(Circulation. 2001;103:2879.)
© 2001 American Heart Association, Inc.
Editorial |
Cell Cycle and Cell Migration
New Pieces to the Puzzle
From the Vascular Biology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.
Correspondence to Elizabeth G. Nabel, MD, NHLBI/NIH, Bldg 10/8C 103, Bethesda, MD 20892. E-mail enabel{at}nih.gov
Key Words: Editorials • cell cycle • cyclins
Formation of a healed arterial wound is a landmark event during the repair of vascular lesions. Adult coronary, peripheral, and cerebral arteries are exposed to multiple stresses, including mechanical trauma, endothelial denudation, damage to intimal and medial smooth muscle, abnormal shear stresses, and oxidation of LDL by macrophages, and they must undergo repair. This wound repair process, like in many other organs, requires that a complex network of molecular signals be regulated within the cytoplasm and nucleus of an endothelial or smooth muscle cell. The cell cycle is a key regulator of these signals and has direct effects on multiple cell processes, including cell proliferation, DNA repair, apoptosis, and cell migration.
Vascular smooth muscle cells (VSMCs) within the media of
adult arteries are normally quiescent, proliferate at low indices, and
exist in the G0 phase of the cell cycle
(Figure 1
). On stimulation by growth factors or after
mechanical trauma, VSMCs exit the quiescent stage
G0 and progress through the
G1 and G1/S transition of
the cell cycle. G1 progression is regulated by
the assembly and phosphorylation of
G1 cyclin/cyclin-dependent kinase (CDK)
complexes, predominantly cyclin D/CDK-4,6 and cyclin E/CDK-2 in VSMCs.
For example, cyclin D is a growth factor sensor whose transcription
initiation, assembly, and nuclear transport is mitogen-dependent.
Endogenous inhibitors of the cyclin/CDK
complexes are termed the CDK inhibitors
(CKIs).1 Two groups of CKIs
are dominant in cardiovascular biology: the
CIP/KIP family, which includes
p21Cip1, p27Kip1,
and p57Kip2, and the INK4 family, which
includes p15Ink4b,
p16Ink4a,
p18Ink4c, and
p19Ink4d. Interestingly, the CIP/KIP
proteins are important for growth regulation in many cell types,
whereas the INK4 proteins are linked to developmental processes and
cell transformation.
|
The G1 cyclin/CDK and CKI complexes are active within the nucleus and serve essential functions of growth regulation, DNA damage and repair, and apoptosis. The seminal event of the cell cycle is the transition from the G1 into the S phase, where DNA replication occurs, thus leading to mitosis or cell division. The G1/S phase is a tightly regulated series of molecular checks and balances. Cyclin/CDK complexes, on activation by CDK-activating kinase, phosphorylate the retinoblastoma gene product; a hyperphosphorylated retinoblastoma gene product releases several sequestered proteins, including the transcription factor E2F. After the withdrawal of growth factors or under quiescent conditions, cyclin D levels decrease, and free p27Kip1 effectively blocks the kinase activity of cyclin E/CDK-2 and arrests cells in G0. Although many proteins regulating the G1/S phase transition have been characterized, the full details of the transition, including the discovery of novel genes and their function, have yet to be elucidated.
A commonly accepted model of the response to arterial injury suggests that growth factors and other endogenous, biologically active agents are released after injury, thereby changing the composition of the extracellular matrix and triggering a proliferation and migration program.2 VSMCs initially proliferate in the media, migrate from the media into the intima, undergo further rounds of proliferation, and subsequently form the neointima. Growth factor regulation of VSMC proliferation through different signal transduction pathways has been well described. Processes that initiate VSMC migration are also well understood.3 A central question in vascular biology has been whether cell cycle control and cell migration are linked. Whether the cell cycle regulates cell migration and the mechanisms by which it does so have not been determined or characterized. Mitogenic stimulation of VSMCs initiates cell cycle progression and cell migration. However, cells in the late S or G2/M phase do not migrate.4 There is a window of opportunity in the G1 to G1/S transition where VSMCs are able to migrate in response to mitogenic stimuli. Therefore, proteins like the CKIs that regulate G1 cell cycle progression are likely candidates to regulate the initiation of migration. p27Kip1 is one important candidate, given that it is tightly regulated by mitogens.5
The study by Sun et al6 in the present issue of Circulation presents important new data regarding a role for p27Kip1 in regulating VSMC migration, and as such, adds new pieces to the puzzle of the diverse functions of the cell cycle in cardiovascular biology. Previous work by this group and others has demonstrated that rapamycin inhibits VSMC proliferation in vitro and in vivo by blocking the cell cycle at the G1/S transition7 8 and, furthermore, that rapamycin treatment of VSMCs inhibited platelet-derived growth factor–induced migration in a Boyden chamber.9 Rapamycin is an antibiotic that prevents growth factor–dependent downregulation of p27Kip1 through inhibition of the serine/threonine kinase p70S6K, a mitogen-stimulated kinase.10 Rapamycin’s antiproliferative properties are dependent, in part, on p27Kip1; thus,p27Kip1 is a likely candidate for mediating rapamycin’s inhibitory effects on cell migration as well.7 11
Sun et al6 demonstrate that rapamycin inhibits the migration of mouse VSMCs containing p27Kip1 (p27Kip+/+ and p27Kip1+/–), but not mouse VSMCs lacking p27Kip1 (p27Kip1–/–) in 2 different models: a Boyden chamber that measures the migration of VSMCs in vitro and an aortic explant method that indicates the migration of VSMCs in vivo. Interestingly, the inhibition of cell migration by rapamycin was dose-dependent in both models. At lower doses of rapamycin (IC50, 2 nmol/L in vitro, 4 mg · kg–1 · d–1 in vivo), p27Kip1-dependent effects were observed. However, at higher doses of rapamycin (IC50, 200 nmol/L in vitro, 9 mg · kg–1 · d–1 in vivo), inhibition of cell migration was observed in the presence and absence of p27Kip1, indicating a p27Kip1-independent mechanism as well. This observation was confirmed by examining p27Kip1-dependent and -independent migration in the presence of a C3 exoenzyme. The C3 exoenzyme inhibits Ras/RhoA proteins, which are steps in the signal transduction pathway from growth factor receptors to actin-myosin assembly and cell migration, and increases p27Kip1 levels (see Figure 6 in the article by Sun et al6 ). Again, a C3 exoenzyme inhibited the migration of p27Kip1+/+ but not p27Kip1–/– VSMCs, suggesting that both rapamycin and C3 exoenzyme inhibit VSMC migration through p27Kip1-dependent and -independent pathways.
Does p27Kip1 regulate VSMC migration? This intriguing question raises the issue of whether p27Kip1 indirectly regulates VSMC migration by blocking G1/S transition via an inhibition of cyclin/CDK activity. Or does p27Kip1 target a novel signal transduction pathway of cyclin/CDK activity? Or does p27 target a novel signal transduction pathway independent of its cell cycle regulatory function? The authors imply the latter; however, the experimental settings they employ do not permit distinction between these 2 possibilities. Although the experiments are performed with synchronized VSMCs that exit quiescent G0 stage after growth factor stimulation, it should be noted that pretreatment of cells with rapamycin will differentially alter p27Kip1+/+ and p27Kip1–/– VSMCs. That is, pretreatment with rapamycin increases p27Kip1 levels in wild-type p27Kip1+/+ cells but not in p27Kip1–/– cells. This induction of p27Kip1 slows G1/S progression in wild-type p27Kip+/+ cells relative to p27Kip–/– cells and might account, in part, for differences in migration after rapamycin treatment, because wild-type p27Kip+/+ and p27Kip1–/– cells would be in different phases of the cell cycle.
However, the major point to note is that signal transduction pathways downstream of p27Kip1 link cell cycle progression and cell migration. The signal transduction pathways upstream of p27Kip1 are only partially understood. VSMCs in the media of arteries are surrounded by different matrix components, such as type I and type III fibrillar collagen. Degradation of the matrix after vascular injury induces changes in actin-myosin assembly and focal adhesion kinases. Koyama et al12 reported that polymerized type1 collagen fibrils, which are found in intact arteries, upregulate p27Kip1 through integrin signaling and inhibition of p70S6k, whereas monomeric collagen fibrils support VSMC proliferation. These finding suggest that extracellular cues, such as matrix composition, can signal the nucleus, altering the cell cycle. Further studies are required to determine the mechanism of signaling from the cell surface in toward the nucleus and, in turn, from the nucleus to the cell surface that coordinately regulates VSMC migration. For example, cell migration requires cytoskeletal reorganization mediated by mitogen-activated protein kinases and small GTPases.13 Cell migration also requires focal adhesions that involve activation of the focal adhesion kinases.14 These events take place in the cytoplasm, and there is recent intriguing evidence to suggest that early after growth factor induction, p27Kip1 is shuttled out of the nucleus into the cytoplasm without further degradation, where it may carry out diverse functions. These hypotheses require testing in future studies.
The study by Sun et al6 has tremendous clinical application. A major limitation of stent therapy is the development of a cellular response within the stent (ie, in-stent restenosis). Several promising approaches are in clinical investigation, including radiation. However, the development of a therapeutic modality that would reduce the potential toxicities of radiation would have great clinical appeal. Stents coated with antiproliferative and/or antimigratory agents, like sirolimus, are undergoing experimental clinical testing, and early results look very promising.15 In addition to rapamycin, paclitaxel-coated stents also show considerable promise.16 Paclitaxel disrupts microtubules within the cytoskeleton, also preventing cell migration.17 We will eagerly await the results of phase II and III trials of both agents. In the future, one could imagine stent technology progressing from protein coatings to plasmid DNA coatings that might permit longer regulated expression of antiproliferative and/or antimigratory genes.
We now recognize that cell cycle proteins regulate multiple, diverse cellular functions in the cardiovascular system. These are proliferation, DNA damage and repair, and apoptosis; now, with the findings of Sun et al, migration can be added to the list. Studies such as the one by Sun et al6 open up broad new areas for research into the molecular regulation of cell migration by signaling pathways "outward in" and "inward out." Importantly for our patients, the contributions of Sun et al6 and others suggest dynamic new therapeutic applications for agents that modulate the cell cycle and its diverse functions for a variety of vascular diseases.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
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