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Published on: September 26, 2013
Type IV collagenases in human amniotic fluids and amnion epithelial cells
1Department of Pathology, University of Helsinki, Finland.
Insights
Human amniotic fluid contains a specific M(r) 66,000 gelatinase, also found in serum. Amnion cells produce this enzyme, suggesting local production within the amniotic fluid for potential physiological roles.
Area of Science:
- Biochemistry
- Cell Biology
- Reproductive Biology
Background:
- Gelatinases are matrix metalloproteinases involved in tissue remodeling.
- Human amniotic fluid contains various enzymes, including proteases.
- The role of specific gelatinases in pregnancy and fetal development is not fully understood.
Purpose of the Study:
- To identify and characterize gelatinases present in human second-trimester amniotic fluid.
- To investigate the cellular origin and regulation of these gelatinases.
- To compare the concentration of identified gelatinases in amniotic fluid and maternal serum.
Main Methods:
- Gelatin zymography to detect proteolytic activity.
- Immunoblotting with antiserum against human fibroblast gelatinase/type IV collagenase.
- Cell culture of human amnion epithelial cells.
- Immunoprecipitation to confirm protein identity.
- Quantitative zymography to assess enzyme concentrations.
Main Results:
- A constant M(r) 66,000 gelatinase was detected in amniotic fluid, recognized by antiserum to human fibroblast gelatinase.
- Human amnion epithelial cells produced both M(r) 66,000 and M(r) 92,000 gelatinases.
- Transforming growth factor-beta enhanced M(r) 66,000 production, while tetradecanoyl phorbol acetate enhanced M(r) 92,000 production.
- The M(r) 66,000 proteinase in amniotic fluid was identical to that produced by amnion cells and corresponded to human fibroblast gelatinase.
- The M(r) 92,000 gelatinase corresponded to human macrophage/granulocyte gelatinase.
- Concentrations were 5-10 µg/ml (0.1-0.2% of total protein) in amniotic fluid and 30-40 µg/ml (0.03-0.06%) in serum, indicating a 3-4 fold higher relative concentration in amniotic fluid.
Conclusions:
- Human amnion epithelial cells are a likely source of the M(r) 66,000 gelatinase found in amniotic fluid.
- The higher relative concentration in amniotic fluid suggests local production.
- Amnion cells may also produce M(r) 92,000 gelatinase under specific physiological stimuli.
Abstract:
Gelatin zymograms revealed in human second trimester amniotic fluids a constant M(r) 66,000 proteolytically active polypeptide that in immunoblotting was recognized by antiserum against the human fibroblast gelatinase/type IV collagenase. Isolated cultured human amnion epithelial cells produced the M(r) 66,000 proteinase and frequently a M(r) 92,000 one. The production of the former was enhanced by transforming growth factor-beta and that of the latter by tetradecanoyl phorbol acetate. Immunoprecipitation experiments indicated that the M(r) 66,000 amnion epithelial cell proteinase was identical with that found in the amniotic fluids and that the M(r) 92,000 one corresponded to the human macrophage/granulocyte gelatinase/type IV collagenase. Approximate quantifications of the M(r) 66,000 proteinase in amniotic fluids and sera from same individuals were assessed by comparison of dilution series of the samples and standard enzyme preparations in zymography. These measurements indicated that the amount of the proteinase in the amniotic fluids was about 5-10 micrograms/ml representing 0.1-0.2% of total proteins. In sera the corresponding figures were 30-40 micrograms/ml and 0.03-0.06%, respectively. Thus the relative concentration of the M(r) 66,000 proteinase in amniotic fluids was approx. 3-4-fold that in sera, suggesting local production of the protein. The proteinase may be produced by amnion epithelial cells that under so far unknown physiologic stimulus also may start secreting the M(r) 92,000 enzyme.
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