Expression, regulation and functional characterization of matrix metalloproteinase-3 of human trophoblast.

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1beta, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1beta-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1beta-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1beta failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.

Human chorionic gonadotrophin attenuates NF-kappaB activation and cytokine expression of endometriotic stromal cells.

Recently, a clinical study provided evidence that treatment of endometriotic women with human chorionic gonadotrophin (hCG) alleviates disease-related pain and sleeplessness suggesting therapeutic effects of the hormone. Since endometriosis is associated with aberrant concentrations of inflammatory mediators in the peritoneal fluid, we investigated whether hCG may affect cytokine-dependent activation of the key-regulatory transcription factor NF-kappaB and expression of two nuclear factor kappa B (NF-kappaB)-inducible genes, tumour necrosing factor (TNF-alpha) and interleukin (IL)-1beta, in stromal cells isolated from ectopic endometriotic tissues. Electrophoretic mobility shift assay revealed that treatment of these cultures with the urinary preparation hCG-A suppressed TNF-alpha- or IL-1beta-induced NF-kappaB DNA-binding activity, whereas another urinary hCG preparation (hCG-B) was less effective. Recombinant alphahCG or epidermal growth factor (EGF), a contaminant of some urinary hCG preparations, did not alter cytokine-dependent NF-kappaB activation. Immunofluorescene of its p65 subunit revealed that pre-incubation with hCG-A strongly decreased TNF-alpha-dependent nuclear expression of NF-kappaB. Accordingly, hCG-A diminished IL-1beta-induced TNF-alpha transcript levels and protein release measured by quantitative real-time PCR and enzyme-linked immunosorbent assay. The hormone also attenuated TNF-alpha-dependent mRNA expression of IL-1beta. Western blot analyses revealed that hCG-A impaired TNF-alpha-mediated phosphorylation and degradation of the inhibitor IkappaBalpha suggesting that the hormone may reduce nuclear import of NF-kappaB by stabilizing its inhibitor. The data suggest that hCG attenuates inflammation-dependent NF-kappaB activation and cytokine expression that could provide one explanation for the beneficial role of the hormone in endometriotic patients.

Effects of different human chorionic gonadotrophin preparations on trophoblast differentiation.

Recent evidence from the literature suggested that hCG preparations purified from urine of pregnant women, which are widely used in in vitro studies and IVF programs, may contain contaminants such as EGF. To determine the putative biological effects of the contaminating growth factor, we here investigated distinct trophoblast differentiation processes in the presence of various hCG compounds. Western blot analyses indicated that treatment of trophoblastic SGHPL-5 cells and purified term trophoblasts with potentially EGF-contaminated hCG (hCG-A) resulted in auto-phosphorylation of the EGF receptor at tyrosine 1173 whereas supplementation of another urine-purified hCG preparation (hCG-B), recombinant holo-hCG or recombinant alphahCG had no effects. Phosphorylation was specifically blocked by the EGF receptor inhibitor PD153035. Urinary hCG-A was most effective in promoting invasion of SGHPL-5 cells through Matrigel-coated transwells, but increased invasiveness was also observed in the presence of hCG-B or recombinant holo-hCG. Similarly, the extent of syncytialisation of term trophoblasts, quantitated by nuclei in desmoplakin-negative areas, was highest upon addition of hCG-A or recombinant EGF as a control. PD153035 reduced invasion and fusion of trophoblasts supplemented with hCG-A, but did not diminish the effects provoked by hCG-B. In conclusion, the data suggest that the EGF contamination of hCG considerably affects trophoblast function. Experiments using EGF-free hCG preparations demonstrate that the hormone increases trophoblast invasion and syncytialisation.

Cadmium tolerance and co-tolerance in Silene vulgaris (Moench.) Garcke [=S. cucubalus (L.) Wib.].

This paper is the first to report cadmium tolerance in a dicotyledonous species, Silene vulgaris (Moench.) Garcke. The response to cadmium of five populations originating from one uncontaminated and various heavy-metal contaminated sites was examined under standardized conditions for three weeks. The tolerance index (TI), based on the mean relative growth rate (R), was determined. Populations originating from cadmium-contaminated sites showed a distinct tolerance to cadmium. A population from a site enriched only with copper also exhibited a marked co-tolerance to cadmium. A clear difference in biomass production between the sensitive and tolerant populations was attained at 1 µM cadmium. An optimum biomass production in tolerant populations at a metal concentration higher than in the control, as demonstrated for zinc and copper, could not be established for cadmium. The pattern of cadmium uptake and translocation differed between tolerant and sensitive populations. All tolerant populations accumulated cadmium in the roots and showed some degree of restricted transport to the shoots. The effect of cadmium on the elemental distribution in roots and shoots was population-independent for some elements (copper, zinc, potassium) and population-specific for others (phosphorus, magnesium and sodium). The phenomenon of co-tolerance to cadmium is discussed in relation to possible tolerance mechanisms, especially with regard to metal-binding compounds (metallothioneins, phytochelatins).