Binding of alpha2 monoclonal antibody to human cervical tumor cell (SiHa) surface alpha2beta1 integrin modulates MMP-2 activity.
OBJECTIVE
The purpose was to study the interrelationship between cell surface integrin receptor (alpha2beta1) and matrixmetalloproteinases.


METHODS
Immunoprecipitation and cell adhesion assay were done to assay alpha2beta1 and alpha3beta1 on SiHa cell surface. Zymogram was developed to assay secreted MMP activity of cells grown in presence of alpha2 monoclonal antibody. Immunoblot was developed to assay expression of MMP-2, FAK, and p-FAK. Plasma membrane-dependent activation of MMP2 was performed by incubating pure MMP-2 with membrane-enriched fraction isolated from SiHa cells.


RESULTS
Immunoprecipitation and cell adhesion assay results confirmed the presence of alpha2beta1 receptor on SiHa cells. Zymographic analysis of serum-free media collected at different time points from SiHa cells grown on alpha2 monoclonal antibody-coated culture dishes showed the expression and activation of MMP-2 within 2-4 h, confirmed by immunoblot. Western blot of cells grown on alpha2-coated dishes for 30 min-4 h showed increased phosphorylation of FAK. Membrane-enriched fraction isolated from SiHa cells was found to specifically activate proMMP-2 to its activated forms within 30 min.


CONCLUSIONS
The experimental findings strongly indicate that SiHa cell surface alpha2beta1 regulates MMP-2 expression. Increased phosphorylation of focal adhesion kinase (FAK) strongly indicates the possible role of FAK in signaling cascade. Incubation of SiHa cell membrane fraction with pure MMP-2 strongly confirms the cell membrane-dependent activation of proMMP2.
Mitra
Aparna
A
Department of Receptor Biology and Tumor Metastasis, Chittaranjan National Cancer Institute, Kolkata 700 026, India.
Chakrabarti
Jayati
J
Banerji
Aniruddha
A
Chatterjee
Amitava
A
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D038982:Integrin alpha2beta1 / Q000276:immunology / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D038982:Integrin alpha2beta1; D019716:Tissue Inhibitor of Metalloproteinase-2; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2