The role of integrin linked kinase (ILK) in human placenta development

Butler, Trina Maxine (2015) The role of integrin linked kinase (ILK) in human placenta development. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

During placenta development, mononuclear villous cytotrophoblast cells differentiate and fuse with the overlying syncytiotrophoblast, which is the epithelial covering of the chorionic villi. Integrin-linked kinase (ILK), is known to downregulate E-cadherin through Poly (ADP-ribose) polymerase (PARP) and Snail in during epithelial to mesenchymal transition (EMT). Therefore, since ILK is expressed in villous cytotrophoblast, the role of ILK in aiding trophoblast syncytialization via the downregulation of E-cadherin was examined using the trophoblast derived BeWo cell model. The temporal/spatial expression of ILK, PARP, Snail and E-cadherin was determined in first and early second trimester chorionic villi and BeWo cells by immunofluorescence and immunoblot analysis. It was found that ILK co-localized with PARP and Snail in villous cytotrophoblast. PARP and Snail expression also persisted in some cells that appeared to be fusing with the overlying syncytiotrophoblast, as indicated by decreased E-cadherin expression. In BeWo cells undergoing syncytialization, ILK increasingly localized to cell nuclei in correlation with increased nuclear Snail localization, an Ecadherin repressor, and PARP nuclear localization. This was coincident with decreased E-cadherin expression. Wildtype ILK or mutant ILK was also transiently overexpressed in BeWo cells or endogenous ILK depleted by siRNA targeting. Subsequently the cell fusion in these transfected cells, grown under syncytialization conditions, was scored by the presence or absence of E-cadherin immunostaining. Furthermore, PARP and Snail promoter activities were assessed in BeWo cells during syncytialization and upon ILK overexpression and syncytialization using luciferase assays. Over-expression of ILK ii significantly elevated syncytialization in BeWo cells grown under syncytialization promoting conditions, while the knockdown of ILK expression significantly reduced syncytialization. Lastly, PARP and Snail promoter activities were significantly higher in BeWo cells during syncytialization. Therefore, the results demonstrated that ILK aids trophoblast syncytialization and differentiation via the downregulation of E-cadherin, likely through an ILK-PARP-Snail pathway.

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