Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum.

Some secreted proteins that assemble into large complexes, such as extracellular matrices or hormones and enzymes in storage granules, must be kept at subaggregation concentrations during intracellular trafficking. We show surfeit locus protein 4 (Surf4) is the cargo receptor that establishes different steady-state concentrations for a variety of soluble cargo proteins within the endoplasmic reticulum (ER) through interaction with the amino-terminal tripeptides exposed after removal of leader sequences. We call this motif the ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif (ER-ESCAPE motif). Proteins that most readily aggregate in the ER lumen (e.g., dentin sialophosphoprotein [DSPP] and amelogenin, X-linked [AMELX]) have strong ER-ESCAPE motifs to inhibit aggregate formation, while less susceptible cargo exhibits weaker motifs. Specific changes in a single amino acid of the tripeptide result in aggregate formation and failure to efficiently traffic cargo out of the ER. A logical subset of 8,000 possible tripeptides starting a model soluble cargo protein (growth hormone) established a continuum of steady-state ER concentrations ranging from low (i.e., high affinity for receptor) to the highest concentrations associated with bulk flow-limited trafficking observed for nonbinding motifs. Human cells lacking Surf4 no longer preferentially trafficked cargo expressing strong ER-ESCAPE motifs. Reexpression of Surf4 or expression of yeast's ortholog, ER-derived vesicles protein 29 (Erv29p), rescued enhanced ER trafficking in Surf4-null cells. Hence our work describes a new way of preferentially exporting soluble cargo out of the ER that maintains proteins below the concentrations at which they form damaging aggregates.

Targeting osteopontin suppresses glioblastoma stem-like cell character and tumorigenicity in vivo.

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.

Efficient trafficking of acidic proteins out of the endoplasmic reticulum involves a conserved amino terminal IleProVal (IPV)-like tripeptide motif.

Most of the proposed extracellular biomineralization processes include the secretion of proteins that interact with mineral ions and/or mineral surfaces. Typically these proteins are acidic or have acidic domains that interact with multivalent cations in the extracellular environment. We propose that most acidic, Ca(2+)-binding proteins challenge the cell's mechanisms for trafficking through the endoplasmic reticulum (ER) lumen due to lumenal mM calcium that cause them to form large aggregates. We have recently shown that >95% of the DSPP mutations that cause non-syndromic genetic dentin diseases start their dominant negative affects by failing to rapidly exit the ER likely by forming complexes that cannot be normally trafficked to the Golgi. The complexes of mutant DSPP then capture more (severe disease) or less (mild disease) of the DSPP translated from the normal allele. After searching genomic databases as well as the published literature, we found the IleProVal (IPV)-like motif at the predicted amino terminus of many acidic proteins made in the mineralizing as well as non-mineralizing tissues of many species including vertebrates, echinoderms, mollusks, and yeast. While we often focused on acidic proteins reported associated with mineralizing structures, proteins associated with hormones and their storage/secretion, digestion, blood functions, as well as milk and other secreted fluids started with variations of the motif. Our hypothesis is that the IPV-like motif interacts with a highly conserved cargo receptor in the ER that efficiently traffics the acidic proteins out of the organelle before they can form harmful aggregates in the Ca(2+)-rich lumen.

Modulation of canonical Wnt signaling by the extracellular matrix component biglycan.

Although extracellular control of canonical Wnt signaling is crucial for tissue homeostasis, the role of the extracellular microenvironment in modulating this signaling pathway is largely unknown. In the present study, we show that a member of the small leucine-rich proteoglycan family, biglycan, enhances canonical Wnt signaling by mediating Wnt function via its core protein. Immunoprecipitation analysis revealed that biglycan interacts with both the canonical Wnt ligand Wnt3a and the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), possibly via the formation of a trimeric complex. Biglycan-deficient cells treated with exogenous Wnt3a had less Wnt3a retained in cell layers compared with WT cells. Furthermore, the Wnt-induced levels of LRP6 phosphorylation and expression of several Wnt target genes were blunted in biglycan-deficient cells. Both recombinant biglycan proteoglycan and biglycan core protein increased Wnt-induced ß-catenin/T cell-specific factor-mediated transcriptional activity, and this activity was completely inhibited by Dickkopf 1. Interestingly, recombinant biglycan was able to rescue impaired Wnt signaling caused by a previously described missense mutation in the extracellular domain of human LRP6 (R611C). Furthermore, biglycan's modulation of canonical Wnt signaling affected the functional activities of osteoprogenitor cells, including the RUNX2-mediated transcriptional activity and calcium deposition. Use of a transplant system and a fracture healing model revealed that expression of Wnt-induced secreted protein 1 was decreased in bone formed by biglycan-deficient cells, further suggesting reduced Wnt signaling in vivo. We propose that biglycan may serve as a reservoir for Wnt in the pericellular space and modulate Wnt availability for activation of the canonical Wnt pathway.

DMP1 and DSPP: evidence for duplication and convergent evolution of two SIBLING proteins.

Since first being proposed as a tandem gene family in 2001, the relatedness of the 5 SIBLING proteins (BSP, DMP1, DSPP, MEPE, and SPP1/OPN) has predominantly depended on arguments involving shared intron/exon properties as well as conserved protein biochemical properties (e.g. unstructured and acidic) and specific peptide motifs (e.g. phosphorylation and integrin-binding RGD). This report discusses the evidence that an ancient DMP1 gene underwent a simple duplication in the common ancestor of mammals and reptiles and then separately evolved into DSPP-like paralogs in the 2 classes. Genomic sequence analyses show that different copies of the original DMP1 duplication process were selected by mammalian and reptilian (anole lizard) classes to acquire genetically different but biochemically similar phosphoserine-rich repeat domains by convergent evolution. Mammals, for example, expanded phosphoserine motifs encoded exclusively using motifs containing AGC/T serine codons while the reptile line's repeats also used TCN-encoding serine codons. A similar analysis of the origins of the other 4 SIBLINGs will require even more detailed analysis as genome sequences of various fish and amphibia become available.

Secreted Frizzled-related protein-2 (sFRP2) augments canonical Wnt3a-induced signaling.

Secreted Frizzled-related proteins (sFRP) are involved in embryonic development as well as pathological conditions including bone and myocardial disorders and cancer. Because of their sequence homology with the Wnt-binding domain of Frizzled, they have generally been considered antagonists of canonical Wnt signaling. However, additional activities of various sFRPs including both synergism and mimicry of Wnt signaling as well as functions other than modulation of Wnt signaling have been reported. Using human embryonic kidney cells (HEK293A), we found that sFRP2 enhanced Wnt3a-dependent phosphorylation of LRP6 as well as both cytosolic ß-catenin levels and its nuclear translocation. While addition of recombinant sFRP2 had no activity by itself, Top/Fop luciferase reporter assays showed a dose-dependent increase of Wnt3a-mediated transcriptional activity. sFRP2 enhancement of Wnt3a signaling was abolished by treatment with the Wnt antagonist, Dickkopf-1 (DKK1). Wnt-signaling pathway qPCR arrays showed that sFRP2 enhanced the Wnt3a-mediated transcriptional up-regulation of several genes regulated by Wnt3a including its antagonists, DKK1, and Naked cuticle-1 homolog (NKD1). These results support sFRP2's role as an enhancer of Wnt/ß-catenin signaling, a result with biological impact for both normal development and diverse pathologies such as tumorigenesis.

Decorin is processed by three isoforms of bone morphogenetic protein-1 (BMP1).

The secreted small proteoglycan, decorin, modulates collagen fibril formation as well as the bioactivity of various members of the transforming growth factor-beta (TGFbeta) superfamily. Indeed, recombinant prodecorin has been used in several gene therapy experiments to inhibit unwanted fibrosis in model diseases of the kidney, heart, and other tissues although the status of the propeptide within the target tissues is unknown. Currently the protease that removes the highly conserved propeptide from decorin is unproven. Using a variety of approaches, we show that three isoforms of the Tolloid-related bone morphogenetic protein-1 (BMP1) can effectively remove the propeptide from human prodecorin resulting in the well-established mature proteoglycan. Classic BMP1, the full-length gene transcript mTLD (BMP1-3), and BMP1-5 (isoform lacking the CUB3 domain thought to be important for efficient type I collagen C-propeptidase activity) all removed the analogous propeptides from both recombinant human prodecorin and murine probiglycan. Furthermore, the timed removal of the propeptide was found to not be necessary for the addition of decorin's single glycosaminoglycan chain. Decorin therefore joins the growing list of matrix and bioactive molecules processed/activated by the BMP1/Tolloid family. Since the third member of the Class I small leucine-rich proteooglycan (SLRP) superfamily, asporin, also contains a similar cleavage motif at the appropriate location, we propose that the removal of these propeptides by members of the BMP1 family is an additional characteristic of Class I SLRP.

Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis.

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth-supporting tissues, alveolar bone, periodontal-ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM(+)), induced in vivo regeneration of all tooth-supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM(+) induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio-temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM(+).

Molecular evolution of dentin phosphoprotein among toothed and toothless animals.

BACKGROUND: Dentin sialophosphoprotein (DSPP) is the largest member of the SIBLING family and is the most abundant noncollagenous protein in dentin. DSPP is also expressed in non-mineralized tissues including metabolically active ductal epithelia and some cancers. Its function, however, is poorly defined. The carboxy-terminal fragment, dentin phosphoprotein (DPP) is encoded predominantly by a large repetitive domain that requires separate cloning/sequencing reactions and is, therefore, often incomplete in genomic databases. Comparison of DPP sequences from at least one member of each major branch in the mammalian evolutionary tree (including some "toothless" mammals) as well as one reptile and bird may help delineate its possible functions in both dentin and ductal epithelia. RESULTS: The BMP1-cleavage and translation-termination domains were sufficiently conserved to permit amplification/cloning/sequencing of most species' DPP. While the integrin-binding domain, RGD, was present in about half of species, only vestigial remnants of this tripeptide were identified in the others. The number of tandem repeats of the nominal SerSerAsp phosphorylation motif in toothed mammals (including baleen whale and platypus which lack teeth as adults), ranged from approximately 75 (elephant) to >230 (human). These repeats were not perfect, however, and patterns of intervening sequences highlight the rapidity of changes among even closely related species. Two toothless anteater species have evolved different sets of nonsense mutations shortly after their BMP1 motifs suggesting that while cleavage may be important for DSPP processing in other tissues, the DPP domain itself may be required only in dentin. The lizard DSPP had an intact BMP1 site, a remnant RGD motif, as well as a distinctly different Ser/Asp-rich domain compared to mammals. CONCLUSIONS: The DPP domain of DSPP was found to change dramatically within mammals and was lost in two truly toothless animals. The defining aspect of DPP, the long repeating phosphorylation domain, apparently undergoes frequent slip replication and recombination events that rapidly change specific patterns but not its overall biochemical character in toothed animals. Species may have to co-evolve protein processing mechanisms, however, to handle increased lengths of DSP repeats. While the RGD domain is lost in many species, some evolutionary pressure to maintain integrin binding can be observed.

The potential functional interaction of biglycan and WISP-1 in controlling differentiation and proliferation of osteogenic cells.

Biglycan (BGN) and WISP-1 are 2 extracellular matrix proteins that bind to each other and colocalize in mineralizing tissue. Here we show that WISP-1 abrogates the repression of proliferation in bone marrow stromal cells induced by BGN. We also demonstrate that WISP-1 and its variant WISP-1va can alleviate the repressed osteogenic differentiation caused by the absence of BGN. These preliminary data suggest that WISP-1 and BGN may functionally interact and control each other's activity, thus regulating the differentiation and proliferation of osteogenic cells.

A comprehensive analysis of normal variation and disease-causing mutations in the human DSPP gene.

Within nine dentin dysplasia (DD) (type II) and dentinogenesis imperfecta (type II and III) patient/families, seven have 1 of 4 net -1 deletions within the approximately 2-kb coding repeat domain of the DSPP gene while the remaining two patients have splice-site mutations. All frameshift mutations are predicted to change the highly soluble DSPP protein into proteins with long hydrophobic amino acid repeats that could interfere with processing of normal DSPP and/or other secreted matrix proteins. We propose that all previously reported missense, nonsense, and splice-site DSPP mutations (all associated with exons 2 and 3) result in dominant phenotypes due to disruption of signal peptide-processing and/or related biochemical events that also result in interference with protein processing. This would bring the currently known dominant forms of the human disease phenotype in agreement with the normal phenotype of the heterozygous null Dspp (-/+) mice. A study of 188 normal human chromosomes revealed a hypervariable DSPP repeat domain with extraordinary rates of change including 20 slip-replication indel events and 37 predominantly C-to-T transition SNPs. The most frequent transition in the primordial 9-basepair (bp) DNA repeat was a sense-strand CpG site while a CpNpG (CAG) transition was the second most frequent SNP. Bisulfite-sequencing of genomic DNA showed that the DSPP repeat can be methylated at both motifs. This suggests that, like plants and some animals, humans methylate some CpNpG sequences. Analysis of 37 haplotypes of the highly variable DSPP gene from geographically diverse people suggests it may be a useful autosomal marker in human migration studies.

Structural requirements for bone sialoprotein binding and modulation of matrix metalloproteinase-2.

Bone sialoprotein (BSP) has been shown to induce limited gelatinase activity in latent matrix metalloproteinase-2 (MMP-2) without removal of the propeptide and to restore enzymatic activity to MMP-2 previously inhibited by tissue inhibitor of matrix metalloproteinase-2 (TIMP2). The current study identifies structural domains in human BSP and MMP-2 that contribute to these interactions. The 26 amino acid domain encoded by exon 4 of BSP is shown by a series of binding and activity assays to be involved in the displacement of MMP-2's propeptide from the active site and thereby inducing the protease activity. Binding assays in conjunction with enzyme activity assays demonstrate that both amino- and carboxy-terminal domains of BSP contribute to restoration of activity to TIMP2-inhibited MMP-2, while the MMP-2 hemopexin domain is not required for reactivation.

Bone sialoprotein binding to matrix metalloproteinase-2 alters enzyme inhibition kinetics.

Bone sialoprotein (BSP) is a secreted glycophosphoprotein normally restricted in expression to skeletal tissue that is also induced by multiple neoplasms in vivo. Previous work has shown that BSP can bind to matrix metalloproteinase-2 (MMP-2). Because of MMP-2 activity in promoting tumor progression, potential therapeutic inhibitors were developed, but clinical trials have been disappointing. The effect of BSP on MMP-2 modulation by inhibitors was determined with purified components and in cell culture. Enzyme inhibition kinetics were studied using a low-molecular weight freely diffusable substrate and purified MMP-2, BSP, and natural (tissue inhibitor of matrix metalloproteinase-2) and synthetic (ilomastat and oleoyl- N-hydroxylamide) inhibitors. We determined parameters of enzyme kinetics by varying substrate concentrations at different fixed inhibitor concentrations added to MMP-2 alone, MMP-2 and BSP, or preformed MMP-2-BSP complexes and solving a general linear mixed inhibition rate equation with a global curve fitting program. Two in vitro angiogenesis model systems employing human umbilical vein endothelial cells (HUVECs) were used to follow BSP modulation of MMP-2 inhibition and tubule formation. The presence of BSP increased the competitive K I values between 15- and 47-fold for natural and synthetic inhibitors. The extent of tubule formation by HUVECs cocultured with dermal fibroblasts was reduced in the presence of inhibitors, while the addition of BSP restored vessel formation. A second HUVEC culture system demonstrated that tubule formation by cells expressing BSP could be inhibited by an activity blocking antibody against MMP-2. BSP modulation of MMP-2 activity and inhibition may define its biological role in promoting tumor progression.

TGF-beta1 and WISP-1/CCN-4 can regulate each other's activity to cooperatively control osteoblast function.

Wnt-induced secreted protein-1 (WISP-1), like other members of the CCN family, is expressed in skeletal tissues. Its mechanism of action remains unknown. Expression of WISP-1 was analyzed in human bone marrow stroma cells (hBMSC) by RT-PCR. We identified two major transcripts corresponding to those of full-length WISP-1, and of the splice variant WISP-1va which lacks a putative BMP/TGF-beta binding site. To investigate the function of WISP-1 in bone, hBMSC cultures were treated with recombinant human (rh)WISP-1 and analyzed for proliferation and osteogenic differentiation. WISP-1 treatment increased both BrdU incorporation and alkaline phosphatase (AP) activity. Considering the known functional synergy found between the TGF-beta super-family and members of the CCN family, we next tested the effect of WISP-1 on TGF-beta1 activity. We found that rhWISP-1 could reduce rhTGF-beta1 induced BrdU incorporation. Similarly, rhTGF-beta1 inhibited rhWISP-1 induction of AP activity. To explore functional differences between the WISP-1 variants, WISP-1 or WISP-1va were transfected into hBMSC. Both variants could strongly induce BrdU incorporation. However, there were no effects of either variant on AP activity without an additional osteogenic stimulus such as TGF-beta1. Taken together our results suggest a functional relationship between WISP-1 and TGF-beta1. To further define this relationship we analyzed the effect of WISP-1 on TGF-beta signaling. rhWISP-1 significantly reduced TGF-beta1 induced phosphorylation of Smad-2. Our data indicates that full-length WISP-1 and its variant WISP-1va are modulators of proliferation and osteogenic differentiation, and may be novel regulators of TGF-beta1 signaling in osteoblast-like cells.

Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer.

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

SIBLING expression patterns in duct epithelia reflect the degree of metabolic activity.

The SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of secreted glycophosphoproteins includes bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), osteopontin (OPN), and matrix extracellular phosphoglycoprotein (MEPE). For many years, they were thought in normal adults to essentially be limited to metabolically active mesenchymal cells that assembled the mineralized matrices of bones and teeth. Over the last decade they have also been upregulated in a variety of tumors. Three of these proteins (BSP, OPN, and DMP1) have been shown to interact with three matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively). Recently, all five SIBLINGs and their MMP partners when known were observed in specific elements of normal ductal epithelia in salivary gland and kidney. We have hypothesized that the SIBLINGs and their MMP partners may be expressed in ductal cells with high metabolic activity. In this paper, we show that all the SIBLINGs (except MEPE) and their MMP partners are expressed in the metabolically active epithelia of human eccrine sweat gland duct but not in the more passive ductal cells of the macaque (monkey) lacrimal gland. It is hypothesized that MEPE expression may be limited to cells involved in active phosphate transport. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Up-regulation of SIBLING proteins and correlation with cognate MMP expression in oral cancer.

Various combinations of the SIBLING family of proteins have been found to be up-regulated in many human cancers and have been linked to different stages of tumor progression, including metastasis. Bone sialoprotein (BSP), osteopontin (OPN) and dentin matrix protein 1 (DMP1) specifically bind and activate MMP-2, MMP-3, and MMP-9, respectively. These proteases have also been shown to play important roles in oral squamous cell carcinoma (OSCC) invasion and metastasis. However, with the exception of OPN, there are no reports on the expression of the family of five SIBLING proteins in OSCC. This study examines the expression patterns of the SIBLING family (and MMP partners when known) in OSCC, correlating expression to outcome variables. Archived paraffin sections of 87 cases of primary OSCC were screened by immunohistochemistry for the SIBLINGs and their MMP partners. Three SIBLINGs (BSP, DSPP, and OPN), were expressed in OSCC, while DMP1 and MEPE expression were never observed. Furthermore, BSP and OPN were always expressed with their known MMP partners, MMP-2 and MMP-3, respectively. Poorly differentiated tumors exhibited reduced or no immunoreactivity for BSP and OPN but increased immunoreactivity for DSPP. Seventy eight (90%) cases were positive for BSP and DSPP, while 79 cases (91%) were positive for OPN. Overall, 91% of the cases were positive for at least one SIBLING. There were no correlations between SIBLING expression and tumor size ("T"; of the Union Internationale Contre le Cancer [UICC]-TNM classification for OSCC), and between SIBLING expression and lymph node spread for the T1/T2 tumors. The levels of DSPP expression for floor of mouth and retromolar region tumors were higher than for tongue tumors. Statistically significant correlations were, however, found between the expression levels of BSP and MMP-2 (p<0.0001), BSP and MMP-3 (p<0.0001), and OPN and MMP-3 (p<0.0024). We conclude that BSP, DSPP, and OPN are highly up-regulated in OSCC. While the production of these SIBLINGs is independent of T, they correlate with oral location of tumor, cognate MMP expression, and for DSPP, the degree of tumor differentiation.

Dentin matrix protein 1 enhances invasion potential of colon cancer cells by bridging matrix metalloproteinase-9 to integrins and CD44.

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 +/- 13.9 versus 22.3 +/- 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, alpha(v)beta(3), or alpha(v)beta(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.

Bone sialoprotein enhances migration of bone marrow stromal cells through matrices by bridging MMP-2 to alpha(v)beta3-integrin.

UNLABELLED: BMSCs migrate through matrix barriers and differentiate into osteoblasts. BSP enhances osteogenic cell migration through basement membrane and collagen matrices in vitro by localizing MMP-2 on the cell surface through alpha(v)beta(3)-integrin. INTRODUCTION: The specific mechanisms by which bone marrow stromal cells (BMSCs) leave their primary sites, move through matrices encountered during homing to their site of final differentiation, and remove preexisting matrices in preparation for bone matrix production are not well understood. MATERIALS AND METHODS: The enhanced migration of human osteoblast precursor cells through matrix barriers by bone sialoprotein (BSP) was studied by a modified Boyden-chamber assay. The bridging of normally soluble matrix metalloproteinase 2 (MMP-2) to the cell surface receptor, alpha(v)beta(3)-integrin, by BSP was analyzed by flow cytometry. RESULTS: BSP enhanced the in vitro passage of BMSCs and pre-osteoblasts through matrix barriers (Matrigel and denatured type I collagen) in a dose-dependent manner. An intact ArgGlyAsp (RGD) was required in the BSP for enhanced migration through the barriers but was not sufficient, as shown by the inactivity of two other SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family members, osteopontin and dentin matrix protein-1. The specificity of the BSP enhancement activity was apparently caused by this molecule's ability to bridge MMP-2 to the cell surfaces. CONCLUSIONS: Pre-osteoblasts and their BMSC precursors may use MMP-2/BSP/integrin complexes to disrupt matrix barriers during migration to their final destinations in vivo.

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

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.