High threshold of ß1 integrin inhibition required to block collagen I-induced membrane type-1 matrix metalloproteinase (MT1-MMP) activation of matrix metalloproteinase 2 (MMP-2).

ABSTRACT

BACKGROUND: Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding ß1 integrins in collagen-stimulated MMP-2 activation. METHODS: Three ß1 integrin siRNAs were used to elucidate the involvement of ß1 integrins in the Col I-induced MMP-2 activation mechanism. ß1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of ß1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography. RESULTS: All three ß1 integrin siRNAs were efficient at ß1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of ß1, but not αV, which is not. All three ß1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of ß1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped ß1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the ß1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by ß1 integrin siRNA knockdown. CONCLUSION: Together, the data reveals that strong abrogation of ß1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of ß1 integrin.