Characterization and expression of netrin-1 and its receptors UNC5B and DCC in human placenta

Abstract

Netrins are a family of proteins that mediate axonal guidance in the central nervous system (CNS). In addition to the CNS, netrins are involved in cell adhesion, motility, proliferation, differentiation, and survival. Because these processes occur in the placenta, we raised the question of whether netrin-1 is expressed by placental cells during development. In the present study, we analyzed the spatial and temporal distribution of netrin-1 and its two receptors, DCC (deleted in colorectal cancer) and UNC5B (uncoordinated-5 homolog) in human placenta using RT-PCR, Western blotting, and immunohistochemistry analysis. We demonstrated the presence of the proteins and transcripts of netrin-1 and its receptors in placenta and cytotrophoblasts. Furthermore, using immunohistochemistry, we localized endogenous netrin-1 protein staining to villous and extravillous cytotrophoblasts, and secreted netrin-1 outside the syncytiotrophoblasts. The DCC receptor was localized to syncytiotrophoblasts and invasive extravillous cytotrophoblasts during the first trimester and at term. On the other hand, the UNC5B receptor was localized to villous and extravillous cytotrophoblasts proximal to anchoring areas during the first trimester. At term, UNC5B was observed in decidual cells and weakly in extravillous cells. The discrete pattern of netrin-1 and netrin-1 receptor distribution suggested that netrin-1 protein functions might vary with its localization in the placenta and probably with time of gestation.

Figure 1

Amplification of netrin-1 and its receptor cDNAs. Gel electrophoresis and ethidium bromide staining of netrin-1 (Lane 1), UNC5B (Lane 2), and DCC (Lane 3) RT-PCR products from purified cytotrophoblast cells. MW, molecular weight marker.

Figure 2

Western blot analysis of the netrin-1 receptor UNC5B. Western blot analysis of the netrin-1 receptor UNC5B was performed with 100 μg protein extracts. Lane 1, trophoblast tissue (10 weeks gestation) plus the UNC5B antigenic peptide UNC5H2; Lane 2, trophoblast tissue (10 weeks gestation); Lane 3, at term; Lane 4, trophoblast tissue (10 weeks gestation); Lane 5, 48-hr-cultured cytotrophoblasts; and Lane 6, UNC5B-transfected 293 T cells. The Western blot is representative of six separate experiments, each performed with samples prepared in different experiments and used only once.

Figure 3

Western blot analysis of the netrin-1 receptor DCC. Western blot analysis of DCC was performed with 100 μg protein extracts, except for Lanes 5 and 6, which were performed with 70 μg and 35 μg, respectively. The 200-kDa band is the DCC receptor; the strong band at 120 kDa is a proteolytic fragment of the receptor. (A) Lane 1, at-term placenta tissue extract; Lane 2, trophoblast tissue extract (10 weeks gestation); Lane 3, 48-hr-cultured cytotrophoblasts; Lane 4, colon used as a positive control; Lane 5, 48-hr-cultured cytotrophoblast protein extract (70 μg); and Lane 6, 48-hr-cultured cytotrophoblast protein extract (35 μg). Lanes 1–4 are from one experiment; Lanes 5 and 6 are from another experiment. The Western blots are representative of six separate experiments, each performed with samples prepared in different experiments and used only once. (B) Closer view of DCC antibody-labeled bands in the 200-kDa area in 48-hr-cultured cytotrophoblasts, 10 weeks placenta, and colon.

Figure 4

Immunohistology of netrin-1, UNC5B, and DCC in first-trimester human placental tissue. (A–F) Histological sections of anchoring villus. (G–I) Histological sections of decidua. Immunolabeling was as follows: (A) Goat anti-netrin-1; immunoreactivity was found in villous and extravillous cytotrophoblasts and in external surface of syncytiotrophoblasts. (B,I) Mouse anti-DCC showing immunoreactivity in syncytiotrophoblasts and in extravillous cytotrophoblasts located at decidua. (C) Goat anti-UNC5B showing immunoreactivity in villous cytotrophoblasts and in extravillous cytotrophoblasts located proximally to anchoring villus. (D,G) Anti-rabbit c-ErbB2 showing immunoreactivity in extravillous cytotrophoblasts and in external surface of syncytiotrophoblasts. (E,H) Anti-mouse cytokeratin 7 showing immunoreactivity in villous and extravillous cytotrophoblasts and in syncytiotrophoblasts. (F) Negative control processed in the absence of primary antibody.

Figure 5

Immunohistochemical localization of netrin-1, UNC5B, and DCC in term human placental tissue. (A–G) Serial histological sections in decidua. (H) Section in villous trophoblasts. Small arrows indicate decidual cells, and large arrows indicate invasive extravillous cytotrophoblasts. Immunohistology of at-term human placental tissue for netrin-1, UNC5B, and DCC and the relevant c-ErbB2, vimentin, and cytokeratin 7 controls were as follows: (A) Anti-rabbit c-ErbB2 immunoreactivity in extravillous cytotrophoblasts. (B) Anti-mouse cytokeratin immunorectivity in extravillous cytotrophoblasts. (C) Negative control processed in the absence of primary antibody. (D) Anti-vimentin immunoreactivity in decidual cells. (E) Anti-goat UNC5B immunoreactivity in decidual cells and weak staining in extravillous cytotrophoblasts. (F) Anti-mouse DCC immunoreactivity in extravillous cytotrophoblasts and (H) in syncytiotrophoblasts. (G) Anti-goat netrin-1 immunoreactivity in decidual cells, and (I) in syncytiotrophoblasts.