P38 MAPK dependent pathway is responsible for N-LDL and Ox-LDL Cox-2 induction in human endothelial cells

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

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Roles of calcium and kinases in regulation of thrombin-stimulated preproendothelin-1 transcription

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.5.h1918 ◽

1996 ◽

Vol 271 (5) ◽

pp. H1918-H1925 ◽

Cited By ~ 2

Author(s):

T. A. Marsen ◽

M. S. Simonson ◽

M. J. Dunn

Keyword(s):

Endothelial Cells ◽

Inhibitory Effect ◽

Ionophore A23187 ◽

Tetraacetic Acid ◽

Human Endothelial Cells ◽

Mrna Induction ◽

Significant Inhibitory Effect ◽

Calmodulin Kinase ◽

Dependent Signal ◽

Dependent Pathway

The release of the vasoactive peptide endothelin-1 (ET-1) is Ca2+ dependent after thrombin stimulation; however, little is known about the pathways involved. We studied the importance of Ca(2+)-dependent signal transduction pathways on preproET-1 mRNA induction in human endothelial cells. Thrombin-mediated preproET-1 mRNA induction was inhibited after clamping of cytosolic free CA2+ concentration ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Chelation of extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also had a significant inhibitory effect on the induction of preproET-1 mRNA. The Ca2+ ionophore A23187 induced constitutive as well as thrombin-stimulated preproET-1 mRNA expression. Mobilization of Ca2+ stores into the cytosol by inhibition of endoplasmic reticulum Ca(2+)-adenosinetriphosphatase with thapsigargin was effective also in inducing preproET-1 mRNA. Calmodulin antagonists W-7 and calmidazolium, as well as Ca2+/calmodulin-dependent kinase II inhibitor KN-62, significantly reduced thrombin-induced preproET-1 mRNA. Inhibition by cyclosporin A of the Ca(2+)-calmodulin-dependent phosphatase calcineurin potentiated constitutive preproET-1 mRNA. These data suggest that, in human endothelial cells, thrombin-mediated preproET-1 gene induction is regulated by a stimulatory Ca2+/calmodulin kinase II-dependent pathway.

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