Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype

0 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University , Sakyo-ku, Kyoto, 606-8507 , Japan - At the human feto-maternal interface, trophoblasts differentiate towards extravillous trophoblasts (EVTs) and form the cell column. EVTs acquire invasive activity in the distal part of the cell column and begin to migrate into the maternal tissue. We previously reported that dipeptidyl peptidase IV (DPPIV) is expressed on EVTs in the proximal part of cell column and is involved in the inhibition of their migration. Because DPPIV has been shown to degrade several chemokines, we examined possible roles of chemokines in EVT migration. Immunohistochemistry demonstrated that C-C chemokine receptor 1 (CCR1) was hardly detected on cytotrophoblasts and syncytiotrophoblast but was expressed on EVTs in the cell column. In vitro, CCR1 protein was also present on the surface of EVTs that grew out from chorionic villous explants cultured under 20% O2. Chemokines that can bind to CCR1 (CCR1 ligands), such as regulated on activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein-1a (MIP-1a ), were confirmed in the decidual tissues by RT-PCR and immunohistochemistry. These CCR1 ligands promoted the migration of the EVTs that In human placenta, cytotrophoblasts show two distinct patterns of differentiation. In floating villi, cytotrophoblasts differentiate into syncytiotrophoblast and form the syncytial layer. By contrast, at villus-anchoring sites, cytotrophoblasts differentiate into EVTs and form the stratified structure that is called the cell column (Aplin, 1991). EVTs acquire invasive activity in the distal part of the cell column (Damsky et al., 1992) and begin to migrate towards maternal tissue. EVTs that migrate into the decidual tissue are called interstitial trophoblasts and those that migrate along the maternal vessels are called endovascular trophoblasts. Several molecules have been proposed to regulate EVT invasion. Matrix metalloproteinases and serine proteinases, which degrade the extracellular matrix, are secreted by EVTs to make the decidua preferable for their migration (Bischof et al., 1995). The importance of cell adhesion molecules, such as integrins, in EVT migratory processes has also been suggested (Burrows et al., 1996). At the villus-anchoring sites, EVTs gain integrin a 5b 1 in the proximal part of the cell column and integrin a 1b 1 were isolated from the explant cultures in vitro. These results indicate that CCR1 is expressed on trophoblasts as they differentiate to EVTs and that CCR1 ligands produced from the decidual tissue induce EVT migration. By contrast, CCR1 was scarcely expressed on EVTs that grew out from villous explants cultured in 1% O2, indicating that a relatively high oxygenic environment is needed to induce CCR1 expression. Moreover, CCR1 expression on the isolated EVTs was significantly reduced in the presence of decidua-conditioned medium. Such regulation of CCR1 by surrounding oxygenic and decidual environments supports a close correlation between EVT invasion and their expression of CCR1. This study demonstrates that trophoblasts acquire CCR1 as they differentiate to an invasive phenotype at the villus-anchoring sites and indicates a novel role for the chemokine-CCR1 system in the initial step of trophoblastic invasion towards the maternal tissue. at the distal end (Damsky et al., 1992). Because functional antibodies against these integrins affected trophoblastic migration in vitro, this so-called integrin switching was considered to be important for trophoblasts to become invasive (Damsky et al., 1994). However, the precise mechanism(s) involved in the acquisition of invasive activity has not been clarified. We previously reported a characteristic expression profile of DPPIV (EC.3.4.14.5) on EVTs (Sato et al., 2002). DPPIV, a membrane-bound peptidase, can metabolize its substrates and regulate their bioactivity on the cell surface. DPPIV was expressed intensely on EVTs in the proximal part of the cell column, but it was downregulated in the distal part where EVTs acquired invasive activity. This suggests that DPPIV modulated the bioactivity of some molecule(s) that affect trophoblastic migration in situ. Supporting this idea, in JEG-3 cells (a DPPIV-positive choriocarcinoma cell line), inhibition of cellsurface DPPIV activity enhanced invasive activity without affecting proliferation. Recently, DPPIV was demonstrated to metabolize several chemokines in vitro (Van et al., 1999). Fig. 1. Expression of CCR1 protein and mRNA on human EVTs at 9-weeks gestation. (A-E) Serial sections of placental tissue from therapeutic hysterectomy at 9-weeks gestation were doubled-immunostained with anti-CCR1 mAb followed by rhodamine-conjugated secondary antibody plus either FITC-conjugated anti-cytokeratin 7 mAb or FITC-conjugated anti-von Willebrand factor pAb to visualize trophoblasts (A-D) and blood vessels (E), respectively. (C) is a higher magnification of the area indicated in (B). (D,E) The maternal arterial wall is traced with a dashed line. EVTs locating from the cell column (Column) through the trophoblastic shell (Shell) (A,B) and from the trophoblastic shell into the maternal artery (D,E) are shown. CCR1 expression is detected clearly on EVTs in the cell column (B,C) and trophoblastic shell (B), but is scarcely detected on cytotrophoblasts (CT) and syncytiotrophoblast (ST) (C). CCR1 expression is diminished on interstitial trophoblasts (Int TB) (B,C), whereas it is maintained on endovascular trophoblasts (Endov TB) that migrate from the trophoblastic shell into the maternal artery (E). (F) 35-cycle PCR detects a specific band that corresponds to CCR1 in cDNA derived from the microdissected cell columns at 9 weeks of gestation. No band is observed in the negative control in which total RNA from the cell columns was not reverse-transcribed. AV, anchoring villus; IVS, intervillous space; Gl, decidual gland. Scale bars: 200 m m. RANTES, a member of the chemokine family, is a representative substrate of DPPIV and the chemotactic activity of RANTES is abrogated by digestion with DPPIV (Oravecz et al., 1997). Because RANTES is produced in human placental tissues (Denison et al., 1998) and can induce the migration of leukocytes and other cell types (Youngs et al., 1997), we speculated that this chemokine is a candidate for a DPPIV substrate that can affect trophoblastic migration. To substantiate this possibility, we initially examined the expression of RANTES receptors on trophoblasts. C-C chemokine receptor 1 (CCR1), CCR3 and CCR5 are reported to be receptors for RANTES (Murphy et al., 2000), and immunohistochemical experiments revealed that EVTs expressed CCR1. Several chemokines, including CCR1 ligands, have been demonstrated in human placental tissue (Denison et al., 1998; Drake et al., 2001; Red-Horse et al., 2001). They are considered to be involved in either recruiting specific le (...truncated)