Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation.
BACKGROUND
A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the alphavbeta3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface.


METHODS
We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model.


RESULTS
Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-alphavbeta3 integrin interaction does not induce the expression of MT1-MMP.


CONCLUSIONS
These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.
Hwang
Queena
Q
CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Canada. queena.hwang@utoronto.ca
Cheifetz
Sela
S
Overall
Christopher M
CM
McCulloch
Christopher A
CA
Sodek
Jaro
J
D000818:Animals; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D053511:Matrix Metalloproteinase 14 / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D017077:Culture Media, Conditioned; D004792:Enzyme Precursors; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D012795:Sialoglycoproteins; D014364:Tryptophan; D020778:Matrix Metalloproteinase 2; D053511:Matrix Metalloproteinase 14