Soluble latent membrane-type 1 matrix metalloprotease secreted by human mesangial cells is activated by urokinase.
BACKGROUND
Matrix metalloprotease 2 (MMP2) is secreted in a latent inactive form (pro-MMP2) that is activated on the cell surface by a membrane-type 1 MMP (MT1-MMP) in the presence of the tissue inhibitor of MMP (TIMP2). In spite of evidence for the synthesis of MT1-MMP shown by immunoblotting, immunocytochemistry and RT-PCR, and of TIMP2, MMP2 was found exclusively in a latent form in human mesangial cells (HMC) serum-free culture medium.


RESULTS
On purified membranes of HMC, MT1-MMP was found in a 63 kD latent form and as a faint band of 55 kD. The 55 kD band was also present in the ultracentrifuged conditioned medium and likely represented MT1-MMP cleaved from its transmembrane domain, since Northern blot analysis showed only one transcription product. The addition of urokinase plasminogen activator (uPA, 100 nM) to HMC membranes induced the activation of pro-MMP2 via the activation of latent membrane-associated MT1-MMP as reflected by the cleavage of the 63 and 55 kD forms. In addition, when the conditioned medium was successively incubated with uPA and alpha 2-macroglobulin and analyzed by immunoblotting, MT1-MMP decreased, indicating that the soluble MT1-MMP was in a latent form and was activated by uPA.


CONCLUSIONS
Our results provide the first evidence, to our knowledge, of the existence of a soluble latent form of MT1-MMP secreted by primary human cells in culture, confirming that MT1-MMP is an ectoenzyme, and show that uPA can regulate MT1-MMP activity in a soluble phase.
Kazes
I
I
INSERM U 489, Hôpital Tenon, Paris, France.
Delarue
F
F
Hagège
J
J
Bouzhir-Sima
L
L
Rondeau
E
E
Sraer
J D
JD
Nguyen
G
G
D002462:Cell Membrane / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000378:metabolism; D018093:Gelatinases / Q000187:drug effects / Q000378:metabolism*; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000187:drug effects / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D012995:Solubility; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*
D017077:Culture Media, Conditioned; D004792:Enzyme Precursors; D011498:Protein Precursors; D014568:Urokinase-Type Plasminogen Activator; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase; D020778:Matrix Metalloproteinase 2