␤ig-h3 is a TGF-␤-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that all four of the fas-1 domains in ␤ig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking monoclonal antibody specific for the ␣v␤5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell adhesion motif (denoted the YH motif) is located in amino acids 548 -614. Experiments with substitution mutants showed that tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved amino acids could effectively block MRC-5 cell adhesion to ␤ig-h3. Using HEK293 cells stably transfected with the ␤5 integrin cDNA, we confirmed that the ␣v␤5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new ␣v␤5 integrininteracting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing proteins may perform their biological functions by interacting with integrins. ␤ig-h3 is an extracellular matrix protein whose expression in several cell types, including fibroblasts, is strongly induced by TGF-␤.1 The gene encoding ␤ig-h3 was first identified by Skonier et al. (1), who isolated it by screening a cDNA library made from a human lung adenocarcinoma cell line (A549) that had been treated with TGF-␤. The ␤ig-h3 protein comprises 683 amino acids containing four homologous internal repeat domains. These domains are homologous to similar motifs in the Drosophila protein fasciclin-I and thus are denoted fas-1 domains. The fas-1 domain has highly conserved sequences found in secretory and membrane proteins of several species, including mammals, insects, sea urchins, plants, yeast, and bacteria (2).Mutations in ␤ig-h3 have been shown to be responsible for 5q31-linked human autosomal dominant corneal dystrophies. It has a fibrillar structure and interacts with several extracellular matrix proteins such as fibronectin and collagen (3). In addition, ␤ig-h3 has been reported to be involved in cell growth and differentiation, wound healing, and cell adhesion (4 -9). ␤ig-h3 mediates the adhesion of many different cell types, including corneal epithelial cells, chondrocytes, and fibroblasts (8 -10). We reported previously that ␤ig-h3 mediates corneal epithelial cell adhesion by binding to ␣3␤1 integrin. Two motifs interacting with the ␣3␤1 integrin were located within the second and the fourth fas-1 domains of ␤ig-h3. Interestingly, however, we found that these two motifs are not involved in ␤ig-h3-mediated fibroblastic cell adhesion. Furthermore, all four fas-1 domains of ␤ig-h3 mediate fibroblastic cell adhesion, whereas corneal epithelial cell adhesion is supported by just the second and the fourth fas-1 domains. This suggests that ␤ig-h3 has additional motifs that can mediate the ...

␤ig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether ␤ig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only ␤ig-h3 itself but also all four fas-1 domains of ␤ig-h3 mediate endothelial cell adhesion and migration through interaction with the ␣ v ␤ 3 integrin. We found that the ␣ v ␤ 3 integrin-interacting motif of the four fas-1 domains of ␤ig-h3 is the same YH motif that we reported previously to interact with ␣ v ␤ 5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that ␤ig-h3 bears ␣ v ␤ 3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.The ␤ig-h3 is an extracellular matrix protein whose expression is induced by transforming growth factor-␤ in several cell types (1). The ␤ig-h3 protein is composed of 683 amino acids containing four homologous internal repeat domains. These domains are homologous to similar motifs in the Drosophila protein fasciclin-I and thus are denoted fas-1 domains. The fas-1 domain has highly conserved sequences found in the secretory and membrane proteins of many organisms, including mammals, insects, sea urchins, plants, yeast, and bacteria. The fas-1 domain interacts with other matrix proteins such as fibronectin, collagen, and laminin (2) and mediates cell adhesion and migration through interaction with integrins (3, 4). We have reported previously that it bears motifs interacting with the integrins ␣ 3 ␤ 1 (3) and ␣ v ␤ 5 (4). The fas-1 domain is also known to be involved in cell growth, differentiation, tumorigenesis, wound healing, and apoptosis (5-8).Although ␤ig-h3 mediates the adhesion of many different cell types, including corneal epithelial cells, chondrocytes, and fibroblasts, it is not known whether ␤ig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that ␤ig-h3 mediates endothelial cell adhesion and migration through ␣ v ␤ 3 integrin and that the responsible motif is the YH motif, which has been reported to interact with the ␣ v ␤ 5 integrin. In addition, we show that the YH peptide inhibits endothelial tube formation and reduces the number of blood vessels in a Matrigel plug assay. Collectively, the YH motif of the fas-1 domains of the ␤ig-h3 protein interacts with ␣ v ␤ 3 integrin and is an effective inhibitor of angiogenesis. Our data suggest that the peptide fragment containing the YH motif could be a drug candidate for the treatment of diseases dependent on angiogenesis. EXPERIMENTAL PROCEDURESDNA Constructs and Synthetic Peptides-Bacterial expression vectors for wild-type ␤ig-h3, each fa...

big-h3 is a transforming growth factor-b (TGF-b)-induced cell-adhesive molecule and has an RGD sequence at its Cterminus. A previous report suggested that big-h3 normally undergoes carboxy-terminal processing that results in the loss of the RGD sequence. RGD peptides appear to play various roles in cell function. Here we show that the RGD peptides released from big-h3 may facilitate TGF-b-induced apoptosis. We found that carboxy-terminal cleavage of big-h3 occurred after its secretion, and that overexpression of the wild-type big-h3 induced apoptosis, unlike the C-terminal deleted but RGDcontaining mutant big-h3, which is resistant to C-terminal processing. The big-h3-induced apoptosis was abolished by either deletion of the RGD sequence or mutation of RGD to RAE. Synthetic peptides of ERGDEL and GRGDSP derived from big-h3 and fibronectin, respectively, also induced apoptosis, unlike ERGEEL and GRGESP. Culture supernatants of cells overexpressing big-h3 filtered to isolate molecules smaller than 3 kDa also induced apoptosis. A fusion protein composed of the N-terminal 100 amino acids of fibronectin and the RGDcontaining C-terminal part of big-h3 was also subjected to C-terminal cleavage and overexpression resulted in apoptosis. The anti-big-h3 antibody blocks TGF-binduced apoptosis. Thus, big-h3 may be important in regulating cell apoptosis by providing soluble RGD peptides.

We previously reported that the FAS1 domains of Big-h3 bear motifs that mediate endothelial cell adhesion and migration via interactions with Av B3 integrin and regulate angiogenesis. In the present study, we show that the fourth FAS1 domain, designated fastatin, inhibits endothelial adhesion and migration, not only to Big-h3, but also fibronectin and vitronectin, in a RGD-dependent manner. Fastatin and other FAS1 domains suppress endothelial cell tube formation and in vivo neovascularization in a Matrigel plug assay. The antiangiogenic activity of fastatin is associated with antitumor activity in mouse tumor models. Fastatin additionally induces apoptosis in several cells expressing Av B3 integrin, including endothelial cells. Binding of fastatin to Av B3 integrin inhibits phosphorylation of focal adhesion kinase, Raf, extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin. Fastatin is thus the first endogenous angiogenesis regulator identified that inhibits both endothelial cell migration and growth by binding to AvB3 integrin. Our data suggest that FAS1 domains from all possible forms of the four human FAS1 family proteins are potential endogenous regulators for pathologic angiogenesis. Moreover, FAS1 domains such as fastatin may be developed into drugs for blocking tumor angiogenesis. (Cancer Res 2005; 65(10): 4153-61)

betaig-h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF-beta1). We previously demonstrated that betaig-h3 has two alpha3beta1 integrin-interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both betaig-h3 and TGF-beta1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF-beta1 enhances keratinocyte adhesion and migration toward betaig-h3 through the alpha3beta1 integrin. TGF-beta1 did not increase the amount of the alpha3beta1 integrin on the cell surface, but rather increased its affinity for betaig-h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF-beta1-enhanced cell migration but not adhesion to betaig-h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to betaig-h3. The PI3K pathway is also not associated with the affinity of the alpha3beta1 integrin to betaig-h3. TGF-beta1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF-beta1 increases affinity of the alpha3beta1 integrin to betaig-h3, resulting in enhanced adhesion and migration of keratinocytes toward betaig-h3. TGF-beta1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the alpha3beta1 integrin or its adhesion to betaig-h3.