Isolation and culture of term human cytotrophoblast cells and in vitro methods for studying human cytotrophoblast cells' calcium uptake.

Human primary cytotrophoblast cell culture is a very useful model to study the endocrine and immunological functions of syncytiotrophoblasts, as well as the ion exchange between the mother and her fetus, like calcium. In this chapter, we expose the procedure to (1) isolate and purify the cytotrophoblast cells from human term placenta and (2) study syncytiotrophoblast calcium uptake. First, the methodology is based on the enzymatic dissociation of villous placental tissue, followed by Percoll gradient separation. Purity is assessed by flow cytometry using staining against cytokeratin-7, protein specific for trophoblast cells. Cell proliferation is evaluated by a Thiazolyl Blue Tetrazolium Bromide (MTT) assay, hormonal secretion is measured by enzyme-linked immunosorbent assay (ELISA), and fusion is estimated by immunofluorescence using staining against desmosomal proteins. Second, we describe the calcium uptake experiment using the cytotrophoblast cells in culture.

Differences in MAP kinase phosphorylation in response to mechanical strain in asthmatic fibroblasts.

BACKGROUND: Mechanical strain alters protein expression. It results in phosphorylation of MAP kinases and up-regulation of extracellular matrix proteins. We investigated whether phosphorylation of MAP kinase family members was increased in response to mechanical strain in fibroblasts from asthmatic patients (AF) and normal controls (NF), and whether phosphorylation of these signaling molecules would be different in the two cell populations. METHODS: Fibroblasts were obtained from mild, atopic asthmatics and non-atopic volunteers using endobronchial biopsy. Cells were grown on flexible, collagen I-coated membranes, and subjected to mechanical strain (Flexercell). MAP kinase phosphorylation was measured at baseline, and during one hour of strain. We also examined the effect of strain on proteoglycan production. RESULTS: At baseline, there was increased phosphorylation of ERK1/2 and p38, and decreased phosphorylation of JNK in AF vs NF. During strain in NF, p38 phosphorylation was increased. Conversely in AF, strain resulted in an increase in JNK phosphorylation, had no effect on phosphorylation of p38, and resulted in a decrease in ERK1/2 phosphorylation. There was a significant increase in versican protein production after 24 h strain in both AF and NF. JNK inhibition reversed the strain-induced increase in versican in NF, but had no effect in AF. CONCLUSION: These results show that there are phenotypic differences in MAP kinase phosphorylation in AF vs NF, and that different signaling pathways are involved in transducing mechanical stimuli in these two populations of cells.

Integrins in the ovary.

Integrins are the major receptors mediating adhesion to the extracellular matrix. Following ligand binding, conformational changes of integrins induce the recruitment of multiple signaling and scaffolding proteins that connect integrin tails to the actin cytoskeleton and permit activation of signaling pathways regulating cell proliferation, apoptosis, differentiation, and migration. In the ovary, extracellular matrix components present in the follicular basement membrane, around follicular cells, and in the follicular fluid participate in the regulation of follicular development, and a role of integrins in this process is strongly suggested. We discuss available data on integrin expression in ovary, integrin function in granulosa cells, and the possible involvement of integrins in follicular growth, atresia, and luteinization. We also discuss the potential role of oocyte integrins as receptors for sperm ADAMs (a disintegrin and metalloproteinase) in fertilization and the cooperation of integrins with proteinases in regulating proliferation, adhesion, migration, and invasion of ovarian surface epithelium cells in ovarian tumor development.

Cytoskeleton reorganization mediates alpha6beta1 integrin-associated actions of laminin on proliferation and survival, but not on steroidogenesis of ovine granulosa cells.

BACKGROUND: Laminin (LN) is one of the most abundant extracellular matrix components of the basal lamina and granulosa cell layers of ovarian follicles. Culture of ovine granulosa cells (GC) on LN substratum induces cell spreading, enhances cell survival and proliferation, and promotes luteinization. Previous investigations have shown that these effects are mostly mediated by the alpha6beta1 integrin, but its signalization pathways have not been investigated. This study aimed to assess the importance of the cytoskeleton in the alpha6beta1 integrin-mediated actions of laminin on survival, proliferation and steroidogenesis of ovine GC. METHODS: The relationships between morphology and functions of ovine GC cultured on substrata containing LN or/and RGD peptides were investigated. The effects of (1) cytochalasin D, an actin cytoskeleton-disrupting drug, (2) a specific function-blocking antibody raised against alpha6 integrin subunit (anti-alpha6 IgG), and (3) an inhibitor of the ERK1/2 signalization pathway (PD98059) were assessed for GC shape, pyknosis and proliferation rates, oestradiol and progesterone secretions. RESULTS: Cytoskeleton disruption by cytochalasin D induced cell rounding, inhibited proliferation, promoted pyknosis, inhibited progesterone secretion and enhanced oestradiol secretion by GC cultured on LN. When GC were cultured on various substrata containing LN and/or RGD peptides in the presence or absence of anti-alpha6 IgG, both the existence of close correlations between the percentage of round cells, and the GC proliferation rate (r = -0.87) and pyknotic rate (r = 0.76) were established, but no relationship was found between cell shape and steroidogenesis. Inhibition of the ERK1/2 signalization pathway by PD98059 had no effect on GC shape, proliferation or pyknotic rates. However, it dramatically reduced progesterone secretion, expression of cytochrome P450 cholesterol side-chain cleavage and 3beta-hydroxysteroid deshydrogenase enzymes, and enhanced oestradiol secretion, thereby reproducing all the effects of the anti-alpha6 IgG on steroidogenesis of GC cultured on LN. CONCLUSION: LN may participate in the paracrine control of follicular development through different mechanisms. It could enhance proliferation and survival of GC through its alpha6beta1 integrin-mediated actions on cytoskeleton. In contrast, its stimulating action on GC luteinization could be partly mediated by the ERK1/2 pathway, irrespective of cell shape.

Imbalance of receptor-regulated and inhibitory Smads in lung fibroblasts from bleomycin-exposed rats.

Transforming growth factor (TGF)-beta plays a central role in lung fibrosis, stimulating extracellular matrix deposition. Intracellular signaling of TGF-beta is mediated by Smad proteins. We questioned whether the expression and activation of Smads would be altered in lung fibroblasts from rats exposed to bleomycin, an agent used to provoke an experimental model of lung fibrosis. Fibroblasts were isolated from rat lungs 14 d after intratracheal instillation of bleomycin (BLF) or saline (NLF), and cell cultures established. Whole cell lysates were obtained at baseline, and after stimulation with TGF-beta1 (10 ng/ml). Western blot analysis was performed to measure levels of phosphorylated Smad3 (p-Smad3) and Smad7. Real-time PCR was used to determine changes in Smad7 mRNA after TGF-beta stimulation. We found increased baseline levels of p-Smad3 in BLF versus NLF (P < 0.05). In contrast, baseline levels of Smad7 were comparable. The ratio of stimulatory to inhibitory Smads was increased in BLF compared with NLF (P < 0.05). After stimulation with TGF-beta, levels of p-Smad3 were increased in both groups, with maximal responses at 30 min (P < 0.01). While Smad7 mRNA levels were significantly upregulated (at 1 h) after TGF-beta in both groups, the increase in Smad7 protein was significant in NLF only. We conclude there is sustained activation of Smad signaling in lung fibroblasts isolated from bleomycin-exposed rats, with an imbalance between the levels of p-Smad3 and Smad7. Insufficient levels of the inhibitory Smad7 at baseline, and inadequate response to TGF-beta, may contribute to the fibrotic phenotype characteristic of BLF.