P70S6K MODULATES THE ANGIOGENIC EFFECTS OF ANGIOPOIETIN-1 Zongxian Cao*, and Bing-Hua Jiang Mary Babb Randolph Cancer Center, Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA *
[email protected] INTRODUCTION. Angiopoietin-1 (Ang1) is an endothelial specific growth factor, which is critical for vessel maturation and stabilization during angiogenesis (1). We found that Ang1 markedly induces the activation of the serine/threonine kinase p70S6K in human umbilical vein endothelial cells (HUVECs). p70S6K has emerged as an important regulator of cell cycle progression and cell growth (2). However, little is known about the role of p70S6K in angiogenesis. In this study, we genetically modified p70S6K activity in HUVECs using adenoviral vector-mediated gene transfer, and examined the role of p70S6K in Ang1-induced endothelial cell migration, invasion, survival, and tube formation. METHOD. Recombinant adenoviruses expressing a constitutively active p70S6K (p70S6K-CA) or a dominant-negative p70S6K (p70S6K-KD) were generated by using the AdEasy system (3). Alterations of p70S6K activity in the cells were determined by in vitro p70S6K kinase assay. Actin filaments were stained with rhodamine-phalloidin and visualized by confocal microscopy. Migration of HUVECs was assayed using a 6.5 mm Transwell® device (Corning Incorporated). In vitro invasion assay was performed using BD Biocoat™ Matrigel invasion chamber (BD Biosciences). MMP2 secretion and activation were assayed by gelatin zymography. Cell viability was determined by trypan blue dye exclusion. Endothelial tube formation assay was performed in 96-well plates precoated with growth factor-reduced Matrigel (BD Biosciences). RESULTS. Ang1 stimulates p70S6K activation in HUVECs in a PI3K and MAPK-dependent manner. The cells transduced with p70S6K-KD exhibit remarkable morphological changes, characterized by disruption of stress fiber formation, cell rounding, and loss of cell polarity and motile structures. Overexpression of p70S6K-KD completely blocks Ang1-induced cell migration,
whereas expression of p70S6K-CA enhances Ang1-induced cell migation. Ang1 treatment significantly increases cell invasion through the Matrigel. Inhibition of p70S6K activity markedly reduces cell invasion induced by Ang1. Correlated with its effect on cell invasion, p70S6K also regulates Ang1-induced MMP-2 secretion and activation. The transduction of 70S6K-KD completely abrogates the effect of Ang1 on cell viability, whereas p70S6K-CA enhances Ang1-mediated cell survival. The effect of p70S6K on cell survival is further corroborated by its modulation of PARP cleavage in the cells. Strikingly, p70S6K-KD completely inhibits Ang1-induced endothelial tube formation. In contrast, cells tranduced with 70S6K-CA form more tube structures in the presensce or absence of Ang1. DISCUSSION. This study examined the regulation and functional significance of p70S6K in endothelial cells (ECs) in response to Ang1 stimulation. We provide first evidence that p70S6K regulates Ang1-induced angiogenic responses of EC, including cell migration, invasion, survival, and cappillary morphogenesis, which are critical cellular events for angiogenesis. Notably, modulation of p70S6K activity in ECs has significant effects on basal EC behavior in addition to the effects on Ang1-induced cellular responses. Furthermore, Ang1 induces p70S6K activation through PI3K and MAPK signaling pathways. These two signaling cascades, which are critical regulators of the angiogenic phenotypes in ECs, can aslo be activated by many other angiogenic factors, such as bFGF and VEGF. Therefore, our data suggest that p70S6K may function at a converging point of multiple signaling pathways to modulate EC responses to angiogenic stimulation. ACKNOWLEDGEMENT. This work was supported by National Cancer Institute Grant R01CA109460, and by American Cancer Society Research Scholar Grant 04-076-01-TBE (to B.H. Jiang). REFERENCES 1. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. (1996) Cell. 87, 1171-1180 2. Thomas G. (2002) Biol. Res. 35, 305-313 3. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. (1998) PNAS. 95, 2509-2514
