Expression of CD82 in human trophoblast and its role in trophoblast invasion

Abstract

Background: Well-controlled trophoblast invasion at maternal-fetal interface is a critical event for the normal development of placenta. CD82 is a member of transmembrane 4 superfamily, which showed important role in inhibiting tumor cell invasion and migration. We surmised that CD82 are participates in trophoblast differentiation during placenta development.

Methodology/principal findings: CD82 was found to be strongly expressed in human first trimester placental villous and extravillous trophoblast cells as well as in trophoblast cell lines. To investigate whether CD82 plays a role in trophoblast invasion and migration, we further utilized human villous explants culture model on matrigel and invasion/migration assay of trophoblast cell line HTR8/SVneo. CD82 siRNA significantly promoted outgrowth of villous explants in vitro (P<0.01), as well as invasion and migration of HTR8/SVneo cells (P<0.05), whereas the trophoblast proliferation was not affected. The enhanced effect of CD82 siRNA on invasion and migration of trophoblast cells was found associated with increased gelatinolytic activities of matrix metalloproteinase MMP9 while over-expression of CD82 markedly decreased trphoblast cell invasion and migration as well as MMP9 activities.

Conclusions/significance: These findings suggest that CD82 is an important negative regulator at maternal-fetal interface during early pregnancy, inhibiting human trophoblast invasion and migration.

Figure 1. Expression of CD82 in human placental villi and cell lines.

(A) Immunostaining of CD82 in normal human placental villi in the first trimester, maternal decidua, second trimester and third trimester. (a) CD82 was strongly expressed in trophoblast columns (TC) and moderate in CTB of human placental villi during the first trimester. (c) CD82 was highly expressed in decidual cells (DC). (e) CD82 was highly expressed in the maternal decidua, not detected in EVT and very faint in anchoring villous during the early second trimester. Boxed region is enlarged on the upper panel. (g) CD82 was absent in villous and very faint in EVT during the late second trimester. (i) CD82 was moderate expressed in cytotrophoblast cells invaded into the maternal decidua and highly expressed in syncytiotrophoblast in the third trimester. (b, f, h, j) Immunohistochemical staining with anti-cytokeratin7 (CK7) as a marker of CTB, TC in the first trimester placental vill, EVT in the maternal decidua; (d) Immunohistochemical staining with anti-vimentin as a maker of decidual cells. (k, l, m, n) negative controls (NEG) on sections in which normal IgG was used in place of primary antibody. CTB: cytotrophoblast; STB: syncytiotrophoblast; TC: trophoblast column; EVT: extravillous trophoblast; MD: maternal decidua, AV: anchoring villous W: weeks of pregnancy; Bar represents 100 µm. (B) Expression of CD82 in different trophoblast cell lines determined by semiquantitative RT-PCR and Western blotting, respectively. GAPDH was used as an internal control for RT-PCR or loading control for Western blotting. HTR8/SVneo: a human invasive extravillous trophoblast cell line derived from immortalized first trimester trophoblast; B6Tert: immortalized cytotrophoblast cell line; JEG-3: human choriocarcinoma cell lines. (C) Immunofluorescence of CD82 in HTR8/SVneo cell lines. Fluorescence signals specific to CD82 antibody were visualized as green, and the nuclei were shown by DAPI staining (blue).

Figure 2. Silencing of CD82 promote trophoblast outgrowth and migration in villous explant cultures.

(A) Villous explants from 5 to 8 weeks of gestation were maintained in culture for 5 days on matrigel under low (3% O₂) oxygen tension. Villous explants of CD82 siRNA significantly increase budding and outgrowth of EVT from the distal end of the villous tips comparing with negative control. Serial pictures of villous explants were taken under the light microscope after 24, 48, and 72 h of culture in vitro. (B) Villi (a) transfected with FITC-tagged siRNA, showing the transfection efficiency (b). Villi (c) as a control transfected nothing, showing the villi background fluorescence (d). (C) Three experiments as in B were quantified by measuring the the migration distance of villous tip relative to CON siRNA transfected for 24 h. (t-test).

Figure 3. Silencing of CD82 promoted invasion and migration of HTR8/SVneo cells.

(A, B) Representative images of filters containing invaded cells in Matrigel invasion assay and transwell cell migration assay are shown. The statistical bar graphs show the summary of three independent experiments. (t-test) (C) Confirmation of RNA interference of CD82 was shown by RT-PCR and Western blotting. GAPDH was used as an internal control in RT-PCR and a loading control in Western blotting. (D) MTT assay showed no significant difference on proliferation. (t-test) (E) CD82 siRNA-1,-2 had no significant effect on apoptosis of HTR8/SVneo cells. (t-test).

Figure 4. Over-expression of CD82 inhibited invasion and migration of HTR8/SVneo cells.

(A, B) Representative images of filters containing invaded cells in Matrigel invasion assay and transwell cell migration assay are shown. The statistical bar graphs show the summary of three independent experiments. (t-test) (C) Confirmation of over-expression of CD82 was shown by RT-PCR and Western blotting. GAPDH was used as an internal control in RT-PCR and a loading control in Western blotting. (D) MTT assay showed no significant difference on proliferation. (t-test) (E) Overexpression CD82 had no significant effect on apoptosis of HTR8/SVneo cells. (t-test) Mock: pFLAG-CMV4 empty vector, CD82: pFLAG-CMV4-CD82 vector.

Figure 5. The effect of CD82 on the activities of MMP2, MMP9 and expression of TIMP1, TIMP2.

(A, C) HTR8/SVneo cells were transfected with CD82 siRNA-1,-2 or pFLAG-CMV4-CD82 plasmid. Total proteins were extracted and western blotting (WB) was performed to detect expression of CD82. Serum-free culture medium was collected for gelatin zymography assay and for Western blotting assay. CD82 siRNA decreased, while over-expression CD82 increased protein levels of both TIMP1 and TIMP2. (B, D) Statistical assay of the zymographic results in A or C. CD82 siRNA increased, while over-expressed CD82 decreased the activity of pro-MMP9. Activity of pro-MMP2 was not significantly affected.