Culture of human A375 melanoma cells in the presence of fibronectin causes expression of MMP-9 and activation of MMP-2 in culture supernatants.
Interactions between tumor cell surface integrin receptors and extracellular matrix (ECM) ligands play an important role in tumor development, affecting cell survival, proliferation, and migration. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen-activated protein kinase (MAPK) pathways. It has been reported that integrins also regulate expression and function of matrix metalloproteinases (MMPs). In this present work, we cultured human A375 melanoma cells in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity.


METHODS
A375 cells were cultured in serum-free culture medium (SFCM) in the presence of fibronectin (25 microg/0.75 ml), SFCM was collected and gelatin zymography was performed. Western blot and RT-PCR were performed with A375 cells cultured in the presence of fibronectin.


RESULTS
Culture of A375 cells in the presence of fibronectin led to expression of MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) or PI-3K inhibitor (LY294002) and grown in the presence of fibronectin, MMP-9 expression and MMP-2 activation was inhibited. Tyrosine phosphorylation of FAK and ERK were increased in A375 cells grown in the presence of fibronectin. Increased MMP-9 mRNA expression and processing of MT1-MMP were also observed in A375 cells grown in the presence of fibronectin.


CONCLUSIONS
Our findings indicate culture of A375 cells in SFCM in the presence of fibronectin perhaps generates a signaling cascade that leads to expression of MMP-9 and activation of MMP-2 in culture supernatants within 2 h. The signaling pathway activated is probably the FAK/ERK pathway.
Department of Receptor Biology and Tumor Metastasis, Chittaranjan National Cancer Institute, 37 S.P. Mukherjee Road, Kolkata-700 026, India.
Aniruddha
A
Banerji
Shamik
S
Das
Amitava
A
Chatterjee
D016895:Culture Media, Serum-Free; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005353:Fibronectins / Q000494:pharmacology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism*; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured
D016895:Culture Media, Serum-Free; D005353:Fibronectins; D051416:Focal Adhesion Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9