The tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity required for cell migration/invasion associated with cancer progression and angiogenesis. TIMPs also modulate cell proliferation in vitro and angiogenesis in vivo independent of their matrix metalloproteinase inhibitory activity. Here, we show that TIMP-2 mediates G₁ growth arrest in human endothelial cells through de novo synthesis of the cyclin-dependent kinase inhibitor p27^Kip1. TIMP-2-mediated inhibition of Cdk4 and Cdk2 activity is associated with increased binding of p27^Kip1 to these complexes in vivo. Protein-tyrosine phosphatase inhibitors or expression of a dominant negative Shp-1 mutant ablates TIMP-2 induction of p27^Kip1. Finally, angiogenic responses to fibroblast growth factor-2 and vascular endothelial growth factor-A in “motheaten viable” Shp-1-deficient mice are resistant to TIMP-2 inhibition, demonstrating that Shp-1 is an important negative regulator of angiogenesis in vivo.

TGF-β induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-β1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1α/β (HIF-1α/β). To examine the transcriptional regulation of VEGF by TGF-β1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1α/β or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-β1, whereas cotransfection of HIF-1α/β and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-β1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1α/β and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-β1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-β1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-β1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.

We investigated a possible role of reactive oxygen species (ROS) in p70^S6k activation, which plays an important role in the progression of cells from G₀/G₁ to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H₂O₂ generated extracellularly by glucose/glucose oxidase led to the activation of p70^S6k and p90^Rsk and to phosphorylation of p42^MAPK/p44^MAPK. The activation of p70^S6k and p90^Rsk was dose-dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70^S6kusing specific inhibitors for p70^S6k signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin-sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70^S6k activity. In addition, Ca²⁺ chelation also inhibited ROS-induced activation of p70^S6k, indicating that Ca²⁺ is a mediator of p70^S6k activation by ROS. However, down-regulation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70^S6kby ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70^S6k activity by H₂O₂ in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H₂O₂, phosphorylation, and activation of p70^S6k, which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70^S6k signaling pathway.