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Downregulation of caveolin-1 enhances fusion of human BeWo choriocarcinoma cells.

Collett GP, Linton EA, Redman CW, Sargent IL - PLoS ONE (2010)

Bottom Line: Assessment of cell fusion by desmosomal protein immunostaining revealed that cells transfected with caveolin-1 siRNA showed significantly enhanced fusion in response to treatment with dibutyryl cyclic AMP compared with cells transfected with a non-silencing control.In addition, biochemical differentiation, assessed by expression of placental alkaline phosphatase, was upregulated in caveolin-1 siRNA-transfected cells, with or without dbcAMP treatment.Assessment of Akt phosphorylation showed that caveolin-1 knockdown resulted in a significant reduction in phosphorylation at Thr(308).

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom. Gavin.Collett@obs-gyn.ox.ac.uk

ABSTRACT

Background: Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. Caveolin-1 has been shown to be expressed in human villous cytotrophoblast and to be downregulated during fusion into syncytiotrophoblast but it is unclear whether it plays a role in this process.

Methodology/principal findings: We used RNA interference to determine whether caveolin-1 plays a role in differentiation and fusion in the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Assessment of cell fusion by desmosomal protein immunostaining revealed that cells transfected with caveolin-1 siRNA showed significantly enhanced fusion in response to treatment with dibutyryl cyclic AMP compared with cells transfected with a non-silencing control. Furthermore, caveolin-1 knockdown alone was sufficient to promote spontaneous fusion. In addition, biochemical differentiation, assessed by expression of placental alkaline phosphatase, was upregulated in caveolin-1 siRNA-transfected cells, with or without dbcAMP treatment. Assessment of Akt phosphorylation showed that caveolin-1 knockdown resulted in a significant reduction in phosphorylation at Thr(308).

Conclusions/significance: Taken together, these results suggest that caveolin-1 regulates BeWo cell differentiation and fusion, possibly through a mechanism involving modulation of Akt activity.

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Related in: MedlinePlus

siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.A, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. B, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).
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pone-0010529-g001: siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.A, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. B, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).

Mentions: BeWo cells were transfected with siRNA duplexes targeted to human caveolin-1 mRNA (Cav-1 siRNA) or a non-silencing control which has no homology to any known mammalian gene. Caveolin-1 expression was efficiently downregulated in a dose-dependent manner by transfection (48 h) with Cav-1 siRNA, as demonstrated by an immunoblot of BeWo cell lysates (Fig. 1A). Transfection with the non-silencing control did not affect caveolin-1 expression compared with mock-transfected controls. Levels of β-actin were unaffected by transfection with either Cav-1 or non-silencing siRNA. Densitometric analysis revealed that transfection with 50 nM Cav-1 siRNA resulted in a 72% knockdown of caveolin-1 expression compared with the non-silencing control (Fig. 1B). For all further experiments, cells were transfected with siRNA duplexes at 50 nM.


Downregulation of caveolin-1 enhances fusion of human BeWo choriocarcinoma cells.

Collett GP, Linton EA, Redman CW, Sargent IL - PLoS ONE (2010)

siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.A, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. B, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865536&req=5

pone-0010529-g001: siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.A, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. B, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).
Mentions: BeWo cells were transfected with siRNA duplexes targeted to human caveolin-1 mRNA (Cav-1 siRNA) or a non-silencing control which has no homology to any known mammalian gene. Caveolin-1 expression was efficiently downregulated in a dose-dependent manner by transfection (48 h) with Cav-1 siRNA, as demonstrated by an immunoblot of BeWo cell lysates (Fig. 1A). Transfection with the non-silencing control did not affect caveolin-1 expression compared with mock-transfected controls. Levels of β-actin were unaffected by transfection with either Cav-1 or non-silencing siRNA. Densitometric analysis revealed that transfection with 50 nM Cav-1 siRNA resulted in a 72% knockdown of caveolin-1 expression compared with the non-silencing control (Fig. 1B). For all further experiments, cells were transfected with siRNA duplexes at 50 nM.

Bottom Line: Assessment of cell fusion by desmosomal protein immunostaining revealed that cells transfected with caveolin-1 siRNA showed significantly enhanced fusion in response to treatment with dibutyryl cyclic AMP compared with cells transfected with a non-silencing control.In addition, biochemical differentiation, assessed by expression of placental alkaline phosphatase, was upregulated in caveolin-1 siRNA-transfected cells, with or without dbcAMP treatment.Assessment of Akt phosphorylation showed that caveolin-1 knockdown resulted in a significant reduction in phosphorylation at Thr(308).

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, United Kingdom. Gavin.Collett@obs-gyn.ox.ac.uk

ABSTRACT

Background: Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. Caveolin-1 has been shown to be expressed in human villous cytotrophoblast and to be downregulated during fusion into syncytiotrophoblast but it is unclear whether it plays a role in this process.

Methodology/principal findings: We used RNA interference to determine whether caveolin-1 plays a role in differentiation and fusion in the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Assessment of cell fusion by desmosomal protein immunostaining revealed that cells transfected with caveolin-1 siRNA showed significantly enhanced fusion in response to treatment with dibutyryl cyclic AMP compared with cells transfected with a non-silencing control. Furthermore, caveolin-1 knockdown alone was sufficient to promote spontaneous fusion. In addition, biochemical differentiation, assessed by expression of placental alkaline phosphatase, was upregulated in caveolin-1 siRNA-transfected cells, with or without dbcAMP treatment. Assessment of Akt phosphorylation showed that caveolin-1 knockdown resulted in a significant reduction in phosphorylation at Thr(308).

Conclusions/significance: Taken together, these results suggest that caveolin-1 regulates BeWo cell differentiation and fusion, possibly through a mechanism involving modulation of Akt activity.

Show MeSH
Related in: MedlinePlus