ABSTRACT
We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.
Subject(s)
Apolipoproteins/pharmacology , Glycoproteins/pharmacology , Membrane Transport Proteins/pharmacology , Muscle, Smooth, Vascular/drug effects , Platelet-Derived Growth Factor/pharmacology , Animals , Aorta/cytology , Apolipoproteins/biosynthesis , Apolipoproteins/genetics , Apolipoproteins/physiology , Apolipoproteins D , Becaplermin , Cattle , Cell Movement/drug effects , Cells, Cultured/cytology , Cells, Cultured/drug effects , Culture Media, Serum-Free/pharmacology , DNA, Complementary/genetics , Drug Synergism , Ductus Arteriosus/cytology , Enzyme Activation/drug effects , Fetal Blood/chemistry , Glycoproteins/genetics , Glycoproteins/physiology , Humans , MAP Kinase Signaling System/drug effects , Membrane Transport Proteins/genetics , Membrane Transport Proteins/physiology , Muscle, Smooth, Vascular/cytology , Proto-Oncogene Proteins c-sis , Pulmonary Artery/cytology , RNA, Messenger/biosynthesis , Rats , Recombinant Fusion Proteins/physiology , Sheep , Signal Transduction , Transfection , rac1 GTP-Binding Protein/physiologyABSTRACT
OBJECTIVE: Elevated apolipoprotein D (apoD) levels are associated with reduced proliferation of cancer cells. We therefore investigated whether apoD, which occurs free or associated with HDL, suppresses vascular smooth muscle cell (VSMC) proliferation, which is related to the pathobiology of disease. METHODS AND RESULTS: Intense immunoreactivity for apoD was observed in human atherosclerotic plaque but not in normal coronary artery. However, an increase in apoD mRNA was seen in quiescent relative to proliferating fetal lamb aortic VSMCs, and in the rat aortic VSMC line (A10), we demonstrated uptake of apoD from serum. Stable transfection of apoD in A10 cells in the absence of serum did not influence VSMC proliferation assessed by [3H]-thymidine incorporation. ApoD, administered at a dose of 100 ng/mL, completely inhibited basal as well as platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation (P<0.01) but had no effect on fibroblast growth factor-induced VSMC proliferation. ApoD did not suppress PDGF-BB or fibroblast growth factor-2-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 but selectively inhibited PDGF-BB-mediated ERK1/2 nuclear translocation. CONCLUSIONS: Our data suggest that apoD selectively modulates the proliferative response of VSMC to growth factors by a mechanism related to nuclear translocation of ERK1/2.
