Butler Trina M, Elustondo Pia A, Hannigan Greg E, MacPhee Daniel J
Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's NLA1B3V6, Canada.
Reprod Biol Endocrinol. 2009 May 22;7:51. doi: 10.1186/1477-7827-7-51.
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.
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.
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.
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.
在滋养层细胞分化的融合途径中，绒毛干细胞滋养层细胞增殖，子代细胞分化并与现有的合体滋养层细胞融合以维持多核层。整合素连接激酶（ILK）在孕早期和中期的绒毛细胞滋养层细胞中高表达，但在合体滋养层细胞中几乎检测不到，因此我们研究了ILK在促进滋养层细胞融合中的潜在作用。
通过免疫印迹和免疫荧光分析，确定在进行合体化的BeWo细胞中ILK的时空表达和活性。还用含有野生型或两种突变ILK cDNA构建体的pEGFP表达载体转染BeWo细胞。然后根据E-钙黏蛋白免疫染色的有无，对在合体化条件下生长的转染细胞中的细胞融合发生率进行评分。还同样评估了转染细胞中β-hCG的表达，β-hCG是合体滋养层细胞激素分化的标志物。
在合体化过程中，ILK催化活性增加，并且ILK开始越来越多地定位于BeWo细胞核，这与pAkt和Snail蛋白表达增加相关。与含有空载体或dn-ILK的细胞相比，表达组成型活性（ca）-ILK的BeWo细胞中的合体化也显著升高（p < 0.05）。此外，在表达wt-ILK和ca-ILK的细胞中，细胞质β-hCG表达明显增加（p < 0.05）。
ILK促进的合体化至少部分取决于ILK催化活性，而激素分化似乎取决于与ILK相关的蛋白质相互作用和催化活性。这项研究表明，ILK在BeWo细胞的合体化和分化中起新作用，可能是通过ILK-Akt-Snail途径，并提示ILK在体内绒毛细胞滋养层细胞的同一过程中起作用。
