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oxytocinase during pregnancy
Biochimica et Biophysica Acta 1751 (2005) 19 – 25 http://www.elsevier.com/locate/bba
Review
Gene regulation and physiological function of placental leucine aminopeptidase/oxytocinase during pregnancy Seiji Nomura*, Tomomi Ito, Eiko Yamamoto, Seiji Sumigama, Akira Iwase, Mayumi Okada, Kiyosumi Shibata, Hisao Ando, Kazuhiko Ino, Fumitaka Kikkawa, Shigehiko Mizutani Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine Nagoya, 466-8550, Japan Received 7 May 2004; received in revised form 21 March 2005; accepted 12 April 2005 Available online 2 May 2005
Abstract Human pregnancy serum and placenta have the ability to degrade uterotonic peptide oxytocin (OT). Placental leucine aminopeptidase (P-LAP), which is also called cystine aminopeptidase, is the only membrane aminopeptidase known to functionally degrade OT as oxytocinase (OTase). P-LAP/OTase hydrolyzes several peptides other than OT including vasopressin and angiotensin III. P-LAP/OTase predicted from cDNA sequence is a type II integral membrane protein, which is converted to a soluble form existing in maternal serum by metalloproteases, possibly ADAM (a disintegrin and metalloproteinase) members. P-LAP/OTase activity increases with normal gestation, while decreases in the patients with preterm delivery and severe preeclampsia. In placenta, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts. Activator protein-2 (AP-2) and Ikaros transcription factors play significant roles in exerting high promoter activity of P-LAP/OTase in the trophoblastic cells. Moreover, P-LAP/OTase is transcriptionally regulated in a trophoblast-differentiation-dependent fashion via up-regulation of AP-2, putatively AP-2a. P-LAP/OTase may be involved in maintaining pregnancy homeostasis via metabolizing peptides such as OT and vasopressin. D 2005 Elsevier B.V. All rights reserved. Keywords: Aminopeptidase; Oxytocin; Placenta; Pregnancy; Transcriptional regulation; Trophoblast differentiation
1. Introduction During pregnancy, oxytocin (OT) is the most potent uterotonic peptide hormone and plays a critical role in the regulation of labor. Human pregnancy serum and placenta are known to have the ability to degrade OT [1], which should be involved in regulating OT action, because local concentrations of OT in the feto-placental –maternal unit depend upon a balance between synthesis and degradation. OT has a cystine ring structure at its N-terminus that is essential for exerting its uterotonic activity. Of two types of peptidases that metabolize OT, post-proline endopeptidase and aminopeptidase, aminopeptidase is able to open the ring structure and destroy OT activity, but whether post-proline
* Corresponding author. Tel.: +81 52 744 2261; fax: +81 52 744 2268. E-mail address: [email protected] (S. Nomura). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.04.006
endopeptidase has similar potential remains unclear [2– 4]. Thus, aminopeptidase should be regarded as oxytocinase (OTase) [5]. This enzyme is named cystine aminopeptidase, cystinyl aminopeptidase, CAP and placental leucine aminopeptidase (P-LAP; EC 3.4.11.3) after a chemical method to measure the enzymatic activity. For the remainder of this review, I will use the term P-LAP/OTase. The major protein in insulin-responsive glucose transporter isotype GLUT4 vesicles in rat adipocytes, which cotranslocates with GLUT4 to the cell surface in response to insulin, was found to have aminopeptidase activity [6,7]. Its cDNA cloning and characterization confirmed that the protein is a member of zinc-dependent membrane aminopeptidase family [8], hence it has been named insulinregulated membrane aminopeptidase (IRAP). Interestingly, independent cDNA cloning of human P-LAP/OTase [9] showed that rat IRAP and human P-LAP/OTase were 87% identical at amino acid levels, indicating that IRAP is a rat
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homologue of human P-LAP/OTase. As suggested by this finding, contrary to the initial hypothesis that P-LAP is a placenta-specific enzyme, P-LAP/OTase has a widespread tissue distribution [9,10]. In addition, success in cDNA cloning of human P-LAP/OTase further led us to isolate human P-LAP genome [11] and investigate the regulatory mechanisms of human P-LAP/OTase gene. In this review article, we have overviewed biochemical characteristics and functions of human P-LAP/OTase, especially with focusing on the possible physiological roles and gene regulation of P-LAP/OTase during pregnancy.
2. Biochemical characteristics of P-LAP/OTase We and another group have cloned the cDNA encoding human P-LAP/OTase from human placental cDNA library [9,12]. P-LAP/OTase cDNA cloning enabled us to deduce protein structure of P-LAP/OTase and prepare recombinant proteins. The finding that P-LAP/OTase is a type II membrane protein raised the question how P-LAP/OTase is released into the serum of pregnant women.
addition, Ser-80 and Ser-91 of this domain are the major phosphorylation sites by protein kinase C-~, which may be involved in P-LAP/OTase trafficking [15]. A large extracellular domain contains the GAMEN motif and HEXXH(18X)-E consensus sequence of zinc-binding site, which constitute the active site of gluzincin aminopeptidase family [16]. Based on the cDNA of human P-LAP/OTase, a largescale production system of recombinant human P-LAP/ OTase was established [17], which confirmed and newly provided enzymatic characteristics of P-LAP/OTase using molecular defined proteins. P-LAP/OTase preferentially hydrolyzes small peptides which possess an N-terminal ring structure, such as OT and vasopressin [17,18]. However, P-LAP/OTase dose not hydrolyze all the hormones with this structure, such as endothelins and calcitonin [17], indicating that whether or not one peptide hormone is cleavable by P-LAP/OTase may depend on the PV1 site and/ or the molecular size of the peptide. Additionally, P-LAP/ OTase also releases an N-terminal neutral or basic, not acidic, amino acids of small peptides including angiotensin III, somatostatin, Lys-bradykinin, Met-enkephalin, dynorphin A and nurokinin A [17 – 20].
2.1. Structural and enzymatic characteristics of P-LAP/OTase
2.2. P-LAP/OTase secretase
The predicted P-LAP/OTase contains three domains; an N-terminal 108 amino acid cytoplasmic domain, a 23-amino acid single transmembrane domain and an 893-amino acid extracellular domain (Fig. 1). Intracellular trafficking of PLAP/OTase in response to insulin and OT was demonstrated in adipocytes and vascular endothelial cells, respectively [8,13]. The N-terminal cytoplasmic domain of P-LAP/ OTase is thought to interact with a retention/sorting protein that also regulates the distribution of GLUT4 [14]. In
The presence of P-LAP/OTase activity in the serum of pregnant women has been known for a half century, which is consistent with the finding that P-LAP/OTase cDNA was cloned using P-LAP/OTase purified from retroplacental serum [9]. However, predicted structure of P-LAP/OTase from cDNA is a type II integral membrane protein, suggesting that P-LAP/OTase exists as a soluble form in maternal serum and a membrane-bound form in placenta. Soluble P-LAP/OTase in maternal serum is truncated
Fig. 1. Domain structure of P-LAP/OTase predicted from cDNA. Black box represents a putative transmembrane domain. Positions of the consensus sequence of the zinc-binding site and the N-terminal end of soluble P-LAP/OTase are also shown.
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between Phe154 and Ala155 and lacks cytoplasmic and transmembrane domains [9] (Fig. 1). It is generally accepted that P-LAP/OTase in maternal serum originates from placenta because serum P-LAP/OTase activity increases with gestation and disappears after delivery, and because PLAP/OTase is released into conditioned medium of placental organ culture [21]. Since P-LAP/OTase genome analysis revealed no alternative splicing sites around the N-terminal end of soluble P-LAP/OTase [11,22], soluble form appeared to be derived from the native membrane-bound form by post-translational processing. Initial attempts to elucidate the mechanisms of generating soluble P-LAP/OTase demonstrated that chelating agents such as EDTA and 1,10phenanthroline suppressed P-LAP/OTase release, indicating a possible involvement of metalloproteases in P-LAP/OTase shedding [23], which are distinct from angiotensin converting enzyme (ACE) secretase [24]. Further efforts to identify the metalloproteases demonstrated that ADAM (a disintegrin and metalloproteinase) members, at least including ADAM12, would be involved in P-LAP/OTase shedding in human placenta [25]. However until now, changes in PLAP/OTase secretase activity, including ADAM12, during pregnancy remain unknown, which seems to affect the PLAP/OTase activity in maternal serum.
3. Physiological properties of P-LAP/OTase during pregnancy Synthesized by both mother and fetus [26] as well as by placenta [27], OT concentrations in feto-placental-maternal unit would be affected by fetal growth and various environmental stresses during pregnancy. Since increased OT synthesis will induce myometrial contraction and labor onset, P-LAP/OTase is thought to play an important role in balancing degradation against production of OT to maintain the homeostasis of pregnancy (Fig. 2). Sequential measurements of P-LAP/OTase activity in maternal serum may serve as a good marker for monitoring diseases peculiar to
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pregnancy. Expression levels and location of P-LAP/OTase in placenta were also investigated. 3.1. P-LAP/OTase in maternal serum P-LAP/OTase activity in maternal serum increases during gestation to a maximum at near term, from 10 IU/ml at 10 weeks to 80 IU/ml at 38 weeks of gestation [21,28]. Some cases showed that the detailed measurements of P-LAP/ OTase activity reached a plateau just prior onset of labor [29,30]. Since OT production from fetus is increased with his growth, the findings may suggest that P-LAP/OTase increases to balance between the synthesis and degradation of OT, and that labor is established when OT synthesis overcomes the degradation. Thus, it may be a fascinating possibility that P-LAP/OTase is useful for predicting the timing of labor onset, which should be examined by further clinical studies. In accordance with this possibility, maternal serum P-LAP/OTase activity decreased in patients with spontaneous preterm delivery [31]. In addition, short-term feedback mechanism of regulating OT action is also postulated. OT stimulation to umbilical vascular endothelial cells induced translocation of P-LAP/OTase from the cytosol to the plasma membrane, which would degrade OT at the cell-surface [13]. Preeclampsia is a disorder that is peculiar to pregnancy typically with hypertension, proteinuria and edema. In preeclamptic patients, P-LAP/OTase activity was rather increased above the normal range when preeclampsia was still mild, but then decreased progressively with deterioration of preeclampsia [32]. Vasopressin, another good substrate peptide of P-LAP/OTase [17], is associated with hypertension in preeclampsia. Vasopressin enhances the intracellular cAMP levels via vasopressin V2 receptors [33], which may lead to inducing P-LAP expression in trophoblasts [34]. It is tempting to speculate that increased vasopressin may initially up-regulate P-LAP/OTase levels, which could counteract the hypertensive activity of vasopressin, and that the failure to induce a further rise in PLAP/OTase activity will worse preeclampsia. 3.2. P-LAP/OTase in placenta
Fig. 2. P-LAP/OTase functions as a barrier at the interface between mother and fetus. P-LAP/OTase regulates uterine contraction and vasoconstriction via degrading the oxytocin and vasopressin produced by the fetus in placenta.
P-LAP/OTase mRNA levels in human placenta are increased with advancing gestation, but the change is small compared with that observed in maternal serum [21]. A great increase in P-LAP/OTase in maternal serum may reflect the net effects of an increase in P-LAP/OTase synthesis in placenta, enlargement of placental volume and enhancement of P-LAP/OTase secretase activity. Villous trophoblasts in human placenta are composed of two types of cells, cytotrophoblasts and syncytiotrophoblasts. Cytotrophoblasts differentiate and fuse to syncytiotrophoblasts, which exist in the external site of villi exposed to maternal blood. At light microscopic levels, P-LAP/OTase was predominantly localized to syncytiotro-
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phoblasts in human placenta, while scarcely to cytotrophoblasts throughout the gestation [10,21]. In accordance with this observation, ultrastructurally, P-LAP/OTase was expressed on the surface of apical microvilli of syncytiotrophoblasts and, to a lesser extent, on the basal infoldings [35]. Moreover, in situ hybridization also demonstrated that P-LAP/OTase mRNA was mostly expressed in syncytiotrophoblasts in human placenta [36]. Similarly in mouse placenta, the trophoblast cells of the placental labyrinth facing the maternal blood space had a strong PLAP/OTase expression [37]. The predominant expression of P-LAP/OTase on the apical surface of syncytiotrophoblasts would be reasonable because P-LAP/OTase could function as a barrier at a site of interaction between the mother and fetus.
4. P-LAP/OTase gene regulation As mentioned above, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts, suggesting that P-LAP/OTase expression may depend on the trophoblast differentiation. Similar differentiation-dependent expression can be observed on human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) genes [38,39]. Differentiation from cytotrophoblasts requires the prior increase in intracellular cAMP levels, and also cAMP or its analogues stimulate trophoblast differentiation [40,41]. Moreover, numbers of hormones and cytokines act on placental gene regulation during pregnancy. Isolating P-LAP/OTase genomic clones is essential for elucidating the regulatory mechanism of P-LAP/OTase gene. 4.1. P-LAP/OTase gene regulation in trophoblast under basal condition We isolated genomic clones containing the 5V-upstream region of the P-LAP/OTase gene and determined the transcriptional initiation sites [11]. Human P-LAP/OTase gene was assigned to 5q14.2 –q15 of human chromosome, but up to now, this area is not associated with known genetic disorders. Rasmussen et al. further analyzed human P-LAP/ OTase gene and demonstrated that the human P-LAP/OTase gene contains 18 exons and 17 introns and that the gluzincin aminopeptidase motif, GAMEN-(31X)-HEXXH-(18X)-E, is encoded by exons 6 and 7, which is similar to other family members such as aminopeptidase N and aminopeptidase A [22]. Regulatory mechanisms of human P-LAP/OTase gene in trophoblasts have been intensely investigated [42,43]. Primary reporter assays showed the significance of promoter region from 297 to +49 in expressing high promoter activity in trophoblastic choriocarcinoma cells. Footprinting analysis using nuclear extracts from those cells demonstrated at least four footprinting sites (FP1 to FP4) in this
region, among which FP3 ( 214 to 183) was critical for high promoter activity. Electrophoretic mobility shift assay (EMSA) to identify the proteins interacting with DNA at FP3 revealed three retarded bands, one of which was generated by Activator protein-2 (AP-2) binding and another one (or two) of which was generated by Ikaros binding. Reporter assays combined with site-directed mutagenesis demonstrated that AP-2 was a main activator, and that Ikaros functioned with AP-2 cooperatively for maximal expression of human P-LAP/OTase gene. Ikaros, which was first identified as a factor that binds to the T cell receptor CD3y gene [44], is now reported to be required for the development of all lymphoid lineages [45,46]. However, despite numbers of observations in lymphocytes, little is known about Ikaros in other tissues. P-LAP/OTase gene regulation by Ikaros is the first report to show that Ikaros functions in trophoblast cells, although whether Ikaros is actually involved in P-LAP/OTase gene regulation in vivo remains unclear. Interestingly, the promoter region of aminopeptidase A, which is highly expressed in human placenta, also contains a DNA-binding motif for Ikaros [47]. AP-2 composes a family of at least three members, encoded by independent genes: AP-2a, AP-2h and AP-2g [48 – 50]. Among the members, AP-2g is abundantly and AP-2a is moderately expressed, while AP-2h is either not present or present in extremely low amounts in placenta and choriocarcinoma trophoblasts [51,52]. As suggested by the result that AP-2a and AP-2g recognize the same consensus DNA sequence, both AP-2a and AP-2g bound to FP3 site of P-LAP/OTase promoter region [43]. 4.2. P-LAP/OTase gene regulation with trophoblast differentiation In addition to the basal expression, mechanisms of trophoblast differentiation-dependent expression of P-LAP/ OTase were studied [34] using BeWo choriocarcinoma cells exposed to adenylate cyclase activator forskolin (FSK) as a model for trophoblast differentiation [53]. In this cell model, P-LAP/OTase activity and mRNA levels were increased along with FSK-induced differentiation. It should be noted here that the 5V-region up to 1.1 kb of the P-LAP gene contains no cAMP response elements (CREs; 5VTGACGTCA-3V) and that hCG-h subunit gene, which also lacks consensus CREs in the promoter region, responds to cAMP via AP-2 binding sites [40]. Whether or not similar mechanism associated with AP-2 is involved in P-LAP/ OTase gene regulation was examined in two ways. First, deletion or mutation of AP-2 binding site in the FP3 region abrogated the stimulatory effects of FSK on luciferase activity of the construct 214/+49 in BeWo cells. Second, in AP-2-deficient Hep-G2 cells, FSK failed to stimulate luciferase activity of the construct 214/+49. These results indicated that AP-2 binding to the FP3 region of the PLAP/OTase promoter was involved in up-regulating P-
S. Nomura et al. / Biochimica et Biophysica Acta 1751 (2005) 19 – 25
Fig. 3. P-LAP/OTase gene regulation with trophoblast differentiation. PLAP/OTase gene expression is up-regulated during the process of trophoblast differentiation, which requires increase in intracellular cAMP concentrations.
LAP/OTase expression with trophoblast differentiation. Moreover, an attempt to identify the key AP-2 family members for differentiation-dependent regulation of PLAP/OTase gene was made by immunocytochemical method. FSK induced remarkably high immunoreactivity of AP-2a, especially in the morphologically changed cells with enlarged and lobed nuclei, compared with non-treated cells. AP-2g was also expressed in nuclei and cytoplasm in non-treated cells, while FSK showed no stimulatory effects on its immunoreactivity. AP-2h expression neither under basal state nor after FSK-treatment was observed in BeWo cells. Therefore, it is strongly postulated that P-LAP/OTase is transcriptionally regulated in a differentiation-dependent fashion during placental development via up-regulation of AP-2, putatively AP-2a (Fig. 3). 4.3. P-LAP/OTase gene regulation by peptide and cytokine stimulants Patients with spontaneous preterm delivery have higher serum concentrations of inflammatory cytokines including interleukin-1h (IL-1h), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) [54,55] and lower P-LAP/OTase activities than those with normal delivery [31]. In addition, the P-LAP/OTase promoter region contains putative binding sites for cytokine-associated transcription factors. Therefore, the possibility that inflammatory cytokines suppress P-LAP/ OTase expression, which may cause spontaneous preterm delivery, was postulated. However, contrary to this possibility, IL-1h up-regulated P-LAP/OTase expression both at the mRNA and protein levels. For the induction of P-LAP/ OTase by IL-1h, de novo additional protein synthesis was required [56]. In addition to stimulating glucose transport [57], the binding of insulin to cell surface receptors alters the expression of numerous genes in a variety of tissues [58]. In trophoblastic cells, insulin has no stimulatory effects on glucose uptake or glycogen synthesis [59], while insulin is involved in increasing synthesis of hPL [60] and hCG [61]. Insulin exerts its positive or negative effects on gene expression at the transcriptional levels via insulin responsive elements (IREs) in the promoter region [55]. Since P-LAP/ OTase promoter region contains two analogous sites to IRE,
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we could postulate that insulin regulates P-LAP/OTase gene expression via these sites. In fact, insulin increased P-LAP/ OTase activity and mRNA expression, which was abrogated by prior exposure of cycloheximide to trophoblasts. However, luciferase assays showed no regulatory regions within 1.1 kb upstream of the P-LAP/OTase gene containing two analogous sites to IRE, indicating other mechanisms of the insulin-induced P-LAP/OTase mRNA accumulation [62].
5. Conclusion and perspective We overviewed biochemical and physiological properties of P-LAP/OTase as well as its gene regulation in trophoblasts. P-LAP/OTase is involved in the various pathophysiological processes including the maintenance of pregnancy homeostasis. In addition to the applications as a marker for monitoring disorders such as threatened premature delivery and preeclampsia, P-LAP/OTase may stand as a novel therapeutic target. Stimulants that increase cAMP levels could suppress uterine contraction during pregnancy via the up-regulation of P-LAP/OTase. Otherwise, recombinant PLAP/OTase could directly attenuate OT actions. Contrary to the initial idea that P-LAP/OTase is limited to placenta, P-LAP/OTase is widely distributed in human and mouse normal tissues [9,10,36] as well as malignant cells [63,64]. Physiological functions of P-LAP/OTase in the organs other than placenta have not been fully understood, while several lines of evidence about this query are accumulating.
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S. Nomura et al. / Biochimica et Biophysica Acta 1751 (2005) 19 – 25 [44] K. Georgopoulos, D.D. Moore, B. Derfler, Ikaros, an early lymphoidspecific transcription factor and a putative mediator for T cell commitment, Science 258 (1992) 808 – 812. [45] K. Georgopoulos, M. Bigby, J.H. Wang, A. Molnar, P. Wu, S. Winandy, A. Sharpe, The Ikaros gene is required for the development of all lymphoid lineages, Cell 79 (1994) 143 – 156. [46] C.A. Klug, S.J. Morrison, M. Masek, K. Hahm, S.T. Smale, I.L. Weissman, Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 657 – 662. [47] J. Wang, H. Walker, Q. Lin, N. Jenkins, N.G. Copeland, T. Watanabe, P.D. Burrows, M.D. Cooper, The mouse BP-1 gene: structure, chromosomal localization, and regulation of expression by type I interferons and interleukin-7, Genomics 33 (1996) 167 – 176. [48] P.J. Mitchell, C. Wang, R. Tjian, Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen, Cell 50 (1987) 847 – 861. [49] M. Oulad-Abdelghani, P. Bouillet, C. Chazaud, P. Dolle, P. Chambon, AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during differentiation of P19 embryonal carcinoma cells, Exp. Cell Res. 225 (1996) 338 – 347. [50] J.A. Williamson, J.M. Bosher, A. Skinner, D. Sheer, T. Williams, H.C. Hurst, Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors, Genomics 35 (1996) 262 – 264. [51] C. LiCalsi, S. Christophe, D.J. Steger, M. Buescher, W. Fischer, P.L. Mellon, AP-2 family members regulate basal and cAMP-induced expression of human chorionic gonadotropin, Nucleic Acids Res. 28 (2000) 1036 – 1043. [52] C. LiCalsi, S. Christophe, D.J. Steger, M. Buescher, W. Fischer, P.L. Mellon, AP-2 family members regulate basal and cAMP-induced expression of human chorionic gonadotropin, EMBO J. 28 (2000) 1036 – 1043. [53] B. Wice, D. Menton, H. Geuze, A.L. Schwartz, Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro, Exp. Cell Res. 186 (1990) 306 – 316. [54] S.L. Hillier, S.S. Witkin, M.A. Krohn, D.H. Watts, N.B. Kiviat, D.A. Eschenbach, The relationship of amniotic fluid cytokines and preterm
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Review
Gene regulation and physiological function of placental leucine aminopeptidase/oxytocinase during pregnancy Seiji Nomura*, Tomomi Ito, Eiko Yamamoto, Seiji Sumigama, Akira Iwase, Mayumi Okada, Kiyosumi Shibata, Hisao Ando, Kazuhiko Ino, Fumitaka Kikkawa, Shigehiko Mizutani Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine Nagoya, 466-8550, Japan Received 7 May 2004; received in revised form 21 March 2005; accepted 12 April 2005 Available online 2 May 2005
Abstract Human pregnancy serum and placenta have the ability to degrade uterotonic peptide oxytocin (OT). Placental leucine aminopeptidase (P-LAP), which is also called cystine aminopeptidase, is the only membrane aminopeptidase known to functionally degrade OT as oxytocinase (OTase). P-LAP/OTase hydrolyzes several peptides other than OT including vasopressin and angiotensin III. P-LAP/OTase predicted from cDNA sequence is a type II integral membrane protein, which is converted to a soluble form existing in maternal serum by metalloproteases, possibly ADAM (a disintegrin and metalloproteinase) members. P-LAP/OTase activity increases with normal gestation, while decreases in the patients with preterm delivery and severe preeclampsia. In placenta, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts. Activator protein-2 (AP-2) and Ikaros transcription factors play significant roles in exerting high promoter activity of P-LAP/OTase in the trophoblastic cells. Moreover, P-LAP/OTase is transcriptionally regulated in a trophoblast-differentiation-dependent fashion via up-regulation of AP-2, putatively AP-2a. P-LAP/OTase may be involved in maintaining pregnancy homeostasis via metabolizing peptides such as OT and vasopressin. D 2005 Elsevier B.V. All rights reserved. Keywords: Aminopeptidase; Oxytocin; Placenta; Pregnancy; Transcriptional regulation; Trophoblast differentiation
1. Introduction During pregnancy, oxytocin (OT) is the most potent uterotonic peptide hormone and plays a critical role in the regulation of labor. Human pregnancy serum and placenta are known to have the ability to degrade OT [1], which should be involved in regulating OT action, because local concentrations of OT in the feto-placental –maternal unit depend upon a balance between synthesis and degradation. OT has a cystine ring structure at its N-terminus that is essential for exerting its uterotonic activity. Of two types of peptidases that metabolize OT, post-proline endopeptidase and aminopeptidase, aminopeptidase is able to open the ring structure and destroy OT activity, but whether post-proline
* Corresponding author. Tel.: +81 52 744 2261; fax: +81 52 744 2268. E-mail address: [email protected] (S. Nomura). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.04.006
endopeptidase has similar potential remains unclear [2– 4]. Thus, aminopeptidase should be regarded as oxytocinase (OTase) [5]. This enzyme is named cystine aminopeptidase, cystinyl aminopeptidase, CAP and placental leucine aminopeptidase (P-LAP; EC 3.4.11.3) after a chemical method to measure the enzymatic activity. For the remainder of this review, I will use the term P-LAP/OTase. The major protein in insulin-responsive glucose transporter isotype GLUT4 vesicles in rat adipocytes, which cotranslocates with GLUT4 to the cell surface in response to insulin, was found to have aminopeptidase activity [6,7]. Its cDNA cloning and characterization confirmed that the protein is a member of zinc-dependent membrane aminopeptidase family [8], hence it has been named insulinregulated membrane aminopeptidase (IRAP). Interestingly, independent cDNA cloning of human P-LAP/OTase [9] showed that rat IRAP and human P-LAP/OTase were 87% identical at amino acid levels, indicating that IRAP is a rat
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homologue of human P-LAP/OTase. As suggested by this finding, contrary to the initial hypothesis that P-LAP is a placenta-specific enzyme, P-LAP/OTase has a widespread tissue distribution [9,10]. In addition, success in cDNA cloning of human P-LAP/OTase further led us to isolate human P-LAP genome [11] and investigate the regulatory mechanisms of human P-LAP/OTase gene. In this review article, we have overviewed biochemical characteristics and functions of human P-LAP/OTase, especially with focusing on the possible physiological roles and gene regulation of P-LAP/OTase during pregnancy.
2. Biochemical characteristics of P-LAP/OTase We and another group have cloned the cDNA encoding human P-LAP/OTase from human placental cDNA library [9,12]. P-LAP/OTase cDNA cloning enabled us to deduce protein structure of P-LAP/OTase and prepare recombinant proteins. The finding that P-LAP/OTase is a type II membrane protein raised the question how P-LAP/OTase is released into the serum of pregnant women.
addition, Ser-80 and Ser-91 of this domain are the major phosphorylation sites by protein kinase C-~, which may be involved in P-LAP/OTase trafficking [15]. A large extracellular domain contains the GAMEN motif and HEXXH(18X)-E consensus sequence of zinc-binding site, which constitute the active site of gluzincin aminopeptidase family [16]. Based on the cDNA of human P-LAP/OTase, a largescale production system of recombinant human P-LAP/ OTase was established [17], which confirmed and newly provided enzymatic characteristics of P-LAP/OTase using molecular defined proteins. P-LAP/OTase preferentially hydrolyzes small peptides which possess an N-terminal ring structure, such as OT and vasopressin [17,18]. However, P-LAP/OTase dose not hydrolyze all the hormones with this structure, such as endothelins and calcitonin [17], indicating that whether or not one peptide hormone is cleavable by P-LAP/OTase may depend on the PV1 site and/ or the molecular size of the peptide. Additionally, P-LAP/ OTase also releases an N-terminal neutral or basic, not acidic, amino acids of small peptides including angiotensin III, somatostatin, Lys-bradykinin, Met-enkephalin, dynorphin A and nurokinin A [17 – 20].
2.1. Structural and enzymatic characteristics of P-LAP/OTase
2.2. P-LAP/OTase secretase
The predicted P-LAP/OTase contains three domains; an N-terminal 108 amino acid cytoplasmic domain, a 23-amino acid single transmembrane domain and an 893-amino acid extracellular domain (Fig. 1). Intracellular trafficking of PLAP/OTase in response to insulin and OT was demonstrated in adipocytes and vascular endothelial cells, respectively [8,13]. The N-terminal cytoplasmic domain of P-LAP/ OTase is thought to interact with a retention/sorting protein that also regulates the distribution of GLUT4 [14]. In
The presence of P-LAP/OTase activity in the serum of pregnant women has been known for a half century, which is consistent with the finding that P-LAP/OTase cDNA was cloned using P-LAP/OTase purified from retroplacental serum [9]. However, predicted structure of P-LAP/OTase from cDNA is a type II integral membrane protein, suggesting that P-LAP/OTase exists as a soluble form in maternal serum and a membrane-bound form in placenta. Soluble P-LAP/OTase in maternal serum is truncated
Fig. 1. Domain structure of P-LAP/OTase predicted from cDNA. Black box represents a putative transmembrane domain. Positions of the consensus sequence of the zinc-binding site and the N-terminal end of soluble P-LAP/OTase are also shown.
S. Nomura et al. / Biochimica et Biophysica Acta 1751 (2005) 19 – 25
between Phe154 and Ala155 and lacks cytoplasmic and transmembrane domains [9] (Fig. 1). It is generally accepted that P-LAP/OTase in maternal serum originates from placenta because serum P-LAP/OTase activity increases with gestation and disappears after delivery, and because PLAP/OTase is released into conditioned medium of placental organ culture [21]. Since P-LAP/OTase genome analysis revealed no alternative splicing sites around the N-terminal end of soluble P-LAP/OTase [11,22], soluble form appeared to be derived from the native membrane-bound form by post-translational processing. Initial attempts to elucidate the mechanisms of generating soluble P-LAP/OTase demonstrated that chelating agents such as EDTA and 1,10phenanthroline suppressed P-LAP/OTase release, indicating a possible involvement of metalloproteases in P-LAP/OTase shedding [23], which are distinct from angiotensin converting enzyme (ACE) secretase [24]. Further efforts to identify the metalloproteases demonstrated that ADAM (a disintegrin and metalloproteinase) members, at least including ADAM12, would be involved in P-LAP/OTase shedding in human placenta [25]. However until now, changes in PLAP/OTase secretase activity, including ADAM12, during pregnancy remain unknown, which seems to affect the PLAP/OTase activity in maternal serum.
3. Physiological properties of P-LAP/OTase during pregnancy Synthesized by both mother and fetus [26] as well as by placenta [27], OT concentrations in feto-placental-maternal unit would be affected by fetal growth and various environmental stresses during pregnancy. Since increased OT synthesis will induce myometrial contraction and labor onset, P-LAP/OTase is thought to play an important role in balancing degradation against production of OT to maintain the homeostasis of pregnancy (Fig. 2). Sequential measurements of P-LAP/OTase activity in maternal serum may serve as a good marker for monitoring diseases peculiar to
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pregnancy. Expression levels and location of P-LAP/OTase in placenta were also investigated. 3.1. P-LAP/OTase in maternal serum P-LAP/OTase activity in maternal serum increases during gestation to a maximum at near term, from 10 IU/ml at 10 weeks to 80 IU/ml at 38 weeks of gestation [21,28]. Some cases showed that the detailed measurements of P-LAP/ OTase activity reached a plateau just prior onset of labor [29,30]. Since OT production from fetus is increased with his growth, the findings may suggest that P-LAP/OTase increases to balance between the synthesis and degradation of OT, and that labor is established when OT synthesis overcomes the degradation. Thus, it may be a fascinating possibility that P-LAP/OTase is useful for predicting the timing of labor onset, which should be examined by further clinical studies. In accordance with this possibility, maternal serum P-LAP/OTase activity decreased in patients with spontaneous preterm delivery [31]. In addition, short-term feedback mechanism of regulating OT action is also postulated. OT stimulation to umbilical vascular endothelial cells induced translocation of P-LAP/OTase from the cytosol to the plasma membrane, which would degrade OT at the cell-surface [13]. Preeclampsia is a disorder that is peculiar to pregnancy typically with hypertension, proteinuria and edema. In preeclamptic patients, P-LAP/OTase activity was rather increased above the normal range when preeclampsia was still mild, but then decreased progressively with deterioration of preeclampsia [32]. Vasopressin, another good substrate peptide of P-LAP/OTase [17], is associated with hypertension in preeclampsia. Vasopressin enhances the intracellular cAMP levels via vasopressin V2 receptors [33], which may lead to inducing P-LAP expression in trophoblasts [34]. It is tempting to speculate that increased vasopressin may initially up-regulate P-LAP/OTase levels, which could counteract the hypertensive activity of vasopressin, and that the failure to induce a further rise in PLAP/OTase activity will worse preeclampsia. 3.2. P-LAP/OTase in placenta
Fig. 2. P-LAP/OTase functions as a barrier at the interface between mother and fetus. P-LAP/OTase regulates uterine contraction and vasoconstriction via degrading the oxytocin and vasopressin produced by the fetus in placenta.
P-LAP/OTase mRNA levels in human placenta are increased with advancing gestation, but the change is small compared with that observed in maternal serum [21]. A great increase in P-LAP/OTase in maternal serum may reflect the net effects of an increase in P-LAP/OTase synthesis in placenta, enlargement of placental volume and enhancement of P-LAP/OTase secretase activity. Villous trophoblasts in human placenta are composed of two types of cells, cytotrophoblasts and syncytiotrophoblasts. Cytotrophoblasts differentiate and fuse to syncytiotrophoblasts, which exist in the external site of villi exposed to maternal blood. At light microscopic levels, P-LAP/OTase was predominantly localized to syncytiotro-
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phoblasts in human placenta, while scarcely to cytotrophoblasts throughout the gestation [10,21]. In accordance with this observation, ultrastructurally, P-LAP/OTase was expressed on the surface of apical microvilli of syncytiotrophoblasts and, to a lesser extent, on the basal infoldings [35]. Moreover, in situ hybridization also demonstrated that P-LAP/OTase mRNA was mostly expressed in syncytiotrophoblasts in human placenta [36]. Similarly in mouse placenta, the trophoblast cells of the placental labyrinth facing the maternal blood space had a strong PLAP/OTase expression [37]. The predominant expression of P-LAP/OTase on the apical surface of syncytiotrophoblasts would be reasonable because P-LAP/OTase could function as a barrier at a site of interaction between the mother and fetus.
4. P-LAP/OTase gene regulation As mentioned above, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts, suggesting that P-LAP/OTase expression may depend on the trophoblast differentiation. Similar differentiation-dependent expression can be observed on human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) genes [38,39]. Differentiation from cytotrophoblasts requires the prior increase in intracellular cAMP levels, and also cAMP or its analogues stimulate trophoblast differentiation [40,41]. Moreover, numbers of hormones and cytokines act on placental gene regulation during pregnancy. Isolating P-LAP/OTase genomic clones is essential for elucidating the regulatory mechanism of P-LAP/OTase gene. 4.1. P-LAP/OTase gene regulation in trophoblast under basal condition We isolated genomic clones containing the 5V-upstream region of the P-LAP/OTase gene and determined the transcriptional initiation sites [11]. Human P-LAP/OTase gene was assigned to 5q14.2 –q15 of human chromosome, but up to now, this area is not associated with known genetic disorders. Rasmussen et al. further analyzed human P-LAP/ OTase gene and demonstrated that the human P-LAP/OTase gene contains 18 exons and 17 introns and that the gluzincin aminopeptidase motif, GAMEN-(31X)-HEXXH-(18X)-E, is encoded by exons 6 and 7, which is similar to other family members such as aminopeptidase N and aminopeptidase A [22]. Regulatory mechanisms of human P-LAP/OTase gene in trophoblasts have been intensely investigated [42,43]. Primary reporter assays showed the significance of promoter region from 297 to +49 in expressing high promoter activity in trophoblastic choriocarcinoma cells. Footprinting analysis using nuclear extracts from those cells demonstrated at least four footprinting sites (FP1 to FP4) in this
region, among which FP3 ( 214 to 183) was critical for high promoter activity. Electrophoretic mobility shift assay (EMSA) to identify the proteins interacting with DNA at FP3 revealed three retarded bands, one of which was generated by Activator protein-2 (AP-2) binding and another one (or two) of which was generated by Ikaros binding. Reporter assays combined with site-directed mutagenesis demonstrated that AP-2 was a main activator, and that Ikaros functioned with AP-2 cooperatively for maximal expression of human P-LAP/OTase gene. Ikaros, which was first identified as a factor that binds to the T cell receptor CD3y gene [44], is now reported to be required for the development of all lymphoid lineages [45,46]. However, despite numbers of observations in lymphocytes, little is known about Ikaros in other tissues. P-LAP/OTase gene regulation by Ikaros is the first report to show that Ikaros functions in trophoblast cells, although whether Ikaros is actually involved in P-LAP/OTase gene regulation in vivo remains unclear. Interestingly, the promoter region of aminopeptidase A, which is highly expressed in human placenta, also contains a DNA-binding motif for Ikaros [47]. AP-2 composes a family of at least three members, encoded by independent genes: AP-2a, AP-2h and AP-2g [48 – 50]. Among the members, AP-2g is abundantly and AP-2a is moderately expressed, while AP-2h is either not present or present in extremely low amounts in placenta and choriocarcinoma trophoblasts [51,52]. As suggested by the result that AP-2a and AP-2g recognize the same consensus DNA sequence, both AP-2a and AP-2g bound to FP3 site of P-LAP/OTase promoter region [43]. 4.2. P-LAP/OTase gene regulation with trophoblast differentiation In addition to the basal expression, mechanisms of trophoblast differentiation-dependent expression of P-LAP/ OTase were studied [34] using BeWo choriocarcinoma cells exposed to adenylate cyclase activator forskolin (FSK) as a model for trophoblast differentiation [53]. In this cell model, P-LAP/OTase activity and mRNA levels were increased along with FSK-induced differentiation. It should be noted here that the 5V-region up to 1.1 kb of the P-LAP gene contains no cAMP response elements (CREs; 5VTGACGTCA-3V) and that hCG-h subunit gene, which also lacks consensus CREs in the promoter region, responds to cAMP via AP-2 binding sites [40]. Whether or not similar mechanism associated with AP-2 is involved in P-LAP/ OTase gene regulation was examined in two ways. First, deletion or mutation of AP-2 binding site in the FP3 region abrogated the stimulatory effects of FSK on luciferase activity of the construct 214/+49 in BeWo cells. Second, in AP-2-deficient Hep-G2 cells, FSK failed to stimulate luciferase activity of the construct 214/+49. These results indicated that AP-2 binding to the FP3 region of the PLAP/OTase promoter was involved in up-regulating P-
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Fig. 3. P-LAP/OTase gene regulation with trophoblast differentiation. PLAP/OTase gene expression is up-regulated during the process of trophoblast differentiation, which requires increase in intracellular cAMP concentrations.
LAP/OTase expression with trophoblast differentiation. Moreover, an attempt to identify the key AP-2 family members for differentiation-dependent regulation of PLAP/OTase gene was made by immunocytochemical method. FSK induced remarkably high immunoreactivity of AP-2a, especially in the morphologically changed cells with enlarged and lobed nuclei, compared with non-treated cells. AP-2g was also expressed in nuclei and cytoplasm in non-treated cells, while FSK showed no stimulatory effects on its immunoreactivity. AP-2h expression neither under basal state nor after FSK-treatment was observed in BeWo cells. Therefore, it is strongly postulated that P-LAP/OTase is transcriptionally regulated in a differentiation-dependent fashion during placental development via up-regulation of AP-2, putatively AP-2a (Fig. 3). 4.3. P-LAP/OTase gene regulation by peptide and cytokine stimulants Patients with spontaneous preterm delivery have higher serum concentrations of inflammatory cytokines including interleukin-1h (IL-1h), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) [54,55] and lower P-LAP/OTase activities than those with normal delivery [31]. In addition, the P-LAP/OTase promoter region contains putative binding sites for cytokine-associated transcription factors. Therefore, the possibility that inflammatory cytokines suppress P-LAP/ OTase expression, which may cause spontaneous preterm delivery, was postulated. However, contrary to this possibility, IL-1h up-regulated P-LAP/OTase expression both at the mRNA and protein levels. For the induction of P-LAP/ OTase by IL-1h, de novo additional protein synthesis was required [56]. In addition to stimulating glucose transport [57], the binding of insulin to cell surface receptors alters the expression of numerous genes in a variety of tissues [58]. In trophoblastic cells, insulin has no stimulatory effects on glucose uptake or glycogen synthesis [59], while insulin is involved in increasing synthesis of hPL [60] and hCG [61]. Insulin exerts its positive or negative effects on gene expression at the transcriptional levels via insulin responsive elements (IREs) in the promoter region [55]. Since P-LAP/ OTase promoter region contains two analogous sites to IRE,
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we could postulate that insulin regulates P-LAP/OTase gene expression via these sites. In fact, insulin increased P-LAP/ OTase activity and mRNA expression, which was abrogated by prior exposure of cycloheximide to trophoblasts. However, luciferase assays showed no regulatory regions within 1.1 kb upstream of the P-LAP/OTase gene containing two analogous sites to IRE, indicating other mechanisms of the insulin-induced P-LAP/OTase mRNA accumulation [62].
5. Conclusion and perspective We overviewed biochemical and physiological properties of P-LAP/OTase as well as its gene regulation in trophoblasts. P-LAP/OTase is involved in the various pathophysiological processes including the maintenance of pregnancy homeostasis. In addition to the applications as a marker for monitoring disorders such as threatened premature delivery and preeclampsia, P-LAP/OTase may stand as a novel therapeutic target. Stimulants that increase cAMP levels could suppress uterine contraction during pregnancy via the up-regulation of P-LAP/OTase. Otherwise, recombinant PLAP/OTase could directly attenuate OT actions. Contrary to the initial idea that P-LAP/OTase is limited to placenta, P-LAP/OTase is widely distributed in human and mouse normal tissues [9,10,36] as well as malignant cells [63,64]. Physiological functions of P-LAP/OTase in the organs other than placenta have not been fully understood, while several lines of evidence about this query are accumulating.
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