insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Regulatory Peptides 117 (2004) 187 – 193 www.elsevier.com/locate/regpep

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells Masayuki Nakata a, Seiji Nomura a,*, Yoko Ikoma a, Seiji Sumigama a, Fumi Shido a, Tomomi Ito a, Mayumi Okada a, Fumitaka Kikkawa a, Masafumi Tsujimoto b, Shigehiko Mizutani a a

Department of Obstetrics and Gynecology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa, Nagoya, 466-8550, Japan b Laboratory of Cellular Biochemistry, RIKEN (Institute of Physical and Chemical Research), Wako, 351-0148, Japan Received 25 April 2003; received in revised form 26 September 2003; accepted 2 October 2003

Abstract Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10 7 M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10 7 M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis. D 2003 Elsevier B.V. All rights reserved. Keywords: Gene regulation; Oxytocin; Placenta; Promoter; Protease

1. Introduction In addition to stimulating glucose transport [1,2], the binding of insulin to cell surface receptors alters the expression of numerous genes in a variety of tissues [3,4]. As suggested by the observation that placenta is an ideal tissue from which to purify insulin receptors (IR) [5], insulin likely plays a functional role in placenta. In placental and trophoblastic cells, insulin has no stimulatory effect on glucose uptake or glycogen synthesis [6,7], while insulin is involved in enhancing synthesis of human placental lactogen (hPL) [8] and human chorionic gonadotropin (hCG) [9] as well as in the regulation of 3h-hydroxysteroid dehydrogenase [10]. Although a detailed molecular mechanism through which * Corresponding author. Tel.: +81-52-744-2261; fax: +81-52-7442268. E-mail address: [email protected] (S. Nomura). 0167-0115/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.10.015

insulin mediates these effects in placenta remains unknown, insulin primarily regulates gene expression at the transcriptional level. Several responsive elements associated with gene regulation by insulin, insulin responsive elements (IREs), have been established, and these include the 10-bp sequence (TGGTGTTTTG) that is present in phosphoenolpyruvate carboxinase (PEPCK) and other insulin-regulated genes [4]. Placental leucine aminopeptidase (P-LAP), which is identical to cystine aminopeptidase [11], is the enzyme responsible for the complete inactivation of endocrine or paracrine oxytocin (OT) activity in placenta [12,13]. The finding that P-LAP activity increases with gestation [14,15] and decreases in patients with spontaneous preterm delivery [16] suggest that P-LAP plays a critical role in the maintenance of pregnancy. To explore the molecular mechanisms underlying P-LAP gene regulation, we isolated genomic clones containing the 5V -upstream region of the P-LAP gene

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[17]. The P-LAP promoter contains several putative nucleotide consensus sequences, such as binding sites for activator protein-2 (AP-2) and Ikaros, the functional roles of which in P-LAP gene transcription have since been confirmed in trophoblastic cells [18,19]. Interestingly, within 1.1 kb of the P-LAP promoter region, two analogous sites for 10-bp-IRE were identified. Therefore, we postulated that insulin might regulate P-LAP gene expression via these sites. Previous studies provide further evidence for the relationship between P-LAP and insulin. Several groups, including our own, have isolated P-LAP cDNA clones [20,21] and demonstrated that this enzyme is a homologue of rat insulin-regulated membrane aminopeptidase (IRAP) [22], which is present in the glucose transporter isoform GLUT4 vesicles of rat adipocytes [23,24]. Because insulin co-translocates IRAP/P-LAP from the cytosol to the cell membrane with GLUT4 in adipocytes and skeletal muscle cells, P-LAP may also be involved in glucose homeostasis via insulininduced trafficking of GLUT4 vesicles. Insulin is known to regulate expression of important glucose homeostasis genes that encode proteins such as PEPCK [25], glucose-6-phosphate dehydrogenase [26] and insulin receptor [27]. These results indicate the possibility that insulin may regulate PLAP gene expression, but to date, this possibility has not been confirmed. In the present study, we examined the effects of insulin on P-LAP expression in human choriocarcinoma BeWo cells, which have retained several placental properties and are therefore considered to be a suitable model of placental trophoblastic cells [28]. We investigated the relationship between insulin and P-LAP protein and mRNA levels. In addition, we functionally analyzed the P-LAP promoter region in an attempt to elucidate the molecular mechanisms of insulin-mediated P-LAP gene regulation.

2. Materials and methods 2.1. Cell culture Monolayer BeWo (ATCC CCL-98) human choriocarcinoma cells were maintained in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 Ag/ml). Human insulin was purchased from Sigma. 2.2. P-LAP enzymatic activity and Western blot analysis P-LAP activity was measured using L-leucine-p-nitroanilide (Sigma) as a substrate at 405 nm as previously described [14]. Cells were manually detached from dishes using a scraper and sonicated in lysis buffer (phosphatebuffered saline (PBS) containing 1% Triton-X, and protease inhibitor cocktail tablets; Complete Mini, EDTA-free

(Roche, Mannheim, Germany). Lysates were clarified by centrifugation at 8000  g for 15 min. The concentration of cellular proteins was measured using a BCA Protein Assay Kit (PIERCE, Rockford, IL, USA). Protein extracts (20 Ag) were resolved on 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). Anti-P-LAP immunoreactive proteins were visualized using the ECL plus Western blotting detection kit (Amersham Pharmacia Biotech, NJ, USA) with a rabbit anti-P-LAP polyclonal antibody (1:1000 dilution) [29]. 2.3. RNA isolation and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot procedure Total RNA was isolated from the cells using an RNeasy kit (QIAGEN K.K., Tokyo, Japan) according to the manufacturer’s instructions. RT-PCR/Southern blot procedure was performed as previously described [30]. Briefly, Total RNA (1 Ag) was reverse-transcribed with 2.5 AM random hexamers (Applied Biosystems, Foster City, CA, USA) in a total volume of 20 Al. Aliquots (1 Al) of RT reaction samples were amplified by PCR for P-LAP using the following primers in 50 Al mixtures: P-LAP sense; 5V-GGGCACAGATCAGGCTTCCCACT-3V, P-LAP anti-sense; 5VGATCTCAGCTTGTTTTTCTTGGCTTG-3V. RT-PCR for h-actin was performed using sense (5V-AACCGCGAGAAGATGACCCAG-3V) and anti-sense (5V-CTCCTGCTTGCTGATCCACAT-3V) primers. The number of PCR cycles for each product was established in preliminary experiments to ensure that each reaction remained within the exponential phase of amplification. PCR products were resolved by electrophoresis on 1.0% agarose gels, then transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) using a vacuum blotting system and cross-linked to the membrane by ultraviolet irradiation. Southern hybridization proceeded using 32P-P-LAP cDNA and 32P-h-actin cDNA as probes. P-LAP mRNA levels were normalized gainst h-actin expression measured with a BAS 2000 Bioimage Analyzer (Fuji Photo Film, Kanazawa, Japan) after autoradiography. 2.4. Construction of luciferase reporter plasmids, transfection and luciferase assay We prepared P-LAP promoter-luciferase constructs by subcloning PCR-derived fragments of the P-LAP 5V-flanking region into the pGL3-Basic vector (Promega, Madison, WI, USA) at the KpnI site as previously described [18,30]. PCR was performed using primers, of which a KpnI restriction site was added to the 5V-portion. The PCR fragments were digested with KpnI and subcloned into a similarly digested pGL3-Basic vector. BeWo cells were plated (0.8  106 cells/6-well plate) 24 h before transfection. Firefly luciferase reporter plasmid DNA (1 Ag) and 0.1 Ag of pRL-TK plasmid DNA as an internal control to standardize

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transfection efficiency were transiently co-transfected into cultured cells for 3 h using the LipofectAMINE PLUSk Reagent (Life Technologies, Gaithersburg, MD, USA). Twenty-one hours after transfection, cells were incubated with or without 10 7 M of insulin for a further 12 or 24 h in serum-free medium and were then lysed with 500 Al of passive lysis buffer. Firefly and renilla luciferase activities were measured using dual-luciferase reporter assay systems (Promega). 2.5. Statistical analysis All experiments were repeated at least three times in triplicate. Data are expressed as mean F SD. Because data did not have a normal distribution, we employed nonparametric statistics. Comparisons within groups were made by Kruskal-Wallis one-way analysis of variance, while those between groups were made by Mann –Whitney U-test for two independent samples, and Boneferroni correction was used for multiple comparisons. Differences were considered significant when the P value was < 0.05.

3. Results 3.1. Effects of insulin on P-LAP protein in BeWo cells We incubated BeWo cells with insulin for 24 h at physiological concentrations from 10 8 to 10 6 M in order to assess the effects of insulin on P-LAP activity (Fig. 1). To exclude the possibility that insulin might alter cell growth, we adjusted P-LAP activity for cellular protein concentrations. Although exposure to 10 8 insulin did not result in significant changes in P-LAP activity, 10 7 and 10 6 M insulin increased P-LAP activity significantly. Maximal

Fig. 1. Dose-dependent effects of insulin on P-LAP activity. BeWo cells were cultured for 24 h with various concentration of insulin. Data represent mean F SD of triplicate determinations from three independent experiments. *P < 0.05 vs. control by Mann – Whitney U-test with Boneferroni correction.

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Fig. 2. P-LAP protein expression determined by Western blotting. P-LAP signals with expected molecular weight (175 kD) obtained after incubation with indicated concentrations of insulin are shown on representative films.

enhancement of P-LAP activity was observed using 10 7 M insulin (181.3 F 22.7%) ( P < 0.05). We confirmed this by Western blotting analysis using anti-P-LAP polyclonal antibody (Fig. 2). The signal intensity of a single band at the expected molecular weight of 175 kD obviously increased in the presence of 10 7 M insulin. Band intensity at 10 6 M insulin was less than that at 10 7 M insulin. We determined the density of the bands using scanning densitometry, which demonstrated a significant increase only at 10 7 M, with a small but non-significant increase at 10 6 M (data not shown). We therefore concluded that 10 7 M insulin increased P-LAP protein levels most efficiently and then investigated the time-course effects of 10 7 M insulin treatment on P-LAP activity (Fig. 3). Incubation with insulin did not significantly stimulate P-LAP activity up to 12 h, while caused a significant increase in activity after 24 h. Insulin treatment for 48 h increased P-LAP activity to 201.3 F 23.2% ( P < 0.05), which was not significantly different from that at 24 h. 3.2. Effects of insulin on P-LAP mRNA We examined whether induction of P-LAP protein by insulin was associated with an increase in P-LAP mRNA

Fig. 3. Time-course of insulin effects on P-LAP activity. BeWo cells were cultured either in the absence (control, open columns) or presence of 10 7 M insulin (closed columns) for the indicated periods. The medium in each well was changed daily. Data are expressed as mean F SD of triplicate determinations from at least three independent experiments. *P < 0.05 vs. control by Mann – Whitney U-test.

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Fig. 4. Effects of insulin and cycloheximide on P-LAP mRNA levels in BeWo cells. Cells were incubated for 24 h in serum-free medium with insulin. Total RNA was amplified by RT-PCR, then analyzed by Southern blotting. Signals after autoradiography are shown on representative films. Upper and lower panels show P-LAP and h-actin, respectively. Induction of P-LAP mRNA by 10 7 M insulin was abrogated by cycloheximide (CHX) pretreatment.

and whether 10 7 M insulin had a more potent effect on P-LAP mRNA induction than 10 6 M insulin. BeWo cells were incubated with 10 7 M and 10 6 M insulin for 12 or 24 h, then mRNA was extracted and P-LAP mRNA levels were determined by semiquantitative RT-PCR followed by Southern blotting. Preliminary experiments demonstrated no apparent changes in P-LAP mRNA levels after 12h insulin treatment (data not shown). We then determined

the range of PCR cycles required to provide linearity for 24 h-insulin treatment, which indicated that 22– 25 and 14– 17 cycles were appropriate to reach the linear non-saturating increase phase for P-LAP and for h-actin, respectively (data not shown). The representative Southern blots of PCR products after 24 cycles for P-LAP and 16 cycles for hactin shown in Fig. 4 demonstrated that 10 7 and 10 6 M insulin increased P-LAP mRNA expression and that 10 7 M insulin was superior to 10 6 M in stimulating P-LAP mRNA. We also evaluated whether de novo protein synthesis was required for this P-LAP accumulation. Cells were pre-incubated for 30 min with cycloheximide (10 Ag/ml), an inhibitor of translational elongation, then stimulated with 10 7 M insulin. Cycloheximide abrogated insulin enhanced P-LAP expression, suggesting that induction of P-LAP transcription by insulin requires synthesis of additional proteins (Fig. 4). 3.3. Effects of insulin on luciferase activity The 1.1 kb 5V-flanking region of P-LAP contains two possible IREs. As shown in Fig. 5A, IRE1 located from 421 to 412 and IRE2 located from 261 to 252

Fig. 5. Effects of insulin on luciferase activity in deletion constructs of P-LAP promoter transfected into BeWo cells. (A) Sequence comparison between IREs in P-LAP promoter and the 10-bp-IRE of PEPCK gene. (B) Relative luciferase activity. P-LAP deletion fragments were linked to the luciferase reporter gene in a PGL-3 basic vector, and constructs were transiently transfected into BeWo cells. After transfection, cells were incubated in the presence or absence of 10 7 M insulin for 12 h. Induction of luciferase activity after 12-h incubation with insulin on the construct 164/ + 49 was set at 1. Results are expressed as fold activation of relative luciferase activity for individual constructs exposed to insulin. Data are presented as means F SD of three independent transfection experiments performed in triplicate and were statistically compared by the Kruskal – Wallis test.

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have a 7/10 and 8/10 match with the 10-bp-IRE sequence in the PEPCK gene (TGGTGTTTTG), respectively. To determine whether insulin enhances P-LAP transcription via these elements, we investigated the effects of insulin on the luciferase activity of P-LAP deletion constructs in BeWo cells. We incubated transfected BeWo cells with insulin for 12 and 24 h. Induction of luciferase activity after 12h insulin incubation on the construct 164/ + 49, which does not include IRE1 and IRE2, was set at 1, and the relative luciferase activities (the ratio of luciferase activity in the presence of insulin to that in the absence of insulin) for the other constructs are shown in Fig. 5B. Deletion from 752 to 411, which removed the IRE1, did not result in any significant differences in luciferase activity between cells incubated in the presence or absence of insulin. Further deletion to 251, which removed the IRE2, had no effect on the relative luciferase activity after exposure to insulin. These results suggested that neither IRE1 nor IRE2 participates in insulin-induced P-LAP mRNA expression. The results after 24-h insulin incubation were similar (data not shown).

4. Discussion The action of OT depends on metabolism in addition to synthesis and its receptor levels. P-LAP functions as an oxytocinase by opening the ring structure of OT [29], the critical motif for OT uterotonic bioactivity. Our sequencing of the P-LAP promoter region [17] suggested the possible involvement of insulin as a P-LAP gene regulator. We used BeWo choriocarcinoma cells in this study due to their several trophoblastic properties, which ensure that these cells are able to act as a suitable model of the placenta [28]. In addition, transcription factors that regulate P-LAP gene expression under basal conditions have been identified in BeWo [18,19]. Here, we demonstrated that insulin up-regulates P-LAP expression both at the mRNA and protein levels. However, mechanisms other than the predicted pathway via the 10-bpIRE, and involving de novo synthesis of additional proteins appeared to mediate P-LAP up-regulation by insulin. We observed that insulin treatment increased P-LAP activity and that insulin concentrations of 10 7 M resulted in more potent enhancement than was observed at 10 6 M. To confirm these results and to examine the possibility that endogenous activators or inhibitors of P-LAP activity were mediated by insulin, we performed Western blot analysis, which showed a similar pattern. These results of dose – response experiments seem reasonable, because the physiological concentrations of insulin are thought to range from 10 10 to 10 7 M. Our results also agreed with the finding that 10 7 M insulin exerts maximal stimulatory effects on 3h-hydroxysteroid dehdrogenase activity in cytotrophoblasts [10]. We then examined the change in P-LAP mRNA in insulin-treated BeWo cells to determine whether an increase

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in P-LAP proteins is associated with increased accumulation of mRNA. RT-PCR and Southern blot showed an increase in P-LAP mRNA after insulin incubation, and more potent effects at 10 7 M than 10 6 M insulin, which is consistent with the findings for P-LAP activity. This suggests that the increase in P-LAP protein after insulin treatment is regulated at the transcriptional level. We also observed that the increase in P-LAP mRNA caused by 10 7 M insulin was suppressed by cycloheximide treatment, suggesting that de novo proteins synthesis is required for this increase. It has been generally accepted that insulin exerts its positive or negative effects on gene expression at the transcriptional level. Substantial effort has been devoted to identifying the DNA sequences and the trans-acting factors responsible for mediating these effects, particularly on genes that have a metabolic connection with insulin. Promoter analysis of the PEPCK gene, a key enzyme for gluconeogenesis, demonstrated that the 10-bp sequence between 416 and 407 (TGGTGTTTTG) sufficiently explains the repressive effects of insulin on PEPCK gene transcription [31,32]. This 10-bp sequence, or analogous sequences, exist in a number of other genes that are known to be regulated by insulin, and these sequences have both positive and negative effects on transcription, irrespective of orientation [4]. We isolated genomic clones containing the promoter regions for P-LAP [17], the sequences of which included two putative IREs of apparent homology with the 10-bp-sequence. Therefore, we postulated that insulin influences P-LAP transcriptional activity via these elements located in the P-LAP 5V-flanking region. However, the present study found that none of the luciferase-P-LAP constructs investigated were significantly affected after exposure to insulin, despite an increase in P-LAP mRNA. The presence of mismatched nucleotides in the IREs in the P-LAP promoter could not account for their no responsiveness to insulin, because the functional IREs of glucokinase, insulin-like growth factor binding ptotein-1, alpha-amylase have 8/10, 7/10 and only 6/10 match with the 10-bpsequence, respectively [33 – 35]. Proteins that bind to the 10-bp-IRE have not been clearly identified, while interaction with other transcription factors, such as hepatic nuclear factor-3 (HNF-3), has been demonstrated in hepatocytes [36,37]. The 10-bp-sequence resembles and frequently overlaps the binding sites for HNF-3. Therefore, the observation that putative IREs in the P-LAP promoter region did not confer insulin responsiveness in trophoblasts would not exclude the possibility of their function in other cells such as hepatocytes, although further studies are required. Failure to prove our postulation suggests the involvement of other as yet unidentified mechanisms through which insulin may increase P-LAP mRNA. It is possible that different IREs may be involved in this mechanism, because multiple IREs have been reported to exist [38]. However, we did not observe significant insulin-induced effect on the activity of any luciferase-P-LAP constructs investigated, suggesting that the responsible elements accounting for

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the insulin-dependent increase in P-LAP mRNA may be located in regions further upstream than 752 or in introns. The finding that insulin-induced P-LAP transcription was suppressed by cycloheximide indicates the involvement of intermediary transcription factors. The human placenta has IR on the membrane of the syncytiotrophoblast microvilli [39]. Interestingly, P-LAP is also expressed on the apical membrane in syncytiotrophoblasts [15,40] and ultrastructurally on the microvilli [41], indicating the co-existence of P-LAP and IR. Therefore PLAP gene regulation by insulin appears to be very likely. However, placental P-LAP expression increases throughout normal pregnancy [15], while the number of IR on syncytiotrophoblasts decreases after mid-gestation [42]. Although insulin might participate in the regulation of P-LAP in early gestation, insulin does not appear to be the principal stimulator of P-LAP gene in mid-or late gestation. What are the clinical implications of the P-LAP upregulation by insulin? Since insulin is not known to regulate glucose transfer from mother to fetus, one major biological effect of insulin in placenta is the stimulation of mitogenesis [43]. This notion is supported by the evidence that placental IR are present in areas of high proliferative activity in the first trimester. OT, target peptide of P-LAP, promotes trophoblast cell growth [44]. Therefore, induction of P-LAP by insulin, which leads to attenuating OT activity, may regulate the trophoblast growth accompanied by the mitogenic effects of insulin. P-LAP and insulin may also be related to the increased incidence of spontaneous preterm delivery among women with diabetes mellitus (DM) [45]. Pregnant women with DM have lower insulin sensitivity than those with normal glucose tolerance. Although speculative, insufficient induction of P-LAP due to insulin resistance in women with DM, which results in relative increase in local OT levels, may be associated with the unsuccessful prevention of preterm uterine contraction. In conclusion, we observed that exposure to insulin stimulated BeWo trophoblastic cells to express P-LAP, which required de novo synthesis of other proteins, but the sequences responsible for regulating P-LAP gene expression by insulin were not found within the 1.1 kb upstream region. If further studies using adipose or skeletal muscle cells, in which insulin is known to increase cellsurface P-LAP activity by stimulating the translocation of PLAP vesicles, could demonstrate the results similar to those in this study, insulin would enhance cell-surface P-LAP activity via short-and long-term effects in those tissues.

Acknowledgements The work was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from the Ministry of Public management, Home affairs, Posts and Telecommunications

of Japan for specific medical research (in collaboration with Nagoya Teishin Hospital).

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