Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis.

Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1.

Modulation of cell spreading and migration by pp125FAK phosphorylation.

We provide evidence for both matrix-dependent and pp60v-src tyrosine kinase-dependent modulation of cell migration via tyrosine phosphorylation of pp125FAK, a focal adhesion kinase, thought to be involved in integrin-mediated signaling. Enhanced pp125FAK tyrosine phosphorylation and cell spreading was associated with decreased migration. Cells plated on type I collagen were less spread and exhibited lower levels of pp125FAK tyrosine phosphorylation and faster migration rates compared with cells on fibronectin that were well spread, which exhibited enhanced levels of pp125FAK tyrosine phosphorylation and slower migration rates. Inside-out signaling via expression of pp60v-src or its kinase-negative mutant caused a decrease in cell migration by changing the extent of pp125FAK tyrosine phosphorylation to above or below the levels obtained with control cells plated on fibronectin. Hence, pp125FAK tyrosine phosphorylation appears to play a role in the signaling cascade pathway involved in regulation of extracellular matrix-modulated, integrin-mediated cell migration.

Expression of transforming growth factor type III receptor in vascular endothelial cells increases their responsiveness to transforming growth factor beta 2.

Bovine aortic endothelial cells (BAECs) express both type I and type II receptors for transforming growth factor beta (TGF beta). These cells respond to TGF beta 1 but are relatively refractory to another isoform of TGF beta, termed TGF beta 2. TGF beta s are thought to signal through receptor complexes composed of type I and/or type II receptors, both of which appear to be functional serine-threonine kinases. The TGF beta type III receptor, on the other hand, does not seem to have any direct signaling capacity. We have now stably transfected BAECs with the type III receptor cDNA. These cells displayed surface expression of the type III receptor protein, as determined by cross-linking with iodinated TGF beta 1 and immunoprecipitation with antibodies to the type III receptor protein. Transfected BAECs exhibit increased responsiveness to TGF beta 2 by several different criteria including an increase in plasminogen activator inhibitor-1 protein and inhibition of migration and proliferation. Thus, the type III receptor protein may play a role in presenting TGF beta 2 to the type II receptor and increase responsiveness to TGF beta 2 to a level comparable to that of TGF beta 1.