The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis.
Angiogenesis, the formation of new blood vessels, is a critical step for tumor growth and metastasis and an integral component of the pathologic inflammatory response in arthritis and the proliferative retinopathies. The CD13/aminopeptidase N (CD13/APN) metalloprotease is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. Here, we show that cytokine induction of CD13/APN in endothelial cells is regulated by distinct Ras effector pathways involving Ras/mitogen-activated protein kinase (MAPK) or PI-3K. Signals transduced by activated Ras, Raf, and mitogen-induced extracellular kinase (MEK) stimulate transcription from the CD13/APN proximal promoter. Inhibition of these pathways and extracellular signal-regulated serine/threonine kinase (ERK-2) and PI-3K by expression of dominant-negative proteins or chemical inhibitors prevented induction of CD13/APN transcription in response to basic fibroblast growth factor (bFGF). We show that Ras-induced signal transduction is required for growth factor-induced angiogenesis, because inhibition of downstream mediators of Ras signaling (MEK or PI-3K) abrogated endothelial cell migration, invasion, and morphogenesis in vitro. Reintroduction of CD13/APN, a shared downstream target of these pathways, overrode the suppressive effect of these inhibitors and restored the function of endothelial cells in migration/invasion and capillary morphogenesis assays. Similarly, inhibition of MEK abrogated cell invasion and the formation of endothelial-lined capillaries in vivo, which was effectively rescued by addition of exogenous CD13/APN protein. These studies provide strong evidence that CD13/APN is an important target of Ras signaling in angiogenesis and is a limiting factor in angiogenic progression.
Bhagwat
Shripad V
SV
Departments of Pathology, and Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN, USA.
Petrovic
Nenad
N
Okamoto
Yasuhiro
Y
Shapiro
Linda H
LH
D000818:Animals; D018826:CD13 Antigens / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002196:Capillaries / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology*; D004790:Enzyme Induction / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005799:Genes, Dominant; D011905:Genes, ras; D006390:Hemangioendothelioma / Q000473:pathology; D006801:Humans; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D009024:Morphogenesis; D009389:Neovascularization, Pathologic / Q000201:enzymology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D018826:CD13 Antigens; D016222:Fibroblast Growth Factor 2; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D016283:Proto-Oncogene Proteins p21(ras); D011993:Recombinant Fusion Proteins; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; C501009:HRAS protein, human