Elsevier

Placenta

Volume 28, Issues 2–3, February–March 2007, Pages 118-126
Placenta

Expression and Distribution of Notch Protein Members in Human Placenta Throughout Pregnancy

https://doi.org/10.1016/j.placenta.2006.03.010Get rights and content

Abstract

Notch signaling is an evolutionarily conserved mechanism used by invertebrates and vertebrates to control cell fates through close-range cell interactions. Four Notch receptors have been identified in vertebrates and different ligands, divided into Delta-like and Serrate-like (Jagged). Several studies have demonstrated that Notch signaling is involved in different branches of the cell fate decision tree: differentiation, proliferation and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and a correct fetal growth. Moreover, Notch and its ligands participate in the vascular remodelling and stabilization, other two processes much important and ticklish in human placenta. So, we decided to investigate the pattern of expression of Notch-1, Notch-4 and Jagged-1, together with two members related to Notch pathway and involved in angiogenesis: VEGF and p21, in human placenta during gestation by immunoblotting and immunohistochemistry. We showed a modulation of Notch proteins throughout the pregnancy; in particular we showed a slight decrease of Notch-1 throughout pregnancy, with a decreased cytoplasmic staining from the first to the third trimester of gestation in cytotrophoblast and syncytiotrophoblast. In contrast Jagged-1 showed an increase throughout pregnancy especially in syncytiotrophoblast and stroma during the third trimester of gestation. In addition, we found by immunoblotting an increase of VEGF expression from the first to the third trimester and an intense VEGF expression inside endothelial cells throughout the gestation as also confirmed by immunohistochemistry. We also showed a decrease of p21 expression during the pregnancy both through immunoblotting and immunohistochemistry assays. Moreover, we observed Notch localization in extravillous trophoblast cells that are able to invade the decidualized endometrium. Our results suggest an involvement of Notch signaling in regulation of placental cell fate decision and in angiogenesis that are dramatically important to maintain a normal physiology of this organ during pregnancy.

Introduction

Notch proteins are a transmembrane receptor family that is structurally and functionally conserved from worms to humans. Notch was first identified in Drosophila as a gene involved in neuronal cell fate decision, but this family of receptors is now known to regulate the fate decisions of developing cells in various tissues during embryogenesis as well as in postnatal stages [1], [2], [3], [4], [5], [6].

The mammalian family of Notch proteins consists of four genes encoding Notch receptors (N1–N4). Each one is synthesized as a single transmembrane polypeptide which is subsequently cleaved and transported to the cell surface as a heterodimer. The extracellular domains contain several (29–36) tandem epidermal growth factor (EGF)-like repeats that bind ligands. Notch ligands are classified into two structurally related groups: Delta-like ligands (Dll-1, -3, and -4) and Serrate-like ligands (Jagged-1 and -2). Both types of ligands are transmembrane proteins with a number of tandem EGF-like repeats in their extracellular domain and a unique Delta, Serrate and Lag-2 (DSL)-binding domain in the amino terminus necessary for the receptor interaction.

The signal transduction induced by Notch pathway is initiated when the extracellular domain of Notch binds to its ligand on adjacent cells, resulting in a sequential proteolytic cleavage of the transmembrane subunit, with release of the intracellular domain of Notch (NIC). NIC translocates to the nucleus and binds to a transcriptional repressor (CSL, CBF1/Su(H)/Lag-1), converting it into an activator, thereby activating target genes [7].

Most of the Notch target genes encode transcription regulators, which in turn modulate cell fate by affecting the function of tissue-specific basic helix-loop-helix transcription factors or through other molecular targets [8], [9]. Moreover, it has been hypothesized a probable implication of Notch receptors and their ligands in the regulation of vascular formation [9]. Growing evidence suggests involvement of Notch signaling in the regulation of vascular formation [9]. In particular, it has been demonstrated that combined deletion of both Notch-1 and Notch-4 results in albeit variable defects in arterial development [10]. In addition, mice lacking Jagged-1 develop some of the vascular defects seen in the Notch mutants, implying that it might be a ligand for these Notch receptors during vascular development [10], [11], [12].

The placenta is a complex organ that is composed of maternal and fetal material and that plays an integral role not only in the supply of nutrients to the fetus but also in the maintenance of pregnancy [13]. Moreover, human placental vascularization is the result of local de-novo formation of capillaries out of pluripotent mesenchymal precursor cells in placental villi [14]. The human placenta is able to synthesize and secrete a variety of hormones and molecules that regulate the fine balance between proliferation, differentiation and invasion of trophoblastic cells. The trophoblast is differentiated into the villous trophoblast (syncytiotrophoblast and cytotrophoblast), which covers the placental villi, and the extravillous trophoblast (EVT), consisting of cell islands and cell columns [15], [16], [17], [18], [19], [20]. The extravillous trophoblastic cells of cell columns located proximally to the villous stroma have been identified as proliferating cells, whereas extravillous trophoblastic cells located distally to the villous stroma show invasive characteristics and are no longer proliferative [15], [16], [17].

The purpose of this study was to investigate the cellular localization of Notch-1, Notch-4 and Jagged-1 in human placenta during gestation, because these proteins are able to regulate proliferation, differentiation and embryonic development, all processes that are particularly relevant to the physiology of human placenta. In addition, in order to clarify the role of Notch signaling in angiogenesis, we have studied, throughout the pregnancy, the pattern of expression of VEGF and p21 that have been, respectively, supposed members upstream and downstream of Notch signaling function in angiogenesis.

Section snippets

Samples

Human placental samples were obtained with informed consent from patients undergoing surgery such as cesarean section for normal placenta and uterine evacuation for normal chorionic villi. A total of 30 samples, 15 full-term placentae and 15 chorionic villous samples from first trimester, were used and the gestation period ranged from 5 to 40 weeks. The specimens were immediately fixed in formalin for immunohistochemistry.

Immunohistochemistry

Immunohistochemistry was carried out essentially as described previously

Distribution of Notch-1, Notch-4 and Jagged-1 in human placenta during gestation

We investigated the localization and distribution of Notch-1, Notch-4 and Jagged-1 in human placenta during gestation by immunohistochemistry. We observed that in the first trimester, Notch-1 was localized in the cytoplasm of cytotrophoblast at moderate level of expression (Fig. 1a); in addition Notch-1 had an intense membrane immunopositivity in the syncytiotrophoblast (Fig. 1a1). During the third trimester of gestation, Notch-1 immunopositivity slightly decreased in the cytoplasm of

Discussion

Notch signals in combination with other cellular factors, influence differentiation, proliferation and apoptotic events at all stages of development. In particular, Notch signaling seems to function as a general developmental tool used to direct cell fate and to build an organism [5]. Notch receptors have a common structural organization that is conserved throughout evolution, with some variation on the general theme [25]. Similarly, multiple Notch ligands have been identified in vertebrates

Acknowledgements

We thank Dr. Pia Furno for editing the manuscript and Mr. Giuseppe Falcone for his contribution to the image elaboration. This work was supported in part by Second University of Naples, and AIRC funds (A.D.L.). We thank the I.S.S.C.O. (President H. E. Kaiser) for the continuous support.

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