Elsevier

Placenta

Volume 26, Issue 6, July 2005, Pages 476-483
Placenta

Human Placental Hofbauer Cells Express Sprouty Proteins: a Possible Modulating Mechanism of Villous Branching

https://doi.org/10.1016/j.placenta.2004.08.008Get rights and content

The development of the chorionic villous tree into a complex and organized ramified tubular network can be termed branching morphogenesis. Studying the molecular mechanisms involved in this process may contribute to the understanding of pregnancy complications such as preeclampsia. Sprouty (Spry) proteins are important regulators of branching morphogenesis and growth factor signaling. We analyzed the expression of Spry genes in human placenta. RT-PCR and immunohistochemistry were employed to detect placental Spry expression. Quantitative RT-PCR was used to assess the effect of FGF and reduced oxygen fraction on Spry gene expression. Spry 1, 2 and 3 expression was observed in placental tissue from all three trimesters. Our results reveal for the first time that Spry proteins are localized in the stroma of the chorionic villi, adjacent to cytotrophoblasts in areas of villous sprouting. Immunofluorescent double staining with anti-Spry and anti-CD68 confirmed that placental macrophages (Hofbauer cells) express Spry. Reduced oxygen fraction, FGF-4 and FGF-10 stimulated Spry-2 expression. Hofbauer cells also expressed c-Cbl, a protein that interacts with Spry. Placental expression of Spry and c-Cbl implies an important role for Hofbauer cells in placental development, possibly through a mesenchymal–epithelial interaction with trophoblasts. Regulation of Spry-2 expression by FGF-4 and FGF-10 suggests an orchestrated regulatory system that modulates villous branching.

Introduction

Adequate maternal–fetal nutrient supply and gas exchange is dependent upon normal placental development [1], [2]. Maldevelopment of the chorionic villous tree has been associated with preeclampsia and intrauterine growth restriction (IUGR), conditions that contribute greatly to maternal and fetal morbidity and mortality. Poorly branched villi detected by morphologic examination are found in placentas from pregnancies complicated by fetal asphyxia. Scanning electron microscopy reveals that the terminal villi are long and largely unbranched [3], [4]. The placenta, like other branched organs such as the lung and kidney, and in particular vascular systems, is composed of a network of tubes. The formation of this three-dimensional structure involves a coordination of several cellular processes including specification of cell fate, changes in cell shape, migration, proliferation and cell–matrix interaction. Many of the molecular mechanisms that underlie this complex process, termed branching morphogenesis, have been elucidated by studying the development of the Drosophila fly trachea. Critical genes for tracheal branching are branchless, a homologue of mammalian fibroblast growth factor (FGF), and Sprouty (Spry). In addition to their role in branching morphogenesis, members of the Sprouty gene family are important regulators of growth factor signaling [5], [6], [7].

Sprys are membrane-associated proteins that modulate receptor tyrosine kinase (RTK) signaling [8]. To date, four mammalian Sprys have been identified. These proteins consist of conserved carboxy-terminal cysteine-rich domains and highly divergent amino-terminal domains. Stimulation of Spry proteins is associated with their translocation from the microtubules to the membrane and results in an autocrine effect [9]. Initially, members of the Spry family were considered as negative regulators of the RTK-induced mitogen activated protein kinase (MAPK) pathway, that is induced by growth factors such as FGF, epidermal growth factor (EGF), platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). The exact points at which Spry intercepts the MAPK pathway remain controversial. Possible explanations for MAPK pathway regulation include binding of Spry to Grb2 or Raf. Among the four isoforms of Spry, the biological role of Spry-2 has been characterized as a regulator of FGF signaling pathways in modeling different varieties of branching tissues during development [5], [6], [10]. Recently, it has been learned that Spry-2 proteins can both positively and negatively regulate RTK signaling, and the switch between the two different outcomes is attributed to the presence or absence of c-Cbl. c-Cbl, a ubiquitin ligase that plays a prominent role in mediating ligand-dependent down regulation of RTK, binds to phosphorylated tyrosines on the activated RTK, and leads to its ubiquitination, endocytosis and subsequent degradation through the proteasomal pathway [11]. Since phosphorylated Spry proteins can also interact with c-Cbl, they are in direct competition with activated RTKs for binding to this protein. Because c-Cbl binds preferentially to Sprys and less to RTKs, Sprys are degraded, whereas RTKs exhibit prolonged surface exposure and sustained signaling. c-Cbl related degradation of Spry-2 has been observed through activated EGFR and FGFR signaling (for review, see Refs. [5], [10]).

While important for branching morphogenesis and regulation of FGF as well as other tyrosine-kinase mediated pathways, the role of placental Spry proteins is currently unknown. We hypothesize that Spry proteins play an important role in normal human placental development. We therefore sought to characterize the expression and localization of Spry in first, second and third trimester placentas using RT-PCR and immunohistochemistry. We determined that Spry is expressed throughout the three trimesters, and that placental villous macrophages (Hofbauer cells) are the primary cells that express Spry proteins. In addition, we found that FGF-4 and FGF-10 stimulate Spry expression by Hofbauer cells.

Section snippets

Tissue preparation and cell culture

Normal first and second trimester placentas were obtained from legal interruptions of pregnancy. Third trimester placentas were taken from normal deliveries. The study was approved by the institutional review board. Villous macrophages (Hofbauer cells) were isolated according to the following protocol: we initially isolated placental cells from first trimester placentas as described by Kliman et al. [12], with some modifications [13]. Briefly, the villous tissue was rinsed with PBS. Then, it

Expression of Spry genes and proteins in first, second and third trimester placentas

Using RT-PCR we found that Spry 1, 2 and 3 are expressed in whole placental tissue from first, second and third trimesters of pregnancy. A representative gel of first trimester placental tissue is shown in Figure 1. The expected product size for Spry 1, 2 and 3 were 438 bases, 471 bases and 290 bases, respectively.

We analyzed placental tissue using immunohistochemistry with anti-sprouty-2 antibody. We initially used positive antigen control and verified that the placental protein identified by

Discussion

We determined that placental Hofbauer cells are the major cells that express Spry and c-Cbl proteins at the maternal–fetal interface. This is the first time that placental expression of Spry proteins is reported, and the first time that Spry expression is described in macrophages. Hofbauer cells belong to the family of mononuclear phagocytes. During physiological and pathological processes they are recruited to sites of tissue injury, inflammation, atherogenesis and tumor development, where

Acknowledgements

This work was supported in part by Israel's Ministry of Health, Chief Scientist Grant and by the Joint Hebrew University-Hadassah Grant.

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