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MSX2 Induces Trophoblast Invasion in Human Placenta.

Liang H, Zhang Q, Lu J, Yang G, Tian N, Wang X, Tan Y, Tan D - PLoS ONE (2016)

Bottom Line: Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin.Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells.Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Center, Chongqing Medical University, Chongqing, China.

ABSTRACT
Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

No MeSH data available.


Related in: MedlinePlus

Expression of MSX2 in human placental villi and cell lines.(A) Immunohistochemical localization of MSX2 in normal human placental villi at different stages of pregnancy (a) a placental villi from the first trimester. (b) a placental villi from the second trimester. (c) a placental villi from the second trimester. EVTs invaded into the maternal decidua at the second and third trimester immunostained with MSX2 (b,c) and CK7, a marker of EVT (e,f). (g,h,i) 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; AV: anchoring villi MD: maternal decidua; Bar = 200 μm. (B) (a) Western blotting of MSX2 in the human placental villi from the first (n = 3) and the third (n = 3) trimesters. GAPDH was used as a loading control(Also C). (b) Three experiments as in a were quantified by measuring the intensity of MSX2 protein bands relative to GAPDH controls (Mann Whitney test; *, P<0.05). (C) Expression of MSX2 in different trophoblast cell lines determined by Western blotting. HTR8/SVneo: a human invasive extravillous trophoblast cell line derived from immortalized first trimester trophoblast; JEG-3, JAR, and BeWo: human choriocarcinoma cell lines.
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pone.0153656.g001: Expression of MSX2 in human placental villi and cell lines.(A) Immunohistochemical localization of MSX2 in normal human placental villi at different stages of pregnancy (a) a placental villi from the first trimester. (b) a placental villi from the second trimester. (c) a placental villi from the second trimester. EVTs invaded into the maternal decidua at the second and third trimester immunostained with MSX2 (b,c) and CK7, a marker of EVT (e,f). (g,h,i) 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; AV: anchoring villi MD: maternal decidua; Bar = 200 μm. (B) (a) Western blotting of MSX2 in the human placental villi from the first (n = 3) and the third (n = 3) trimesters. GAPDH was used as a loading control(Also C). (b) Three experiments as in a were quantified by measuring the intensity of MSX2 protein bands relative to GAPDH controls (Mann Whitney test; *, P<0.05). (C) Expression of MSX2 in different trophoblast cell lines determined by Western blotting. HTR8/SVneo: a human invasive extravillous trophoblast cell line derived from immortalized first trimester trophoblast; JEG-3, JAR, and BeWo: human choriocarcinoma cell lines.

Mentions: To characterize the putative role of MSX2 in placentation, we first examined the location of MSX2 expression in human placental villi by immunohistochemistry. As shown in Fig 1A, MSX2 protein intensely expressed in both the STB, CTBs and trophoblast column(TC) of first trimester placental villi (5 weeks; Fig 1Aa), however predominantly expressed in STB and in invading interstitial EVTs in mid-pregnancy (15 weeks; Fig 1Ab). Meanwhile, in term placentas (38 weeks), the expression of MSX2 protein was detected in villous and EVTs that invaded into the maternal decidua (Fig 1Ac). These findings demonstrate that MSX2 is dynamically expressed in trophoblast lineages throughout gestation. The identification of trophoblast cells were confirmed cytokeratin 7 (CK7) and staining respectively on a separate serial placental sections (Fig 1Ad, 1Ae and 1Af). In addition, expression of MSX2 in term placental villi was significantly lower than that in the first trimester based on Western blotting (Fig 1Ba and 1Bb; P<0.05). Furthermore, we examined the expression of MSX2 in human trophoblast cell lines by western blotting MSX2 proteins were present in HTR8/SVneo cells (normal trophoblast cell lines), were high in JEG-3 cells (human choriocarcinoma cell lines) and JAR cells (human choriocarcinoma cell lines), but low in BeWo cells (human choriocarcinoma cell lines) (Fig 1C).


MSX2 Induces Trophoblast Invasion in Human Placenta.

Liang H, Zhang Q, Lu J, Yang G, Tian N, Wang X, Tan Y, Tan D - PLoS ONE (2016)

Expression of MSX2 in human placental villi and cell lines.(A) Immunohistochemical localization of MSX2 in normal human placental villi at different stages of pregnancy (a) a placental villi from the first trimester. (b) a placental villi from the second trimester. (c) a placental villi from the second trimester. EVTs invaded into the maternal decidua at the second and third trimester immunostained with MSX2 (b,c) and CK7, a marker of EVT (e,f). (g,h,i) 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; AV: anchoring villi MD: maternal decidua; Bar = 200 μm. (B) (a) Western blotting of MSX2 in the human placental villi from the first (n = 3) and the third (n = 3) trimesters. GAPDH was used as a loading control(Also C). (b) Three experiments as in a were quantified by measuring the intensity of MSX2 protein bands relative to GAPDH controls (Mann Whitney test; *, P<0.05). (C) Expression of MSX2 in different trophoblast cell lines determined by Western blotting. HTR8/SVneo: a human invasive extravillous trophoblast cell line derived from immortalized first trimester trophoblast; JEG-3, JAR, and BeWo: human choriocarcinoma cell lines.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835074&req=5

pone.0153656.g001: Expression of MSX2 in human placental villi and cell lines.(A) Immunohistochemical localization of MSX2 in normal human placental villi at different stages of pregnancy (a) a placental villi from the first trimester. (b) a placental villi from the second trimester. (c) a placental villi from the second trimester. EVTs invaded into the maternal decidua at the second and third trimester immunostained with MSX2 (b,c) and CK7, a marker of EVT (e,f). (g,h,i) 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; AV: anchoring villi MD: maternal decidua; Bar = 200 μm. (B) (a) Western blotting of MSX2 in the human placental villi from the first (n = 3) and the third (n = 3) trimesters. GAPDH was used as a loading control(Also C). (b) Three experiments as in a were quantified by measuring the intensity of MSX2 protein bands relative to GAPDH controls (Mann Whitney test; *, P<0.05). (C) Expression of MSX2 in different trophoblast cell lines determined by Western blotting. HTR8/SVneo: a human invasive extravillous trophoblast cell line derived from immortalized first trimester trophoblast; JEG-3, JAR, and BeWo: human choriocarcinoma cell lines.
Mentions: To characterize the putative role of MSX2 in placentation, we first examined the location of MSX2 expression in human placental villi by immunohistochemistry. As shown in Fig 1A, MSX2 protein intensely expressed in both the STB, CTBs and trophoblast column(TC) of first trimester placental villi (5 weeks; Fig 1Aa), however predominantly expressed in STB and in invading interstitial EVTs in mid-pregnancy (15 weeks; Fig 1Ab). Meanwhile, in term placentas (38 weeks), the expression of MSX2 protein was detected in villous and EVTs that invaded into the maternal decidua (Fig 1Ac). These findings demonstrate that MSX2 is dynamically expressed in trophoblast lineages throughout gestation. The identification of trophoblast cells were confirmed cytokeratin 7 (CK7) and staining respectively on a separate serial placental sections (Fig 1Ad, 1Ae and 1Af). In addition, expression of MSX2 in term placental villi was significantly lower than that in the first trimester based on Western blotting (Fig 1Ba and 1Bb; P<0.05). Furthermore, we examined the expression of MSX2 in human trophoblast cell lines by western blotting MSX2 proteins were present in HTR8/SVneo cells (normal trophoblast cell lines), were high in JEG-3 cells (human choriocarcinoma cell lines) and JAR cells (human choriocarcinoma cell lines), but low in BeWo cells (human choriocarcinoma cell lines) (Fig 1C).

Bottom Line: Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin.Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells.Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Center, Chongqing Medical University, Chongqing, China.

ABSTRACT
Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

No MeSH data available.


Related in: MedlinePlus