New regulatory mechanisms for human extravillous trophoblast invasion

Abstract

Human extravillous trophoblasts (EVT) invade maternal deciduas and reconstructed maternal spiral arteries during early placentation. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVT from further invasion have not been well understood. Recently, it was found that EVT that had already ceased their invasion, specifically expressed cluster of differentiation (CD9) and dipeptidyl peptidase IV (DPPIV) on their cell surface. In addition, EVT migrating to maternal spiral arteries expressed CC chemokine receptor type‐1 (CCR‐1), which is a chemokine receptor for regulated on activation normal T cell expressed and secreted (RANTES) and so on. CD9 is associated with integrin molecules on the cell surface and is considered to modulate integrin function. In contrast, DPPIV is a cell surface peptidase that can metabolize RANTES at extracellular sites before its accessing to the chemokine receptors. In vitro functional assay showed that CD9, DPPIV and RANTES are involved in the regulation for EVT invasion. From these findings, it can be proposed that CD9 and DPPIV, including chemokines, are new regulatory factors for human extravillous trophoblasts. (Reprod Med Biol 2005; 4: 189–195)

INTRODUCTION

HUMAN TROPHOBLASTS DIFFERENTIATE to two major cell lineages, that is villous trophoblasts and extravillous trophoblasts (EVT). Villous trophoblasts form chorionic villi, covering the surface portion of the villi and transporting nutrients and oxygen from the mother to the fetus. In contrast, EVT invade the maternal decidual tissues and reconstruct maternal spiral arteries, infiltrating both arterial lumen and muscle layer. This infiltration by EVT causes the loss of arterial contractility and then maintains adequate maternal blood flow into the intervillous spaces to support placental function. The interruption of this invasion will cause subsequent placental dysfunction, leading to various obstetrical disorders such as pre‐eclampsia. ¹ Thus, EVT invasion is an essential process for embryo implantation and placental formation. In contrast to malignant cells, EVT invasion is confined spatially to the uterus and temporally to early pregnancy. However, the molecules that were reported to regulate EVT invasion cannot fully explain the spatiotemporal development and differentiation of EVT, particularly from invasive to non‐invasive phenotypes. EVT also formed a peripheral layer of chorion laeve that is an intermediate component of fetal membrane. Although fetal membrane is considered to play a central role in regulation for homeostasis of amniotic fluid during pregnancy and in initiation of labor, the precise mechanisms and roles of chorion laeve are also largely unknown.

Although EVT directly face the maternal cells in the decidua and in the fetal membrane, despite being a semiallograft, the invading EVT are not attacked by the maternal immune cells during their infiltration toward maternal spiral arteries and fetal membrane. In contrast to other organ constructions in the embryo and placenta, the remodeling that occurs in the maternal spiral arteries requires embryo‐derived cells and maternal cells to cooperatively build tissue, being more complex than those in the organ development in embryogenesis.

To understand these regulatory mechanisms, we investigated the molecules that are specifically expressed on human EVT, which might be involved in the EVT invasion. In the present syudy, new findings concerning the molecules expressed on EVT and their roles in EVT function are described.

Role of CD9 in EVT invasion

Various mechanisms for EVT invasion have been proposed, including growth factors. ² , ³ EVT invasion was reportedly regulated by several molecules such as matrix metalloproteinases (MMP) and serine proteinases, which degrade the extracellular matrix to facilitate EVT migration into the deciduas. ⁴ , ⁵ In addition, cell–cell and/or cell–extracellular matrix interactions mediated by adhesion molecules, such as cadherins and/or integrins, have been considered important for EVT invasion. ⁶ For example, when trophoblasts acquire an invasive phenotype in the cell column, down‐regulation of integrin α6β4 and up‐regulation of integrin α5β1 and integrin α1β1 occur. ⁷ These integrins have also been reported to modulate trophoblast motility. ⁸ , ⁹ However, integrin α5 is equally expressed on interstitial trophoblasts, regardless of the depth of invasion or the stage of pregnancy. Thus, the mechanism(s) that causes cessation of EVT invasion has not yet been thoroughly clarified.

Previously, it was reported that a cell surface molecule, CD9, was predominantly expressed on human EVT that had ceased invasion at a deep site in the maternal decidual tissues. ¹⁰ In contrast, CD9 was not expressed on villous trophoblasts as previously described. ¹¹ In addition, CD9 was weakly expressed on the cell column of the chorion, showing that CD9 is a differentiation‐related molecule for EVT.

CD9 was initially considered to be specific to acute lymphoblastic leukemia cells. ¹² This antigen was also expressed on a variety of tumors and normal human cells, including pre‐B cells, activated T cells, platelets and Schwann cells. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ Although the physiological role of CD9 is unknown, it has been shown that anti‐CD9 mAbs induce the migration of Schwann cells ¹³ and that CD9 regulates the adhesion of pre‐B cells to bone marrow fibroblasts, ¹⁷ suggesting the involvement of CD9 in cell adhesion and migration. Therefore, we postulated a possible role for CD9 in EVT invasion.

In order to investigate the functional role of CD9 on trophoblasts, we used the human trophoblast‐like choriocarcinoma cell line BeWo, because this cell line expresses CD9 on their cell surface and has the characteristics of both villous and extravillous trophoblasts. ¹⁸ , ¹⁹ , ²⁰ For example, this cell line secretes hCG after stimulation by forskolin. The invasion assay showed that the binding of the anti‐CD9 mAb (ALB‐6) to CD9 enhanced the number of invaded BeWo cells without affecting the proliferation of BeWo cells. ²¹

Because CD9 is known to be associated with β1‐related integrins in the other cells, ²² , ²³ CD9 in the extravillous trophoblasts might modulate the function of β1‐related integrins. By Western blotting analysis of the affinity‐purified proteins, both integrin α3 and α5 expressed on EVT in the chorion laeve were associated with CD9, suggesting the functional relationship between CD9 and these integrins. The similar association between CD9 and integrin α3 and α5 was also confirmed in BeWo cells. ²¹

In invasion assay using anti‐integrin α3, α5, and β1 blocking mAbs, these mAbs inhibited BeWo cell invasion in a dose‐dependent manner, indicating that both integrins α3β1 and α5β1 facilitate BeWo cell invasion by interactions with the extracellular matrix (ECM). The anti‐CD9 mAb, ALB‐6, enhanced BeWo cell invasion in the presence of the anti‐integrin α3 mAb, but had no effect on BeWo cell invasion after treatment with the anti‐integrin α5 and β1 mAbs. This indicates that an interaction between integrin α5β1 and the ECM is necessary for the stimulatory effects of the ALB‐6 mAb on BeWo cell invasion, suggesting the involvement of integrin α5β1 in the regulation of BeWo cell invasion by the CD9 molecule. ¹⁰ , ²¹

CD9 molecule is also expressed on endometrial epithelial cells associated with β integrins. It was found that the reverse relationship of CD9 expression between normal endometrial epithelial cells and endometrial cancer cells, and showed that CD9 could regulate endometrial cancer cell invasion, suggesting that the mechanism inducing cessation of EVT invasion at a deep site in the maternal decidual tissues could also be applied to prevent cancer cell invasion. ²⁴ , ²⁵ As speculated from the above findings, the molecules expressed on the EVT that have ceased invasion are important because these molecules might be involved in the regulatory mechanism of EVT invasion as well as cancer cell invasion.

Role of membrane‐bound peptidases, dipeptidyl peptidase IV, carboxypeptidase M and laeverin in EVT invasion

It has been previously reported that human EVT in the chorion leave, which have already ceased invasion, expressed a membrane‐bound aminopeptidase, dipeptidyl peptidase IV (DPPIV). ²⁶ This membrane‐bound peptidase has its catalytic site at an extracellular site and can degrade or activate several biologically active peptides before these peptides access specific cell surface receptors. Through metabolizing the biologically active peptides, membrane‐bound peptidases are considered to regulate cell function and differentiation. ²⁷ In the reproductive organs, several membrane‐bound cell surface peptidases are expressed on various cells according to their specific differentiation stages and are proposed to play an important role in the regulation of the reproductive system. ²⁸ , ²⁹ For example, expressions of DPPIV, carboxypeptidase‐M (CP‐M) and endothelin‐converting enzyme‐1 on human luteinizing granulosa cells that produce progesterone increase after ovulation. ³⁰ , ³¹ , ³² , ³³ In the placenta, a number of membrane‐bound peptidases are reported to be expressed on the villous trophoblasts. These are proposed to metabolize vasoconstractive or dilatative peptides in the maternal serum to regulate maternal blood pressure. ³⁴ However, in contrast to villous trophoblasts, there is little information about peptidase expression on EVT.

Dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPPIV, EC.3.4.14.5) is known as T‐cell activation antigen CD26. ³⁵ DPPIV removes an Xaa (one unspecified amino acid)‐Pro or Xaa‐Ala dipeptide from the N‐termini of polypeptides or proteins. ³⁶ The reported physiological substrates for DPPIV are substance P, β‐casomorphin, endomorphin, neuropeptide Y, peptide YY, glucagon‐like peptide, gastric inhibitory peptide, growth hormone releasing factor, vasostatin, fibrin α‐chain and some chemokines. ³⁷

An immunohistochemical study showed that DPPIV was clearly expressed on cytotrophoblasts in the floating villi and on EVT in the proximal part of the cell column of the anchoring villi early in pregnancy. Its expression rapidly decreased in the distal part of the cell column and its expression was not detected on migrating EVT in the maternal decidual tissues. DPPIV expression, however, appeared again on EVT that migrated more deeply as pregnancy proceeded. In contrast to EVT in the maternal decidual tissues, endovascular trophoblasts that invaded toward spiral arteries, lacked DPPIV expression up to at least 20 weeks of gestation. The EVT located in the proximal part of the cell column and in the deep portion of maternal tissue during the first trimester of pregnancy have been reported to be of the non‐invasive phenotype. ³⁸ In contrast, the EVT migrating in the decidua in the first trimester and endovascular trophoblasts until 20 weeks of gestation are considered the invasive phenotype. Thus, DPPIV expression was detected on the EVT that belong to the non‐invasive phenotype, but not observed on EVT showing an invasive phenotype. Furthermore, when the villous explant culture system was used, migrating EVT from villous tips that correspond to cell column rapidly lose DPPIV expression during the change of their phenotype from proliferative EVT to invasive EVT. From these findings, it is speculated that DPPIV expression on EVT is associated with their invasive property and coincides with the spatiotemporal differentiation of EVT. ³⁹

To clarify the role of DPPIV in EVT invasion, we carried out a matrigel invasion assay using a human choriocarcinoma‐derived cell line, DPPIV‐positive JEG‐3 cells. The cell surface expression of DPPIV on JEG‐3 cells was confirmed by flow cytometry and its enzyme activity was shown to be inhibited by diprotin A, DPPIV competitive inhibitor. ⁴⁰ In the presence of diprotin A, the invasion of JEG‐3 cells was significantly enhanced in a dose‐dependent manner without affecting cell proliferation. These results indicate that the inhibition of DPPIV enzyme activity promoted JEG‐3 cell invasion, suggesting that some substrates for DPPIV stimulate JEG‐3 cell invasion and that diprotinA supports the effects of these invasion‐promoting substrates. The key peptides responsible for JEG‐3 invasion should be produced by JEG‐3 cells or present in the fetal calf serum used by the culture medium. Accordingly, it is speculated that at the embryo implantation site, some invasion‐promoting peptides are produced by endometrial stroma or invading EVT and the effects of these biologically active peptides at the proximal sites of the cell column are attenuated by DPPIV, and that these peptides promote EVT invasion at the distal site of the cell column and maternal decidual tissues, where the expression of DPPIV rapidly disappears. At deep sites in the maternal decidual tissues, the increased DPPIV inhibit invasion‐promoting peptides to protect further invasion of EVT, suggesting a novel regulatory mechanism of EVT invasion by membrane‐bound peptidases.

Carboxypeptidase M

It was also found that carboxypeptidase M (CP‐M) (EC 3.4.17.12) is expressed on EVT. This enzyme is a membrane‐bound peptidase that removes arginine or lysine from the carboxy terminal of several peptides. ⁴¹ CP‐M was reported to be expressed on tissue macrophages. Because this molecule is not expressed on monocytes, it has been widely accepted as a differentiation marker for monocyte‐macrophage lineage cells. ⁴² It has been previously reported that CP‐M is a new differentiation marker for the granulosa‐large luteal cell lineage. ³² CP‐M was known to metabolize several biologically active peptides, such as bradykinin and dynorphin A. However, key substrates that are concerned with function or differentiation for macrophages and large luteal cells have not yet been clarified. Although CP‐M has also been reported to be expressed in various other organs such as syncytiotrophoblasts in the placenta, lungs, kidney, intestine, brain, peripheral nerve and so on, ⁴¹ , ⁴³ , ⁴⁴ the physiological role of CP‐M has not yet been thoroughly elucidated.

Immunohistochemical analysis showed that immunoreactive CP‐M is expressed on syncytiotrophoblasts, but not on cytotrophoblasts in the floating chorionic villi. At villus‐anchoring sites, CP‐M was hardly detected on EVT in the proximal part of the cell column, but weakly detected on some EVT in the distal part. In the decidual tissue, almost all interstitial trophoblasts expressed CP‐M. Notably, CP‐M was clearly expressed on EVT in the trophoblastic shells and in the maternal vessels including endovascular trophoblasts. These findings indicate that CP‐M expression is induced on human trophoblasts during their differentiation process toward EVT. ⁴⁵

CP‐M was expressed on JEG‐3 cells and DL‐ mercaptomethyl‐3‐guanidino‐ethyltiopropanoic acid (MGTA), an inhibitor of CP‐M, enhanced the number of invading JEG‐3 cells in the matrigel invasion assay, supporting the belief that CP‐M enzyme activity is involved in the regulation of JEG‐3 cell invasion. Undefined substrates for CP‐M might contribute to promoting JEG‐3 cell invasion. Recently, arginine that is removed from the carboxy‐terminal of various peptides by carboxypeptidase was reported to be used as a substrate for nitric oxide (NO) synthesis, suggesting that CP‐M can promote local NO production. ⁴⁶ This might contribute to maternal vessel dilatation around endovascular trophoblasts to supply sufficient blood circulation within intervillous spaces.

Laeverin

To identify molecules that regulate EVT function, we raised monoclonal antibodies (mAbs) that reacted with human EVT named CHL1 and CHL2. CHL1 was shown to detect a melanoma cell adhesion molecule (MCAM/CD146) by panning analysis of COS cells transfected with human chorion laeve‐derived cDNA library. ⁴⁷ , ⁴⁸ In contrast, CHL2 antigen was shown to be a novel membrane‐bound aminopeptidase. ⁴⁹ The molecular mass of CHL2 antigen purified from placental tissues was 160 kDa. Although the N‐terminal partial amino acid sequence and one internal sequence are still unreported, the other three internal sequences matched those deduced from the coding region of the estimated sequence tag (1672 bp, AK075131). Based on this information, the full‐length of the coding cDNA sequence of CHL2 antigen (2970 bp) was determined by 5′ rapid amplification of cDNA ends, showing that CHL2 antigen is a novel protein homologous with membrane‐bound aminopeptidases that has not been reported elsewhere. ⁴⁹

Because this protein was isolated from chorion laeve, this protein was named ‘laeverin’. Laeverin contains a long region corresponding to the peptidase M1 motif (98–506). In this region, laeverin has a highly conserved coordination motif, that is, His‐Glu‐Xaa‐Xaa‐His‐18 amino acids‐Glu (415–438). This zinc‐binding motif is shared by a variety of enzyme activities. This structure has been named gluzincin aminopeptidase. ⁵⁰ Near the N‐terminus, laeverin contains a highly hydrophobic region (14–36, 23 residues), which corresponds to a transmembrane domain, suggesting that laeverin is a membrane‐bound protein with a short cytoplasmic tail. The amino acid sequence of laeverin shows homology with aminopeptidase N. ⁵¹ Although the substrates or enzyme characterization of laeverin are still unknown, its specific expression on EVT strongly suggests the physiological involvement of laeverin in EVT function.

Differential expressions of membrane‐bound peptidases at human implantation sites

In contrast to CP‐M, DPPIV was detected on cytotrophoblasts in the chorionic villi and EVT at proximal sites in the cell column, but its expression on EVT diminished from the distal part of the cell column to invading interstitial trophoblasts and EVT in the shell during early pregnancy. ³⁹ In contrast, laeverin was specifically expressed on EVT that belong to more limited populations than those bearing CP‐M. This indicates that physiological switching of the expressions of the membrane‐bound peptidases occurs during the EVT differentiation process. In maternal decidual tissues, aminopeptidase‐N was expressed on the cell surface of endometrial stromal cells and its expression increased during the decidualization process. ⁵² The inhibition of this enzyme activity attenuated progesterone‐induced decidualization of cultured endometrial stromal cells. ⁵³ Taken together with these findings, it is suggested that membrane‐bound peptidases expressed on EVT and decidual cells cooperationally regulate EVT function at the feto‐maternal interface. ²⁸ , ²⁹

Role of chemokine–chemokine receptor system in EVT invasion

Recently, several chemokines such as regulated on activation normal T cell expressed and secreted (RANTES), stromal cell derived factor‐1 (SDF‐1), eotaxin and macrophage‐derived chemokines were reported to be substrates for DPPIV. ⁵⁴ By reverse transcription polymerase chain reaction (RT–PCR) analysis, expression of CCR1, CCR10 and XCR1 were shown on EVT isolated from primarily villous implant culture. An immunohistochemical study showed that CCR1 expression was reversely correlated with those of DPPIV. CCR1 was detected on the EVT at the distal site of the cell column and a high expression was maintained on the endovascular and perivascular EVT migrating into maternal spiral arteries through the shell that covers the maternal decidual tissues in front of the intervillous space. In contrast, CCR1 expression rapidly disappeared on EVT that migrated into peripheral maternal decidual tissues.

The expression of ligands for CCR1 such as RANTES, inflammatory protein‐1α (MIP‐1α) and monocyte chemotactic protein‐2 (MCP‐2) in the maternal decidual tissues were confirmed by RT–PCR and immunohistochemistry. Because RANTES is a ligand for CCR1 and a substrate for DPPIV, the effects of RANTES on the invasive property of EVT were examined using isolated EVT from primary villous explant culture. By invasion assay, RANTES promoted invasion of EVT and similar effects were also observed in other chemokine ligands for CCR1, MIP‐1α and MCP‐2. These findings indicate that chemokines can induce EVT migration in vitro and suggest their involvement in the regulation of EVT invasion in vivo. ⁵⁵

However, the immunohistochemical study did not detect dominant localization of chemokine‐expressing cells around or within the maternal vessels. Recently, human platelets have been shown to release several chemoattractants including chemokines. ⁵⁶ Because platelets lose their stored substances immediately after activation, it is no wonder that we could not detect immunoreactive chemokines in platelets at the feto‐maternal interface. By immunohistochemistry, maternal platelets were localized among endovascular trophoblasts within the lumen of spiral arteries. ECM were also detected among endovascular trophoblasts and platelets, suggesting that the platelets in these arteries were activated by ECM. In vitro, platelets attached to EVT isolated from human villous explant cultures and expressed P‐selectin on the cell surface, showing that platelets had been activated. Platelets significantly enhanced migration of EVT without affecting proliferation of EVT. The invasion‐enhancing effect of platelet‐derived culture medium on EVT was neutralized by anti‐CCR1 antibody. These findings suggest that maternal platelets activated in the spiral arteries can regulate trophoblastic vascular infiltration by releasing various soluble factors and support the belief that chemokines are involved in EVT invasion toward maternal arteries. ⁵⁷

CONCLUSION

IN THE FIELD of reproductive and perinatal medicine, it is important to elucidate how maternal uterine tissues regulate EVT invasion while allowing their infiltration of spiral arteries. It is also important to clarify how EVT differentiate and change their phenotype during invasion. As shown in the present review, CD9, membrane‐bound peptidases and chemokine‐receptors are differentially expressed on EVT along with their differentiation processes and stages, and these molecules were shown to affect EVT invasion. To identify key substrates for these peptidases or to detect factors that influence expressions of these molecules will contribute to clarifying the mechanism of human EVT invasion.