Download Regulation of the endothelial cell cycle by the ubiquitin

SPOTLIGHT REVIEW
Cardiovascular Research (2010) 85, 272–280
doi:10.1093/cvr/cvp244
Regulation of the endothelial cell cycle
by the ubiquitin-proteasome system
Pasquale Fasanaro, Maurizio C. Capogrossi, and Fabio Martelli*
Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, Rome, Italy
Received 21 April 2009; revised 17 June 2009; accepted 8 July 2009; online publish-ahead-of-print 17 July 2009
Time for primary review: 30 days
Degradation of poly-ubiquitinated proteins by the 26S-proteasome complex represents a crucial quantitative control mechanism. The
ubiquitin-proteasome system (UPS) plays a pivotal role in the complex molecular network regulating the progression both between and
within each cell-cycle phase. Two major complexes are involved: the SKP1-CUL1-F-box-protein complex (SCF) and the anaphase-promoting
complex/cyclosome (APC/C). Notwithstanding structural similarities, SCF and APC/C display different cellular functions and mechanisms of
action. SCF modulates all cell-cycle stages and plays a prominent role at G1/S transition mainly through three regulatory subunits: Skp2,
Fbw7, and b-TRCP. APC/C, regulated by Cdc20 or Cdh1 subunits, has a crucial role in mitosis. In this review, we will describe how the
endothelial cell cycle is regulated by the UPS. We will illustrate the principal SCF- and APC/C-dependent molecular mechanisms that modulate cell growth, allowing a unidirectional cell-cycle progression. Then, we will focus our attention on UPS modulation by oxidative stress, a
pathogenic stimulus that causes endothelial dysfunction and is involved in numerous cardiovascular diseases.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Cell cycle † Endothelium † Oxidative stress † Proteasome † Ubiquitin
----------------------------------------------------------------------------------------------------------------------------------------------------------This article is part of the Spotlight Issue on: The Role of the Ubiquitin-Proteasome Pathway in Cardiovascular Disease
1. Introduction
The post-translational regulation of the cell cycle is predominantly
achieved by two types of protein modifications—phosphorylation
and ubiquitination, for a qualitative and a quantitative control,
respectively. Indeed, ubiquitination regulates multiple aspects of
the cell cycle in all eukaryotes: it controls the progression
between and within the phases, through the checkpoints and coordinates the growth of the cell with the surrounding environment.
The cyclin-dependent kinase family members (CDKs) are crucial
for the progression through each phase of the cell cycle. The activity
of the CDKs is dependent on the availability of a cyclin partner and
can be negatively regulated by the expression of specific CDK inhibitors (CKIs).1 Cyclin levels are finely modulated by the ubiquitinproteasome system (UPS), allowing the unidirectionality of the
cell-cycle progression. The UPS also controls CKI levels, yielding
both quantitative and qualitative regulation of the CDK activity.
Transcription factors are another target, allowing a less direct, but
pivotal modulation of cell proliferation. Given the role of the UPS
in the regulation of cell-cycle progression and arrest, it is not surprising that a deregulation of this system is involved in the pathogenesis
of many cardiovascular and non-cardiovascular diseases.2 In this
review, we will focus on endothelium. First, we will describe the
principal UPS-dependent molecular mechanisms that modulate cellcycle progression. In many circumstances, these mechanisms have
not been studied in endothelial cells, specifically. However, since
they have been observed in many different cell systems, they are
likely present in endothelial cells as well. Then, we will describe
the UPS-dependent modulation of the cell cycle by oxidative
stress that induces endothelial dysfunction and plays a causal role
in numerous cardiovascular diseases.
2. The UPS molecular machinery
Protein degradation by the UPS can be divided in two phases: (i)
covalent attachment of ubiquitin (Ub), an 8 kDa protein, to the
substrate protein, and (ii) degradation of the ubiquitinated
protein by the 26S-proteasome complex (see references 3–5
for extensive reviews on proteasome structure and on the ubiquitination machinery). Three enzyme families are involved in protein
ubiquitination: E1, E2, and E3. One Ub moiety is first loaded on the
active-site cysteine of E1 enzymes. Charging with Ub induces a
conformational change in E1, allowing the recruitment of one
* Corresponding author. Tel: þ39 06 6646 2431/33/34, Fax: þ39 06 6646 2430, Email: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009. For permissions please email: [email protected].
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Ubiquitin-proteasome system and cell-cycle regulation
conjugating enzyme E2 that receives the activated Ub. The specificity of the ubiquitination process is achieved by about 1000 E3
Ub-protein ligases. These enzymes are categorized into two
major classes, HECT or RING, on the basis of their catalytic
domain. HECT-domain-E3s are charged with Ub by an E2
enzyme and then they transfer Ub to their substrate; in contrast,
RING-domain-E3s allow the direct transfer of the Ub moiety
from E2 to the target protein. Substrates can be modified with a
single Ub or with Ub chains, but only poly-ubiquitination addresses
proteins for degradation by the 26S-proteasome. In fact, monoubiquitination rather modulates growth factor endocytosis,
PCNA activity during DNA-repair, and is involved in chromatin
modification during the S phase.6 Once covalently linked to its
protein target, a Ub can accept another Ub in seven lysine residues, yielding different chains that induce different events, according to the lysine residue preferentially used. Lysine-48-linked chains
are a canonical signal for degradation.7 They are recognized by the
26S-proteasome complex, a compartimentalized protease that
unfolds and degrades ubiquitinated proteins in an ATP-dependent
manner. The proteasome is composed by a 20S core and several
regulatory components. The 26S-proteasome is abundant and ubiquitously expressed within eukaryotic cells, both in the nucleus
and in the cytoplasm, showing an intracellular distribution dependent on cell type and differentiation stage. Before the degradation
of the substrate, the covalent bond between Ub and the protein is
hydrolysed by a de-ubiquitinating enzyme (DUB) resident in the
26S-proteasome, allowing the release of the Ub moieties and the
omeostasis of the Ub cell pool.8 Lysine-11-linked chains are the
product of a specific class of ubiquitination complexes (anaphasepromoting complex/cyclosome—APC/C, see what follows) and
trigger proteasomal degradation as well.9 Conversely, lysine-63linked chains are not recognized by the 26S-proteasome and
rather have a regulatory role, i.e. in DNA damage response10 or
in lysosomal targeting.11 The roles of poly-ubiquitin chains linked
through K6, K11, K27, K29, or K33 are less well understood.
However, quantitative proteomics in yeast show that these linkages are abundant in vivo and that they all target proteins for
degradation with only partially redundant functions.12 Besides
ubiquitination, proteins could also be modified by the small
Ub-like modifier SUMO.13 SUMOylation is reversible and
regulates key components of the cell cycle, such as the mitotic
checkpoint.14
In this review, we will focus on two major classes of RING-E3
Ub ligases that are essential in cell-cycle regulation and were
best characterized: the SKP1-CUL1-F-box-protein complex (SCF)
and the APC/C (extensively reviewed in references 15,16).
These complexes display a similar structure: they are composed
of three invariable subunits—a catalytic RING protein, a scaffold
protein and an adaptor protein—and a variable component, an
F-box protein for SCF and the activators Cdh1 and Cdc20 for
APC/C, which confer substrate specificity (Figure 1). The APC/C
complex also includes at least 11 other components, still poorly
characterized.17 Despite the structural similarities, these complexes have both different mechanisms of action and different cellular functions. SCF activity is involved in all cell-cycle stages and
plays a prominent role in G1/S transition, whereas APC/C regulates the progression through mitosis and G1.
Figure 1 Structural similarities between SCF and APC/C E3
Ub-ligase complexes. Both complexes are composed of a catalytic RING protein (blue), a scaffold protein (red), and an adaptor
protein (green). Variable components (yellow) give substrate
specificity to the complexes: about 70 F-box proteins have
been identified in humans, whereas APC/C is modulated by
Cdh1 or Cdc20.
2.1 The SCF E3-ligase
The SCF complex is always active and its action is dependent on
post-translational modifications of the substrates, such as phosphorylation. Once a substrate is phosphorylated, it is recognized
by a specific F-box protein and addressed to ubiquitination. To
date, about 70 F-box proteins have been identified in humans
and three of them, Skp2, Fbw7, and b-TRCP, have a relevant
role in cell-cycle regulation (Figure 2; reviewed in reference 18).
Skp2 F-box protein allows the SCF complex to target many key
regulators of the cell cycle (Figure 2A), such as Cyclin-D1, Cyclin-E,
p130Rb2, E2F1 as well as the CKIs p27kip1 (p27), p21Waf1/Cip1/Sdi1
(p21), and p57kip2 (p57). Skp2-deficient mice show elevated
levels of p27 protein,19 suggesting that p27 is a primary target of
Skp2. In keeping with this notion, the expression of Skp2 is inversely related to p27 expression during the differentiation of human
embryonic stem cells20 and in many human tumours.16 It was
recently discovered that Akt-mediated phosphorylation of Skp2
promotes its accumulation in the cytoplasm and increases SCF
complex formation,21,22 establishing Skp2 as a relevant link
between PI3K-Akt signalling and cell-cycle progression.
Fbw7 is an F-box protein that allows the degradation of crucial
cell-cycle regulators such as c-myc, Jun, Cyclin-E, and Notch
(Figure 2B). Mice lacking Fbw7 die at embryonic day 10.5,
showing a combination of defects in vascular and haematopoietic
development and heart chamber maturation.23,24 Fbw7 is involved
in mammalian vascular development via Notch activity:
Fbw7-deficient embryos display increased Notch protein levels
and a consequent deregulation of the transcriptional repressor
Hey1, key factor of cardiovascular development.
b-TRCP has emerged as a pivotal factor in the control of two of
the main CDK regulators, the kinase Wee125 and the phosphatase
Cdc25,26 that down- and up-modulate CDK activity, respectively
(Figure 2C). Degradation of Cdc25 is an integral part of the S-phase
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P. Fasanaro et al.
Figure 2 Selection of SCF and APC/C substrates. The approximate E3 time of activity during the cell cycle is indicated by grey shadows. Skp2
(A), Fbw7 (B), and b-TRCP (C) modulate SCF activity. Cdc20 (D) and Cdh1 (E) modulate APC/C activity.
checkpoint: in response to DNA damage or stalled DNA replication,
DNA damage sensors, such as ATR-Chk1, phosphorylate Cdc25 at
several residues, allowing the recognition by b-TRCP, followed by
Cdc25 degradation and the consequent inhibition of the CDKs. Furthermore, b-TRCP-null fibroblasts display defects in the regulation
of cell division, showing polyploidy, centrosome over-duplication,
impaired progression through mitosis, and reduced growth rate.27
These effects are likely due to b-TRCP-mediated regulation of the
APC/C inhibitor Emi1 that ensures a proper activation of the APC/
C complex in mitosis.28 Another relevant substrate of b-TRCP is
b-catenin: indeed, stabilization of b-catenin by mutation of the phosphorylation sites recognized by SCFb-TRCP de-regulates the cell cycle
and induces cancer.29
2.2 The APC/C E3-ligase
Unlike SCF targets, usually, APC/C substrates are not modified,
whereas the APC/C complex activity is regulated during cell-cycle
progression.30 APC/C is activated by phosphorylation during
mitosis31 and is down-modulated in late G1, by auto-degradation
of its specific E2.32 The activators Cdh1 and Cdc20 can also be
inhibited or degraded, allowing a fine-tuning of the APC/C activity
through the cell cycle. Coherently with its mitotic activity, APC/C
localizes on the centrosome and the spindle during mitosis, i.e. in
hotspots with an elevated concentration of APC/C substrates.33
Cdc20 is active from anaphase to late mitosis (Figure 2D), when
it is replaced by Cdh1, which allows the targeting of different substrates. Cdc20 is a crucial component of the spindle checkpoint: it
is necessary for Securin degradation, an essential event for chromosomal separation.34 Furthermore, Cdc20 targets the mitotic
cyclins during anaphase and regulates the stability of the kinase
Nek2A, implicated in regulating centrosome structure at G2/M
transition.35 During G2 phase, Cdc20 is bound and inhibited by
members of the mitotic checkpoint complex, such as Mad2 and
BubR1,36 and by the DUB USP44.37 Once all kinetochores are correctly attached to the spindle, Cdc20 is released and APC/CCdc20 is
consequently activated. The lack of attachment between the
spindle and the kinetochores or the absence of a correct tension
between sister chromatid microtubules activates the spindle
checkpoint that maintains Cdc20 inhibition and allows the correction of erroneous chromosome attachments.38
Cdh1 activity is regulated by phosphorylation, whereas its
protein level remains constant through the cell cycle. Cdh1 is
Ubiquitin-proteasome system and cell-cycle regulation
275
Figure 3 Interplay between SCF and APC/C complexes. (A) During G1, APC/CCdh1 ubiquitinates Skp2, contributing to the post-mitotic
down-modulation of SCF. (B) At G1/S boundary, SCFSkp2 activity increases, and CKIs substrates are ubiquitinated and degraded. This, in
turn, activates cyclin/CDKs complexes that phosphorylate Cdh1, contributing to the down-modulation of the APC/C complex activity. (C )
At the onset of mitosis, SCFb-TRCP induces the degradation of the APC/CCdc20 inhibitor Emi1, increasing APC/C activity.
present in its dephosphorylated/active form from late mitosis to
late G1; its activity is extinguished at G1/S transition and remains
low up to early M (Figure 2E). Cyclin-CDKs can phosphorylate
Cdh1, inhibiting its binding to the core APC/C subunits.39 Thus,
Cdh1 activity is low when cyclin/CDKs activity is high and vice
versa. Indeed, the G1 phase is characterized by a low activity of
cyclin/CDKs complexes. Cdh1 contributes to inhibit these complexes: APC/CCdh1 mediates the degradation of mitotic cyclins40
and SKP2,41,42 which, in turn, targets for the degradation the
CKIs such as p27, p21, and p57. The maintenance of a low
cyclin/CDKs activity is essential for the formation of pre-replicative
complexes at DNA origins. However, Cdh1 also regulates the formation of pre-replicative complexes directly, inducing the degradation of the inhibitor geminin.43 Furthermore, APC/CCdh1
induces the degradation of other mitotic kinases during G1, such
as Aurora A and B, Plk1, and Nek2A.44 – 47
2.3 SCF/APC cross-regulation
Not only APC and SCF cooperate in the regulation of common
substrates, but also their activities are regulated by feedback
loops. As already mentioned, APC/CCdh1 ubiquitinates the F-box
protein Skp2 during G1, contributing to the post-mitotic downmodulation of SCF (Figure 3A).41,42 Furthermore, at the G1/S
boundary, the SCF-dependent degradation of CKIs leads to the
activation of Cyclin-A/Cdk2 complex that, in turn, phosphorylates
Cdh1, inducing its dissociation from APC/C (Figure 3B).48 Finally, in
the early M phase, SCFb-TRCP induces the degradation of the APC/
CCdc20 inhibitor Emi1,28 consequently activating APC/C (Figure 3C).
3. Regulation of cell-cycle
progression by the UPS
The UPS, through ubiquitination and de-ubiquitination, coordinates
cell-cycle progression between each phase, ensuring a
unidirectional way to the cell cycle. The UPS also plays a role in
the exit from the cell cycle that can occur in response to starvation, in quiescent cells49,50 and, in an irreversible manner,
during terminal differentiation.51
As already mentioned, APC/CCdh1 plays a crucial role in the exit
from mitosis, regulating the degradation of mitotic cyclins40 and
inhibiting consequently CDK1, the main mitotic kinase. Furthermore, APC/C mediates the degradation of other key mitotic
factors. Given the abundance of substrates that have to be
degraded in a time-definite manner, the APC/C discriminates
between them, showing different degrees of processivity that
reflect APC/C regulation of the cell cycle.52 Cyclin-B1 and
Securin, for example, have a high rate of APC/C-mediated ubiquitination and consequently are among the first proteins to be
degraded after APC/C activation in anaphase. The processivity
rule, although useful, does not account for the whole degradative
temporal succession, indicating that other regulatory mechanisms
are in place as well: for instance, the CKI p21 and Cyclin-A are
degraded earlier than expected.53,54
Mitosis progression is also modulated by other E3 complexes
besides APC/C. Indeed, HECT-domain-E3s have been emerging as
crucial players.55 For instance, depletion of HECT-domain-E3
HECTD3 leads to multipolar spindle formation,56 and the
HECT-domain-E3 Smurf2 modulates the localization and stability
of Mad2 during the spindle checkpoint.57 SUMOylation plays a role
in mitosis as well: SUMO-E3-ligase RanBP2 is required to ensure
proper microtubule –kinetochore attachment58 and for the resolution of sister centromeres.59 Furthermore, it was recently demonstrated that the Cul3/KLHDC5-dependent degradation of the
microtubule-severing protein p60/Katanin is required for normal
mitosis in mammalian cells.60 A complex UPS regulation seems to
be at play, since Katanin is also targeted by the EDVP E3 ligase
complex that uses the dual-specificity kinase DYRK2 as a scaffold.61
Regulation of CKI p27 levels by the UPS is instrumental in the
irreversibility of the G1/S transition. During G1, Src and Abl
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kinases are activated and they phosphorylate p27.62 In this form,
p27 is displaced from CDK2 and is phosphorylated again by
CDK2 itself, allowing its recognition by the F-box protein Skp2.
The SCFSkp2-dependent degradation of p27 up-regulates CDK2
activity, with the consequent release of E2Fs transcription factors
from pRb and cell entry in S phase.63 E2F-1, which is crucial in
the induction of S phase during the transition from quiescence
to proliferation, is a short-lived protein and is degraded by the
UPS.64 – 66 Binding of underphosphorylated pRb,64 – 66 as much as
of Mdm2,67 can protect E2F-1 from degradation. Conversely,
p14ARF binding increases E2F-1 turnover, establishing a negative
feed-back loop, since E2F-1 activates p14ARF transcription.68,69
SCFSkp2 can trigger E2F-1 ubiquitination and proteasomal degradation in S/G2 phase70; however, other ligases are likely involved
in different phases of the cell cycle.
The transcription factor NF-kB is a mediator of the immune
response, inflammation, and vascular remodelling. NF-kB is also
implicated in the modulation of cell growth, activating the
expression of genes such as Cyclin-D1, c-myc, and the F-box
Skp2.71 In the cytoplasm, NF-kB is inhibited by the interaction
with IkB-a, that, in turn, is regulated by the UPS. IkB-a can be
stabilized by many DUBs, such as A20, UCHL1, and Cezanne,72 – 74
inducing an attenuation of NF-kB translocation to the nucleus
and of the consequent activation. Furthermore, it was recently
demonstrated that another DUB that targets and down-modulates
the NF-kB pathway, namely CYLD, inhibits the proliferation of
vascular cells.75 Cyclin-D1 expression and the E2F pathway are
inhibited by the over-expression of CYLD, and it is worth
noting that NF-kB induces CYLD transcription, creating an
auto-regulatory feedback loop.76
The constant degradation of hypoxia-inducible factor-1a
(HIF-1a) in normoxic conditions is critical to modulate vascular
growth, mainly owing to the HIF-dependent regulation of two
key angiogenic factors: VEGF and angiopoietin-2. This topic is
beyond the scope of this review and was extensively reviewed in
the past.77,78 However, it is worth noting that the ubiquitination
of HIF-1a is mediated by VHL, which forms a complex with elongins B and C, cullin 2, and RBX1, playing a role analogous to that of
an F-box protein.79,80
To ensure that the growth of each cell is integrated with the surrounding cells, G1/S transition is also dependent on environmental
signals. The UPS plays a role in this regulation, since many SCFFbw7
substrates, such as Cyclin-E and c-myc, promote cell entry in S
phase. The ubiquitination of these proteins by SCFFbw7is triggered
by GSK3 kinase, which, in turn, is regulated by mitogenic signalling.81 Specifically, GSK3 is active in cytostatic conditions, resulting
in the phosphorylation and the consequent degradation of SCFFbw7
substrates. Conversely, in response to mitogenic signals, GSK3 is
down-modulated, Fbw7 substrates are stabilized, and the cell can
enter the S phase.
4. Role of the UPS in oxidative
stress-induced cell-cycle regulation
Oxidative stress82 plays a causal role in ageing as well as in numerous diseases, including tissue ischaemia and reperfusion, diabetic
P. Fasanaro et al.
vasculopathy, hypertension, and atherosclerosis. Because of the
action of oxidants, proteins, nucleic acids, and membrane lipids
can be modified and damaged. The formation of reactive oxygen
species (ROS) can be also induced during many physiological cellular processes, including the regulation of signal transduction,
gene expression, and proliferation. Therefore, depending on the
concentration, the molecular species, and the subcellular localization, ROS exert two physio-pathological effects: damage to
various cell components and activation of specific signalling
pathways.
About 90% of oxidized proteins are degraded with a
Ub-independent mechanism by the 20S-proteasome, the catalytic
core of the proteasome system.83 Generally, proteasomal degradation increases due to mild oxidation, whereas it decreases at
higher oxidant levels owing to proteasome inhibition by protein
aggregates. In fact, high oxidative stress rates induce massive
protein cross-linking and the formation of indegradable and nonexocytosable aggregates, also known as lipofuscin, that bind and
inhibit the 20S-proteasome, further increasing the oxidative
damage.84 In these conditions, cells undergo permanent growth
arrest, followed by apoptosis or necrosis, and the role of ubiquitination in oxidized protein removal seems to be marginal. Conversely, at sublethal levels of oxidative stress, the UPS plays a pivotal
role in mounting the complex array of cell adaptations that include
increase in expression of antioxidant enzymes, activation of protective genes, cell-cycle arrest, and apoptosis. Depending on the
severity of the damage, the cell type, and the genotype, oxidative
stress can either up- or down-modulate the degradation by the
UPS of specific substrates. Furthermore, a direct effect of oxidative
stress on 26S-proteasome has been described as well. Oxidative
stress induced by a prostaglandin-D2 metabolite increases
protein carbonylation of S6 ATPase, a 26S-proteasome regulatory
subunit, likely altering recognition and degradation of proteasomal
substrates.85
Finally, not only oxidative stress modulates the UPS, but the
opposite is true as well: numerous studies indicate that proteasomal inhibition induces oxidative stress, thus activating a signalling
cascade leading to cell death and apoptosis.86,87
4.1 UPS activation by oxidative stress
Endothelial cell treatment with hydrogen peroxide up-regulates
the levels of specific Ub mRNAs, such as Uba52 and UbB, and
increases the Ub-conjugating activity to numerous endogenous
substrates.88 As a consequence, many cell-cycle relevant proteins,
such as Nrf2, p53, FOXOs, and pRb, are affected.
4.1.1 NRF2
A link between the general response to oxidative stress and the
UPS is represented by Keap1. This stress sensor is an inhibitor
of the transcription factor Nrf2 that, in turn, is a major transcriptional regulator of the antioxidant response genes.89 Indeed, Nfr2
was recently indicated as a possible therapeutic target to restore
antioxidant defences in diabetes90 and a key transcriptional regulator of the vasoprotective effects of shear stress.91 In normal conditions, Nrf2 is bound in the cytoplasm and inhibited by Keap1,
which functions as an adaptor subunit of the SCF/Cul3-based E3
ligase system to promote a rapid degradation of Nrf2.92 In
Ubiquitin-proteasome system and cell-cycle regulation
response to oxidative stress, Keap1 releases Nrf2 from inhibition,
allowing the stabilization and the nuclear translocation of Nrf2,
with the consequent expression of antioxidant response genes.
Nrf2 affects the cell-cycle progression and induces growth arrest
directly, modulating the glutathione-mediated signalling93 and, at
least in part, upregulating the CKI p27.94
4.1.2 p53
p53 transcription factor represents a crucial mediator for the cellular response to oxidative stress. p53 is an integral component of
multiple cell-cycle checkpoints that modulates many cellular functions, such as transcription, DNA synthesis and repair, cell cycle
arrest, senescence, and apoptosis. A very large body of literature
is present on p53-elicited cell responses, mainly growth arrest
and apoptosis, that are stimulus- and cell type-specific. p53 regulation is similarly complex and articulated (see references 95,96
for extensive reviews on p53 regulation and function). Under
normal conditions, p53 levels are maintained low, but in the presence of cellular stress, p53 is rapidly stabilized, dramatically increasing its half-life. The main effect of p53 on cell cycle is mediated by
the transcriptional activation of CKI p21. CDKs inhibition contributes to the stress-induced growth arrest, allowing the cells to
repair the damages on cellular components and to set up an adaptive response to counteract further damage. In contrast, when
levels of stress reach a situation beyond repair, p53 activates an
apoptotic pathway.
p53 is regulated through a plethora of post-translational modifications, including phosphorylation, methylation, and acetylation,
but precise governing of its function seems to rely on Mdm2, an
E3 ligase that induces p53 ubiquitination.97 Moreover, p53 regulates mdm2 gene transcription, forming a negative feedback loop
with its main E3 ligase.98 Mdm2 binds and inhibits p53 at least by
two mechanisms: by inducing its degradation and blocking its transcriptional activation. Depending on protein levels, Mdm2 can
induce both mono- and poly-ubiquitination of p53.97 In the presence of low levels of Mdm2, p53 is usually mono-ubiquitinated,
and this modification induces the export from the nucleus to the
cytoplasm, where p53 can be further modified. When Mdm2
levels are high, such as in non-stressed cells or in the latter
stages of the DNA damage response, p53 is rapidly polyubiquitinated and degraded by the 26S-proteasome. Furthermore,
Mdm2 can ubiquitinate itself and this may be the predominant
mechanism for maintaining its short half-life. It was also demonstrated that DNA-damage kinases increase the rate of Mdm2 selfubiquitination and that this auto-degradation is required for p53
activation.99 Similar to other cell-damaging agents, ROS downregulate Mdm2, both in vitro and in vivo, triggering p53 stabilization.100,101 Mdm2/p53 complex is also modulated by seladin-1,
which, following oxidative stress, binds to p53 and displaces
Mdm2, contributing to p53 accumulation in response to
stress.102 The UPS can modulate p53 by other E3 ligases, but
the elicited responses have not been investigated in detail thus
far.95,96
4.1.3 FOXO
FOXO is a transcription factor family that controls cell-cycle progression, DNA repair, defence against oxidative damage, and
277
apoptosis. Indeed, FOXO plays a role in angiogenesis, cardiovascular development, and hypertension, and it has been shown that
FOXO dysregulation is involved in several cardiovascular diseases.103 The overall effect of FOXO on cell-cycle control is G1
arrest, by positive modulation of CKIs p21 and p27 and downmodulation of D-cyclins. Cytoplasmic FOXO1 interacts with the
F-box protein Skp2 and is targeted for degradation. Following oxidative stress, FOXO1 is phosphorylated by Jnk kinase and translocated into the nucleus, where its transcriptional activity
contributes to growth arrest. An additional complexity layer is represented by the fact that not only phosphorylation, but also monoubiquitination can induce the nuclear localization of FOXO in
response to oxidative stress.104 Furthermore, it was recently
demonstrated that Mdm2 can act as an E3 Ub ligase for FOXO
as well.105
4.1.4 The pRb pathway
The pRb pathway represents a key system of the cell-cycle machinery.63 In proliferating endothelial cells, oxidative stress induces a
rapid cell-cycle arrest dependent on pRb de-phosphorylation
mediated by PP2A/PR70 phosphatase.106,107 After this event,
other pathways that induce pRb de-phosphorylation are activated,
probably to maintain and reinforce the pRb-dependent growth
arrest. Specifically, D-cyclins are ubiquitinated and degraded by
the proteasome with a Ca2þ/CamKII-dependent mechanism,
leading to the consequent CDK4/6 inhibition and to an increase
of endothelial cell survival upon oxidative stress.108 Indeed,
Cyclin-D1 degradation is also induced by growth factor deprivation, hypoxia, osmotic stress, and ultraviolet irradiation;109 – 112
so it seems that this event represents a common adaptive response
to stress conditions.
4.2 UPS down-modulation by oxidative
stress
Oxidative stress can inhibit specific components of the UPS. For
instance, hydrogen peroxide treatment oxidizes APC11, the catalytic RING protein of the APC/C complex (Figure 1), preventing
the contact with the E2-conjugating enzyme Ubc4, thus inhibiting
the Ub transfer to APC/C substrates.113 This event prevents the
degradation of Cyclin-B and Securin and contributes to the
growth arrest induced by oxidative stress. Furthermore, TGFb, a
potent inhibitor of cell growth, down-modulates specific
proteasome-associated proteolitic activities, and this event correlates with the production of hydrogen peroxide induced by
TGFb.114
Among all ROS, nitric oxide (NO) modulates many physiopathological aspects of blood vessels.115 In vascular smooth
muscle cells, NO-induced cell-cycle arrest is mediated, at least in
part, by the stabilization of both CKI p21 protein and mRNA.
Specifically, NO prevents p21 protein ubiquitination and degradation.116 Recently, a direct regulation of UPS by NO was demonstrated.117 It was found that NO not only reversibly inhibits the
26S-proteasome through its S-nitrosylation, but also modulates
both the expression and the localization of specific proteasomal
subunits.
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5. Concluding remarks
The UPS plays a prominent role in the complex systems that allow
the unidirectional way of the cell cycle, controlling the progression
between and within the phases. SCF and APC/C complexes play a
prominent role in such aspects. However, other still insufficiently
investigated E3 ligases and de-ubiquitinating complexes modulate
the cell cycle as well, adding more complexity and further levels
of regulation to the cell growth machinery. The identification and
the careful characterization of these E3 ligases and de-ubiquitinases
represent the challenge for the years to come.
Another aspect that is worth noting is that the involvement of
the UPS in cell-cycle regulation has been studied mainly in
non-endothelial systems. Since the endothelium is composed of
non-transformed, genetically intact cells, the molecular mechanisms elucidated so far are likely at work in endothelial cells as
well. However, revisiting UPS-dependent cell-cycle regulation in
a vascular context, both in vitro and in vivo, may allow us to
uncover possible unique regulatory mechanisms and to assess
the prevalence of each pathway.
In conclusion, our understanding of the role of UPS in cardiovascular diseases is just at the beginning. The use of proteasome
inhibitors is entering the clinical arena in oncology,118,119 pointing
our attention towards a clinically oriented understanding of the
UPS in the vascular field as well.
Acknowledgements
We apologize to the many researchers whose work was not cited
in this review owing to space limitations. We thank Marco Crescenzi and Alessandra Magenta for their critical reading of the
manuscript.
Conflict of interest: none declared.
Funding
The authors are supported by Ministero della Salute (Italian Ministry of
Public Health).
Glossary
20S-Proteasome: the core component of the protease that unfolds
and degrades proteins.
26S-Proteasome: the 20S core and the 19S regulator component
together form the 26S proteasome, responsible for the degradation of poly-ubiquitinated proteins.
APC/C: the anaphase-promoting complex/cyclosome is an E3 ubiquitin ligase that regulates the progression through mitosis and
G1 phase. APC/C activity and substrate specificity depend on
the activators Cdh1 and Cdc20.
CDKs: the cyclin-dependent kinases are the catalytic subunits of a
family of heterodimeric serine/threonine kinases. CDKs regulate
the progression through each phase of the cell cycle.
CKIs: inhibitors of cyclin-CDK activity. CKIs are divided in two
families: the Cip/Kip family, which comprises p21waf1/cip1/sdi1,
p27kip1, and p57kip2, and the Ink4 family, which includes
p16INK4a, p18INK4c, and p19INK4d.
P. Fasanaro et al.
Cyclins: proteins that bind and modulate CDK activity. Cyclin
protein levels are finely modulated by degradation during the
cell cycle.
E2F: a transcription factor family that modulates genes regulating
DNA synthesis, G2 phase, and apoptosis.
SCF: the SKP1-CUL1-F-box-protein complex is an E3 ubiquitin
ligase essential in cell-cycle regulation, playing a prominent
role in G1/S transition. SCF substrate specificity is regulated
by the F-box component. Three F-box proteins Skp2, Fbw7,
and b-TRCP play a prominent role in cell-cycle regulation.
Ubiquitin: an 8 kDa protein that can be covalently attached to proteins. Three enzymes families are involved in ubiquitination: activation enzyme (E1), conjugation enzyme (E2), and ligation
enzyme (E3)
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