Integrin-linked kinase can facilitate syncytialization and hormonal differentiation of the human trophoblast-derived BeWo cell line

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

The expression and role of integrin-linked kinase during human trophoblast differentiation

A critical phase of placental development is the differentiation of cytotrophoblasts into invasive extravillous trophoblasts. Alterations of this process can lead to serious pregnancy pathologies such as preeclampsia and intrauterine growth restriction. Unfortunately it is poorly understood which physiological and molecular mechanisms are involved in the regulation of trophoblast differentiation. -- lntegrin-Linked Kinase (ILK) is a ubiquitous protein able to regulate outside-inside signaling by binding to integrins. Since ILK is overexpressed in tumors that become invasive, it was hypothesized that ILK is expressed in human placenta and that it is necessary for the differentiation of trophoblasts into invasive cells. To test the hypothesis, spatial and temporal patterns of ILK expression were studied in normal human and mouse placentas by immunohistochemistry, immunofluorescence analysis, and immunoblot analysis. In human placenta, ILK was present in cytotrophoblast and extravillous trophoblast in the first trimester (n=17), early second trimester (n=6) and at term (n=10). By comparison, ILK was present in the three layers of the mouse placenta: labyrinth, junctional zone, and trophoblast giant cells. -- HTR8-SVneo cells were also used to determine the role of ILK in migration and invasion. When ILK function was impaired in HTR8-SVneo cells with a dominant negative form of ILK, migration and invasion were inhibited. Furthermore, HTR8-SVneo and human chorionic villous explant cultures were utilized to study the effect of the oxygen environment on ILK expression and activity. Exposure of HTR8-SVneo cells to a low oxygen environment produced a transient increase in the expression of ILK mRNA, but did not affect the level of protein expression nor its kinase activity. In contrast to these results, placental explants showed lower expression of ILK under 3% O₂ environment compared to placental ex plants exposed to 8% and 20% O₂ . Placentas from preeclamptic patients also showed lower detection of ILK in the fetal capillaries compared to age-matched controls. -- In total these novel results showed that ILK is present in human and mouse placenta in cells that invade the maternal uterus during pregnancy and that it plays a critical role in the migration and invasion of trophoblasts. -- The results contribute to a better understanding of the mechanisms involved in the control of placental invasion and could assist in the production of future tools to improve the early diagnosis and treatment of gestational trophoblastic diseases such as preeclampsia

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore

AbstractThe permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) — sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix

Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle