Culture of human fibrosarcoma HT-1080 cells in presence of fibronectin activates MMP-2.
The importance of tumor cell surface integrin receptors in regulation of matrix metalloproteinase (MMP) expression and function has been reported. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase (MAPK) pathways. In this present study, we cultured human fibrosarcoma cells, HT-1080, in presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity.


METHODS
HT-1080 cells were cultured in serum free medium (SFCM) in presence of fibronectin, SFCM was collected, and gelatin zymography was performed. Western blot and immunocytochemistry were performed with HT-1080 cells cultured in presence of fibronectin.


RESULTS
Culture of HT-1080 cells in presence of 50 microg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. When HT-1080 cells were treated with PI-3K inhibitor (LY294002) and grown in presence of fibronectin, MMP-2 activation was partially inhibited, but when cells were treated with ERK inhibitor (PD98059) and grown in presence of fibronectin, MMP-2 activation was almost completely inhibited. Tyrosine phosphorylation of FAK and ERK were increased in HT-1080 cells grown in presence of fibronectin. Processing of MT1-MMP was also observed in HT-1080 cells grown in presence of fibronectin. The reorganization of actin filaments in fibronectin treated HT-1080 cells was also noticeable.


CONCLUSIONS
Our findings indicate that culture of HT-1080 cells in SFCM in presence of fibronectin perhaps generates a signaling cascade that leads to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. The signaling pathway activated seems to be the FAK/ERK pathway.
Chakrabarti
Jayati
J
Department of Receptor Biology and Tumor Metastasis, Chittaranjan National Cancer Institute, 37 S.P. Mukherjee Road, Kolkata-700 026, India.
Mitra
Aparna
A
Banerji
Aniruddha
A
Chatterjee
Amitava
A
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000032:analysis; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D003470:Culture Media; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism / Q000502:physiology; D005353:Fibronectins / Q000494:pharmacology*; D005354:Fibrosarcoma / Q000201:enzymology*; D005419:Flavonoids / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism / Q000502:physiology; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D009025:Morpholines / Q000494:pharmacology; D015398:Signal Transduction
D000199:Actins; D002867:Chromones; D003470:Culture Media; D005353:Fibronectins; D005419:Flavonoids; D039081:Integrin alpha5beta1; D061965:Matrix Metalloproteinase Inhibitors; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D048049:Extracellular Signal-Regulated MAP Kinases; D020778:Matrix Metalloproteinase 2; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one