Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells.
BACKGROUND AND PURPOSE
Homocysteine is an intermediate product formed during the metabolism of methionine, and is increased in cells with folate deficiency. Patients with hyperhomocysteinemia tend to develop cardiovascular disease. Here, we have examined the molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells (RASMCs).


EXPERIMENTAL APPROACH
Cultures of RASMC were challenged with homocysteine and then incubated with folate added. Changes in p21/p27, AKT and RhoA were followed by RT-PCR, Western blotting and immunocytochemistry. Transfection and anti-sense techniques were also used. Cell viability, growth and migration were measured.


KEY RESULTS
Folate up-regulated p21/p27 through a Src/ERK-dependent mechanism that accounted for its anti-proliferative effects on RASMC. Folate protected RASMC from the effects of homocysteine by reducing AKT1, focal adhesion kinase (FAK), paxillin, and p190RhoGAP activation/phosphorylation, along with cytosolic levels of p21 and p27, and increasing RhoA activation. Overexpression of AKT1, but not of AKT2, induced p21/p27 phosphorylation and increased cytosolic p21/p27 levels, as did homocysteine treatment. By contrast, and similarly to folate treatment, transfection with dominant negative (DN) AKT1 counteracted these effects. Additionally, AKT was shown to be an upstream target of FAK activation. In RASMC overexpressing constitutively active RhoA, activation of RhoA mediated the anti-migratory effects of folate. Addition of Y27632 (a RhoA inhibitor) and DNRhoA counteracted the anti-migratory effects, confirming RhoA involvement.


CONCLUSION AND IMPLICATIONS
Folate was anti-proliferative and anti-migratory in homocysteine-challenged RASMC. Mechanisms underlying folate-mediated protection against the proatherosclerotic effects of homocysteine have been delineated.
['Chou|Ying|Y|', 'Lin|Hui-Chen|HC|', 'Chen|Kuan-Chou|KC|', 'Chang|Chi-Cheng|CC|', 'Lee|Wen-Sen|WS|', 'Juan|Shu-Hui|SH|']
D000818:Animals D001011:Aorta D002465:Cell Movement D049109:Cell Proliferation D002478:Cells, Cultured D050759:Cyclin-Dependent Kinase Inhibitor p21 D005492:Folic Acid D005786:Gene Expression Regulation D006710:Homocysteine D008297:Male D032389:Myocytes, Smooth Muscle D010766:Phosphorylation D051057:Proto-Oncogene Proteins c-akt D051381:Rats D017207:Rats, Sprague-Dawley D015398:Signal Transduction D020742:rhoA GTP-Binding Protein
folate homocysteine AKT RhoA p190RhoGAP cell migration p21/cip1 p27/kip1