Ha-Won Jeong

RGD peptides released from beta ig-h3, a TGF-beta-induced cell-adhesive molecule, mediate apoptosis.

βig-h3 is a transforming growth factor-β (TGF-β)-induced cell-adhesive molecule and has an RGD sequence at its C-terminus. A previous report suggested that βig-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 βig-h3 may facilitate TGF-β-induced apoptosis. We found that carboxy-terminal cleavage of βig-h3 occurred after its secretion, and that overexpression of the wild-type βig-h3 induced apoptosis, unlike the C-terminal deleted but RGD-containing mutant βig-h3, which is resistant to C-terminal processing. The βig-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 βig-h3 and fibronectin, respectively, also induced apoptosis, unlike ERGEEL and GRGESP. Culture supernatants of cells overexpressing βig-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 RGD-containing C-terminal part of βig-h3 was also subjected to C-terminal cleavage and overexpression resulted in apoptosis. The anti-βig-h3 antibody blocks TGF-β-induced apoptosis. Thus, βig-h3 may be important in regulating cell apoptosis by providing soluble RGD peptides.

Regulation of Tumor Angiogenesis by Fastatin, the Fourth FAS1 Domain of βig-h3, via αvβ3 Integrin

We previously reported that the FAS1 domains of βig-h3 bear motifs that mediate endothelial cell adhesion and migration via interactions with αvβ3 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 βig-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 αvβ3 integrin, including endothelial cells. Binding of fastatin to αvβ3 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 αvβ3 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.

TGF-β1 enhances βig-h3-mediated keratinocyte cell migration through the α3β1 integrin and PI3K

βig‐h3 is an extracellular matrix (ECM) protein whose expression is highly induced by transforming growth factor beta1 (TGF‐β1). We previously demonstrated that βig‐h3 has two α3β1 integrin‐interacting motifs, which promote adhesion, migration, and proliferation of human keratinocytes. Both βig‐h3 and TGF‐β1 have been suggested to play important roles in the healing of skin wounds. In this study, we demonstrate that TGF‐β1 enhances keratinocyte adhesion and migration toward βig‐h3 through the α3β1 integrin. TGF‐β1 did not increase the amount of the α3β1 integrin on the cell surface, but rather increased its affinity for βig‐h3. LY294002, an inhibitor of PI3K, blocked the basal and TGF‐β1‐enhanced cell migration but not adhesion to βig‐h3. A constitutively active mutant of PI3K stimulated cell migration but not adhesion to βig‐h3. The PI3K pathway is also not associated with the affinity of the α3β1 integrin to βig‐h3. TGF‐β1 induced phosphorylation of AKT and FAK. Taken together, these data suggest that TGF‐β1 increases affinity of the α3β1 integrin to βig‐h3, resulting in enhanced adhesion and migration of keratinocytes toward βig‐h3. TGF‐β1 also enhances migration through PI3K, but PI3K is not associated with either the binding affinity of the α3β1 integrin or its adhesion to βig‐h3.