Homocysteine stimulates MAP kinase in bovine aortic smooth muscle cells.
BACKGROUND
Hyperhomocysteinemia is recognized as a risk factor for atherosclerotic disease. However, the mechanism of homocysteine effects on smooth muscle cell proliferation, which is a hallmark of atherosclerosis, is unknown. The object of this study was to test the effects of homocysteine on smooth muscle cell proliferation, and to examine the mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase 1 and 2, that are known to be involved in cell proliferation.


METHODS
For the proliferation study, bovine aortic smooth muscle cells (BASMC, 10, 000/well) were allowed to grow for 2 days before 2 mmol/L D,L -homocysteine was added for 2, 4, 6, and 8 days to simulate the clinical hyperhomocysteinemic condition. For the MAP kinase study, quiescent BASMC were exposed to 2 mmol/L D,L -homocysteine for 1.5, 5, 10, 20, 30, and 60 minutes, and the active forms of MAP kinase were detected with Western immunoblotting. The degree of phosphorylation of MAP kinase was determined by densitometry.


RESULTS
D,L -homocysteine stimulated BASMC proliferation by 20% by day 8. MAP kinase phosphorylation was activated as much as six fold by D,L -homocysteine, with a peak at 30 minutes. PD98059, an inhibitor of MAP kinase phosphorylation, inhibited the homocysteine-induced MAP kinase phosphorylation and attenuated the increase in BASMC proliferation.


CONCLUSIONS
These data are consistent with the hypothesis that D,L -homocysteine stimulation of BASMC proliferation involves MAP kinase activation.
Woo
D K
DK
Department of Surgery, Yale University School of Medicine, New Haven, CT 06510, USA.
Dudrick
S J
SJ
Sumpio
B E
BE
D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006710:Homocysteine / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D010766:Phosphorylation
D004791:Enzyme Inhibitors; D005419:Flavonoids; D006710:Homocysteine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one