P277	The effect of endostatin is mediated by PP2A.
1A.Schmidt, 2D.Wenzel, 2B.K.Fleischmann, 1W.Bloch
1German Sport University Cologne, Dept. of Molecular and Cellular Sport Medicine, Cologne, DE; 2University Bonn, Institute for Physiology I, Bonn, DE.

The proteolytic fragment of collagen XVIII, endostatin is described as an anti-angiogenic and tumour inhibitory protein. Endostatin also influences signal transduction in endothelial cells and differentiation mechanisms which are important for angiogenesis as for example morphogenesis. Recently we were able to show that endostatin lead to a dephosphorylation of the MAP-kinase ERK1/2 and induces retraction of endothelial cells and vessels during wound healing as well as in endothelial tubes of in vitro differentiated embryonic stem cells. Both effects are inhibited by 8bromo-cGMP, a cell permeable derivat of cGMP. Recently it was shown that endostatin influences the NO/cGMP pathway by inhibition of endothelial NO-synthase (eNOS) activity via Protein Phosphatase 2A (PP2A). From the mentioned endostatin-mediated effects we conclude that endostatin influences the VEGF/Flk-1/NO/eNOS/sGC/cGMP/ERK signalling pathway. This rises the question, if the observed endostatin-mediated dephosphorylation of ERK1/2-kinase is influenced by PP2A. Therefore we investigated endothelial tube formation in embryonic stem cell culture (EB) in vitro and analysed the role of PP2A for endostatin-mediated effects. The PP2A inhibitor ocadaic acid is able to block endostatin-mediated dephosphorylation of ERK1/2-kinase in endothelial tubes of EBs. This shows that the endostatin effects are mediated by PP2A. To answer the question if the PP2A-induced dephosphorylation of ERK1/2 is directly mediated by cGMP, we analysed if the sGC inhibitor ODQ is also able to dephosphorylate ERK1/2-kinase. But by treating EBs with ODQ neither we observed dephosphorylation of ERK1/2 nor a decrease of sGC in endothelial tubes was found. Summarising these observations we conclude that the endostatin-mediated downregulation of cGMP by reduction of eNOS activity via PP2A can only boost the PP2A effect for dephosphorylation of ERK1/2. The PP2A-mediated dephosphorylation of ERK1/2 in endothelial cells is induced by a further pathway.

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