Hydroxychloroquine restores trophoblast fusion affected by antiphospholipid antibodies.

BACKGROUND: Obstetric antiphospholipid syndrome (APS) is defined by pregnancy complications associated with antiphospholipid antibodies (aPL). The mechanisms of the pathogenic effects of aPL in pregnancy are poorly understood. Toll-like receptors (TLR) have been implicated previously in APS. OBJECTIVES: The aims of our study were (1) to determine aPL effects on trophoblastic cell fusion and differentiation, (2) to identify which TLR is involved in this process, and (3) to evaluate the efficacy of hydroxychloroquine (HCQ) to counteract the effects of aPL. METHODS: BeWo cells are a model for trophoblast fusion and differentiation. Fusion index was assessed by immunocytochemical examination, and biochemical differentiation by using ELISA-measured ß-human choronic gonadotropin hormone (ß-hCG) secretion. We used three types of aPL to study their effect on cell fusion and differentiation: aPL derived from obstetric APS patients and affinity purified and polyclonal rabbit anti-ß2-glycoprotein-1 (anti-ß2GP1) antibodies. Experiments on fusion were confirmed using primary cytotrophoblastic cells. RESULTS: All of the types of aPL used decreased the fusion index in BeWo and primary trophoblastic cells (64%, 52%, and 41% for BeWo cells and 67% and 62% for primary cells, respectively), and anti-ß2GP1 antibodies decreased hCG secretion in BeWo cells (41%). To block TLR4 antibodies or to abolish TLR4 cell surface expression restored fusion index in both cell types and ß-human choronic gonadotropin hormone excretion in BeWo cells. HCQ treatment induced the same effect and decreased TLR4 mRNA (40% and 35%, respectively) and protein expressions (62% and 42%, respectively) in BeWo cells. CONCLUSION: Anti-ß2GP1 antibodies decrease trophoblastic differentiation via TLR4. This effect is restored by HCQ, suggesting its therapeutic interest in APS pregnancies.

Regulation of MMP-9 by p53 in first trimester cytotrophoblastic cells.

BACKGROUND: The matrix metalloproteinase (MMP) family is known to play a key role in tissue remodelling during embryonic development and in pathological conditions, such as cardiovascular disease, arthritis and cancer metastasis. It has been shown previously that p53 regulates positively or negatively the expression of different MMPs. Because of p53 overexpression in trophoblastic cells, and its potential role in regulating MMP-2 and MMP-9 expression in different cell lines, we hypothesized that the expression of MMP-9 could also be regulated by p53 in first trimester cytotrophoblasts (CTB). METHODS AND RESULTS: Transfection experiments in CTB demonstrated that wild-type p53 down-regulates the -670 (P < 0.001) but not the -531 and -90 human MMP-9 promoter/CAT reporter plasmid activity, whereas p53 mutants partially lost this repressive activity. However, endogenous p53 is not able to regulate MMP-9 expression in CTB. The presence of high molecular weight complexes of p53 in CTB suggests a potential mechanism of inactivation of p53 transcriptional activity towards MMPs in these cells. CONCLUSIONS: Although p53 is mutated in trophoblast, it is functionally incompetent towards MMPs in these cells.

Trophoblastic p53 is stabilised by a cis-trans isomerisation necessary for the formation of high molecular weight complexes involving the N-terminus of p53.

P53 is a transcription factor also called the "cellular gatekeeper of genome" because it can induce cell cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. Wild type p53 is localised in both the cytoplasm and nucleus of first trimester trophoblastic cells (CTB). Immunoblotting of CTB with different p53 antibodies led us to suggest that the N-terminus of p53 could be involved in the formation of high molecular weight complexes (HMWC), leading to the stabilisation of p53 in these cells. Here, we demonstrate that the N-terminus of p53 is involved in the formation of HMWC. Post-translational modifications of p53 seem to be responsible for its stabilisation and inactivation in CTB. We demonstrate that cis-trans isomerisation of proteins by the prolyl isomerase Pin1 is indispensable for the formation of these HMWC and stabilisation of p53. In contrast to observations made in other cells, in CTB, interaction of Pin1 and p53 does not involve phosphorylation of residues ser33, thr81 and ser315 of p53; on the contrary, phosphorylation of p53 leads to the rapid disappearance of some HMWC and destabilises p53. Moreover, decreasing HMWC or inhibiting Pin1 activity increases p53 activity towards its target genes MMP-9 and MMP-2, thus confirming the role of Pin-1 and these HMWC in the regulation of trophoblast invasiveness.