Elsevier

Placenta

Volume 25, Issue 1, January 2004, Pages 20-28
Placenta

Differential Expression of the nm23 Genes in the Developing Human Trophoblast

https://doi.org/10.1016/j.placenta.2003.08.003Get rights and content

Abstract

NDP kinases are the non-specific enzymes which catalyse the synthesis of the NTPs through a transfer reaction using ATP as phosphoryl donor. In addition to their enzymatic activity, they display other not yet explained functions related to cell growth, differentiation and apoptosis, embryonic development, tumour progression and metastasis. In this study, the expression patterns of the three highly related NDP kinases A, B and C isoforms were investigated in the developing human trophoblast. Both NDP kinase A and B were found to be primarily present in the villous and extravillous cytotrophoblasts, while NDP kinase C was found almost exclusively in the syncytiotrophoblast layer. This suggests that NDP kinase A and B could be a marker for the mononuclear stage of differentiation of villous trophoblasts, while NDP kinase C could be a marker of the syncytiotrophoblast layer.

Introduction

In all eutherian mammals, placenta formation derives from two basic elements: the embryonic mesoderm which gives rise to a vascular network and stroma; and on the other hand, the trophoblast cell lineage which produces an outer epithelium. Trophoblast cell derivatives interact with the underlying stroma and develop into the fetal vascular exchange unit. In humans, this exchange unit is a villous tree that floats in a large blood-filled space. The cytotrophoblast cells of individual villi are in contact with stroma and are considered to be a progenitor cell population: they differentiate into syncytiotrophoblast as villi grow and mature. In addition, at the tipof a small proportion of villi which are called anchoring villi and attach to the decidua, cytotrophoblasts can also differentiate into specialized cells that migrate out of the villi, forming columns of cytotrophoblasts that enter into contact with the endometrium and invade deep into the maternal decidua and myometrium under a strict temporal and spatial control ([1], for review). Thus, the cytotrophoblasts, which are thought to be the trophoblastic stem cells ([2], for review), may be considered good models for studies on processes controlling cell differentiation and/or invasiveness, such as regulation of proto-oncogenes and tumour suppressor gene expression [3], [4]or control of matrix metalloproteinases and their inhibitors [5].

Nucleoside diphosphate (NDP) kinase has been primarily described as the non-specific enzyme which catalyses the synthesis of nucleoside triphosphates (NTPs) through a transfer reaction using adenosine triphosphate (ATP) as phosphoryl donor and any NDP as acceptor. Until now, eight different NDP kinases isoforms have been found in humans ([6], for review). The first four are named NDP kinase A, B, C and D and are respectively encoded by the nm23-H1, nm23-H2, DR-nm23 (nm23-H3), and nm23-H4 genes. The products of these four nm23 genes are highly related and remarkably well conserved in residues which are crucial for nucleotide binding and enzymatic reaction. In addition to their enzymatic activity, NDP kinases display other functions related to growth, cell differentiation, embryonic development, tumour progression, metastasis and apoptosis [6], [7]. The mechanism of action of these additional functions is not yet understood and is a controversial issue.

The nm23-H1 gene, which encodes the NDP kinase A isoform, is thought to play a role in the regulation of cellular differentiation, motility or invasiveness (see for reviews [8], [9], [10]). A pioneering study on human cytotrophoblasts [11]suggested that in these cells and in culture conditions, NDP kinase A expression is controlled by growth factors and may be coupled with cell proliferation. We subsequently showed by immunocytochemical studies that in normal placenta, NDP kinase A is present in the cytotrophoblasts but is absent in the syncytiotrophoblast multinucleated layer [12]. These results were confirmed by Okamoto's group [13], [14]. In order to throw new light on the relative functions of the different NDP kinase isoforms, we describe here the expression patterns of NDP kinases A, B and C in the developing human trophoblast. The localization of the nm23 gene transcripts was evaluated at different stages of pregnancy by in situ hybridization, and the distribution pattern of the corresponding proteins was studied by immunohistochemistry. The proliferation potencies of the different cellular populations were assessed by checking the cell immunoreactivity of Ki-67, a protein whose expression is strictly correlated with the active phases of the cell cycle [15].

Section snippets

Tissue sampling

In situ hybridization and immunohistochemistry studies were performed on two different tissue collections. A first group of normal placental samples (n=6) came from legal abortions performed at 7–12 weeks of pregnancy at the Maternity Unit of the University of Geneva. A second group of 56 samples consisted of chorionic villi biopsies removed for prenatal diagnosis at the Hospital of the Bordeaux Medical School (France). The gestational age ranged from 10 to 35 weeks of pregnancy and the samples

In situ hybridization

In situ hybridization studies were performed on 1st trimester floating villi. By using 35S-labelled probes, signals were found for nm23-H1, nm23-H2 and nm23-H3 (Figure 1a, c, e). Counterstaining clearly showed that nm23-H1 (Figure 1b) and -H2 (Figure 1d) mRNAs were expressed in trophoblast structures. In contrast, the stroma staining remained near the background level (Figure 1b, d). However, the exact localization of nm23-H1, -H2 and -H3 transcripts either in the cytotrophoblast or in the

Discussion

NM23/NDP kinases form a ubiquitous family of highly homologous proteins which are thought to be involved in the regulation of normal cell functions. Although the first three isoforms are expressed in every cell, it is not known whether they carry out unique or redundant cellular functions. Moreover, and depending on the cell type considered, they generally differ in their expression pattern. Therefore, the overlapping but individual patterns of tissue expression of the different isoforms

Acknowledgements

This work was supported by the Comité Départemental des Pyrénées Atlantiques of the French Ligue Nationale Contre le Cancer. We are grateful to Pr. Ioan Lascu (IBGC, Bordeaux, France) and to Dr Dominique Carles for fruitful discussion. We would also like to thank Catherine Robinet and Hélène Roberteau for their technical assistance, and Dr Marcie Kritzik for help in editing the manuscript.

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    F. Guyon and B. Marnet contributed equally to this study.

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