Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and -independent pathways.
Endothelial expression of matrix metalloproteinase-9 (MMP-9), which degrades native type IV collagen, was implicated as a prerequisite for angiogenesis. Therefore, the aim of this study was to determine signaling requirements that regulate MMP-9 expression in endothelial cells. Both, primary and permanent human umbilical vein endothelial cells (HUVEC and ECV304, respectively) were stimulated with phorbol 12-myristate 13-acetate (PMA) and the cytokine tumor necrosis factor-(alpha) (TNF(alpha)) to induce MMP-9 expression. While both cell types responded to PMA at the protein, mRNA and promoter level by induction of MMP-9, TNF(alpha) caused this response only in ECV304. Inhibitors specific for mitogen-activated protein/ERK kinase 1/2 (MEK1/2), protein kinase C (PKC), and Ras and co-transfections of wild-type and mutant Raf were used to elucidate the signaling cascades involved. Thus, we could show that the Raf/MEK/ERK cascade is mainly responsible for MMP-9 induction in endothelial cells and that this cascade is regulated independently of PKC and Ras subsequent to TNF(alpha) stimulation and in a PKC-dependent manner as a result of PMA treatment. In addition, PMA triggers a Ras-dependent signal transduction pathway bypassing the phosphorylation of ERK. Finally, we provide evidence that sustained phosphorylation of ERK1/2 is necessary but not sufficient for expression of MMP-9.
Genersch
E
E
Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Wiltberg Strasse 50, Germany. elke.genersch@medkem.lu.se
Hayess
K
K
Neuenfeld
Y
Y
Haller
H
H
D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology; D018631:ras Proteins / Q000378:metabolism*
D002273:Carcinogens; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D020780:Matrix Metalloproteinase 9; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate