play important roles in the rat placenta. Coincident with the plateau of Pit-l transcription after pregnant day 16 (the present study), transcriptional activities and serum levels of several PLs such as PL-II, PLP-A and PLP-C reach the peak in the same late gestation (Soares et al., 1991; Hamlin et al., 1994). These data suggest that Pit-l may be involved in the synthesis of several PLs. RT-PCR amplification also revealed the transcription of Pit-l gene in the rat placenta (Fig. 2). The RT-PCR procedure reproducibly generated a discrete Pit-l cDNA band with 616 bp in size that was expected on the base of the rat Pit-l cDNA sequence (Ingraham et al.. 1988). Size of the PCR product from 12- and l&day placenta was exactly the same with that from AP. The negative control tissue of lung and Pit-l primers

M

B

L

AP

12

18

Fig. 2. RT-PCR analysis of Pit-l mRNA (upper panel) and Southern blot hybridization of PCR products with ECL-labeled Pit-l cDNA probe (lower panel). RNAs from placentas of 12 and 18 day of pregnancy (12 and 18), anterior pituitary (AP). and lung (L) were reverse transcribed and amplified with 30 cycles of PCR. PCR of primers alone (blank, B) and lung did not amplify any detectable PCR products. DNA marker (M) is HaeIIl digested +6X 174 DNA.

B.J. Lee et al. I Molecular and Cellular Endocrinology 118 (1996) 9-14

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L

AP

PL

Fig. 3. Western blot analysis of immunoreactive Pit-l protein. Tissue lysates (50 fig) from lung (L), anterior pituitary (AP) and placenta of l&day pregnancy (PL) were fractionated on SDS-containing 12% polyacrylamide gels and transferred to nitrocellulose membrane. The membrane was hybridized with Pit-l antibody (1:5000 dilution), and incubated with an alkaline phosphatase conjugated anti-rabbit goat IgG. The membrane was visualized by incubation with phosphatase substrates.

alone (blank) did not give rise to any detectable PCR products, which indicates no cross-tissue or cross-tube contaminations in the RT-PCR conditions. Southern blot hybridization of PCR products with the rat Pit-l cDNA probe confirmed the positive signals of Pit-l expression in the rat placenta (Fig. 2, lower panel). Sequencing of the PCR fragments from the rat placenta (about 200 bp with an upstream PCR primer, data not shown) revealed sequence homology of about 99% with the reported rat Pit-l cDNA sequence (Ingraham et al., 1988), which also confirmed the expression of the same Pit-l gene in the rat placenta as in AP. Our RT-PCR condition did not amplify any other Pit-l variant forms described previously (Konzak and Moor, 1992; Voss et al., 1993; Day and Day, 1994b). It may be explained that primer sites were not matched to detect other Pit-l variants and our PCR conditions were adjusted to detect the main splicing form, Pit-la among Pit-l variants. Immunoreactive Pit-l protein of approximately 33 kilodaltons (kDa) was present in the rat placenta as well as in AP (Fig. 3) which coincides well with the Pit-l band reported previously (Voss et al., 1991; Day and Day, 1994a). Moreover, density of the immunoreactive Pit-l band of placenta was very similar to that of AP when the same amounts of nuclear proteins (50 pg) were used. Our Western blot experiment did not detect the 31 kDa band of Pit-l that was reported to originate from different usage of translation initiation sites as suggested by a previous study (Voss et al., 1991). The cellular localization of Pit-l mRNA and protein in the coronal sections of the rat placenta was examined by in situ hybridization and immunohistochemistry (Fig. 4). Numerous positive signals of Pit-l mRNA were observed in AP but not in posterior pituitary (PP) (Fig. 4A). In the 13-day placenta, dense positive signals of Pit-l mRNA were present in the trophoblast cells of junctional zone (Fig. 4B). In higher magnification, signals of Pit-l mRNA were found in the giant cells and spongiotrophoblast cells (Fig. 4C). Immunofluorescence showed positive signals of immunoreactive Pit-l in AP but not in PP (Fig. 4D). The intensive signals of Pit-l

protein were present in the same junctional zone of 13-day placenta (Fig. 4E). Immunofluorescence for the parts of the same serial sections using normal rabbit serum did not generate any detectable immunoreactive Pit-l signals (data not shown). Fluorography of 17-day placenta showed clear positive signals of Pit-l protein in the labyrinth zone (Fig. 4F), but not in the junctional zone (data not shown), which suggests that during gestational progress, the site of Pit-l synthesis shifts from junctional zone to labyrinth zone. The present in situ hybridization and immunohistochemical experiments localized the Pit-l mRNA and protein in the trophoblast cells of the junctional and labyrinth zone in the 13-day and 17-day placenta, respectively. Trophoblast cells of the rat and mouse placenta have been elucidated as a synthetic site for PLs and PRL-like proteins (Campbell et al., 1989; Duckworth et al., 1990; Faria et al., 1991; Yamaguchi et al., 1992). Therefore, Pit-l and PLs seem to be synthesized in the similar region of the rat placenta, which suggests that Pit-l may be involved in the synthesis of PLs. Interestingly, the present immunohistochemical study and our unpublished data, showing the site of Pit-l expression migrates from junctional zone to labyrinth zone during gestational progress, are consistent with the previous report that the site of PL-II expression shifted from the junctional zone to the labyrinth zone during the second half of pregnancy (Campbell et al., 1989). Pit-l may regulate the gene expression of PLs and/or PRL- and GH-like proteins in the placenta. This hypothesis would be supported by previous reports that the promoter region of hCS-A and hGH-V gene is bound to the same transcription factor Pit-l that is bound to the promoters of PRL and GH genes (Lemaigre et al., 1989; Nachtigal et al., 1989; Nickel et al., 1991). In the hemopoietic and lymphoid tissues, Pit-l was reported to be expressed and thought to regulate local GH and PRL expression (Delhase et al., 1993) suggesting the local regulatory role of Pit-l on the GHand/or PRL-family genes. In addition, phylogenetically conserved Pit-l was found in the salmon pituitary and it was believed to regulate the expression of salmon PRL (Elsholtz et al., 1992; Ono et al., 1994). Therefore, Pit-l seems to be highly conserved as a target gene-specific transcription factor among tissues and phylogenetic linages. On the other hand, the placenta-specific expression of PLs may be due to other regulators such as GATA factors for the trans-activation of mouse PL-I gene (Ng et al., 1994) and transcriptional enhancer factor-l (TEF-1) for hCS genes (Lytras and Cattini, 1994). Further studies are required to elucidate precise mechanisms for the tissue-specific and target gene-specific regulation of PLs. In summary, with the Western blot and immunohistochemical evidence that Pit-l mRNA in the placenta translates into immunoreactive peptide in the same site,

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Fig. 4. In situ hybridization of Pit-l mRNA (A, B and C) and immunofluorescent localization of Pit-l protein (D, E and F) in the rat pituitary and placenta. Tissue sections were hybridized with “S-labeled Pit-l cRNA probe and photographed under a darkfield microscope (A, B and C). Tissue sections were incubated with anti-Pit-l antiserum and followed by fluorescent detection (D, E and F). Widespread intensive signals of Pit-l mRNA were observed in anterior pituitary but not in posterior pituitary (A). Positive signals of Pit-l mRNA were present in the junctional zone of 13-day placenta (B and C). Immunoreactive signals of Pit-l protein were highly concentrated in the junctional zone of 13-day placenta (E) as well as in anterior pituitary (D). Labyrinth zone of 17-day placenta also showed immunoreactive Pit-l proteins (F). AP. anterior pituitary; PP, posterior pituitary; D, decidua; JZ, junctional zone. Bar = 600 pm (A), 300 pm (B), and 100 pm (C, D, E and F).

the present study demonstrated the Pit-l gene expression in the rat placenta. The finding of placental Pit-l mRNA and protein reinforces the potential role of Pit- 1 as a transcriptional activator in the trophoblast cells of the rat placenta.

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