Foxing Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) are essential for the structural integrity and contractile responses of the arterial vessel wall. VSMC proliferation and survival are also implicated in vascular disease including restenosis following angioplasty or stenting, and VSMC apoptosis is an important regulator of plaque stability. Thus, unraveling the molecular mechanisms that govern the survival, proliferation and migration of VSMCs is of great clinical importance. In this issue of Circulation Research Lee et al1 have added another piece to this jigsaw puzzle. They show that the rapid increase in expression of the secreted cysteine-rich angiogenic protein CYR61 following stimulation of VSMCs with either serum growth factors or angiotensin II in vitro, or after mechanical arterial injury in vivo, can be attenuated by FOXO3a. Previous studies have shown that CYR61 is highly expressed in human atherosclerotic plaques, correlating with the degree of stenosis and plaque histopathology.2 Lee et al1 investigated how expression of CYR61 is regulated. The CYR61 promoter contains multiple binding sites for different transcriptional regulators. In particular, Lee et al1 identified a site for Forkhead/winged helix box gene, group O (FOXO) family of transcription factors. Because previous studies had indicated that FOXO3a inhibits neointimal hyperplasia by promoting cell cycle arrest and apoptosis,3,4 Lee et al1 investigated whether FOXO3a influenced the expression of CYR61. They demonstrate that FOXO3a functions as a transcriptional repressor of CYR61. Because the serine/threonine kinase Akt/PKB phosphorylates and inhibits FOXO3a activity, Lee et al1 used a modified allele that contains mutated Akt phosphorylation sites (TM-FOXO3a). Adenovirus-mediated expression of this constitutively active TM-FOXO3a protein in rat VSMCs represses both basal CYR61 expression and expression induced by serum or angiotensin II. This repression seems to be a direct effect because FOXO3a is detected at the CYR61 promoter by chromatin immunoprecipitation (ChIP). Moreover, a reporter assay shows that deletion of the FOXO binding site in the CYR61 promoter abrogates the repression of CYR61 expression by TM-FOXO3a. The kinetics of phosphorylation of endogenous FOXO3a following serum stimulation are consistent with the expression of CYR61. In agreement with this, the authors also show that a dominant negative Akt protein (DN Akt) that is unable to phosphorylate and inactivate endogenous FOXO3a reduces CYR61 expression induced by serum or angiotensin II stimulation. Predictably coexpression of DNFOXO3a restores CYR61 expression. These data indicate that FOXO3a-dependent repression of CYR61 is regulated by Akt. Because the activity of Akt itself is governed by growth factors, this delineates a pathway whereby the expression of CYR61 can be regulated by extracellular signals (see Figure). The FOXO family of transcription factors are involved in the regulation of cell proliferation, migration, differentiation and death.5–8 Because these authors had previously demonstrated that FOXO3a induces apoptosis and cell cycle arrest in VSMC4 they investigated whether either of these properties is mediated by CYR61. Previous reports9,10 show that CYR61 synergizes with other growth factors to promote proliferation and, consistent with this, Lee et al1 demonstrate increased proliferation in VSMC transduced with adenovirusCYR61 (Ad-CYR61). However, in experiments reported in supplemental Table I TM-FOXO3a has little or no effect on VSMC proliferation compared with Ad-GFP control cells, despite a reduction in endogenous CYR61. Although coexpression of TM-FOXO3a partially inhibits the increased proliferation driven by Ad-CYR61, it is difficult to determine the mechanism. TM-FOXO3a is unlikely to repress the adenovirus promoter used to express CYR61. It is also possible that FOXO3a’s ability to inhibit the Ad-CYR61 driven increase in proliferation is related to transcriptional regulation of other cell cycle control genes such as p27,3,4 p2111 or D-type cyclins.7 Lee et al1 also demonstrate that supernatant from AdCYR61 cells (containing secreted CYR61) promotes VSMC migration consistent with reports in endothelial cells.9 VSMC cultures incubated with supernatant from Ad-TM-FOXO3a cells show reduced migration. However, supernatant from cells coexpressing Ad-CYR61 and Ad-TM-FOXO3a promotes migration equivalent to the control. This suggests that FOXO3a-dependent reduction in migration is mediated by repression of CYR61. Whether repression of CYR61 is involved in the induction of apoptosis that occurs as a result of FOXO3a expression is not clear. Whereas CYR61 expression has little effect on TM-FOXO3a–induced apoptosis in vivo, it appears to inhibit FOXO3a-induced cellular detachment and reduced viability in vitro without affecting the proportion of cells in the sub-G1 (presumably apoptotic) population. The study by Lee et al1 adds another link to the pathway leading from growth factors to VSMC proliferation (Figure). Growth factor stimulation results in activation of the phosThe opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Division of Cardiovascular Medicine (T.D.L., M.R.B.), Addenbrooke’s Hospital, Cambridge, UK. Correspondence to Trevor D. Littlewood, the Division of Cardiovascular Medicine, Box 110, ACCI, Addenbrooke’s Hospital Cambridge, CB2 2QQ. E-mail [email protected] (Circ Res. 2007;100:302-304.) © 2007 American Heart Association, Inc.