BOR - Papers in Press, published online ahead of print
June 15, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.039859
BIOLOGY OF REPRODUCTION 73, 695–702 (2005)
DOI: 10.1095/biolreprod.105.039859
© 2005 by the Society for the Study of Reproduction, Inc.
Early Placental Insulin-Like Protein (INSL4 or EPIL) in Placental and Fetal Membrane Growth1
Lynnae Millar 2, 3,
Nicole Streiner 3,
Lisa Webster 3,
Sandra Yamamoto 4,
Rachel Okabe 4,
Tasha Kawamata 3,
Jacqueline Shimoda 3,
Erika Büllesbach 5,
Christian Schwabe 5, and
Gillian Bryant-Greenwood 3,4
Department of Obstetrics and Gynecology,3
John A. Burns School of Medicine, Pacific Biosciences Research Center,4 University of Hawaii, Honolulu, Hawaii 96822
Department of Biochemistry and Molecular Biology,5 Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT
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Early placental insulin-like protein (INSL4 or EPIL) is a member of the insulin superfamily of hormones, which is highly expressed in the placenta. We have confirmed this at term and shown it to be expressed by the maternal decidua. Although an abundance of locally acting growth factors are produced within the uterus during pregnancy, we hypothesized that INSL4 plays an important role in fetal and placental growth. We have demonstrated with cell lines and primary cells that it has a growth-inhibitory effect by causing apoptosis and loss of cell viability. We used primary amniotic epithelial cells for flow cytometry to show that INSL4 caused apoptosis, which was dose-related and significant (P < 0.05) at 50 ng/ml. This was confirmed by measurement of the nuclear matrix protein in the media. In comparison, relaxin treatment (up to 200 ng/ml) had no effect on apoptosis. The addition of INSL4 (3–30 ng/ml) also caused a loss of cell viability, although it had no effect on the numbers of cells at different phases of the cell cycle. Placental apoptosis is an important process in both normal placental development and in fetal growth restriction. Therefore, an in vivo clinical correlate was sought in fraternal twins exhibiting discordant growth. Expression of the INSL4 gene was doubled in the placenta of the growth-restricted twin compared to the normally grown sibling, suggesting that it may be linked to a higher level of apoptosis and loss of cell viability and, therefore, that it may contribute to fetal growth restriction.
apoptosis, early placental insulin-like peptide, growth restriction, placenta
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INTRODUCTION
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Synchronization of the growth of the fetus, uterus, and placenta appears to be coordinated by key growth factors as well as by substrate bioavailability. In recent years, the importance of the insulin-like growth factor (IGF)-1 and IGF2 has become clear. Cord blood levels of IGF1 and IGF-2 correlate with birth and placental weights [1], and their levels are significantly lower in growth-restricted fetuses compared to those in normally grown controls [2, 3]. A chimeric mouse model with disruption of one of the Igf2 alleles had progeny that were smaller than average [4]. Recently, we have shown that relaxin, a member of the insulin hormone superfamily, acts as a growth factor for an amniotic epithelial-like cell line (WISH) and, possibly, as an autocrine/paracrine hormone of the human fetal membranes. We demonstrated that relaxin in vitro probably causes the proliferation of WISH cells by increasing the transcription of the IGF2 gene [5].
The early placental insulin-like peptide (EPIL or INSL4) also is a member of the insulin superfamily. It has the most homology with the human relaxins, RLN1 and RLN2 (44% and 43%, respectively), and only 15% homology wtih insulin [6, 7]. Initially, it was identified from a subtracted cDNA library of a first-trimester placenta [6, 8] and was shown to be expressed most highly in embryonic and trophoblastic tissues [9]. Indeed, its high level of expression in the placental syncytiotrophoblast early during gestation suggested it to be important in fetal and placental growth and development. The INSL4 gene is comprised of two exons and one intron, similar to the other members of its superfamily. The clustering of this gene with the two human relaxin genes, RLN1 and RLN2, on the same chromosome at 9p24 [10] suggests that the INSL4 and RLN1 genes resulted from late gene-duplication events. The INSL4 gene is expressed only in higher primates, probably after the divergence of the New World and Old World monkeys [7]. It recently was shown that a human endogenous retrovirus element is inserted into the human INSL4 gene promoter. Its placental specific expression is mediated by the 3'-LTR (long terminal repeats) of the retroviral element, suggesting that this ancient retroviral infection may have been a major event for the functional evolution of the human placenta [11].
The INSL4 protein appears to be unlike relaxin or insulin, which have their mature peptides fully processed and, therefore, lack the connecting peptides. The IGFs, on the other hand, remain completely unprocessed in their mature forms. Because the INSL4 protein only has dibasic recognition sites for putative enzyme cleavage between the C and A domains, two peptide chains of 13 and 4 kDa would be produced [8]. However, how this peptide is processed in vivo remains unknown. Levels of the pro-INSL4 protein decrease in amniotic fluid with advancing gestation, at the same time that its concentrations in serum rise. The pattern of pro-INSL4 levels in amniotic fluid in both normal and chromosomally abnormal pregnancies correlate strongly with the levels of chorionic gonadotroph and its free subunits, suggesting that the production of these hormones by the syncytiotrophoblast may be controlled by a common regulatory pathway [12, 13]. However, synthetic INSL4 has no hydrophobic core and lacks helical structure at physiological pH [14]. No specific target tissue has been identified, and it lacks binding activity to the relaxin receptor (LGR7), its splice variant, or the INSL3 receptor (LGR8) [7]. Indeed, no biological function has yet been ascribed to this protein. Because of its structural homologies with other hormones involved in growth regulation, we sought biological actions for INSL4 in the human amnion and placenta and compared them to the effects of human relaxin 2 (RLN2). At the same time, an in vivo correlate was sought to link any biological activity with a clinically defined obstetrical problem, because inappropriate fetal growth is a significant risk factor for preterm birth [15, 16]. Both large- and small-for-gestational-age infants have a two- to threefold increased risk of preterm delivery compared to that of normally grown infants [16]. The greater the impairment of fetal growth, the higher the risk of preterm birth. Infants with severe growth impairment have a sixfold increased rate of preterm birth secondary to preterm, premature rupture of membranes compared to that in normally grown newborns [17].
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MATERIALS AND METHODS
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Hormones
The INSL4 was chemically synthesized with an insulin-like disulfide-bonding pattern as previously described [14]. Because of the lack of basic amino acids at the B/C peptide junction, it was synthesized with a 36-amino-acid-residue B chain, with the C-terminal pentapeptide omitted [14]. Recombinant human relaxin 2 was a generous gift from BAS Medical, Inc. (San Mateo, CA), and recombinant IGF2 was obtained from Bachem, Inc. (Torrance, CA).
Patients, Tissues, and Cell Culture
Fetal membranes and placentas were collected from Kapiolani Medical Center for Women and Children (Honolulu, HI) with informed consent and approval from the Institutional Review Board. For the study of INSL4 gene expression in the placenta by reverse transcription-polymerase chain reaction (RT-PCR), a tissue was collected after elective cesarean section at term (n = 1). A sample of placental trophoblast was taken from the center of the tissue, avoiding the chorionic and basal plates, and the full-thickness fetal membrane also was collected. For the quantitative real-time PCR, placental trophoblast (n = 4 different patients) was similarly collected, and the membranes were separated (n = 4), the amnion carefully stripped from the choriodecidua, and the decidua scraped from the chorion. None of these tissues had any signs of histological chorioamnionitis. The placental distribution of INSL4 gene expression was studied by collecting term placental samples from both cesarean sections before labor (n = 5 different patients) and normal spontaneous delivery (NSD) (n = 5 different patients) from the chorionic and basal plates as well as midway between these two sites; trophoblast was collected both from a lobe adjacent to the umbilical cord and from a lobe at the edge of the placenta. These patients had no clinical evidence of infection, but histological chorioamnionitis was not excluded. For an in vivo model of fetal growth restriction, placental samples (n = 3 different patients) and full-thickness membrane (n = 4 different patients) were collected from patients delivering preterm discordant twins (gestational age, 32–37 wk) by elective cesarean section before the onset of labor; the demographic data for these patients are shown in Table 1. These pregnancies were chosen over singleton pregnancies, because one twin of each pair was of normal size and, therefore, was a perfect control for the growth-restricted twin. The intrauterine environment was otherwise identical and decreased the normal variability observed when placentas from singleton pregnancies have been used. In addition, gestational length was identical for each twin in a pair. Discordant twins differ by 25% in weight, and the five pairs used here had a mean discordance of 33%. The samples used were either monoamniotic or diamniotic/ dichorionic. Samples were taken from both the growth-restricted and normally grown placentas by harvesting villous trophoblast as a cross-section of tissue from the peripheral edge. None of these patients had any clinical or histological evidence of infection. Samples were collected into liquid nitrogen and stored at –80°C until use, except those for INSL4 gene expression throughout the placenta, which were stored at 4°C in RNAlater (Qiagen, Valencia, CA) for several days before use. For the primary amnion cell cultures, the fetal membranes were cut from the placentas of women delivering at term by cesarean section (n = 7 different patients). The amniotic epithelial cells and fibroblasts were isolated by two methods as previously described [18, 19]. These cells and fibroblasts were compared, and the yield of fibroblasts was found to be considerably greater using the method described by Casey and MacDonald [19]. In addition, the contamination of each cell type with the other was lower (
2% fibroblasts contaminating the epithelial cells, and 10% epithelial cells contaminating the fibroblasts) as assessed by immunocytochemical staining for cytokeratin (epithelial cells) and vimentin (mesenchymal cells). Therefore, this method [19] was used for further studies. The Mann-Whitney nonparametric two-tailed test was used for analysis of gene expression in the fetal membranes and placentas, with results presented as the mean ± SD.
Culture of Cell Lines: WISH and JAR Cells and Their Treatment
Two cell lines were used; JAR cells (American Type Tissue Culture Collection [ATCC] HTB-144), representative of the placenta, and human amnion-derived WISH (ATCC CCL-25) cells, representative of the fetal membranes. Both were obtained from the ATCC (Manassas, VA). The WISH cells were grown in Dulbecco modified Eagle medium (DMEM)/ F12 supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), penicillin (100 U/ml), and streptomycin (100 µg/ml). The JAR cells were grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics at the same concentrations as used for WISH cells. Both cell types were incubated at 37°C in 5% carbon dioxide and 95% air. After reaching 70–80% confluence, they were either dispersed with 0.25% trypsin and 1.0 mmol/L of EDTA and then plated as described for each experiment or were treated as described below for flow cytometry.
Cell Proliferation Assays
The WISH and JAR cells were plated into 96-well culture plates at a density of 5000 cells per 200 µl of growth medium supplemented with 5% FBS. After the cells attached (48 h), they were washed with minimal media (0.25% FBS in DMEM/F12) and treated with either INSL4 (3, 10, and 30 ng/ml) or IGF2 (30 ng/ml) for 5 days. The dose of IGF2 was predetermined [5]. The INSL4 and IGF2 were diluted in minimal media, which was replaced after 48 h. Cells incubated in media alone were used as controls. The number of viable cells was measured with the CellTiter96 Aqueous One Solution kit (Promega, Madison, WI) as per the manufacturer's instructions (n = 4 experiments for both cell lines). The One Solution reagent was added to the culture media, and the cells were incubated for 30 min at 37°C in 5% carbon dioxide and 95% air, after which absorbance at 490 nm was read in a 96-well microplate reader (Molecular Devices, Sunnyvale, CA). Statistical analysis was performed by the two-tailed Mann-Whitney test and expressed as average percentage of the control value ± SD.
Cell-Cycle Analysis
The WISH cells were plated into 100-mm dishes at a density of 2 x 106 cells per 10 ml of medium supplemented with growth media (5% FBS in DMEM/F12) and grown for 48 h. The media were removed, and the cells were washed in Hanks balanced salt solution (HBSS) and then reincubated for synchronization for 24 h in minimal medium (0.25% FBS in DMEM/F12). They were then treated with INSL4 (30 ng/ml) or staurosporine (100 nM; Sigma, St. Louis, MO) as a positive control [20] for 48 h in growth medium. Cells incubated in growth medium alone acted as negative controls. At the end of the incubation, they were counted, and 2 x 106 cells were thoroughly resuspended in cold PBS, fixed in 70% ethanol, and placed on ice for at least 2 h. These cells were pelleted, resuspended in PBS, allowed to sit for 60 sec, and repelleted. The supernatants were decanted, and the pellets were resuspended in propidium iodide (PI) staining solution (2% PI, 20% DNase-free RNase, and 1% Triton X-100 in PBS) and then incubated for 30 min at room temperature in the dark. Fluorescence was measured by flow cytometry with a Beckman Coulter Epics XL instrument with a 488-nm argon laser, and 10 000 cells per sample were counted (cell doublets were gated out). The flow-cytometric data were analyzed with the Multicycle Program (Phoenix Flow Systems, San Diego, CA) to determine the percentage of cells in the G1/G0 (Gap 1/Gap 0), S (Synthesis), and G2/M (Gap 2/Mitosis) phases of cell growth. Four experiments were performed at different times. Statistical analysis used the two-tailed Mann-Whitney test and results expressed as the average percentage of total cells ± SD.
Analysis of Apoptosis
Several commercial reagents were tested to determine the optimal method for detachment of the primary amniotic epithelial cells for flow cytometry in order to minimize apoptosis. Amniotic epithelial cells were seeded into six-well plates and grown for 7–11 days in DMEM/F12 with 10% FBS. At 90% confluence, the media were removed and the cells washed with HBSS. The cells were then either scraped from the dish with a rubber policeman or incubated in the dish with either 750 µl of Accutase (Innovative Cell Technologies, San Diego, CA) or Accumax (Innovative Cell Technologies) for 10 min at 37°C. Alternatively, other cells were treated with a solution of sodium citrate and 0.135 M KCl for 5 min at 37°C (http://www.medicine.uiowa.edu/flowcytometry/citric_saline.html) or Cell-Stripper (Mediatech, Hearndon, VA) for 5 min at 37°C. Detached cells were collected by centrifugation and analyzed for apoptosis. Accutase preserved the viability of the detached cells, which was so pronounced in comparison to the other treatments that it was used for all further experiments. Staurosporine was used to induce apoptosis as previously described [20]. To determine its correct concentration for apoptosis induction, amniotic epithelial cells and fibroblasts (n = 3, isolated from different patients) were treated with a range of concentrations (25, 50, and 100 nM) for 12 h (Fig. 1) and 24 h (not shown) and then analyzed by flow cytometry. For the epithelial cells, both 50 and 100 nM staurosporine caused an increase in the numbers of apoptotic cells compared to controls, and these concentrations were used for further studies (Fig. 1A). However, staurosporine failed to cause apoptosis in fibroblasts (Fig. 1B); therefore, all further experiments were conducted with epithelial cells.
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FIG. 1. Determination of the effect of staurosporine (STS) treatment (one well/dose) on induction of apoptosis by flow cytometry in (A) primary amniotic epithelial cells and (B) fibroblasts (n = 3, isolated from different patients). The epithelial cells showed increased apoptosis with 50 and 100 nM STS compared to the controls. No effect of STS on the apoptosis of fibroblasts was observed. Results are shown as the mean ± SD
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The effect of INSL4 on apoptosis was compared to that of 100 nM staurosporine, which caused maximal apoptosis. Primary amniotic epithelial cells (n = 4 isolated from different patients) were treated with INSL4 (5, 25, and 50 ng/ml) or staurosporine (100 nM) in 1.5 ml of treatment medium (DMEM/F12 with no FBS or antibiotics) for 24 h at 37°C. The controls were grown in media alone. After treatment, media were removed and centrifuged at 210 x g for 5 min to pellet floating cells. The remaining cells on the plate were washed with HBSS and added to the same tubes. Accutase (750 µl) was added to each well, and the plates were incubated at 37°C for 10 min. The cells were then detached by gentle scraping and pooled with the floating cells. They were centrifuged at 210 x g for 5 min and washed with 1x Annexin Binding Buffer (Molecular Probes, Eugene, OR) and recentrifuged as before, and the supernatant was then removed. Apoptosis was detected with the Vybrant Apoptosis Assay (Molecular Probes) according to the manufacturer's protocol. This assay is based on binding by the apoptotic cells of Annexin V conjugated to the fluorescent dye, Alexa Fluor 488. Propidium iodide was used to monitor necrosis, because these cells stain both orange (PI) and green (Alexa Fluor) whereas viable cells exclude both dyes. Alexa Fluor/PI-staining solution (200 µl; Molecular Probes) was prepared as directed by the manufacturer and then added to each pellet. The cells were resuspended and incubated for 15 min at room temperature in the dark. Next, 1x binding buffer (800 µl) was added to each tube and placed on ice. Cell fluorescence was measured by flow cytometry using a Beckman Coulter Epics XL with a 488-nm argon laser. Statistical analysis was performed using the Mann-Whitney two-tailed test, with results expressed as the mean ± SD.
The effects of INSL4 on late-stage apoptosis also were measured from the amount of nuclear matrix protein in the media from these experiments (n = 4). This protein is only released into the medium by apoptotic and necrotic cells. The protein was measured using an ELISA assay (Calbiochem, Temecula, CA). Significance was determined by the Mann-Whitney two-tailed test, with results expressed as the mean ± SD.
To study the effects of relaxin (RLN2) on apoptosis, amniotic epithelial cells (n = 7, isolated from different patients) were incubated with 50 nM staurosporine either alone or together with 200 ng/ml of human relaxin for 12 h in treatment media. Controls included media alone or relaxin alone. Following treatment, the cells were detached with Accutase. Floating and adherent cells were combined and pelleted by centrifugation at 210 x g for 5 min as described above. Apoptosis was measured using the Vybrant Apoptosis Kit (Molecular Probes) following the manufacturer's protocol and then analyzed by flow cytometry as described above.
Isolation of Total RNA, RT-PCR, and Quantitative Real-Time PCR
The RNA was isolated from samples of separated amnion, chorion, decidua, and placenta as previously described [5]. For the regionalization of expression in the placenta, RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. For the twin placentas and membranes, RNA was extracted using either the RNeasy kit or TRIZOL (Invitrogen). The RNA from WISH and JAR cells was extracted using TRIZOL. All RNA samples used for quantitative real-time PCR were treated with either DNA-Free (Ambion, Austin, TX) or RNase-free DNase (Qiagen) to remove any contamination with genomic DNA. For RT-PCR, samples of total RNA (2 µg) obtained from WISH and JAR cells, fetal membranes, and placenta were reverse transcribed using Moloney murine leukemia virus and random hexamers according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). The PCR amplification was performed using specific primers for INSL4 (GenBank accession no. L34838; forward primer, 5'-AACTCCTTAGAGAAAGCCTAGCA-3'; reverse primer, 5'-TCGTACCTAAGGCTTGTCCATCT-3'). The DNA was denatured for 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec, 49°C for 15 sec, and 72°C for 30 sec. A final extension step was carried out at 72°C for 10 min. The products were visualized by gel electrophoresis.
Quantitative real-time PCR was performed using an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA) or an Opticon (MJ Research, Reno, NV). Specific primers (300 nM each) were made by Integrated DNA Technologies, Inc. (Coralville, IA), and were used in conjunction with QuantiTech SYBR Green PCR kit (Qiagen) for the study of INSL4 gene expression in the amnion, chorion, decidua, and placenta (forward primer, 5'-GCCTGAGAAGACATTCACCA-3'; reverse primer, 5'-TCGTACCTAAGGCTTGTCCA-3'). A melting curve was performed at the end of the PCR reaction to confirm a single product. A FAM-labeled LUX (light upon extension) primer and probe set for INSL4 were designed by Invitrogen and used for quantitation of INSL4 expression in different areas of the placenta (LUX probe, 5'-GAGCTGTTCAGACAGTGGTTTCTTCAGCTC-3'; primer, 5'-GAGGGTGGCTGCTGGAATCT-3'). For the iCycler real-time PCR with SYBR green, DNA samples were amplified for one cycle at 95°C for 15 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec, followed by a final extension step at 72°C for 10 min. For the Opticon with SYBR green, DNA samples were amplified for one cycle at 95°C for 15 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 15 sec, and 72°C for 20 sec, followed by extension as above. The PCR using the LUX probe and primer with Amplitaq Gold (Applied Biosystems) was carried out on the Opticon using these conditions: DNA samples were amplified for one cycle at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. All samples were run in triplicate, and a dilution series of an INSL4 cDNA probe (583 base pairs [bp]) in PCR 2.1 vector (Invitrogen) was used as a standard in all real-time PCR reactions. Statistical analysis was performed with the Mann-Whitney two-tailed test, and results were expressed as the mean ± SD.
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RESULTS
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Expression of the INSL4 gene was seen as a strong, single band of 192 bp in both the WISH and JAR cells and in the term placenta (Fig. 2A, lanes 1, 4, and 3, respectively) but, with these primers, was detectable only as a very faint band in the full-thickness fetal membranes (Fig. 2A, lane 2). The PCR control (Fig. 2A, lane 5) was negative. Quantitative real-time PCR on separated placenta, amnion, chorion, and decidua collected at term cesarean section before labor showed a significantly higher level of INSL4 expression in the placenta (P < 0.05) compared to that in the amnion, chorion, or decidua. For the membranes, the maternal decidua had the highest expression, followed by the amnion and chorion (Fig. 2B). To determine if the expression of the INSL4 gene showed any pattern throughout the placenta, quantitative real-time PCR was used to compare its expression in the central area near the umbilical cord, at its periphery, as well as at three levels of thickness at each of these sites. This was performed on placentas obtained both before and after NSD. No significant difference was found at the different levels of thickness at either site in cesarean section or NSD samples. However, when the expression at each level in the central area was compared to that at the periphery, significantly greater expression of INSL4 was observed at the periphery of the placenta in the basal plate (P < 0.02), the trophoblast (P < 0.03), and the chorionic plate (P < 0.03). Figure 3 shows the placentas obtained at cesarean section before labor. Similar significant differences also were obtained in placentas after NSD (data not shown) compared to the basal plate (P < 0.03), trophoblast (P < 0.008), and chorionic plate (P < 0.03). Therefore, INSL4 gene expression occurs predominantly at the periphery of the placenta.
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FIG. 2. A) INSL4 gene expression by RT-PCR shown as a single band of 192 bp in cell lines and tissues. Lane 1: amniotic epithelial-like WISH cells; lane 2: full-thickness fetal membrane; lane 3: placental trophoblast; lane 4: choriocarcinoma JAR cells; lane 5: negative control. B) INSL4 expression in tissues by quantitative real-time PCR shown as gene expression relative to that in the amnion. Tissues were all collected at term before labor, placenta (n = 4), amnion, chorion, and decidua (n = 4 from different patients). Results are shown as the mean ± SD. The asterisk indicates significantly (P < 0.05) more INSL4 expressed in the placenta compared to the amnion, chorion, or decidua
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FIG. 3. INSL4 gene expression as determined by quantitative real-time PCR in different regions of the term placenta before labor (n = 5 from different patients). Tissue was collected from the central area near the umbilical cord and at the placental periphery at three levels of thickness: basal plate, trophoblast, and chorionic plate. Results are expressed as the mean ± SD of INSL4 copy number. Significantly greater expression of INSL4 was found at the periphery of the placenta in the basal plate (asterisk; P < 0.02), trophoblast (P < 0.03), and chorionic plate (triangle; P < 0.03) compared to the central region of each
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The treatment of primary amniotic epithelial cells with staurosporine caused significant apoptosis (P < 0.05) as measured by flow cytometry (Fig. 4, A, B, and D). Their treatment with INSL4 caused a dose-related induction of apoptosis that almost reached significance at 25 ng/ml (P = 0.057) and that became significant at 50 ng/ml (P < 0.05) (Fig. 4, C and D). The cytograms from the flow cytometric analysis (Fig. 4, A–C) showed that most of the control cells were viable. The cells excluded both dyes and appeared in the lower left quadrant (Fig. 4A). Treatment with staurosporine (100 nM) induced apoptosis, as indicated by a large number of cells staining with Alexa Fluor 488 and moving to the lower right quadrant (Fig. 4B). Treatment with INSL4 also increased the numbers of apoptotic cells (Fig. 4C), although not as many of the cells shifted as occurred with the staurosporine treatment (Fig. 4B). However, INSL4 also caused an increase in the numbers of necrotic cells that stained with both reagents, shifting them to the upper right quadrant (Fig. 4C), but this result was not significant. The effect of INSL4 on apoptosis also was reflected by the increased nuclear matrix protein concentrations in the media (Fig. 4E), which was significant at both 25 ng/ml (P < 0.05) and 50 ng/ml (P < 0.05) of INSL4.
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FIG. 4. Effect of treatment of primary amniotic epithelial cells with INSL4 for 24 h (n = 4, different patients). Cytograms of flow-cytometric analysis are shown. A) Control, untreated cells. B) Staurosporine (STS)-induced apoptosis. C) INSL4 treatment, showing that INSL4 also induced apoptosis and necrosis. D) quantitation of the flow-cytometric data (mean ± SD) showing a dose-related effect of INSL4 on induction of apoptosis. Asterisks show STS significantly (P < 0.05)-induced apoptosis compared to the controls; INSL4 (50 ng/ml) significantly induced apoptosis (P < 0.05). E) Nuclear matrix protein (NMP) measurement in the medium of the experiments shown in D expressed as the mean ± SD, reflecting the effect of INSL4 on causing apoptosis and necrosis. Asterisks show significantly (P < 0.05) increased NMP compared to the controls
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A similar study was carried out with human relaxin, but unlike INSL4, human relaxin had no effect on the numbers of apoptotic cells. The cytograms showed increased numbers of apoptotic cells induced by treatment with staurosporine (Fig. 5B) compared to the control (Fig. 5A) and a lack of an effect from treatment with relaxin (200 ng/ml) (Fig. 5C). However, when the same concentrations of staurosporine and relaxin were used together, relaxin reduced the numbers of apoptotic cells from 61% to 46% (Fig. 5D); the quantitative results are shown in Figure 5E. Although this effect of relaxin failed to reach statistical significance, the results were in marked contrast to the effects of INSL4 as shown in Figure 4. Thus, INSL4 and relaxin appear to have opposite effects on the apoptosis of amniotic epithelial cells, with INSL4 inducing apoptosis and relaxin protecting the cells from an apoptotic stimulus.
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FIG. 5. Effect of relaxin treatment (12 h) on the apoptosis of primary amniotic epithelial cells (n = 7 different patients). Cytograms of flow-cytometric analysis are shown. A) Control, untreated cells. B) Staurosporine (STS)-induced apoptosis. C) Relaxin (RLN2) treatment had no effect on the numbers of apoptotic cells. D) STS and RLN2 together. The RLN2 reduced the numbers of apoptotic cells from 61% to 46% (not significant). E) Results of the flow-cytometric data shown in A–D expressed as the mean ± SD
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Because of the apoptotic effect of INSL4 in these studies, we sought an effect on the proliferation or number of viable WISH and JAR cells (representative of the amnion and placenta, respectively). For these experiments, IGF2 (30 ng/ml) was used as a positive control to induce (P < 0.05) the proliferation of WISH cells (Table 2). Treatment with INSL4 (30 ng/ml) significantly reduced (P < 0.05) the numbers of WISH cells compared to the control. Treatment with INSL4 at 3 and 10 ng/ml significantly (P < 0.05) reduced the numbers of JAR cells (Table 2) compared to the control. The effect of INSL4 was therefore more pronounced on the placental JAR cells than on the amniotic epithelial-derived WISH cells. These results led us to seek an effect of INSL4 on the cell cycle using staurosporine as a positive control (Table 3). Staurosporine had a significant effect on the cell cycle (P < 0.05) as expected, arresting cells in the G2/M phase [20]. Treatment with INSL4 had no effect on the numbers of cells at the different stages of the cell cycle (Table 3).
We sought an in vivo correlate of these in vitro data and measured the expression of INSL4 in the placentas and fetal membranes of discordant twins. Expression of the INSL4 gene was more than twofold greater in the placentas of growth-restricted twins compared to normally grown twins (Fig. 6A), but this difference did not reach statistical significance because of the small sample size of these rare tissues. In contrast, INSL4 gene expression in the full-thickness fetal membranes was similar in the growth-restricted and normally grown twins (Fig. 6B).
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FIG. 6. INSL4 gene expression by quantitative real-time PCR in (A) placentas (n = 3) and (B) fetal membranes (n = 4) obtained from discordant twins (one twin with normal growth and the other with growth restriction [IUGR]. Results are presented as the mean ± SD . Expression of the INSL4 gene was twofold greater in the placentas of the IUGR twins (not significant)
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DISCUSSION
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In the present study, we show that INSL4 is produced by the placenta and, to a much lesser extent, by the maternal decidua at term and that it probably acts locally to inhibit growth by causing apoptosis. It has been shown previously that INSL4 gene expression is highest in the syncytiotrophoblast, with less in the cytotrophoblast, villous stroma, and intermediate trophoblast [9]. Our demonstration that significantly more INSL4 gene expression occurs in the periphery of the placenta, in the basal plate, trophoblast, and chorionic plate, both before and after spontaneous labor and delivery, is important. It suggests that INSL4 is associated with increased apoptosis and necrosis, because the normal human placenta shows 90% of infarctions at its margin [21]. The inhibitory effects of INSL4 on growth also are in agreement with a recent study showing that a subclone of the SKBR3 breast cancer cell line with high levels of INSL4 gene expression had a much lower rate of growth than that of the parental cell line [22]. Considered together, these observations suggest that INSL4 may be produced both to counterbalance rapid growth and to decrease growth as required. An abundance of growth-stimulating peptides is produced by the intrauterine tissues. Several of these, like INSL4, are members of the insulin superfamily of hormones [2, 3, 5]. We compared the effects of relaxin and INSL4 on primary cells and showed that the two peptides had markedly different effects on apoptosis. Relaxin alone had no effect, but it was mildly protective against apoptosis when used together with an apoptosis-inducing agent. However, INSL4 caused apoptosis. Therefore, INSL4 appears to be unique in this family of hormones in terms of its action as an apoptotic agent/growth inhibitor.
Unfortunately, little is known about the structure of endogenous INSL4, and even less is known about its receptor or mode of action. Structurally, the native peptide may be significantly longer in length than the synthetic peptide used in the present study [7, 14]. Whereas the INSL4 molecule indeed appeared to be structureless in solution by circular dichroism spectroscopic criteria [14], such a study is no substitute for examination of biological activity. Therefore, we believe that the results of the present study supersede the idea that INSL4 is structureless and, therefore, has no biological function. A similar synthetic two-chain peptide corresponding to the predicted primary structure of INSL4 was recently shown to lack the ability to interact with LGR7 and LGR8. These leucine-rich repeat G protein-coupled receptor family members bind relaxin and INSL3, respectively [7]. Therefore, INSL4 likely has its own receptor, which may or may not be a member of the same receptor family as LGR7 and LGR8. However, the present study suggests that the synthetic peptide used was, indeed, able to interact with its receptor to cause a biological effect.
Placental apoptosis is an important process in normal placental development and increases as pregnancy advances [23]. An increased rate of apoptosis has been associated with placental and fetal growth restriction in singleton and twin pregnancies [24, 25]. We used flow cytometry and a cell proliferation assay to show that INSL4 affects the number of viable cells by causing apoptosis without affecting the cell cycle. Although the flow-cytometric studies used only primary amniotic epithelial cells or an amniotic epithelial-like cell line (WISH), the cell proliferation study used both WISH cells and a placenta-derived cell line (JAR). The INSL4 had a similar effect on cell proliferation in both cell lines. This suggests that INSL4 plays a similar role during growth in both the placenta and the amniotic epithelium. In the apoptosis study, INSL4 also slightly increased the numbers of necrotic cells, but this was not statistically significant.
We sought an in vivo clinical correlate for this finding by using tissues from fraternal twins with major discordant growth, and we showed twice the level of INSL4 gene expression in the placentas of growth-restricted twins compared to that in those of normally grown twins. This suggests INSL4 may be linked to the increased apoptosis in these tissues. No difference was observed in its expression in the fetal membranes of these patients, suggesting that in vivo, its effects may be greater in the placenta than in the membranes. Unfortunately, the results of the present study failed to reach significance because of the marked variability of expression in different patients and the small number of samples available. Discordant twin samples were optimal for study because of the controlled maternal environment for both the pathological twin and its normally grown control. We additionally excluded tissues from patients who were in labor or had clinical or histological evidence of chorioamnionitis. Such tissues are rarely available, limiting our sample size.
Gene expression can vary in the normal placenta, both within and between different sites [25]. We sampled various areas of the placenta for differences in the expression of INSL4 and showed that it was significantly higher in the peripheral region compared to the central area. We attempted to control for this difference in the collection of the pathological twin samples by sampling the full-thickness tissue at the placental periphery. However, it is possible that in a pathological state, such as major growth restriction, INSL4 gene expression might vary in a different pattern and that the most significant aberrant area of its expression was missed. The identification of a mediator capable of inducing aberrant growth in the placenta is obviously important, and further studies are needed to confirm our findings both in vivo and in vitro. In addition, the precise endogenous structure of this hormone needs to be known and its receptor identified before more firm conclusions can be drawn.
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ACKNOWLEDGMENTS
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We thank the nurses and staff of the labor and delivery ward of Kapiolani Medical Center for Women and Children for their help with the tissue collection for the present study. We also thank Dr. Lynn Iwamoto for help with the cell-cycle study and Tercia Ku for the technical assistance with the flow cytometry.
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FOOTNOTES
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1 Supported by NIH grant HD-24314 (G.B.G.) and by grants to the University of Hawaii and Kapi'olani Medical Center under the Research Centers in Minority Institutions Program of the National Institutes of Health (RR1A1-03061 and RR-11091). 
2 Correspondence: Lynnae Millar, Department of Obstetrics and Gynecology and Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, 1960 East West Rd., Biomed T-510, Honolulu, HI 96822. FAX: 808 956 5361; mill8lynn{at}aol.com
Received: 12 January 2005.
First decision: 19 February 2005.
Accepted: 9 June 2005.
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ABSTRACT
INTRODUCTION
