doi: 10.1161/01.RES.0000259101.39931.d3
© 2007 American Heart Association, Inc.
Editorials |
Foxing Smooth Muscle Cells
FOXO3a–CYR61 Connection
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 +dl2{at}mole.bio.cam.ac.uk
See related article, pages 372–380
Key Words: FOXO3a • CYR61 • neointimal hyperplasia • vascular smooth muscle cell
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 DN-FOXO3a 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).
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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 adenovirus-CYR61 (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 Ad-CYR61 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 phosphotidylinositol-3-kinase–Akt (PI3K–Akt) pathway. The phosphorylation of the Forkhead transcription factors FKHR (FOXO1), FKHR-L1 (FOXO3a) and AFX (FOXO4) by Akt results in their being bound by 14.3.3 proteins. This results in the retention of these transcription factors in the cytoplasm and their ubiquitin-mediated degradation. Growth factor withdrawal leads to FOXO3a dephosphorylation, nuclear translocation and target gene activation. Although Akt is a major regulator of FOXO3a,12 serum/glucocorticoid-regulated kinase (SGK) and IkB kinase (IKK) also phosphorylate and inactivate FOXO3a.13,14 FOXO3a transcriptional activity is also regulated by acetylation15 and its own expression is induced by stimuli including oxidative stress.16
Although interesting, there are many questions that arise from this article. First, why does expression of TM-FOXO3a alone have little effect on basal proliferation despite repression of endogenous CYR61? It is possible that FOXO3a is only able to inhibit proliferation when expression of CYR61 exceeds basal levels, consistent with the low level of CYR61 expression seen in unstimulated VSMC. CYR61 is unlikely to be the only mechanism through which FOXO3a-mediated VSMC growth arrest is achieved. Second, it is not clear how CYR61 repression mediates growth arrest, or how it is regulated independently of FOXO3a, for example after stimulation with angiotensin II. Binding sites for several other transcription factors that affect cell proliferation are also present within the CYR61 promoter. Although the reporter construct used by Lee et al1 contained many of these sites, the deletion construct lacking the FOXO binding site might have also lost other transcription factor binding sites, so it is possible that additional factors might mediate the effect of TM-FOXO3a in an indirect fashion. The interaction of FOXO proteins with accessory proteins may mediate their activity and determine which target genes they regulate.6,17 Although the ChIP assay reported by Lee et al indicates that FOXO3a binds directly to the CYR61 promoter, evidence for direct repression by FOXO proteins is contradictory. Although FOXO1 is reported to bind directly to the cyclin D2 promoter (by ChIP assay), FOXO3a-dependent repression of cyclin D2 appears to be indirect, relying on transcriptional activation of the repressor protein, BCL6.7,18 It would be interesting to know if binding of endogenous FOXO3a to the CYR61 promoter can be detected (either by ChIP or electrophoretic mobility shift assay), and whether binding is lost following Akt activation. The fact that DN FOXO3a (in which the carboxy terminal transactivation domain is deleted) is able to reverse the repression of CYR61 expression by TM-FOXO3a, indicates that the domain responsible for gene repression might overlap with the activation domain. Clearly, precise identification of the region(s) that mediate transcriptional repression by FOXO3a will be extremely important. This region is likely to encompass residues that are responsible for the recruitment of transcriptional cofactors by FOXO3a. As induction of apoptosis by FOXO3a appears to depend on a functional transactivation domain, modified proteins in which the repression domain is incapacitated without affecting the activation domain (and vice versa) would be valuable tools in studying the relative contribution of FOXO3a-dependent VSMC apoptosis and cell cycle inhibition to neointimal growth, atherogenesis and plaque pathology.
Finally, it is not clear whether the failure of CYR61 to suppress FOXO3a-mediated apoptosis in vivo indicates that CYR61 has no effect on apoptosis. FOXO3a regulates the expression of multiple genes involved in apoptosis such as GiLZm, Fas-L, TWEAK, BIM, TRAIL, NOXA, PUMA, and FLIP. In addition FOXO3a also regulates genes that affect oxidative stress (GADD45, HSP70, iNOS) and metabolism (mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS2), human pyruvate dehydrogenase kinase-4, basal glucose-6-phosphatase catalytic subunit gene, manganese superoxide dismutase (MnSOD)), many of which indirectly regulate apoptosis. Although Lee et al1 found that CYR61 could rescue FOXO3a-induced cell detachment and loss of viability, there was no effect on the appearance of hypodiploid peaks and TUNEL positivity. These markers of apoptosis are both notoriously insensitive and dependent on kinetic processing of DNA and other markers of cell death should be examined in this context.
It is clear that the inappropriate proliferation, senescence and apoptosis of VSMC are implicated in several vascular diseases. Therefore, the elucidation of the signaling pathways that govern these processes in VSMCs and how they are altered in the diseased state is essential. The article by Lee et al1 is, therefore, an important contribution to this basic knowledge.
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Acknowledgments |
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Sources of Funding
T.D.L. and M.R.B. are supported by a British Heart Foundation Professorship (to M.R.B.).
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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 Top References |
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1. Lee HY, Chung JW, Youn SW, Kim JY, Park KW, Koo BK, Oh BH, Park YB, Chaqour B, Walsh K, Kim HS The Forkhead transcription factor, FOXO3a, is a negative regulator of angiogenic immediate early gene, CYR61, leading to inhibition of vascular smooth muscle cell proliferation and neointimal hyperplasia. Circ Res. 2007; 100: 372–380.
2. Sigala F, Georgopoulos S, Papalambros E, Chasiotis D, Vourliotakis G, Niforou A, Kotsinas A, Kavantzas N, Patsouris E, Gorgoulis VG, Bastounis E. Heregulin, cysteine rich-61 and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with clinical data. Eur J Vasc Endovasc Surg. 2006; 32: 238–245.[CrossRef][Medline] [Order article via Infotrieve]
3. Abid MR, Yano K, Guo S, Patel VI, Shrikhande G, Spokes KC, Ferran C, Aird WC. Forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia. J Biol Chem. 2005; 280: 29864–29873.
4. Park KW, Kim DH, You HJ, Sir JJ, Jeon SI, Youn SW, Yang HM, Skurk C, Park YB, Walsh K, Kim HS. Activated forkhead transcription factor inhibits neointimal hyperplasia after angioplasty through induction of p27. Arterioscler Thromb Vasc Biol. 2005; 25: 742–747.
5. Accili D, Arden KC. FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell. 2004; 117: 421–426.[CrossRef][Medline] [Order article via Infotrieve]
6. Birkenkamp KU, Coffer PJ. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem Soc Trans. 2003; 31: 292–297.[Medline] [Order article via Infotrieve]
7. Schmidt M, Fernandez de Mattos S, van der Horst A, Klompmaker R, Kops GJ, Lam EW, Burgering BM, Medema RH. Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Mol Cell Biol. 2002; 22: 7842–7852.
8. Van Der Heide LP, Hoekman MF, Smidt MP. The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem J. 2004; 380: 297–309.[CrossRef][Medline] [Order article via Infotrieve]
9. Kireeva ML, Mo FE, Yang GP, Lau LF. Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion. Mol Cell Biol. 1996; 16: 1326–1334.[Abstract]
10. Kolesnikova TV, Lau LF. Human CYR61-mediated enhancement of bFGF-induced DNA synthesis in human umbilical vein endothelial cells. Oncogene. 1998; 16: 747–754.[CrossRef][Medline] [Order article via Infotrieve]
11. Hauck L, Harms C, Grothe D, An J, Gertz K, Kronenberg G, Dietz R, Endres M, von Harsdorf R. Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes. Circ Res. 2007; 100: 50–60.
12. Plas DR, Thompson CB. Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. J Biol Chem. 2003; 278: 12361–12366.
13. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol. 2001; 21: 952–965.
14. Hu MC, Lee DF, Xia W, Golfman LS, Ou-Yang F, Yang JY, Zou Y, Bao S, Hanada N, Saso H, Kobayashi R, Hung MC. IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell. 2004; 117: 225–237.[CrossRef][Medline] [Order article via Infotrieve]
15. Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W, Bultsma Y, McBurney M, Guarente L. Mammalian SIRT1 represses forkhead transcription factors. Cell. 2004; 116: 551–563.[CrossRef][Medline] [Order article via Infotrieve]
16. Kajihara T, Jones M, Fusi L, Takano M, Feroze-Zaidi F, Pirianov G, Mehmet H, Ishihara O, Higham JM, Lam EW, Brosens JJ. Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial decidualization. Mol Endocrinol. 2006; 20: 2444–2455.
17. Birkenkamp KU, Coffer PJ. FOXO transcription factors as regulators of immune homeostasis: molecules to die for? J Immunol. 2003; 171: 1623–1629.
18. Fernandez de Mattos S, Essafi A, Soeiro I, Pietersen AM, Birkenkamp KU, Edwards CS, Martino A, Nelson BH, Francis JM, Jones MC, Brosens JJ, Coffer PJ, Lam EW. FoxO3a and BCR-ABL regulate cyclin D2 transcription through a STAT5/BCL6-dependent mechanism. Mol Cell Biol. 2004; 24: 10058–10071.
Related Article:
Circ. Res. 2007 100: 372-380.
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