Gonatropin-mediated inhibition of proteolytic enzymes produced by human trophoblast in culture
We investigated the effect of gonadotropins on protease that were suggested to be implicated in the invasive activity of the trophoblast. hCG levels ranging from 10 x 10(3) to 333 x 10(3) IU/L produced a dose-dependent inhibition of the in vitro globinolytic activity of the purified proteases trypsin, chymotrypsin, and urokinase, but failed to inhibit plasmin, collagenase, elastase, and tissue-type plasminogen activator. Likewise, FSH inhibited purified trypsin and urokinase, but not plasmin or tissue-type plasminogen activator. Culture medium conditioned with human trophoblast displayed serine protease and urokinase-like activities; exposure of the cultured trophoblast to exogenous hCG markedly suppressed serine protease and urokinase activities in the conditioned medium. A short treatment of the conditioned medium with trypsin abolished the hCG-mediated inhibition of urokinase activity. The present findings offer an explanation for earlier observations that hCG reduced collagenase activity in trophoblasts without affecting the level of collagenase-specific mRNA. The present results are also consistent with the concept that hCG, by its direct ability to inhibit certain serine proteases and urokinase in trophoblast, suppresses a protease-mediated conversion of procollagenase to active collagenase. The ability of hCG to prevent initiation of the collagenolytic cascade suggests that gonadotropins may regulate the transient invasive activity of the trophoblast.
Available from: Alejandro Tapia Pizarro
- "These apparently discordant effects of hCG on trophoblast function may be explained by the fact that the previous studies evaluated only the migratory effects, whereas we have assessed the invasive potential of the cells, including ECM remodelling and cell motility . In addition, other in vitro studies using primary trophoblast cultures have demonstrated that hCG decreases trophoblast cell invasiveness (Milwidsky et al., 1993; Yagel et al., 1993). "
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ABSTRACT: STUDY QUESTIONAre secreted extracellular matrix (ECM) remodelling elements, relevant to embryo implantation and placentation, modified by hCG in endometrial stromal cells (ESCs)?SUMMARY ANSWERhCG decreases tissue inhibitor of metalloproteinase 1 (TIMP-1) secretion in ESCs, thereby facilitating extravillous trophoblast invasion in vitro.WHAT IS KNOWN ALREADYSuccessful embryo implantation and placentation depend on the appropriate invasion of the trophoblast into the maternal endometrial stroma. hCG is one of the earliest embryo-derived secreted signals in the endometrium which abundantly expresses hCG receptors. However, there is little data concerning the effects of hCG on endometrial ECM remodelling with respect to embryo implantation.PARTICIPANTS/ MATERIALS, SETTING, METHODS
This study was conducted in an academic research laboratory within a tertiary-care hospital. Samples were collected from 36 women undergoing benign gynaecological surgery during the mid-secretory phase. ESCs were isolated and stimulated with hCG (10 UI/ml) or vehicle. Conditioned media (CM) were analysed to determine changes in the secreted profile of nine matrix metalloproteinases (MMPs) and three tissue-specific inhibitors of MMPs (TIMPs) using an ELISA array. Data were confirmed by gelatine zymography, western blot and ELISA. The HTR8/SVneo cell line served as a model for trophoblast cells. The invasive potential of trophoblast cells was assessed using Transwell invasion assays under CM or co-culture conditions with ECS and the role of regulated molecules was examined by using immunoprecipitation in CM prior to the assessment of invasive potential.MAIN RESULTS AND THE ROLE OF CHANCEMMP-2 levels increased 30%, whereas TIMP-1 levels decreased 20% in CM from ESCs stimulated with hCG (P < 0.05). Gelatine zymography confirmed an increase in MMP-2 activity (P < 0.05). ELISA and western blotting also confirmed the reduction in TIMP-1 upon hCG treatment (P < 0.05). Invasion assays revealed a ∼50% increase in invading HTR8/SVneo cells in chambers with hCG-stimulated ESCs compared with the control (P < 0.05). Immunodepletion of TIMP-1 from control ESC-CM partially resembled the effect of CM from hCG-stimulated ESCs in the trophoblast invasion assays.LIMITATIONS, REASONS FOR CAUTIONThe assays were performed in vitro and ESCs were not decidualized, therefore they reflected the very early stages of embryo implantation or the advanced stages when decidualization fails.WIDER IMPLICATIONS OF THE FINDINGSOur data suggest that hCG induces endometrial stromal extracellular remodelling by modulating secreted MMP-2 and TIMP-1. This regulation may be physiologically relevant because it increases the invasive potential of trophoblast-derived cells. At present, few data exist concerning the implications of hCG and endometrial ECM remodelling in embryo implantation. Hence, our results should be confirmed by further in vivo studies.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by FONDECYT 11100443, PBCT-PSD51 (IDIMI) and FONDAP 15010006. None of the authors have any conflicts of interest to declare.
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ABSTRACT: The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.
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ABSTRACT: The priority in elucidation of the problems associated with HIV infection has been mainly determined by experimental feasibility
rather than by its importance. Molecular biologists have rapidly produced an impressive body of knowledge about the structure
of HIV, but this understanding is not sufficient to explain complex biological effects accompanying viral pathogenesis. Physicians,
fourteen years into the epidemic have been left with a confused mixture of AZT, CD4 counts, false promises of vaccines, morbidity
charts and nothing else. Immunologists, in their turn, disappointed by the results of their own narrow scope of inquiries
for a direct explanation of the pathological effect of the virus, started to look for alternative concepts such as self-inflicted
immunopathology mediated through the capacity of HIV to mimic host antigens and to trigger autoimmune responses. Although
this idea was proposed even before HIV was implicated as a cause of AIDS, mainstream research has been oriented toward goals
that were easier to grasp. As a result, little progress has been made to adopt the viral ‘kamikaze’ phenomenon to the development
of a meaningful therapeutic strategy.
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