Daniel J. MacPhee

Focal adhesion kinase is a key mediator of human trophoblast development.

Trophoblast differentiation during the first trimester of pregnancy involves cell proliferation and invasion and extracellular matrix (ECM) remodeling. Reports have indicated that, in a variety of cell types, processes such as proliferation, invasion, and ECM remodeling require the turnover of focal adhesions mediated by a cytoplasmic tyrosine kinase named focal adhesion kinase (FAK). Therefore, in the present study we examined the expression and spatial localization of FAK during early human placental development. Immunocytochemical and immunoblot analysis showed that FAK and a focal adhesion-associated protein named paxillin were highly expressed between the 5th and 8th weeks of gestation, specifically in villous cytotrophoblast and extravillous trophoblast (EVT) cells. Activated FAK, phosphorylated on Tyr-397, colocalized with α5 integrin and matrix metalloproteinase-2 (MMP2) expression in EVT cells within a previously characterized intermediate, invasive-restrained region. FAK and paxillin expression dramatically decreased after 10 to 12 weeks of gestation coincident with increasing pO2 levels. Exposure of human villous explants of 5 to 8 weeks to a 3% O2 environment resulted in increased trophoblast outgrowth, cell proliferation, and detection of α5 integrin and MMP2, as well as increased activation of FAK in EVT cells compared with explants grown in a 20% O2 environment. To determine whether FAK was a key requisite for trophoblast differentiation, villous explants of 5 weeks gestation were grown in Matrigel in a 3% O2 environment and incubated with 20-mer antisense FAK oligonucleotides. A dramatic reduction of trophoblast outgrowth was observed in antisense-treated explants compared with missense and control cultures, and, in addition, cell proliferation and MMP2 activity in antisense-treated explants were dramatically reduced. These data suggest that FAK is a key kinase involved in early trophoblast cell differentiation and plays a role in regulating cell proliferation and motility during early placental development.

Methodological considerations for improving Western blot analysis.

The need for a technique that could allow the determination of antigen specificity of antisera led to the development of a method that allowed the production of a replica of proteins, which had been separated electrophoretically on polyacrylamide gels, on to a nitrocellulose membrane. This method was coined Western blotting and is very useful to study the presence, relative abundance, relative molecular mass, post-translational modification, and interaction of specific proteins. As a result it is utilized routinely in many fields of scientific research such as chemistry, biology and biomedical sciences. This review serves to touch on some of the methodological conditions that should be considered to improve Western blot analysis, particularly as a guide for graduate students but also scientists who wish to continue adapting this now fundamental research tool.

Uterine Stretch Regulates Temporal and Spatial Expression of Fibronectin Protein and Its Alpha 5 Integrin Receptor in Myometrium of Unilaterally Pregnant Rats

Abstract The adaptive growth of the uterus during pregnancy is a critical event that involves increased synthesis of extracellular matrix (ECM) proteins and dynamic remodeling of smooth muscle cell (SMC)-ECM interactions. We have previously found a dramatic increase in the expression of the mRNAs that encode fibronectin (FN) and its alpha5-integrin receptor (ITGA5) in pregnant rat myometrium near to term. Since the myometrium at term is exposed to considerable mechanical stretching of the uterine wall by the growing fetus(es), the objective of the present study was to examine its role in the regulation of FN and ITGA5 expression at late gestation and during labor. Using myometrial tissues from unilaterally pregnant rats, we investigated the temporal changes in Itga5 gene expression in gravid and empty uterine horns by Northern blotting and real-time PCR, in combination with immunoblotting and immunofluorescence analyses of the temporal/spatial distributions of the FN and ITGA5 proteins. In addition, we studied the effects of early progesterone (P4) withdrawal on Itga5 mRNA levels and ITGA5 protein detection. At all time-points examined, the Itga5 mRNA levels were increased in the gravid uterine horn, compared to the empty horn (P < 0.05). Immunoblot analysis confirmed higher ITGA5 and FN protein levels in the myometrium, associated with gravidity (P < 0.05). Immunodetection of ITGA5 was consistently high in the longitudinal muscle layer, increased with gestational age in the circular muscle layer of the gravid horn, and remained low in the empty horn. ITGA5 and FN immunostaining in the gravid horn exhibited a continuous layer of variable thickness associated directly with the surfaces of individual SMCs. In contrast to the effects of stretch, P4 does not appear to regulate ITGA5 expression. We speculate that the reinforcement of the FN-ITGA5 interaction: 1) contributes to myometrial hypertrophy and remodeling during late pregnancy; and 2) facilitates force transduction during the contractions of labor by anchoring hypertrophied SMCs to the uterine ECM.

Expression of α5 Integrin (Itga5) Is Elevated in the Rat Myometrium During Late Pregnancy and Labor: Implications for Development of a Mechanical Syncytium

Abstract The underlying mechanisms controlling uterine contractions during labor are still poorly understood. Integrins are heterodimeric, transmembrane receptors composed of α and β subunits that can be found in focal adhesions. Because these structures play an important role in the regulation of smooth muscle contractility and cell adhesion, we hypothesized that α5 integrin mRNA (Itga5) and protein (ITGA5) expression would be induced in the rat myometrium during late pregnancy and labor. Itga5 mRNA expression was significantly increased (P < 0.05) from Day 17 to labor, noticeably decreasing 1 day postpartum (PP). Immunoblot analysis illustrated a continual increase in ITGA5 levels during pregnancy, labor, and PP, with levels reaching significance at labor (P < 0.05). Analysis of ITGA5 expression by immunocytochemistry demonstrated that it is primarily localized to myometrial cell membranes in the longitudinal muscle layer of the myometrium from before pregnancy to Day 6, and in both the longitudinal and circular muscle layers from Day 15 to PP. Treatment of late-pregnant rats with progesterone blocked labor and resulted in sustained expression of Itga5 mRNA expression to Day 24. In addition, immunocytochemistry experiments showed ITGA5 was detectable at higher levels in cell membranes of both myometrial layers in progesterone-treated animals on Days 23 and 24, compared with vehicle controls. We propose that ITGA5, with its sole known partner, ITGB1, may be important in promoting cellular cohesion during late pregnancy. This process may aid the development of a mechanical syncytium for efficient force transduction during the sustained, coordinated, and powerful contractions of labor.

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

BackgroundIn 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.MethodsThe 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.ResultsILK 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.ConclusionILK-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.

Morphological and Electrical Properties of Human Trophoblast Choriocarcinoma, BeWo Cells

The syncytiotrophoblast of the human placenta arises from fusion of stem cells called cytotrophoblasts. The molecular mechanisms associated with cell fusion and syncytiation of cytotrophoblastic cells remain largely unknown. In the present study, we investigated the morphological and electrical properties of BeWo cells, a human choriocarcinoma-derived trophoblast cell model, with several features of the human cytotrophoblast. Cultured cells tended to cluster, but only fused into small, multinucleated syncytia in the presence of cAMP (72 h). The morphological features of both the actin and microtubular cytoskeletons indicated that within 72 h of constant exposure to cAMP, intracellular cortical actin cytoskeleton disappeared, which was the most prominent inducing factor of multi-nucleation. The presence of the cation channel protein, polycystin-2 (PC2), a TRP-type cation channel, associated with placental ion transport in term human syncytiotrophoblast, co-localised with acetylated tubulin in midbodies, but was found non-functional under any conditions. Different electrical phenotypes were observed among control BeWo cells, where only 26% (8 of 31 cells) displayed a voltage-dependent outwardly rectifying conductance. Most quiescent BeWo cells had, however, a low, slightly outwardly rectifying basal whole cell conductance. Acute exposure to intracellular cAMP (<15 min) increased the whole cell conductance by 122%, from 0.72 nS/cell to 1.60 nS/cell, and eliminated the voltage-regulated conductance. The encompassed evidence indicates that the early events in BeWo cell fusion and syncytiation occur by cAMP-associated changes in ionic conductance but not morphological changes associated to chronic exposure to the second messenger. This suggests a tight regulation, and important contribution of cation conductances in cytotrophoblastic cells prior to syncytiation.

Integrin-Linked Kinase (ILK) Is Highly Expressed in First Trimester Human Chorionic Villi and Regulates Migration of a Human Cytotrophoblast-Derived Cell Line

Abstract The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.

Spatiotemporal expression of α1, α3 and β1 integrin subunits is altered in rat myometrium during pregnancy and labour

Integrins are transmembrane extracellular matrix (ECM) receptors composed of alpha- and beta-subunits. Integrins can cluster to form focal adhesions and, because there is significant ECM remodelling and focal adhesion turnover in the rat myometrium during late pregnancy, we hypothesised that the expression of alpha(1), alpha(3) and beta(1) integrin subunits in the rat myometrium would be altered at this time to accommodate these processes. Expression of alpha(1) and beta(1) integrin subunit mRNA was significantly increased on Days 6-23 of pregnancy compared with non-pregnant (NP) and postpartum (PP) time points (P < 0.05). In contrast, alpha(3) integrin subunit mRNA expression was significantly increased on Days 14, 21 and 22 compared with NP, Day 10, 1 day PP and 4 days PP (P < 0.05). A relative gene expression study revealed that, of the integrins studied, the expression of beta(1) integrin mRNA was highest in pregnant rat myometrium. The alpha(1), alpha(3) and beta(1) integrin subunit proteins became immunolocalised to myocyte membranes in situ by late pregnancy and labour in both myometrial muscle layers. Increased alpha(1), alpha(3) and beta(1) integrin gene expression during gestation and the specific detection of these subunits in myocyte membranes during late pregnancy and labour may contribute to the cell-ECM interactions required for the development of a mechanical syncytium.

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.

The focal adhesion protein Hic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK

BackgroundMyometrial growth and remodeling of the cytoskeleton and focal adhesions during late pregnancy may be critical aspects of myometrial activation and thus labour. Yet our understanding of these aspects is inhibited by the paucity of information concerning the components of focal adhesions in the myometrium. The focal adhesion protein h ydrogen peroxide-i nducible c lone-5 (Hic-5) has recently been found in mononuclear smooth muscle but was not examined in the myometrium during pregnancy. Thus, the goal of this study was to characterize Hic-5 mRNA and protein expression in the rat myometrium during pregnancy and labour.MethodsRat myometrium samples were obtained from non-pregnant animals, pregnant animals on days (d) 6, 12, 15, 17, 19, 21, 22, 23 (active labour) and 1 day postpartum (PP). In addition, myometrium samples were collected from rats within a progesterone-delayed labour paradigm. Hic-5 mRNA expression was analyzed by Northern blot analysis while Hic-5 protein expression was examined by immunoblot and immunofluorescence analysis.ResultsHic-5 mRNA expression on d15, d19 and d21 was found to be significantly elevated compared to d6 and d12 of pregnancy and expression on d23 was significantly elevated over d6 (p < 0.05). Immunofluorescence analysis demonstrated that detection of Hic-5 protein in the circular muscle layer appeared to increase from d17 onwards, except PP, and Hic-5 was detectable in the cell cytoplasm and more continuously associated with myometrial cell membranes. In the longitudinal muscle layer Hic-5 was readily detectable by d15 and thereafter and primarily associated at myometrial cell membranes. Co-immunofluorescence analysis of potential Hic-5 and focal adhesion kinase (FAK) association in situ demonstrated a limited level of co-localization on d19, d23 and PP in the circular muscle layer while in the longitudinal muscle layer Hic-5 and FAK were readily co-localized at myometrial cell membranes.ConclusionHic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK in situ. Our results are consistent with a potential role for Hic-5 in focal adhesion remodeling in the rat myometrium during late pregnancy.