Matrix metalloproteinases (MMPs) are critical for development, wound healing, and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small, Integrin-Binding LIgand, N-linked Glycoprotein) family can specifically bind (Kd approximately equal nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence, increased susceptibility to plasmin cleavage, and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP-2 and active MMP-2, while osteopontin binds proMMP-3 and active MMP-3, and dentin matrix protein-1 binds proMMP-9 and active MMP-9. Both pro and active MMP-SIBLING complexes are disrupted by the abundant serum protein, complement Factor H, thereby probably limiting SIBLING-mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin alpha(v)beta3.

Previously we have shown that two members of the newly named SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins, bone sialoprotein, and osteopontin, bound first to a cell surface receptor and then to complement Factor H thereby blocking the lytic activity of the alternative pathway of complement. Another member of this family, dentin matrix protein 1, is shown in this paper to be very similar to osteopontin in that it can bind strongly to Factor H (K a ϳ 1 nM) and block the lytic activity through either the vitronectin receptor (␣ V ␤ 3 integrin) or CD44. Binding of Factor H to SIBLING localized to the cells surface was demonstrated by fluorescence-activated cell sorting. Extensive overlapping fragment analyses suggests that both dentin matrix protein 1 and osteopontin interact with cell surface CD44 through their amino termini. Similar fragments of bone sialoprotein, like the intact protein, did not functionally interact with CD44. All three proteins are shown to act in conjunction with Factor I, a serum protease that, when complexed to appropriate cofactors, stops the lytic pathway by digesting the bound C3b in a series of proteolytic steps. These results show that at least three members of this family confer membrane cofactor protein-like activity (MCP or CD46) upon cells expressing RGD-binding integrins or CD44. The required order of the assembly of the complex suggests that this cofactor activity is limited to short diffusional distances.

The up-regulation of various matrix metalloproteinases (MMP), certain cell receptors such as integrins and CD44, and the SIBLING family of integrin-binding glycophosphoproteins have been reported separately and in various combinations for many types of tumors. The mechanisms by which these different proteins may be interacting and enhancing the ability of a cancer cell to survive and metastasize have become an interesting issue in cancer biology. Dentin matrix protein 1 (DMP1) has been known for a number of years to bind to CD44 and ArgGlyAsp sequence-dependent integrins. This SIBLING was recently shown to be able to specifically bind and activate proMMP-9 and to make MMP-9 much less sensitive to inhibition by tissue inhibitors of metalloproteinases and synthetic inhibitors. In this study, we used a modified Boyden chamber assay to show that DMP1 enhanced the invasiveness of the MMP-9 expressing colon cancer cell line, SW480, through Matrigel in a dose-dependant manner. DMP1 (100 nmol/L) increased invasion 4-fold over controls (86.1 F 13.9 versus 22.3 F 9.8, P < 0.001). The enhanced invasive potential required the presence of MMP-9 and at least one of the cell surface receptors, CD44, A v B 3 , or A v B 5 integrin. The bridging of MMP-9 to the cell surface receptors was shown by both pull-down and fluorescence activated cell sorting experiments. Because all of these proteins were also shown by immunohistochemistry to be expressed in serial sections of a colon adenocarcinoma, we have hypothesized that the MMP-9/DMP1/cell surface complexes observed to enhance cell invasion in vitro may be aiding metastatic events in vivo. (Cancer Res 2005; 65(24): 11545-52)

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.