ABSTRACT
Previously, our in vivo studies demonstrated that folic acid (FA) could inhibit angiogenesis and in vitro studies showed that FA reduced vascular endothelial cell proliferation through activating the cSrc/ERK-2/NFκB/p53 pathway mediated by FA receptor (FR). Here, we further examined the effect of FA on endothelial cell migration. Our results showed that FA (10 µM) inhibited the formation of lamellipodia, migration and capillary-like tube formation of human umbilical venous endothelial cells (HUVEC). These inhibition effects induced by FA treatment were not due to reduction of cell survival and cell adhesion on the collagen-coated plate. Treatment of HUVEC with FA (10 µM) increased the activity of cSrc and p190RhoGAP and decreased the activity of RhoA. Over-expression of the constitutively active RhoA construct (RhoA V14) prevented the FA-induced inhibition of migration and capillary-like tube formation in HUVEC. However, these preventive effects were abolished by pretreatment of HUVEC with a ROCK inhibitor, Y27632. Pretreatment with a cSrc inhibitor, PP2, prevented the FA-induced activation of p190GAP, reduction of the RhoA activity and migration inhibition in HUVEC. Moreover, pre-transfection with p190RhoGAP siRNA abolished the FA-induced reduction in the RhoA activity and migration inhibition in HUVEC. Taken together, our results suggest that FA might inhibit endothelial cell migration through inhibiting the RhoA activity mediated by activating the FR/cSrc/p190RhoGAP-signaling pathway. These findings further support the anti-angiogenic activity of FA.
Subject(s)
Endothelial Cells/drug effects , Folic Acid Transporters/metabolism , Folic Acid/pharmacology , Gene Expression Regulation/drug effects , Genes, src/physiology , Guanine Nucleotide Exchange Factors/metabolism , Repressor Proteins/metabolism , Cell Adhesion , Cell Movement/drug effects , Cells, Cultured , Endothelial Cells/physiology , Folic Acid Transporters/genetics , Genes, src/genetics , Guanine Nucleotide Exchange Factors/genetics , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/physiology , Humans , Pseudopodia/drug effects , Pseudopodia/metabolism , Repressor Proteins/genetics , Signal Transduction , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
We previously showed that progesterone (P4) inhibits the proliferation of rat aortic smooth muscle cells (RASMC). Here, we further demonstrate that P4 at physiologic levels (5-500 nM) concentration-dependently inhibited migration of cultured RASMC. The effect is blocked by pretreatment with progesterone receptor (PR) antagonist, RU486. The P4-induced RASMC migration inhibition was through RhoA inactivation induced by cSrc-enhanced RhoA degradation. The P4-induced increases of phosphorylated Src (pSrc) and PR-pSrc complex in RASMC were observed mainly in the membrane fraction. Pre-treatment with a cSrc inhibitor (PP2) or cSrc antisense oligonucleotides prevented the P4-induced decreases of the protein levels of RhoA, phosphorylated FAK (p-FAK) and paxillin phosphorylaton and migration inhibition in RASMC. These findings expend our knowledge of the basis of P4's effect on vascular smooth muscle cell migration and highlight novel pathways of signaling transduction of P4 through PR-mediated nongenomic mechanisms.