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Trophoblast expression dynamics of the tumor suppressor gene gastrokine 2

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Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology.

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The authors thank the research staff of the Departments of Gynecology and Obstetrics and Pathology at the University of Erlangen-Nürnberg for their kind collaboration. Establishment of staining procedures and respective data acquisition was performed by Hannah Bartunik in fulfillment of the requirements for obtaining the degree “Dr. med.” at the Friedrich-Alexander University of Erlangen-Nürnberg, Department of Pediatrics and Adolescent Medicine, Germany.

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The authors declare that they have no conflict of interest.

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Correspondence to Fabian B. Fahlbusch.

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Immunofluorescence (IF) double-staining of human third-trimester sections from AGA (ae) and HELLP (fj) placentas (n = 5 each). Placental tissues were stained with anti-GKN2 (green) and anti-SMA antibodies (red). Nuclei were stained with DAPI (blue). HELLP sections were chosen to illustrate positive stain of syncytial (SCT) GKN2 expression. While in f only punctual SCT expression is found (see marked areas), gi show strong local GKN2 staining in their SCT. Image j shows a HELLP placenta with no syncytial GKN2 expression. However, intravillous GKN2 expression around a SMA-positive (red) vessel is visible (marked by a star). Extravillous trophoblasts (EVT) of AGA (b, c) and HELLP placentas (j) are GKN2 positive. The bar equals 100 µm (PDF 1294 kb)


Negative control images of human first- (ac) and third-trimester placenta (df) using immunohistochemistry (DAB-IHC; a, d) or immunofluorescence (IF; bc, ef) techniques. Abbreviations: villous trophoblast (VT), extravillous trophoblast (EVT), syncytiotrophoblast (SCT) and decidual stroma cells (DC). The bar equals 100 µm (PDF 599 kb)


Negative control images of rat (a, b) and mouse (c, d) tissues (placenta (ac) and stomach (d)) using immunohistochemistry (DAB-IHC; a, e) or immunofluorescence (IF; b, f) techniques. Placental glycogen cells (b, c) and stomach mucosa (d) remain GKN2 negative. Abbreviations: labyrinth zone (LZ), spongiotrophoblast layer (ST), basal zone (BZ), giant cells (GiC) indicated by a dashed circle. Nuclei are stained blue by hematoxylin (a, e) or DAPI (b, f). The bar equals 100 µm (PDF 572 kb)


Immunofluorescence (IF) double-staining of human third-trimester placental sections. Placental tissues were stained with anti-GKN2 and anti-SMA or –HLA-G antibodies. Single channel pictures are shown in a, d, g (green) and b, e, h (red). c, f and i represent the merged images including DAPI stained nuclei (blue). c shows the co-localization of GKN2 and SMA in a single myofibroblast, f the co-localization of GKN2 and HLA-G and i of GKN2 and GKN1 in extravillous trophoblasts. Yellow immunofluorescence indicates overlap of red and green staining. The bar equals 100 µm (PDF 963 kb)

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Fahlbusch, F.B., Ruebner, M., Huebner, H. et al. Trophoblast expression dynamics of the tumor suppressor gene gastrokine 2. Histochem Cell Biol 144, 281–291 (2015).

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