Detection of matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinase-2, urokinase and plasminogen activator inhibitor-1 within matrigel and growth factor-reduced matrigel basement membrane.
OBJECTIVE
Matrigel (MG) basement membrane is commonly used for in vitro studies of cellular migration, invasion and angiogenesis. It contains structural molecules such as collagen IV and laminin plus several growth factors which are diminished in growth factor-reduced Matrigel (GFR-MG). A less appreciated but important aspect of MG is the presence of matrix enzymes and their inhibitors. For relevant interpretation of data using MG/GFR-MG models, it may be necessary to know the enzymes or inhibitors contributed by these basement membranes themselves.


METHODS
Immunoblot and zymography were used to detect the presence or absence of MMP-1 and 7, tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1, TIMP-2), plasminogen activator activity and plasminogen activator inhibitor-1 (PAI-1). Growth and invasion assays using prostate cancer cells were used to assess the effects of TIMP-2 presence or absence.


RESULTS
We detected MMP-7, urokinase plasminogen activator (uPA) and PAI-1 in both Matrigels, TIMP-2 was detected only in regular Matrigel and no MMP-1 or TIMP-1 was detected in either matrix. Invasion assays comparing regular MG and GFR-MG indicated cell line variability with regard to invasion efficiency as two tested prostate cancer lines were unaffected by the MG type while one was significantly more invasive in regular MG. Growth experiments suggest that the presence of TIMP-2 in regular MG may retard growth but overall proliferation is still greater in regular MG than in GFR-MG.


CONCLUSIONS
These data provide a useful reference for interpretation of in vitro Matrigel assays.
Gillette
Karin M
KM
Center for Protease Research, North Dakota State University College of Pharmacy, Fargo, North Dakota, USA.
Forbes
Katie
K
Sehgal
Inder
I
D001485:Basement Membrane / Q000737:chemistry*; D001672:Biocompatible Materials; D003094:Collagen / Q000737:chemistry*; D004338:Drug Combinations; D006133:Growth Substances; D006801:Humans; D007797:Laminin / Q000737:chemistry*; D020782:Matrix Metalloproteinases / Q000032:analysis*; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis*; D011509:Proteoglycans / Q000737:chemistry*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis*; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis*
D001672:Biocompatible Materials; D004338:Drug Combinations; D006133:Growth Substances; D007797:Laminin; D017395:Plasminogen Activator Inhibitor 1; D011509:Proteoglycans; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; C059485:matrigel; D019716:Tissue Inhibitor of Metalloproteinase-2; D003094:Collagen; D014568:Urokinase-Type Plasminogen Activator; D020782:Matrix Metalloproteinases