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Characterization of the molecular mechanisms underlying the cadherin-mediated differentiation of human trophoblastic cells Kwok, Yin Wan


The majority of the biological functions of the human placenta are performed by the syncytial trophoblast layer, a multinucleated cell that is formed by the terminal differentiation and fusion of the underlying layer of mitotically active, mononucleate cytotrophoblasts. This key step in the formation and organization of the placenta is mediated by a decrease in E-cadherin (E-cad) and a concomitant increase in the levels of Cadherin-11 (cad-11) expressed on the surface of differentiating trophoblastic cells. Similarly, aberrant expression of these two cadherin subtypes has also been associated with the development of persistent trophoblastic disease in vivo. To identify the mechanisms underlying the switch in cadherin expression in human trophoblasts, we examined the mRNA levels and the protein levels of Snail and Slug, 2 zinc finger transcription repressors that regulate E-cad expression, and Twist, a transcription factor associated with tumor metastasis, in BeWo choriocarcinoma cells undergoing differentiation and fusion in response to the intracellular secondary messenger, cAMP. Quantitative Competitive Polymerase Chain Reaction (QC-PCR) analysis revealed significant declines in E-cadherin mRNA levels in BeWo choriocarcinoma cells after 12h of culture in the presence of cAMP. The levels of this mRNA transcript continued to decline until the termination of these studies at 48h. Western blot analysis and immunostaining also demonstrated a significant decrease in the protein levels of Ecadherin in response to cAMP. A significant and continuous increase in Snail mRNA levels and protein levels were detected in these trophoblastic cells over time in culture with cAMP using QC-PCR and Western Blot. Intense immunostaining for Snail expression was also detected in the nuclei of cultured BeWo cells. However, the levels of the mRNA transcript encoding Slug remained relatively low and constant, at least at the time points examined in these studies. There was also a significant increase in Twist mRNA levels and protein levels as the BeWo cells underwent terminal differentiation and fusion. Collectively, these observations suggest that Snail and Twist regulate the switch in cadherin subtypes expressed, on the surface of human trophoblastic cells, a cellular event underlying the formation of multinucleated syncytium in vivo and in vitro. Future studies will examine the effects of loss-, or gain-of-function of Snail and Twist on the terminal differentiation and fusion of human trophoblastic cells in vitro.

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