20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-b

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-b (TGF-b). Incubation of R22D cells with 20-HETE for 24 h attenuated [H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)induced [H]thymidine incorporation in R22D cells and human VSMCs. At 5 mM, 20-HETE reduced [H]thymidine incorporation by 34 6 6%; anti-TGF-b neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serumand PDGFinduced expression of cyclin D1, a downstream effector of TGF-b1, which was reversed by anti-TGF-b antibody. Further studies demonstrated that 20-HETE may increase TGF-b release to a level high enough to inhibit [H]thymidine incorporation without altering the steady-state mRNA level of TGF-b. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-b.—Liang, C-J., H. E. Ives, C-M. Yang, and Y-H. Ma. 20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-b. J. Lipid Res. 2008. 49: 66–73. Supplementary key words 20-hydroxyeicosatetraenoic acid & cytochrome P450 & platelet-derived growth factor & DNA synthesis 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid (1) that plays an important role in the regulation of vascular (2–4) and renal (5–7) function. 20-HETE may act as a potent vasoconstrictor by inhibiting Ca-activated K channels (8) and increasing the conduction of L-type Ca channels (9). In contrast, 20-HETE may be converted by cyclooxygenase to prostaglandins and act as a vasodilator (10–12). In kidney, 20-HETE induces diuresis by inhibiting Na-KATPase in proximal tubules (13) and Na/K/Cl cotransporter in the thick ascending limb of Henle’s loop (5–7). In addition, 20-HETE may regulate cell growth. It has been demonstrated that 20-HETE stimulated DNA synthesis in epithelial cells (14), mesangial cells (15), fibroblasts (16), and vascular smooth muscle cells (VSMCs) (17). In cultured VSMCs from adult rabbit (18) and rat (17) as well as in isolated renal arterioles (3), 20-HETE induces the phosphorylation of extracellular regulated protein kinase 1/2 (ERK 1/2), a mitogen-activated protein kinase that plays a pivotal role in the proliferation induced by the activation of receptor tyrosine kinases and G protein-coupled receptors (19). Blockade of the formation of 20-HETE attenuated norepinephrine-induced (18) and angiotensin II-induced (20) ERK 1/2 phosphorylation, suggesting that 20-HETE may serve as a second messenger of these growth factors. Recent studies indicate that the secretion, activity, and clearance of transforming growth factor-b (TGF-b) can be regulated by long-chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) (21, 22). EPA inhibits plateletderived growth factor (PDGF)-induced DNA synthesis by stimulating the secretion of TGF-b in mesangial cells (21). In addition, fatty acids may attenuate TGF-b clearance and thus increase TGF-b activity (22). Although fatty acids have been shown to modulate the secretion, activity, and clearance of TGF-b, it is not known whether 20-HETE may modulate TGF-b availability and subsequently modulate cell growth. In blood vessel walls, TGF-b1, the major isoform of TGF-b, is secreted in a latent, inactive form that binds to Manuscript received 30 March 2007 and in revised form 10 August 2007 and in re-revised form 3 October 2007. Published, JLR Papers in Press, October 4, 2007. DOI 10.1194/jlr.M700155-JLR200 Abbreviations: CDK, cyclin-dependent kinase; ECM, extracellular matrix; EPA, eicosapentaenoic acid; ERK 1/2, extracellular regulated protein kinase 1/2; FBS, fetal bovine serum; 20-HETE, 20-hydroxyeicosatetraenoic acid; LDH, lactate dehydrogenase; LTBP, latent TGF binding protein; MEK, MAPK/ERK kinase; MMP, matrix metalloprotease; PDGF, platelet-derived growth factor; TGF-b, transforming growth factorb; VSMC, vascular smooth muscle cell. 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright D 2008 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org 66 Journal of Lipid Research Volume 49, 2008 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom latent TGF binding protein (LTBP) with binding sites for extracellular matrix (ECM) (23). The matrix-bound latent molecule may be secreted and/or activated when LTBP is proteolytically cleaved by a protease such as plasmin or matrix metalloprotease (MMP) (23). In VSMCs, TGF-b may exert a growth-inhibitory effect by inducing cell cycle arrest at G1 phase (24), which may be attributable to the attenuation of cyclin D, cyclin E, and cyclindependent kinases (CDKs) 2, 4, and 6 (25, 26). In contrast, TGF-b may stimulate cell growth in VSMCs (27, 28). These discrepancies may be the result of differences in VSMC phenotype (27, 29) and the concentration of TGF-b in VSMCs (28). In this study, we tested the hypothesis that TGF-b may mediate the effect of 20-HETE on modulating cell proliferation in VSMCs. We demonstrated that TGF-b may mediate the inhibitory effect of 20-HETE on the growth of VSMCs from neonatal rat or human. MATERIALS AND METHODS