Proliferation of Intimal Smooth Muscle Cells
2000; Elsevier BV; Volume: 275; Issue: 15 Linguagem: Inglês
10.1074/jbc.275.15.11270
ISSN1083-351X
AutoresN. Eric Olson, Jeff Kozlowski, M A Reidy,
Tópico(s)RNA Research and Splicing
ResumoBasic fibroblast growth factor (FGF2) is a potent mitogen for medial smooth muscle cells and is necessary for their proliferation after balloon catheter injury; however, intimal smooth muscle cells do not require FGF2 for their proliferation, and they respond only weakly to exogenous FGF2. The present study examined the activation of extracellular signal-regulated kinase (ERK) signaling as well as the expression and activity of cell cycle proteins in FGF2-stimulated intimal smooth muscle cells. FGF2 activates ERKs 1 and 2, and Western blot analysis showed that cyclin D, cyclin E, and cyclin-dependent kinase (CDKs) 2 and 4 were expressed in intimal smooth muscle cells after FGF2 infusion. FGF2 stimulation, however, did not lead to phosphorylation of the retinoblastoma protein (Rb), CDK 2 activation, or expression of cyclin A. Western blot analysis showed that intimal smooth muscle cells express elevated levels of the cell cycle inhibitors p15INK4b and p27Kip1, compared with medial smooth muscle cells, and that FGF2 stimulation does not reduce the level of these inhibitors. These studies suggest that despite activation of ERKs 1 and 2 and expression of the cell cycle activators, cyclin D and cyclin E, high levels of cell cycle inhibitors may inhibit cell cycle transit in FGF2-stimulated intimal smooth muscle cells. Basic fibroblast growth factor (FGF2) is a potent mitogen for medial smooth muscle cells and is necessary for their proliferation after balloon catheter injury; however, intimal smooth muscle cells do not require FGF2 for their proliferation, and they respond only weakly to exogenous FGF2. The present study examined the activation of extracellular signal-regulated kinase (ERK) signaling as well as the expression and activity of cell cycle proteins in FGF2-stimulated intimal smooth muscle cells. FGF2 activates ERKs 1 and 2, and Western blot analysis showed that cyclin D, cyclin E, and cyclin-dependent kinase (CDKs) 2 and 4 were expressed in intimal smooth muscle cells after FGF2 infusion. FGF2 stimulation, however, did not lead to phosphorylation of the retinoblastoma protein (Rb), CDK 2 activation, or expression of cyclin A. Western blot analysis showed that intimal smooth muscle cells express elevated levels of the cell cycle inhibitors p15INK4b and p27Kip1, compared with medial smooth muscle cells, and that FGF2 stimulation does not reduce the level of these inhibitors. These studies suggest that despite activation of ERKs 1 and 2 and expression of the cell cycle activators, cyclin D and cyclin E, high levels of cell cycle inhibitors may inhibit cell cycle transit in FGF2-stimulated intimal smooth muscle cells. fibroblast growth factor phosphatidylinositol cyclin-dependent kinase extracellular signal-regulated kinase retinoblastoma protein protein kinase B smooth muscle cell inhibitor of CDK4 Excessive growth of vascular smooth muscle cells is an important component in the development of atherosclerotic lesion and in restenosis. In order to study which factors control the growth of these cells, we and many others have used a model of smooth muscle cell proliferation induced by mechanical injury of the rat carotid artery (1.Clowes A.W. Reidy M.A. Clowes M.M. Lab. Invest. 1983; 49: 327-333PubMed Google Scholar, 2.Clowes A.W. Reidy M.A. Clowes M.M. Lab. Invest. 1983; 49: 208-215PubMed Google Scholar). In this model, an inflated Fogarty balloon catheter is passed into the lumen of the common carotid artery, denuding the artery of its endothelial cell lining and damaging the underlying medial smooth muscle cells. This injury results in a predictable response; within 2 days the medial smooth muscle cells begin proliferating, and after 4 days medial smooth muscle cells migrate into the intima, where they continue to proliferate for up to 2 weeks. This leads to the formation of a thickened neointima comprised primarily of smooth muscle cells and extracellular matrix and results in luminal narrowing. In this model, basic fibroblast factor (FGF2)1 has been shown to be a critical mitogen for the proliferation of medial smooth muscle cells. The addition of FGF2 significantly increases medial smooth muscle cell proliferation when administered after injury of the rat carotid artery (3.Lindner V. Lappi D.A. Baird A. Majack R.A. Reidy M.A. Circ. Res. 1991; 68: 106-113Crossref PubMed Scopus (463) Google Scholar), and medial smooth muscle cell proliferation can be significantly inhibited by neutralizing antibodies to FGF2 (4.Lindner V. Reidy M.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3739-3743Crossref PubMed Scopus (595) Google Scholar). Unlike medial smooth muscle cells, FGF2 does not seem to be involved in regulating the proliferation of the smooth muscle cells that have migrated into the intima; neutralizing antibodies to FGF2 do not inhibit intimal smooth muscle cell proliferation (5.Olson N.E. Chao S. Lindner V. Reidy M.A. Am. J. Pathol. 1992; 140: 1017-1023PubMed Google Scholar) after balloon catheter injury, and the addition of FGF2 to arteries with existing intimal lesions causes only a small increase in proliferation (3.Lindner V. Lappi D.A. Baird A. Majack R.A. Reidy M.A. Circ. Res. 1991; 68: 106-113Crossref PubMed Scopus (463) Google Scholar). These data suggest that, in contrast to medial smooth muscle cells, FGF2 is not necessary for intimal smooth muscle cell proliferation, nor is it a potent mitogen for those cells. The purpose of this study was to determine whether differences in the activation of cytoplasmic signaling pathways and/or cell cycle regulation could be responsible for this apparent attenuation of FGF2-stimulated proliferation in intimal smooth muscle cells.FGF2 signal transduction involves the activation of many different cytoplasmic signaling molecules, including the extracellular signal-regulated kinases 1 and 2 (ERKs 1 and 2) (6.Pages G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar, 7.Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochemistry. 1997; 326: 61-68Crossref Scopus (375) Google Scholar, 8.Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar, 9.Scott P.H. Belham C.M. al Hafidh J. Chilvers E.R. Peacock A.J. Gould G.W. Plevin R. Biochem. J. 1996; 318: 965-971Crossref PubMed Scopus (100) Google Scholar). Activation of the ERKs is required for FGF2-stimulated proliferation in several different cell types, and recently we have shown that the ERK signaling pathway is activated following balloon catheter denudation of the rat carotid artery and that ERK activity is required for smooth muscle cell proliferation following this injury (10.Koyama H. Olson N.E. Dastvan F. Reidy M.A. Circ. Res. 1998; 82: 713-721Crossref PubMed Scopus (122) Google Scholar). FGF2 stimulation also activates the PI 3-kinase pathway (11.Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Abstract Full Text PDF PubMed Google Scholar). Activation of this pathway is required for FGF2-stimulated proliferation in a variety of cell types including smooth muscle cells (12.Weiss R.H. Yabes A.P. Am. J. Physiol. 1996; 270: C619-C627Crossref PubMed Google Scholar), and recent data suggest that this pathway is also activated following balloon catheter injury (10.Koyama H. Olson N.E. Dastvan F. Reidy M.A. Circ. Res. 1998; 82: 713-721Crossref PubMed Scopus (122) Google Scholar).Although FGF2 stimulation requires activation of cytoplasmic signaling molecules such as the ERKs and PI 3-kinase to induce proliferation, the resultant signaling must ultimately lead to activation of the cyclin-dependent kinases (CDKs) in order for cells to progress through the G1 phase of the cell cycle and into S phase. The activity of the CDKs is regulated in part by the controlled expression of the cyclins. The cyclins associate with and activate the CDKs. Recently, it has been shown that both ERK and PI 3-kinase signaling can regulate the expression of cyclin D (7.Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochemistry. 1997; 326: 61-68Crossref Scopus (375) Google Scholar, 13.Lavoie J.N. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 14.Gille H. Downward J. J. Biol. Chem. 1999; 274: 22033-22040Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), the activating partner for CDK 4. Cyclin D-CDK 4 activation is required for the phosphorylation and inactivation of Rb (15.Peeper D.S. Bernards R. FEBS Lett. 1997; 410: 11-16Crossref PubMed Scopus (26) Google Scholar, 16.Planas-Silva M.D. Weinberg R.A. Curr. Opin. Cell Biol. 1997; 9: 768-772Crossref PubMed Scopus (206) Google Scholar). This frees the transcription factor E2F, which stimulates the expression of factors necessary for the initiation of S phase, including cyclin E (17.Ohtani K. DeGregori J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12146-12150Crossref PubMed Scopus (531) Google Scholar, 18.Schulze A. Zerfass K. Spitkovsky D. Middendorp S. Berges J. Helin K. Jansen-Durr P. Henglein B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11264-11268Crossref PubMed Scopus (319) Google Scholar, 19.Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1044) Google Scholar). Cylin E activates CDK 2, and this activity along with that of cyclin D-CDK 4 leads to the increased expression of cyclin A (18.Schulze A. Zerfass K. Spitkovsky D. Middendorp S. Berges J. Helin K. Jansen-Durr P. Henglein B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11264-11268Crossref PubMed Scopus (319) Google Scholar, 20.Chen D. Krasinski K. Sylvester A. Chen J. Nisen P.D. Andrés V. J. Clin. Invest. 1997; 99: 2334-2341Crossref PubMed Scopus (174) Google Scholar, 21.Zerfass-Thome K. Schulze A. Zwerschke W. Vogt B. Helin K. Bartek J. Henglein B. Jansen Durr P. Mol. Cell. Biol. 1997; 17: 407-415Crossref PubMed Scopus (113) Google Scholar), which is necessary for entry into S phase (22.Girard F. Strausfeld U. Fernandez A. Lamb N.J. Cell. 1991; 67: 1169-1179Abstract Full Text PDF PubMed Scopus (741) Google Scholar). Thus, for FGF2 signal transduction to result in proliferation, there must be activation of the ERK and/or PI 3-kinase signaling pathways, and this signaling must result in increased expression of cyclin D as well as activation of the cyclin D-CDK 4 complex.Although the expression of the cyclins are necessary for activation of the CDKs, they are not sufficient; there are specific CDK inhibitors capable of inhibiting the CDKs even in the presence of the cyclins (23.Polyak K. Lee M.H. Erdjument Bromage H. Koff A. Roberts J.M. Tempst P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar, 24.Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar, 25.Matsuoka S. Edwards M.C. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. Genes Dev. 1995; 9: 650-662Crossref PubMed Scopus (903) Google Scholar, 26.Harper J.W. Elledge S.J. Keyomarsi K. Dynlacht B. Tsai L.H. Zhang P. Dobrowolski S. Bai C. Connell-Crowley L. Swindell E. Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (856) Google Scholar). The CIP/KIP family of inhibitors can inhibit both the cyclin D-CDK 4 and cyclin E-CDK 2 complexes (23.Polyak K. Lee M.H. Erdjument Bromage H. Koff A. Roberts J.M. Tempst P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar), while the INK4 family of inhibitors is more specific, only inhibiting CDK 4 activity (27.Serrano M. Exp. Cell Res. 1997; 237: 7-13Crossref PubMed Scopus (269) Google Scholar, 28.Carnero A. Hannon G.J. Curr. Top. Microbiol. Immunol. 1998; 227: 43-55PubMed Google Scholar). Overexpression of these inhibitors can attenuate the proliferative response (20.Chen D. Krasinski K. Sylvester A. Chen J. Nisen P.D. Andrés V. J. Clin. Invest. 1997; 99: 2334-2341Crossref PubMed Scopus (174) Google Scholar, 29.Yang Z.Y. Simari R.D. Perkins N.D. San H. Gordon D. Nabel G.J. Nabel E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7905-7910Crossref PubMed Scopus (216) Google Scholar), while a reduction in their expression increases proliferation (30.Rivard N. L'Allemain G. Bartek J. Pouyssegur J. J. Biol. Chem. 1996; 271: 18337-18341Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 31.Shankland S.J. Pippin J. Flanagan M. Coats S.R. Nangaku M. Gordon K.L. Roberts J.M. Couser W.G. Johnson R.J. Kidney. Int. 1997; 51: 1088-1099Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Therefore, the level of these proteins could be critical in determining whether growth factor stimulation results in proliferation.Our data show that FGF2 stimulation of smooth muscle cells in established intimal lesions activates both the ERKs and PI 3-kinase and increases cyclin D expression but does not lead to phosphorylation of the retinoblastoma protein, activation of CDK 2, or increased expression of cyclin A. In these same arteries, high levels of the cyclin-dependent kinase inhibitors p27Kip1 and p15INK4b were noted, and we believe that their presence is responsible for the attenuation of FGF2-induced proliferation.DISCUSSIONFGF2 is a potent mitogen for medial smooth muscle cells and is required for their proliferation following arterial injury. In contrast, FGF2 does not seem to be required for intimal cell proliferation, since blocking antibodies to FGF2 do not inhibit intimal smooth muscle cell proliferation after injury (5.Olson N.E. Chao S. Lindner V. Reidy M.A. Am. J. Pathol. 1992; 140: 1017-1023PubMed Google Scholar, 37.Koyama H. Reidy M.A. Circ. Res. 1997; 80: 408-417Crossref PubMed Scopus (42) Google Scholar). Further, previous studies have shown that smooth muscle cells in intimal lesions respond weakly to FGF2 stimulation (3.Lindner V. Lappi D.A. Baird A. Majack R.A. Reidy M.A. Circ. Res. 1991; 68: 106-113Crossref PubMed Scopus (463) Google Scholar). This study demonstrates that there are dramatic differences in the ability of FGF2 to function as a mitogen for smooth muscle cells in a normal artery as compared with smooth muscle cells of an established intimal lesion. Although intimal smooth muscle cells respond only weakly to FGF2, they have not lost their ability to replicate, since if subjected to another balloon injury, the resultant replication can exceed 25% (37.Koyama H. Reidy M.A. Circ. Res. 1997; 80: 408-417Crossref PubMed Scopus (42) Google Scholar). These observations suggested that intimal smooth muscle cells suffer from a specific defect in FGF2 responsiveness rather than a general inability to proliferate.The observation that FGF2 stimulates ERK activation in intimal smooth muscle cells to a similar degree as in medial smooth muscle cells suggests that the ability of FGF2 to bind to its receptor and activate cytoplasmic signaling pathways is not compromised in these cells. The activation of ERK 1 and 2 is known to be an early event in the stimulation of FGF receptors by FGF (35.Meloche S. Seuwen K. Pages G. Pouysségur J. Mol. Endocrinol. 1992; 6: 845-854Crossref PubMed Google Scholar, 38.Wang J.K. Gao G. Goldfarb M. Mol. Cell. Biol. 1994; 14: 181-188Crossref PubMed Scopus (205) Google Scholar, 39.Huang J. Mohammadi M. Rodrigues G.A. Schlessinger J. J. Biol. Chem. 1995; 270: 5065-5072Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and ERK activation is required for many FGF2-mediated cellular responses including proliferation (35.Meloche S. Seuwen K. Pages G. Pouysségur J. Mol. Endocrinol. 1992; 6: 845-854Crossref PubMed Google Scholar). ERK activity increases after balloon catheter injury of the rat carotid artery and is, in fact, required for medial smooth muscle cell proliferation after balloon injury (10.Koyama H. Olson N.E. Dastvan F. Reidy M.A. Circ. Res. 1998; 82: 713-721Crossref PubMed Scopus (122) Google Scholar). Our data demonstrate that FGF2 is able to activate a cytoplasmic signaling pathway associated with smooth muscle cell proliferation in intimal smooth muscle cells and that the magnitude and duration of the activation is similar in intimal and medial smooth muscle cells. This observation is important because, although ERK activation is associated with proliferation, ERK activation does not necessarily lead to proliferation, and in some cell types ERK activation is associated with differentiation and inhibition of proliferation (40.Tombes R.M. Auer K.L. Mikkelsen R. Valerie K. Wymann M.P. Marshall C.J. McMahon M. Dent P. Biochem. J. 1998; 330: 1451-1460Crossref PubMed Scopus (167) Google Scholar). How activation of the ERK pathway can result in such diverse cellular responses is not entirely understood, but it has been suggested the duration of ERK signaling can determine the outcome of ERK activation. An example of this is that in CCL39 cells short term activation of ERKs is not sufficient to stimulate proliferation (35.Meloche S. Seuwen K. Pages G. Pouysségur J. Mol. Endocrinol. 1992; 6: 845-854Crossref PubMed Google Scholar). Sustained activation of ERK for at least 6–8 h is necessary for stimulation of proliferation in these cells. We found no significant difference in the duration of ERK activation in intimal and medial smooth muscle cells after FGF2 stimulation, with ERK being activated for up to 24 h in both cases. Despite this prolonged activation of the ERK signaling pathway, FGF2 induced only a small increase in intimal smooth muscle cell proliferation compared with medial smooth muscle cells.FGF2 is capable of activating other signaling pathways that may be necessary for cell proliferation, including the PI 3-kinase pathway (11.Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Abstract Full Text PDF PubMed Google Scholar). To evaluate the effects of FGF2 on PI 3-kinase activity, we measured the phosphorylation of PKB, a downstream target of PI 3-kinase signaling and found that FGF2 stimulated an increase in PKB phosphorylation in both medial and intimal smooth muscle cells. Thus, a difference in PI 3-kinase signaling cannot explain differences in responsiveness to FGF2 in intimal and medial smooth muscle cells. Collectively, these data would therefore suggest that the block in the mitogenic signal in FGF2-stimulated intimal smooth muscle cells is downstream of ERK and PI 3-kinase signaling.To try to identify this downstream inhibition, we examined the expression of cyclin D. Cyclin D expression is regulated by growth factors, increasing early in the G1 phase, and is critical for most proliferative signals (41.Mann D.J. Higgins T. Jones N.C. Rozengurt E. Oncogene. 1997; 14: 1759-1766Crossref PubMed Scopus (51) Google Scholar, 42.Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (405) Google Scholar, 43.Lukas J. Bartkova J. Bartek J. Mol. Cell. Biol. 1996; 16: 6917-6925Crossref PubMed Scopus (294) Google Scholar). Recently, PI 3-kinase (14.Gille H. Downward J. J. Biol. Chem. 1999; 274: 22033-22040Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) and the ERKs (7.Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochemistry. 1997; 326: 61-68Crossref Scopus (375) Google Scholar) have been found to regulate the expression of cyclin D, thus providing a direct link between growth factor-mediated signaling and initiation of the cell cycle. Our data show that cyclin D expression increases following FGF2 stimulation of both intimal and medial smooth muscle cells, suggesting that the FGF2-stimulated signaling is sufficient to increase the expression of cyclin D. This observation also demonstrates that intimal smooth muscle cells enter the cell cycle following FGF2 stimulation. We also noted that both cell types express cyclin E, CDK 4, and CDK 2; however, this appears to be constitutive, since FGF2 did not increase the expression of any of these proteins. Although intimal smooth muscle cells express cyclin D-CDK 4 and cyclin E-CDK 2 following FGF2 stimulation, the absence of cyclin A expression, Rb phosphorylation, and CDK 2 activation suggests that the cyclin-CDK complexes are not active in these cells. Active cyclin D-CDK 4 phosphorylates Rb, freeing active E2F. This, along with activation of cyclin E-CDK 2, leads to increased expression of cyclin A. These events are necessary for cells to progress through the G1 phase and into S phase. Intimal smooth muscle cells showed neither Rb phosphorylation, CDK 2 activation, nor expression of cyclin A following FGF2 stimulation. We believe that these data support the hypothesis that intimal smooth muscle cells did enter the cell cycle following FGF2 stimulation but that progression through the G1 phase into S phase was blocked due to an inability to activate CDK 4 and CDK 2.In this report, we have identified several factors that could account for the inhibition of cyclin D-CDK 4 and cyclin E-CDK 2 activity in the smooth muscle cells from arteries with established intimal lesions. In addition to association with the appropriate cyclin, CDKs require dephosphorylation of inhibitory phosphorylation sites for activation (44.Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2923) Google Scholar). The phosphatase Cdc25A is thought to be responsible for this dephosphorylation, and its activity is necessary for proliferation (45.Jinno S. Suto K. Nagata A. Igarashi M. Kanaoka Y. Nojima H. Okayama H. EMBO J. 1994; 13: 1549-1556Crossref PubMed Scopus (398) Google Scholar). Our results show that while the expression of Cdc25A increases after FGF2 stimulation of medial smooth muscle cells, it does not increase after stimulation of intimal smooth muscle cells. Further, this increased expression of Cdc25A in the medial smooth muscle cells correlates with increased activity of CDK 4 and CDK 2. One possibility, therefore, is that the low level of Cdc25A expression in FGF2-stimulated intimal smooth muscle cells contributed to the lack of CDK 4 and CDK 2 activity in these cells and hence attenuated proliferation.Specific inhibitors can also regulate the activity of CDK 4 and CDK 2. The p15INK4b family of inhibitors binds to CDK 4, preventing its association with cyclin D and thus inhibiting activation of CDK 4 (27.Serrano M. Exp. Cell Res. 1997; 237: 7-13Crossref PubMed Scopus (269) Google Scholar, 28.Carnero A. Hannon G.J. Curr. Top. Microbiol. Immunol. 1998; 227: 43-55PubMed Google Scholar). Interestingly, we found high levels of expression of p15INK4b in intimal smooth muscle cells but not in medial smooth muscle cells, which may explain the apparent lack of CDK 4 activity in intimal smooth muscle cells expressing a high level of cyclin D expression. Another family of CDK inhibitors includes p27Kip1, p21Cip, and p57Kip1. Members of this family are more promiscuous and inhibit the activity of cyclin D-CDK 4, cyclin E-CDK 2, and cyclin A-CDK 2 (23.Polyak K. Lee M.H. Erdjument Bromage H. Koff A. Roberts J.M. Tempst P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar, 24.Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar, 25.Matsuoka S. Edwards M.C. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. Genes Dev. 1995; 9: 650-662Crossref PubMed Scopus (903) Google Scholar, 26.Harper J.W. Elledge S.J. Keyomarsi K. Dynlacht B. Tsai L.H. Zhang P. Dobrowolski S. Bai C. Connell-Crowley L. Swindell E. Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (856) Google Scholar). Surprisingly, the expression of p21Cip increased in intimal smooth muscle cells as well as medial smooth muscle cells after FGF2 stimulation. This result is at first puzzling, since higher expression of this cell cycle inhibitor correlates with higher CDK activity and proliferation in medial smooth muscle cells. Although expression of p21Cip can result in inhibition of proliferation, there is now evidence that low level expression of p21Cip actually promotes CDK 4 activity. We cannot say whether the level of p21Cip expression in smooth muscle cells is inhibitory or stimulatory, but what is clear is that the expression of p21Cip in FGF2-stimulated medial smooth muscle cells correlates with increased activity of CDK 4 and CDK 2 and increased proliferation. In contrast, the level of expression of the inhibitor p27Kip1 did correlate with reduced CDK activity, with intimal smooth muscle cells expressing higher levels of p27Kip1 than medial smooth muscle cells. The inhibitor p27Kip1 is normally expressed in quiescent cells, and its expression is down-regulated upon mitogenic stimulation (31.Shankland S.J. Pippin J. Flanagan M. Coats S.R. Nangaku M. Gordon K.L. Roberts J.M. Couser W.G. Johnson R.J. Kidney. Int. 1997; 51: 1088-1099Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 41.Mann D.J. Higgins T. Jones N.C. Rozengurt E. Oncogene. 1997; 14: 1759-1766Crossref PubMed Scopus (51) Google Scholar). Overexpression of p27Kip1 has been shown to inhibit intimal lesion formation (20.Chen D. Krasinski K. Sylvester A. Chen J. Nisen P.D. Andrés V. J. Clin. Invest. 1997; 99: 2334-2341Crossref PubMed Scopus (174) Google Scholar), and blocking the expression of p27Kip1 increased the sensitivity of cultured cells to mitogenic stimulation, including FGF (31.Shankland S.J. Pippin J. Flanagan M. Coats S.R. Nangaku M. Gordon K.L. Roberts J.M. Couser W.G. Johnson R.J. Kidney. Int. 1997; 51: 1088-1099Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Our finding that arteries with established intimal lesions express high levels of two CDK inhibitors, p15INK4b and p27Kip1, might well explain the lack of CDK activity and the weak proliferative effects of FGF2 in these cells.Collectively, these data suggest that the cytoplasmic signaling elicited by FGF2, while sufficient to up-regulate cyclin D expression in intimal smooth muscle cells, is not sufficient to overcome the elevated levels of CDK inhibitors. Pertinent to these findings are data linking PI 3-kinase (46.Takuwa N. Takuwa Y. Mol. Cell. Biol. 1997; 17: 5348-5358Crossref PubMed Google Scholar, 47.Li D.M. Sun H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15406-15411Crossref PubMed Scopus (432) Google Scholar) and ERK (48.Kawada M. Yamagoe S. Murakami Y. Suzuki K. Mizuno S. Uehara Y. Oncogene. 1997; 15: 629-637Crossref PubMed Scopus (176) Google Scholar, 49.Greulich H. Erikson R.L. J. Biol. Chem. 1998; 273: 13280-13288Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) to p27Kip1regulation in some cell types. Our data, however, show that elevated p27 levels are maintained despite activation of both ERK and PI 3-kinase in intimal smooth muscle cells, suggesting that neither PI 3-kinase nor ERK signaling regulates CDK inhibitor levels in smooth muscle cells.There are several possible explanations for the increased expression of p27Kip1 in intimal smooth muscle cells. Interactions with different extracellular matrices have been shown to affect the levels of cell cycle inhibitors, and interestingly, Koyama et al.found that cultured smooth muscle cells grown on polymerized type 1 collagen were less responsive to the mitogenic effects of platelet-derived growth factor (50.Koyama H. Raines E.W. Bornfeldt K.E. Roberts J.M. Ross R. Cell. 1996; 87: 1069-1078Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). This reduced responsiveness was attributed to increased levels of cell cycle inhibitors in cells grown on polymerized collagen (50.Koyama H. Raines E.W. Bornfeldt K.E. Roberts J.M. Ross R. Cell. 1996; 87: 1069-1078Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). We cannot confirm that changes in extracellular matrix are responsible for the increased expression of p27Kip1, but increased expression of several extracellular matrix components, including type 1 collagen, has been demonstrated after arterial injury (51.Nikkari S.T. Jarvelainen H.T. Wight T.N. Ferguson M. Clowes A.W. Am. J. Pathol. 1994; 144: 1348-1356PubMed Google Scholar). Related to this observation are data showing that integrins can also affect p27Kip1 expression; Murgia et al. have found proliferation defects associated with increased expression of p27Kip1 in mice carrying a targeted deletion of the β4-integrin cytoplasmic domain (52.Murgia C. Blaikie P. Kim N. Dans M. Petrie H.T. Giancotti F.G. EMBO J. 1998; 17: 3940-3951Crossref PubMed Scopus (135) Google Scholar). Differences in the extracellular matrix and/or integrin expression may be responsible for changes in the level of p27Kip1 expression and thus may provide a mechanism by which smooth muscle cells can modulate their response to growth factors.Although the expression of p15INK4b, p27Kip1, and Cdc25A in intimal smooth muscle cells may be regulated by different factors, one factor, TGF-β, could be responsible for regulating the expression of all of these proteins. TGF-β has been shown to increase the expression of p27Kip1 (31.Shankland S.J. Pippin J. Flanagan M. Coats S.R. Nangaku M. Gordon K.L. Roberts J.M. Couser W.G. Johnson R.J. Kidney. Int. 1997; 51: 1088-1099Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 53.Bouchard C. Fridman W.H. Sautes C. J. Immunol. 1997; 159: 4155-4164PubMed Google Scholar) and is well known as an inhibitor of proliferation in many cell types, including smooth muscle cells (54.Owens G.K. Geisterfer A.A. Yang Y.W. Komoriya A. J. Cell Biol. 1988; 107: 771-780Crossref PubMed Scopus (234) Google Scholar). In addition to p27Kip1, TGF-β has been shown to also regulate the expression of p15INK4b and Cdc25A (55.Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1882) Google Scholar, 56.Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 18
Referência(s)