Integrin-linked kinase can facilitate syncytialization and hormonal differentiation of the human trophoblast-derived BeWo cell line.
BACKGROUND
In the fusion pathway of trophoblast differentiation, stem villous cytotrophoblast cells proliferate and daughter cells differentiate and fuse with existing syncytiotrophoblast to maintain the multi-nucleated layer. Integrin-linked kinase (ILK) is highly expressed in 1st and 2nd trimester villous cytotrophoblast cells, yet barely detectable in syncytiotrophoblast, thus we examined the potential role of ILK in aiding trophoblast fusion.


METHODS
The temporal/spatial expression and activity of ILK were determined in BeWo cells undergoing syncytialization by immunoblot and immunofluorescence analyses. BeWo cells were also transfected with pEGFP expression vectors containing wildtype or two mutant ILK cDNA constructs. The incidence of cell fusion in transfected cells grown under syncytialization conditions was then scored by the presence or absence of E-cadherin immunostaining. Beta-hCG expression in transfected cells, a marker of syncytiotrophoblast hormonal differentiation, was also similarly assessed.


RESULTS
ILK catalytic activity increased and ILK began to increasingly localize to BeWo cell nuclei during syncytialization in correlation with increased pAkt and Snail protein expression. Syncytialization was also significantly elevated (p < 0.05) in BeWo cells expressing constitutively active (ca)-ILK vs cells containing empty vector or dn-ILK. Furthermore, cytoplasmic Beta-hCG expression markedly increased (p < 0.05) in cells expressing wt- and ca-ILK.


CONCLUSIONS
ILK-facilitated syncytialization is dependent, at least in part, on ILK catalytic activity while hormonal differentiation appears dependent on both ILK-associated protein interactions and catalytic activity. This study demonstrates that ILK plays a novel role in BeWo syncytialization and differentiation, perhaps through an ILK-Akt-Snail pathway, and implicates ILK in the same process in villous cytotrophoblasts in vivo.
Butler
Trina M
TM
Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's NLA1B3V6, Canada. x76tmk@mun.ca
Elustondo
Pia A
PA
Hannigan
Greg E
GE
MacPhee
Daniel J
DJ
D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D045744:Cell Line, Tumor; D002822:Choriocarcinoma; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015726:Giant Cells / Q000166:cytology* / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014327:Trophoblasts / Q000166:cytology* / Q000201:enzymology*; D014594:Uterine Neoplasms; D000099200:Scaffold Protein ILK
D015820:Cadherins; D018997:Chorionic Gonadotropin, beta Subunit, Human; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D000099200:Scaffold Protein ILK