Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells

Ying Chou, Hui Chen Lin, Kuan Chou Chen, Chi Cheng Chang, Wen Sen Lee, Shu Hui Juan

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5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1447-1460
Number of pages14
JournalBritish Journal of Pharmacology
Volume169
Issue number7
DOIs
Publication statusPublished - Aug 2013

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Homocysteine
Folic Acid
Smooth Muscle Myocytes
Focal Adhesion Protein-Tyrosine Kinases
Transfection
Phosphorylation
Paxillin
Hyperhomocysteinemia
Methionine
Cell Movement
Cell Survival
Cardiovascular Diseases
Western Blotting
Immunohistochemistry
Polymerase Chain Reaction
Therapeutics
Growth

Keywords

  • AKT
  • cell migration
  • folate
  • homocysteine
  • p190RhoGAP
  • p21/cip1
  • p27/kip1
  • RhoA

ASJC Scopus subject areas

  • Pharmacology

Cite this

@article{4fbad07ce19c4649a2ea33300c100905,
title = "Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells",
abstract = "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.",
keywords = "AKT, cell migration, folate, homocysteine, p190RhoGAP, p21/cip1, p27/kip1, RhoA",
author = "Ying Chou and Lin, {Hui Chen} and Chen, {Kuan Chou} and Chang, {Chi Cheng} and Lee, {Wen Sen} and Juan, {Shu Hui}",
year = "2013",
month = "8",
doi = "10.1111/bph.12130",
language = "English",
volume = "169",
pages = "1447--1460",
journal = "British Journal of Pharmacology",
issn = "0007-1188",
publisher = "John Wiley and Sons Inc.",
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TY - JOUR

T1 - Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells

AU - Chou, Ying

AU - Lin, Hui Chen

AU - Chen, Kuan Chou

AU - Chang, Chi Cheng

AU - Lee, Wen Sen

AU - Juan, Shu Hui

PY - 2013/8

Y1 - 2013/8

N2 - 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.

AB - 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.

KW - AKT

KW - cell migration

KW - folate

KW - homocysteine

KW - p190RhoGAP

KW - p21/cip1

KW - p27/kip1

KW - RhoA

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