title: Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan.
authors: Ying
authors: Y
authors: Chou
authors: Hui-Chen
authors: HC
authors: Lin
authors: Kuan-Chou
authors: KC
authors: Chen
authors: Chi-Cheng
authors: CC
authors: Chang
authors: Wen-Sen
authors: WS
authors: Lee
authors: Shu-Hui
authors: SH
authors: Juan
mesh_terms: D000818:Animals; D001011:Aorta / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects* / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D005492:Folic Acid / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006710:Homocysteine / Q000378:metabolism*; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D006710:Homocysteine; D005492:Folic Acid; D051057:Proto-Oncogene Proteins c-akt; D020742:rhoA GTP-Binding Protein
keywords: AKT; RhoA; cell migration; folate; homocysteine; p190RhoGAP; p21/cip1; p27/kip1