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. 2013 Feb 21;4(2):e502.
doi: 10.1038/cddis.2013.1.

CUL1 promotes trophoblast cell invasion at the maternal-fetal interface

Q Zhang  1 , Q Chen, X Lu, Z Zhou, H Zhang, H-Y Lin, E Duan, C Zhu, Y Tan, H Wang
Affiliations

CUL1 promotes trophoblast cell invasion at the maternal-fetal interface

Q Zhang et al. Cell Death Dis. .

Erratum in

Abstract

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE VSports手机版. .

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Figures

Figure 1
Figure 1
Expression of CUL1 in human placental villi at the first and the third trimesters. (A) Immunostaining of CUL1 and cytokeratin 7 (CK7) in normal human placental villi from the first and the third trimesters. (a) A placental villi from the first trimester. (b) A placental villi from the third trimester. (c, d) EVT invaded into the maternal decidua at the third trimester immunostained with CUL1 (c) and CK7, a marker of EVT (d). (e, f) Negative controls (NEG) in which normal IgG were used in place of primary antibody. CTB: cytotrophoblast cells; STB: syncytiotrophoblast; TC: trophoblastic column; EVT: extravillous trophoblast; MD: maternal decidua; Bar=100 μm. (B) Western blotting of CUL1 in the human placental villi from the first (n=3) and the third (n=3) trimesters. GAPDH was used as a loading control (here and after). (C) Three experiments as in (B) were quantified by measuring the intensity of CUL1 protein bands relative to GAPDH controls (t-test; **P<0.01)
Figure 2
Figure 2
Silencing of CUL1 suppresses trophoblast outgrowth in extravillous explant cultures. (A) Extravillous explants from 5–8 weeks of gestation were maintained in culture on matrigel under low oxygen (3% O2) tension. Serial pictures of explants incubated with CUL1 siRNA or control (CON) siRNA were taken under the light microscope after 24, 48, and 72 h of culture in vitro. Bar=100 μm. (B) A statistical assay of the migration distance of villous tips. Data are presented as mean±S.D. of three independent experiments (t-test; *P<0.05; **P<0.01). (C) Serum-free culture medium of the explant transfected with indicated siRNAs was collected for gelatin zymography assay. (D) Examination of CUL1 knockdown efficiency after siRNA transfection. (a, f) Hematoxylin and eosin (HE) staining of CON or CUL1 siRNA-treated explants. Bar=100 μm. (b, g) Immunofluorescent staining using integrin α5 antibody (green) identifies the EVT. Bar=50 μm (c, h) Immunofluorescent staining using CK7 antibody (green) identifies the cytotrophoblast and EVT. Bar=50 μm (d, i) Immunofluorescent staining using CUL1 antibody (green) shows that CUL1 protein is lower in CUL1 siRNA-treated EVT (i) as compared with CON siRNA treated ones (d) at the same exposure time. Bar=50 μm. (e, j) DAPI staining of sections d and i to visualize nuclei. Bar=50 μm. a, b, c, and d are serial sections, so are f, g, h, and i. The region of EVT outgrowths is above the dashed lines
Figure 3
Figure 3
CUL1 siRNA inhibits invasion and migration of HTR8/SVneo cells. (a) Confirmation of RNA interference of CUL1 was shown by western blotting. (b, c) Upper panels: representative images of filters containing invaded cells in matrigel invasion assay (b) and transwell cell migration assay (c) are shown; Middle panels: the number of invaded or migrated cells in a 100 × random field; Lower panels: Statistical bar graphs showing the summary of three independent experiments (t-test; *P<0.05; **P<0.01)
Figure 4
Figure 4
The effect of CUL1 siRNA on apoptosis and proliferation of HTR8/SVneo cells. (a) Ratio of apoptotic cells in a HTR8/SVneo cell population transfected with indicated siRNAs (t-test; P>0.05). (b) HTR8/SVneo cells transfected with indicated siRNAs were subjected to western blotting using indicated antibodies. (c) Proliferation assay of HTR8/SVneo cells transfected with indicated siRNAs over 3 days of culture (t-test; **P<0.01)
Figure 5
Figure 5
CUL1 siRNA inhibits the gelatinolytic activities of pro-MMP-9 and increased the expression of TIMP-1 and TIMP-2. (a) HTR8/SVneo cells transfected with indicated siRNAs were subjected to total proteins extraction and western blotting to detect expression of CUL1 (also in c). Serum-free culture medium was collected for gelatin zymography assay. (b) Statistical analysis of the zymographic results in (a) (t-test; **P<0.01). (c) Serum-free culture medium (for the detection of TIMP-1 and -2) or the whole cell proteins (for the detection of CUL1) of HTR8/SVneo cells transfected with indicated siRNAs were used for western blotting using indicated antibodies
Figure 6
Figure 6
CUL1 protein level is decreased during syncytialization. (a) Real-time quantitative PCR analysis of CUL1, β-hCG, and GCM1 in the primary cultured cells treated with indicated siRNAs. The transcript level is expressed as percentage of CON siRNA, after normalization with β-actin (t-test; **P<0.01). (b) Western blotting showing the expression of CUL1 and β-hCG in the primary trophoblast cells cultured for 0 h to 48 h. (c) Statistical analysis of the western blotting results in (b) (one-way ANOVA; *P<0.05; **P<0.01). (d) Western blotting showing the expression of CUL1 in BeWo cells treated with or without forskolin by using indicated antibodies. (e) Twenty-four hours after transfection with siRNAs (CON and CUL1), BeWo cells were treated with 100 μM forskolin or methanol for 48 h. Multinucleated cells were counted as indicated in ‘Materials and Methods'. Statistical bar graph showed that CUL1 enhanced forskolin-induced syncytialization of BeWo cells (NS, P>0.05; *P<0.05; **P<0.01; ANOVA)
Figure 7
Figure 7
CUL1 is significantly downregulated in the human placental villi from PE patients. (a) Western blotting showing the expression of CUL1 proteins in the placental villi from normal pregnant (n=3) or pre-eclamptic (n=3) women. Three groups representing the 11 groups utilized in this study was shown. (b) Statistical analysis of the western blotting results in (a) (n=11; t-test; **P<0.01)
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References

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