J.O.Humtsoe and S.Feng contributed equally to this workWe identi®ed vascular endothelial growth factor and type I collagen inducible protein (VCIP), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis. VCIP/PAP2b exhibits an Arg±Gly±Asp (RGD) cell adhesion sequence. Immunoprecipitation and¯uorescence-activated cell sorting analyses demonstrated that VCIP-RGD is exposed to the outside of the cell surface. Retroviral transduction of VCIP induced cell aggregation/cell± cell interactions, modestly increased p120 catenin expression and promoted activation of the Fak, Akt and GSK3b protein kinases. Furthermore, expression of recombinant VCIP promoted adhesion, spreading and tyrosine phosphorylation of Fak, Shc, Cas and paxillin in endothelial cells. GST±VCIP-RGD, but not GST±VCIP-RGE, speci®cally interacted with a subset of integrins, and these interactions were effectively blocked by anti-a v b 3 and anti-a 5 b 1 integrin antibodies, and by PAP2b/VCIP-derived peptides. Interestingly, PAP2b/VCIP is expressed in close proximity to vascular endothelial growth factor, von Willebrand factor and a v b 3 integrin in tumor vasculatures. These ®ndings demonstrate an unexpected function of PAP2b/VCIP, and represent an important step towards understanding the molecular mechanisms by which PAP2b/VCIP-induced cell±cell interactions regulate speci®c intracellular signaling pathways.

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa) n that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an ␣ 2 ␤ 1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-␣ 2 ␤ 1 and anti-␤ 1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing ␣ 2 ␤ 1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-␣ 2 ␤ 1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125 FAK of C2C12 cells expressing ␣ 2 ␤ 1 integrin, whereas parental C2C12 cells do not. Furthermore, adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125 FAK , p130 CAS , and p68 Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the ␣ 2 integrin interacts with p176 with a relatively high affinity (K D ‫؍‬ 17 nM). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.The collagens are a family of extracellular matrix (ECM) 1 proteins that provide structural support to all multicellular animals (1). The repeating sequence Gly-Xaa-Yaa (GXY), where X is often proline and Y is often hydroxyproline, is a unique feature of the collagen polypeptides (2-4). Long tracks of repeated GXY sequences fold into left-handed polyproline type II-like chains, and three such chains cooperatively twist around a central axis to form a right-handed rope-like superhelix (2, 5-7), considered a defining feature of collagens. The collagens also contain C-and N-terminal noncollagenous domains, which often are proteolyzed during secretion from the cells (7-9). Mature collagen molecules are deposited in the ECM in the form of fibers, networks, and beaded filaments (8). One group of mammalian proteins that fulfill rudimentary host defense functions such as the complement factor C1q (9) and several mammalian lectins (10) also have collagenous domains, but they are not conventional collagens. These proteins form characteristic lollipop-like structures, where the collagenous domains form the stalks, and globular heads correspond to the noncollagenous regions. Collagen-like molecules also have been found in lower eukaryotes such as mussels, worms, and sponges (11), and collagen-like sequences have been identified from analyses of the genomes of prokaryotes (12-15).The streptococcal collagen-like proteins Scl1 and Scl2 (also know...

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates ␤-catenin/lymphoid enhancer binding factor 1 (␤-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via ␤-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated ␤-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of ␤-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of ␤-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate ␤-catenin/ LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated ␤-catenin/ LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.

Evidence shows that Bcl-2 family members play a direct role in the development of some human malignancies. However, the mechanism by which Bcl-2 may influence tumor cell invasion and metastasis remains unclear. Ectopic overexpression of Bcl-2 in the human squamous carcinoma cell line HSC-3 enhanced tumorigenicity and experimental pulmonary metastasis. Interestingly, Bcl-2-expressing cells showed morphologic changes that resembled that of cells with an epithelial-mesenchymal transition phenotype. Analysis revealed increased N-cadherin and vimentin expression in parallel with attenuated E-cadherin level, along with enhanced migration and invasive behavior. Zymography studies confirmed elevated levels of matrix metalloproteinase-9 (MMP-9) in media of Bcl-2-expressing cells. siRNA-mediated suppression of N-cadherin expression not only prevented the enhanced invasion but also blocked the increased MMP-9 expression induced by elevated Bcl-2 expression. Accordingly, pharmacologic inhibition of MMP-9 abrogated the increased tumor cell invasion. Furthermore, the Bcl-2-mediated increase in MMP-9 expression and tumor cell invasion was dependent on fibroblast growth factor receptor-1 or extracellular signal-regulated kinase signaling. Collectively, the data establish that Bcl-2 overexpression in squamous carcinoma cells induces a partial epithelial to mesenchymal transition that promotes not only survival but also invasion and metastasis through the N-cadherin/fibroblast growth factor receptor/extracellular signal-regulated kinase pathway. Mol Cancer Res; 8(2); 170-82. ©2010 AACR.