Identification of a calcium-dependent matrix metalloproteinase complex in rat chorioallantoid membranes during labour.
The induction of the expression of matrix metalloproteinases (MMPs) and their extracellular activation are key processes in connective tissue degradation in the chorioallantoid membrane during rat labour. However, the regulatory mechanisms remain largely unknown. Here, we report the identification of a calcium-dependent high molecular weight complex composed of MMP-9, MMP-3, MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, identified by zymography and western blotting. Molecular sieve chromatography confirmed the presence of a complex of MMPs and TIMPs with an exclusion volume >670 kDa. Differential scanning calorimetry of the complex confirmed the existence of a macromolecular complex that unfolds with a broad transition; it is denatured over a wide range of temperatures and has a T(m) of 72 degrees C in the presence of Ca(2+). When denatured in the absence of Ca(2+), there were at least eight transitions with T(m)s that corresponded to pro-MMP-9, MMP-9, pro-MMP-3, MMP-3, pro-MMP-2, MMP-2, TIMP-1 and TIMP-2. Co-localization of the same molecular components was demonstrated by confocal microscopy using cell-depleted chorioallantoid membranes. The assembly and disassembly of the complex can be reproduced at physiological concentrations of Ca(2+). This complex provides a potential mechanism for the enzymatic regulation of MMPs, which may participate in connective tissue degradation leading to the rupture of the fetal membranes during labour.
Meraz-Cruz
N
N
Direccion de Investigacion, Instituto Nacional de Perinatologia Isidro Espinosa de los Reyes, Mexico City, Mexico.
Ortega
A
A
Estrada-Gutierrez
G
G
Flores
A
A
Espejel
A
A
Hernandez-Guerrero
C
C
Vadillo-Ortega
F
F
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002614:Chelating Agents; D049033:Chorioallantoic Membrane / Q000201:enzymology*; D002850:Chromatography, Gel; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007743:Labor, Obstetric / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D053505:Matrix Metalloproteinases, Secreted / Q000032:analysis* / Q000378:metabolism; D018613:Microscopy, Confocal; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D011247:Pregnancy; D011489:Protein Denaturation; D051381:Rats; D017208:Rats, Wistar; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis / Q000378:metabolism
D002614:Chelating Agents; D009097:Multienzyme Complexes; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D004492:Edetic Acid; D053505:Matrix Metalloproteinases, Secreted; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D002118:Calcium