Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.
Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375.


RESULTS
Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay.


CONCLUSIONS
Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.
Banerji
Aniruddha
A
Department of Receptor Biology & Tumor Metastasis, Chittaranjan National Cancer Institute, Kolkata, India.
Chakraborti
Jayati
J
Mitra
Aparna
A
Chatterjee
Amitava
A
D002457:Cell Extracts / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D039302:Integrin alphaVbeta3 / Q000032:analysis; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008545:Melanoma; D008666:Metalloendopeptidases / Q000378:metabolism*
D002457:Cell Extracts; D004792:Enzyme Precursors; D039302:Integrin alphaVbeta3; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2