The aim of this article was to investigate the signalling pathways involved in metalloproteinase-9 (MMP-9) expression induced by tumour necrosis factor-alpha (TNF-alpha) in first-trimester trophoblastic cells. TNF-alpha-induced MMP-9 expression, secretion and activity were completely blocked by stress-activated protein kinase/jun kinase (SAPK/JNK) and Erk inhibitors (SP600 125 and U0126 respectively) but not by p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203 580 and SB202 190). Stimulation of HIPEC 65 cells with TNF-alpha caused phosphorylation of JNK and extracellular signal-regulated kinase 1/2 (Erk1/2), with a peak after 20 min of treatment. Transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1)-binding site were identified as the cis-elements involved in TNF-alpha activation as determined by electromobility shift assays. TNF-alpha-induced transactivation of NF-kappaB was inhibited by U0126, whereas TNF-alpha-induced transactivation of AP-1 was inhibited by SP600 125. Taken together, these results indicate that in trophoblastic cells, TNF-alpha probably activates two different pathways leading to MMP-9 expression: (a) Erk1/2 pathway which in turn initiates NF-kappaB activation and (b) SAPK/JNK pathway that activates AP-1.

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Taken together these results suggest that in vitro LIF inhibits the differentiation of CTB towards an invasive phenotype by inhibiting the secretion of metalloproteinases, by increasing the deposition of fFN into the extracellular matrix and by inhibiting the differentiation of CTB into syncytium.

A solid phase, second antibody radioimmunoassay technique has been developed for the mea

p53 has been called the cellular gatekeeper of the genome because it can induce cell-cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. As p53 has been observed in first-trimester cytotrophoblastic cells (CTB), but its expression in normal cells is generally not detectable because of its short half-life, p53 could play an important role in cellular differentiation and/or in the control of the invasion of trophoblastic cells; therefore, p53 status was investigated in these cells. Using different antibodies recognizing different epitopes of p53 protein, abundant p53 expression was observed both in nuclear and in cytoplasmic compartments of first-trimester CTB. Whereas p53 was detected in the nuclei of few trophoblastic cells with an antibody recognizing the N-terminal epitope of the protein, high expression level of p53 in the cytoplasm of CTB was detected with an antibody recognizing the middle part of p53. The lack of immunoreactivity of p53 with antibodies recognizing the epitopes located at the N-terminus of p53 and the high level of p53 protein observed in the cytoplasm of CTB suggest that the N-terminus of p53 is involved in the formation of complexes. These cytoplasmic complexes were detected under non-reducing conditions in western blot analysis and had apparent molecular weights (MW) of 195, 167 or 125 kDa. These complexes could prolong the half-life of p53 in the cytoplasm of CTBs. By contrast, in the nuclei of CTBs, p53 seems to be present as a tetramer.