Modulation of ezrin and E-cadherin expression by IL-1β and TGF-β1 in human trophoblasts

https://doi.org/10.1016/j.jri.2004.04.005Get rights and content

Abstract

Objectives:

The present study examines the effects of IL-1β and TGF-β1 in modulation of ezrin, E-cadherin, CD44 and β-catenin expression in human trophoblast cells which may lead to their altered cytoskeleton dynamics during cell-to-cell and cell-to-matrix interactions.

Methods:

Trophoblast (extravillous and villous) cells isolated and purified from early and term placentae and human choriocarcinoma cell line JEG-3 used in this study were challenged with either IL-1β or TGF-β1 (10 ng/ml) for 12 h following which RT-PCR was performed for ezrin, E-cadherin, CD44 and β-catenin. Immunolocalization of these proteins was carried out in the chorionic villi as well as in the cultured cells stimulated by the cytokines. Western Blot was also performed to study the regulation of ezrin and E-cadherin in primary extravillous, villous and term trophoblast by these cytokines. Scanning electron microscopy (SEM) and Matrigel Invasion Assay was used to study the effect of these cytokines on cellular morphology and invasion.

Results:

IL-1β induced a down regulation in the expression of ezrin, E-cadherin and β-catenin while upregulation of CD44 message in both primary trophoblast and JEG-3 cells. On the contrary, TGF-β1 exhibited just an opposite effect, i.e. up regulation of ezrin, E-cadherin, β-catenin, and down regulation of CD44. These observations were further corroborated with the immunolocalization findings of the above proteins in first trimester and term villous tissue, the former having predominance of IL-1β and the latter of TGF-β1 [Am. J. Reprod. Immunol. 48 (2002) 210]. Cellular morphology as observed through SEM revealed an enhanced cell-to-matrix adhesion with poor cell–cell interaction following IL-1β challenge and a strong intercellular adhesion with weak cell-to-matrix interaction in presence of TGF-β1. Crystal violet staining and Matrigel invasion revealed a higher invasion index following IL-1β challenge and a low invasion index following TGF-β1 challenge.

Conclusion:

IL-1β mediated increased cell-to-matrix interaction with reduced cell-to-cell adhesion along with reduced ezrin and E-cadherin expression is associated with enhanced invasiveness while TGF-β1 mediated up regulation of cell-to-cell adhesion with reduced cell-to-matrix interaction along with an increased ezrin and E-cadherin expression, is associated with reduced invasiveness, along with an altered cellular morphology. These facts therefore indicate the possible role of the two cytokines during cell motility and invasion through alteration of cell–matrix and cell–cell interaction.

Introduction

Cellular interactions during peri-implantation events seem to require the expression of a variety of cell adhesion molecules (Basak et al., 2002, Brown and Hunt, 2000, Vaheri et al., 1997, Campbell et al., 1995). Integrins and other cytoskeletal components provide linkage between the cell membrane to the actin based cytoskeleton and are thus essential for cell migration, adherence to extra cellular matrix and cell-to-cell attachment during the process of implantation (Sato et al., 1992). Ezrin belonging to a family of intracellular proteins consisting of ezrin, radixin and moesin (ERM) is an important signal transducing protein that undergoes phosphorylation and translocation on stimulation by cytokines (Hirao et al., 1996, Jiang et al., 1995). Ezrin also interacts with cell surface adhesion molecule CD44, ICAM-1 and E-cadherin (Nagafuchi et al., 1994, Hiscox and Jiang, 1999, Tsukita et al., 1997), the latter, a cell surface glycoprotein is linked internally with cytoskeleton components mainly β-catenin and cadherin (Barth et al., 1997). Increased cellular motility and invasion may occur due to altered cell surface E-cadherin expression leading to disruption of cell-to-cell interacting network. The transmembrane glycoprotein CD44 acts as a receptor for a wide variety of molecules like osteopontin, fibronectin, collagen type IV, type I and hyaluronate (Chintala and Rao, 1996) and is reported to play an important role in metastasis (Kim et al., 2002), cellular invasion (Dingemans et al., 2002) and cell motility by interacting with ezrin (Legg et al., 2002). In the present study, we have tried to explore the regulation of ezrin and E-cadherin by IL-1β and TGF-β1 in the first trimester trophoblast cells which are more invasive and term trophoblast cells, which are mostly non-invasive as well as in JEG-3, a human choriocarcinoma cell line.

Section snippets

Specimens

Human term placentae (third trimester) obtained from spontaneous vaginal delivery of healthy women (n = 25) were collected in normal saline. First trimester placental villi were obtained (n = 40) through vacuum aspiration from subjects undergoing medical termination of pregnancy. A portion of intact villous tissue was fixed in formalin and processed for immunohistochemistry. The institutional ethical committee approved collections of the tissues.

First trimester (5–12 weeks) human extravillous

RT-PCR

Ezrin and E-cadherin message was observed to be very less in trophoblast cells isolated from first trimester placental villi as compared to that obtained from term (Fig. 1a). However, among the different subpopulations of trophoblast isolated from the early/first trimester (5–12 weeks) placenta, extravillous trophoblasts (EVT) were found to express significantly less of both ezrin (Fig. 1b) and E-cadherin (Fig. 1c), as compared to the villous (V) or term trophoblast (T). The expression of ezrin

Discussion

Trophoblast invasion seem to be precisely regulated by a host of upstream factors of autocrine or paracrine origin to influence the extra cellular matrix components (Murray and Lessey, 1999, Simons et al., 1999, Das et al., 2002). Ezrin belonging to a group of intracellular proteins like moesin and radixin (collectively known as ERM family) is present in a wide range of cells concentrated at surface protrusions. This microvillous associated protein has been suggested to be possibly involved in

Acknowledgements

This work was supported by Research Grant from the Indian Council of Medical Research, New Delhi. The authors are grateful to Professor Shashi Wadhwa for providing the Electron Microscope Facility at AIIMS. The technical help of Hariom Sharma and Danbir Singh are acknowledged.

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