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The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated β(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.
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LETTER doi:10.1038/nature10368
A stress response pathway regulates DNA damage
through b
2
-adrenoreceptors and b-arrestin-1
Makoto R. Hara
1
, Jeffrey J. Kovacs
1
, Erin J. Whalen
1
, Sudarshan Rajagopal
1
, Ryan T. Strachan
1
, Wayne Grant
2
, Aaron J. Towers
1,3
,
Barbara Williams
1
, Christopher M. Lam
1
, Kunhong Xiao
1
, Sudha K. Shenoy
1
, Simon G. Gregory
1,3
, Seungkirl Ahn
1
,
Derek R. Duckett
2
& Robert J. Lefkowitz
1,4
The human mind and body respond to stress
1
, a state of perceived
threat to homeostasis, by activating the sympathetic nervous system
and secretingthe catecholaminesadrenaline and noradrenalinein the
‘fight-or-flight’ response. The stress response is generally transient
because its accompanying effects (for example, immunosuppression,
growth inhibition and enhanced catabolism) can be harmful in the
long term
2
. When chronic, the stress response can be associated with
disease symptoms such as peptic ulcers or cardiovascular disorders
3
,
and epidemiological studies strongly in dicate that chronic stress leads
to DNA damage
4,5
. This stress-induced DNA damage may promote
ageing
6
, tumorigenesis
4,7
, neuropsychiatric conditions
8,9
and mis-
carriages
10
. However, the mechanisms by which these DNA-damage
events occur in response to stress are unknown. The stress hormone
adrenaline stimulates b
2
-adrenoreceptors that are expressed thr ough-
out the body, including in germline cells and zygotic embryos
11
.
Activated b
2
-adrenoreceptors promote Gs-protein-dependent activa-
tion of protein kinase A (PKA), followed by the recruitment of
b-arrestins, which desensitize G-protein signalling and function as
signal transducers in their own right
12
.Hereweelucidateamolecular
mechanism by which b-adrenergic catecholamines, acting through
both Gs–PKA and b-arrestin-mediated signalling pathways, trigger
DNA damage and suppress p53 levels respectively, thus synergi-
stically leading to the accumulation of DNA damage. In mice
and in human cell lines, b-arrestin-1 (ARRB1), activated via b
2
-
adrenoreceptors, facilitates AKT-mediated activation of MDM2
and also promotes MDM2 binding to, and degradation of, p53, by
acting asa molecularscaffold. Catecholamine-induced DNA damage
is abrogated in
Arrb1
-knockout (
Arrb1
2/2
) mice, which show pre-
served p53 levels in both the thymus, an organ that responds promi-
nently to acute or chronic stress
1
, and in the testes, in which paternal
stress may affect the offspring’s genome. Our results highlight the
emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and
reveal how DNA damage may accumulate in response to chronic
stress.
As a model of chronic stress and prolonged stimulation of b
2
-
adrenoreceptors
7,13
, wild-type mice were infused for four weeks with
either saline or the b
2
-adrenoreceptor-agonist isoproterenol, a syn-
thetic analogue of adrenaline. First, we tested whether this regimen
affects DNA damage by examining phosphorylation of histone H2AX
(c-H2AX), one of the earliest indicators of DNA damage
14
.
Isoproterenol infusion leads to DNA damage in the thymus (Fig. 1a,
left panel). Accumulation of DNA damage indicates compromised
genome maintenance. To investigate the potential mechanism, we
examined p53 levels in the thymus and found that isoproterenol infu-
sion leads to decreased levels of p53 (Fig. 1a, right panel). Consistent
with the effects of isoproterenol in vivo, chronic stimulation of b
2
-
adrenoreceptors with b-adrenergic catecholamines (isoproterenol,
adrenaline or noradrenaline) leads to accumulation of DNA damage
and a decrease in p53 levels in cultured U2OS cells (Supplementary
Fig. 1a–c), which endogenously express wild-type p53 and only the
b
2
-subtype of b-adrenoreceptors (SupplementaryFig. 2a–c). Moreover,
the p53 in these cells, as wellas in all other cell lines used in these studies
(fibroblasts and HEK-293 cells), was demonstrated to be functional by
a variety of techniques (Supplementary Fig. 3a–k), and all cell lines
endogenously expressed only the b
2
-subtype of b-adrenoreceptors
(Supplementary Fig. 2a–c).
The isoproterenol-induced reduction in p53 levels results from p53
degradation, and is abolished by proteasome inhibition (Supplementary
1
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
2
Translational Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, USA.
3
Center for
Human Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
4
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
Iso
LMB
+
+
––
+
+
WB:p53
WB:FOXO3a
Nucleus
WB:p53
WB:LDH
WB:histone
Iso +–+
ICI –+
Nucleus
c
b
d
WB:p-MDM2
(Ser 166)
WB:MDM2
Iso +–+
ICI –+
fe
Arrb1–/– Arrb2–/–
WT
WB:p-MDM2
(Ser 166)
WB:MDM2
IsoNs
H-89
+
Iso IsoNs IsoNs
123 4 5 6 7
a
p53 protein levels (arbitrary units)
Saline
Iso
WB:p53
WB:β-tubulin
0
0.2
0.4
0.6
0.8
1.0
1.2
P = 0.023
γ-H2AX (arbitrary units)
WB:histone
WB:γ-H2AX
DNA damage p53 levels
Mouse thymus
0
0.5
1.0
1.5
2.0
2.5 P = 0.015
LY294002 –+
Iso –++
Nucleus
WB:LDH
WB:p53
WB:histone
Figure 1
|
Chronic catecholamine stimulation leads to p53 degradation and
accumulation of DNA damage via ARRB1/AKT-mediated activation of
MDM2. a, Isoproterenol infusion leads to accumulation of DNA damage and
decreased p53 levels. Mice (n53–5 for each condition) were infused with
saline or isoproterenol (30 mg kg
21
d
21
) for 4 weeks. All bars represent
mean 6s.e.m. Histone, histone H2B; Iso, isoproterenol; WB, western blot.
b, Isoproterenol-induced p53 reduction is dependent on nuclear export. This
effect is specific to p53, in that another nuclear–cytosol shuttling molecule,
FOXO3a, is not affected. LMB, leptomycin B. c, Preincubation with the b
2
-
adrenoreceptor-selective antagonist ICI118,551 (ICI) blocks isoproterenol-
induced nuclear export of p53. Lactate dehydrogenase (LDH) is a cytosolic
marker and histone is a nuclear marker. d, Isoproterenol stimulation leads to
MDM2 phosphorylation at Ser 166, and is blocked by preincubation with
ICI 118,551. e, Inhibition of the PI3K/AKT cascade abolishes isoproterenol-
stimulated decreases in p53 levels in U2OS cells. LY294002 is a PI3K inhibitor.
f, Isoproterenol stimulation leads to Gs-independent, ARRB1-dependent
MDM2 phosphorylation at Ser 166. Ns, not stimulated.
00 MONTH 2011 | VOL 000 | NATURE | 1
Macmillan Publishers Limited. All rights reserved
©2011
Fig. 1d). Because nuclear exportof p53 has been shown to be involved in
its degradation
15
, we examined p53 localization. Subcellular fractiona-
tion shows that isoproterenol stimulation leads to a decrease in nuclear
p53 and an increase in cytosolic p53 (Supplementary Fig. 1e, lower
panels), thus, isoproterenol stimulation leads to p53 nuclear export.
Immunocytochemical examination also shows increased levels of cyto-
solic p53 after isoproterenol stimulation (Supplementary Fig. 1e, upper
panels). Isoproterenol concentrations as low as 1nM lead to p53 nuc-
lear export, resulting in a decrease in total p53 levels (Supplementary
Fig. 1f). The importance of nuclear export in modulating p53 levels was
investigated bytreating cells with leptomycin B, an inhibitor of nuclear
export. Leptomycin B pretreatment reverses isoproterenol-induced
nuclear export of p53 (Fig. 1b).
To examine whether isoproterenol-induced effects were specifically
mediated by b
2
-adrenoreceptors, U2OS cells were stimulated with iso-
proterenol in the presence or absence of the subtype-selective b
2
-
adrenoreceptor antagonist ICI 118,551. Preincubation with ICI 118,551
abrogates the isoproterenol-induced decrease in p53 levels (Fig. 1c).
During in vivo experiments, isoproterenol infusion leads to accumula-
tion of DNA damage in the cerebellum, where b
2
-adrenoreceptors are
the major subtype of b-adrenoreceptor
16
(Supplementary Fig. 1g).
Furthermore, targeted disruption of the Adrb2 gene in mice markedly
reduces accumulation of DNA damage upon isoproterenol infusion
(Supplementary Fig. 1h). Taken together, these data indicate that
stimulation of the b
2
-adrenoreceptor results in the nuclear export
and degradation of p53 in a specific manner.
The E3 ligase MDM2 has been shown to have an important role in
the regulation of p53 nuclear export and degradation
15
. Consistent with
this, leptomycin B abrogates the ability of MDM2 to degrade p53
(ref. 15). Before MDM2-mediated ubiquitination of p53, the phos-
phoinositide 3-kinase (PI3K)/AKT cascade phosphorylates MDM2,
activating its E3 ligase function
17
. To examine whether stimulation
of b
2
-adrenoreceptors leads to MDM2 phosphorylation via the PI3K/
AKT cascade, wild-type mouse embryonic fibroblasts (MEFs)
were stimulated with isoproterenol in the presence or absence of
ICI 118,551. Isoproterenol stimulation leads to MDM2 phosphor-
ylation at Ser 166, an AKT phosphorylation site, and the effect is
antagonized by ICI 118,551 (Fig. 1d and Supplementary Fig. 1i). To
confirm that MDM2 is phosphorylated by the PI3K/AKT cascade
upon isoproterenol stimulation, U2OS cells were stimulated with iso-
proterenol in the presence of either the PI3K inhibitor wortmannin or
the AKT inhibitor AKTi. MDM2 phosphorylation is abolished by
either wortmannin or AKTi (Supplementary Fig. 1j). Furthermore, a
PI3K inhibitor also abolishes catecholamine-induced lowering of p53
levels in the nucleus (Fig. 1e and Supplementary Fig. 1k). The import-
ance of MDM2 phosphorylation at Ser 166 was demonstrated by the
overexpression of a phosphomimetic mutant at Ser 166 (MDM2-
S166D)
17
, which facilitates the degradation of p53 when compared
to wild-type MDM2 (Supplementary Fig. 1l). These data implicate
the PI3K/AKT cascade downstream of the b
2
-adrenoreceptor as a
mediator of p53 stability through the phosphorylation of MDM2.
Upon activation of b
2
-adrenoreceptors, the PI3K/AKT cascade
can be stimulated by both the Gs–PKA
18
and b-arrestin-mediated
signalling pathways
19,20
. To elucidate which pathway was involved,
we examined the effect of b
2
-adrenoreceptor stimulation in wild-
type MEFs in the presence of H-89, a PKA inhibitor, or in Arrb1
2/2
or Arrb2-knockout (Arrb2
2/2
) MEFs. In wild-type MEFs, H-89 does
not inhibit isoproterenol-stimulated MDM2 phosphorylation (Fig. 1f,
lane 3). In contrast, the isoproterenol effect is abrogated in Arrb1
2/2
(Fig. 1f, lane 5), but not in Arrb2
2/2
, MEFs (Fig. 1f, lane 7).
Furthermore, rescuing Arrb1 expression in Arrb1
2/2
MEFs restores
the effects of isoproterenol stimulation (Supplementary Fig. 1m), and
the effects of isoproterenol on MDM2 phosphorylation are abrogated
in Arrb1
2/2
mice (Supplementary Fig. 1n). These data elucidate a Gs-
independent, ARRB1-dependent signalling pathway that regulates the
activation state of MDM2 through the PI3K/AKT cascade.
b-Arrestins can serve as adaptors for E3 ligases and their sub-
strates
21
. Because we found that MDM2 activation by the PI3K/AKT
cascade is an ARRB1-dependent event, we examined the binding
between b-arrestins and p53, a known MDM2 substrate. In HEK-
293 cells stably overexpressing ARRB1 or ARRB2, p53 binds preferen-
tially to ARRB1, an isoform localized to both the cytosol and nucleus
22
,
but not to ARRB2, which predominantly localizes to the cytosol
22–25
(Supplementary Fig. 4a). Binding between these two molecules at
GST–ARRB1
GST
GST
GST–
ARRB1
WB:p53
a
b
IgG ARRB1
WB:ARRB1
WB:p53
c
d
IP:
WB:GST
IP:MDM2
WB:p53
WB:MDM2
ARRB1 + +
Iso +
e
f
WB:MDM2
WB:p53
Lysate
IP:MDM2
pcDNA
Arrb1
Arrb1-
Q394L
WB:ARRB1
WB:p53
Iso +
Lysate
IP:MDM2
WT
Arrb1–/–
Lysate
IP:MDM2
WB:p53
Nucleus p53
ARRB1 Merged
Figure 2
|
ARRB1 functions as an E3 ligase adaptor for MDM2 and p53
upon catecholamine stimulation. a, Endogenous binding of p53 and ARRB1.
Cell lysates from HEK-293 cells were used for immunoprecipitation (IP) with
an anti-ARRB1 (K-16) antibody or normal IgG, and analysed by
immunoblotting with anti-p53 (DO-1) antibody. b,In vitro binding of p53 and
ARRB1. Purified p53 was incubated with either GST or GST–ARRB1 and
precipitated with glutathione beads. Precipitates were analysed by
immunoblotting with an anti-p53 (DO-1) antibody. c, Confocal analysis of
co-localization of p53 and ARRB1 in non-treated RAW264.7 macrophages,
which endogenously express high levels of both p53 and ARRB1. Scale bar,
10 mm. d, Isoproterenol stimulation facilitates the binding of MDM2 to p53 in
ARRB1 overexpressed cells. e, Nuclear ARRB1 facilitates MDM2 binding to
p53. U2OS cells were transfected with either empty vector (pcDNA), Arrb1 or
Arrb1-Q394L.f, ARRB1 facilitates isoproterenol-induced MDM2 binding to
p53. WT, wild-type.
RESEARCH LETTER
2 | NATURE | VOL 000 | 00 MONTH 2011
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©2011
endogenous levels is also observed in HEK-293 cells and brain homo-
genates (Fig. 2a and Supplementary Fig. 4b). The binding seems to be
direct, because purified ARRB1 tagged with glutathione-S-transferase
(GST–ARRB1) binds to p53 in vitro (Fig. 2b). To examine the effect of
b
2
-adrenoreceptor stimulation on this complex, we treated untrans-
fected HEK-293 cells, which endogenously express only the b
2
subtype
of b-adrenoreceptors (Supplementary Fig. 2b, c), with isoproterenol.
Stimulation of b
2
-adrenoreceptors does not affect the binding of
ARRB1 to p53 (Supplementary Fig. 4c). Subsequently, we mapped
the binding sites in ARRB1 and p53 by performing sequential dele-
tions, followed by immunoprecipitation from HEK-293 cells (Sup-
plementary Fig. 4d, e). We identified the amino terminus of ARRB1
(amino acids 1–186) as critical for binding to p53. In p53, a domain
comprising amino acids 101–186 is required for binding to ARRB1.
Consistent with these results, a synthetic ARRB1-binding peptide
(ARRB-BP), which binds to the N terminus of ARRB1 and induces
a conformational change
26
, disrupts the interaction between ARRB1
and p53 (Supplementary Fig. 4f).
Co-immunoprecipitation experiments after subcellular fractiona-
tion show that more than 90% of the binding between ARRB1 and
p53 occurs in the nucleus (Supplementary Fig. 4g). Additionally, con-
focal analysis reveals that endogenous ARRB1 and p53 co-localize in
the nucleus (Fig. 2c) and a ternary complex between ARRB1, MDM2
and p53 was observed in p53-null NCI-H1299 cells transfected with
wild-type p53 (Supplementary Fig. 4h). The potential effects of nuclear
ARRB1 on MDM2 binding to p53 were investigated in U2OS cells
transfected with either Arrb1 or Arrb1-Q394L, in which a single
amino acid, Gln 394, has been mutated to Leu to create a nuclear
export signal in ARRB1
22,23
. Overexpression of ARRB1 facilitates
the binding of MDM2 to p53, enhancing basal b
2
-adrenoreceptor-
stimulated ARRB1 signalling (Fig. 2d); however, the effect is abolished
with ARRB1-Q394L (Fig. 2e). This result indicates that ARRB1 facili-
tates an MDM2–p53 interaction in the nucleus. The role of endogen-
ous ARRB1 as a facilitator of MDM2–p53 complex formation under
isoproterenol-stimulated conditions is further demonstrated in
Arrb1
2/2
MEFs (Fig. 2f), in which loss of ARRB1 prevents the
increased interaction of MDM2 and p53 after isoproterenol stimu-
lation, when compared to wild-type cells.
Next we examined whether ARRB1 expression affects p53 levels by
comparing different clonal populations of wild-type and Arrb1
2/2
MEFs. Arrb1
2/2
MEFs show increased p53 levels (Supplementary
Fig. 5a). Furthermore, rescuing ARRB1 expression in Arrb1
2/2
MEFs decreases p53 levels in a dose-dependent manner (Fig. 3a).
Differences in p53 levels under basal conditions seem to be the result
of decreased p53 ubiquitination in Arrb1
2/2
MEFs (Fig. 3b, lanes 1
and 3). Furthermore, consistent with b
2
-adrenoreceptor-induced
degradation of p53, isoproterenol stimulation promotes p53 ubiquiti-
nation, but the effect is markedly decreased in Arrb1
2/2
MEFs (Fig. 3b,
lanes 2 and 4). To address further whether ARRB1 facilitates the ubi-
quitination of p53 by MDM2, we conducted in vitro ubiquitination
assays (Supplementary Fig. 5b). Addition of ARRB1 facilitates
MDM2-mediated ubiquitination of p53 and the effect is abolished with
ARRB-BP. Together, these data indicate that upon catecholamine
stimulation, ARRB1 promotes the interaction of MDM2 and p53 by
acting as an E3 ligase adaptor that facilitates ubiquitination of p53.
Cytosolic ARRB1 mediates catecholamine-induced activation of
AKT and MDM2, whereas nuclear ARRB1 serves as an adaptor for
MDM2-dependent ubiquitination of p53. Consistent with these
results, isoproterenol stimulation leads to a lowering of p53 levels
(Fig. 3c, lane 2). By contrast, p53 levels remain constant in Arrb1
2/2
MEFs (Fig. 3c, lane 4). Furthermore, rescuing ARRB1 expression in
Arrb1
2/2
MEFs by transient transfection restores isoproterenol-
stimulated degradation of p53 (Fig. 3c, lane 6), and suppression of
ARRB1 by RNA interference results in suppression of isoproterenol-
stimulated MDM2–p53 complex formation, and a lowering of p53
levels in U2OS cells (Fig. 3d, e).
To differentiate the cytosolic (catecholamine-induced MDM2 phos-
phorylation) and nuclear (E3 ligase adaptor) functions of ARRB1 in
this cascade, a phosphomimetic mutant of MDM2 (MDM2-S166D)
was co-transfected with either Arrb1 or Arrb1-Q394L into Arrb1
2/2
MEFs. This allowed us to focus on the nuclear function of ARRB1.
Restoring ARRB1 expression with the wild type, but not with the
Q394L mutant, facilitates the degradation of p53 (Supplementary
Fig. 5c). Consequently, it seems that although the cytoplasmic pool
of ARRB1 is sufficient to activate MDM2 through the PI3K/AKT
pathway, the nuclear pool of ARRB1 is required to act as an E3 ligase
adaptor for MDM2 towards p53.
To examine this cascade in vivo, we examined the effects of cate-
cholamine on p53 levels and accumulation of DNA damage in the
thymus of Arrb1
2/2
mice. The mice were infused for four weeks with
either saline or isoproterenol. In contrast to wild-type mice (Fig. 1a),
p53 levels are maintained upon isoproterenol infusion in Arrb1
2/2
mice, and accumulation of DNA damage is abrogated (Fig. 3f).
We have observed that isoproterenol infusion leads to lowering of
p53 levels, and have elucidated a molecular mechanism whereby
a
Iso +–+
Arrb1–/–
WB:p53
WB:ARRB1
ARRB1 rescue
–+
WT
1234 56
WB:p53
WB:β-tubulin
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 Saline
Iso
p53 protein levels (arbitrary units)
P = 0.86
c
WB:p53
WB:ARRB1
0 0.5 1.512Arrb1(μg)
Arrb1–/–
Arrb1–/– mice
0
0.2
0.4
0.6
0.8
1.0
1.2
γ-H2AX (arbitrary units)
WB:histone
WB:γ-H2AX
P = 0.46
f
DNA damage p53 levels Mouse thymus
64
97
191
IP:p53
WB:Ubiquitin
WB:MDM2
Iso +–+
WT Arrb1–/–
Lysate
Ubiquitinated
p53
1234
b
(kDa)
WB:MDM2
WB:p53
d
Iso
WB:p53
WB:MDM2
IP:MDM2
–+
+
Lysate
WB:ARRB1
WB:β-tubulin
ARRB1 shRNA
CTL shRNA
–+
+–
Iso
ARRB1 shRNA
Control shRNA
e
Figure 3
|
ARRB1 facilitates catecholamine-induced p53 degradation by
MDM2. a, Rescuing ARRB1 expression in Arrb1
2/2
MEFs decreases p53 levels.
Arrb1
2/2
MEFs were transiently transfected with Arrb1 and cell lysates were
examined by immunoblotting. b, Isoproterenol stimulation leads to
ubiquitination of p53 in a ARRB1-dependent manner. Wild-type and Arrb1
2/2
MEFs were stimulated with 10 mM isoproterenol for 24 h. Cell lysates were
immunoprecipitated with an anti-p53 antibody (FL-393) and analysed by
immunoblotting with an anti-ubiquitin (P4D1) antibody. c, Isoproterenol
stimulation leads to ARRB1-dependent p53 degradation.d, Reduc tion in ARRB1
levels induced by small hairpin RNA (shRNA) in U2OS cells. e, Suppression of
ARRB1 suppresses isoproterenol-inducedbinding of MDM2 to p53 and restores
p53 levels in U2OS cells. U2OS cells were treated with either control shRNA or
ARRB1 shRNA for 72h, followed by 24h stimulation with 10 mM isoproterenol.
f, Levels of p53 in isoproterenol-infused Arrb1
2/2
mice remain constant and
there is no accumulation of DNA damage. Arrb1
2/2
mice (n53–4 for each
condition) were treated as in Fig. 1a. All bars represent mean6s.e.m.
LETTER RESEARCH
00 MONTH 2011 | VOL 000 | NATURE | 3
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ARRB1 regulates MDM2-dependent degradation of p53 upon b
2
-
adrenoreceptor stimulation. Next, we investigated further the effects
of catecholamine-dependent p53 degradation on accumulation of
DNA damage. To visualize the prevalence of DNA damage, we ana-
lysed the formation of c-H2AX foci in both wild-type and Arrb1
2/2
MEFs. After chronic stimulation with isoproterenol, there is an
increase in the formation of c-H2AX foci (sevenfold) in wild-type
MEFs, which is significantly reduced in Arrb1
2/2
MEFs (Fig. 4a,
panels 2 and 4, and Supplementary Fig. 6a). Moreover, rescuing
ARRB1 expression in Arrb1
2/2
MEFs restores the accumulation of
isoproterenol-induced c-H2AX foci (Fig. 4b, panels 5–8, and Sup-
plementary Fig. 6a).
To examine how exposure to stress hormones initiates DNA
damage, p53-null NCI-H1299 cells were chronically stimulated with
isoproterenol. This leads to accumulation of DNA damage (Fig. 4c,
panels 1–8), indicating that DNA damage is triggered by p53-
independent mechanisms after isoproterenol stimulation. One of the
prominent cascades leading to DNA damage is the generation of
reactive oxygen species through Gs–PKA signalling
27
. Accordingly,
accumulation of isoproterenol-induced DNA damage is suppressed
by inhibition of PKA (Fig. 4d, lanes 1 and 3). Consistent with the idea
that ARRB1-mediated effects on DNA damage are due to altered p53
levels, rescuing p53 expression (Supplementary Fig. 3l) decreases iso-
proterenol-induced c-H2AX foci (Fig. 4c, panels 9–16; Fig. 4d, lanes 1
and 2) and the p53 effect is antagonized by co-expression of ARRB1
(Fig. 4c, panels 17–20, and Supplementary Fig. 6b). These G-protein-
mediated and ARRB1-mediated pathways may synergistically affect
the accumulation of isoproterenol-induced DNA damage. Thus,
combining PKA inhibition with rescue of p53 expression abrogates
accumulation of DNA damage (Fig. 4d, lanes 1 and 4). Catecholamine-
induced lowering of p53 levels may lead to increased survival of cells
containing DNA damage, owing to an impaired DNA damage check-
point and repair cascade
28
. This would then facilitate accumulation of
DNA damage. Accordingly, U2OS cells were irradiated with ultra-
violet light after isoproterenol stimulation. Chronic stimulation leads
to increased FOS expression, an indicator of cell survival and prolif-
eration (Supplementary Fig. 6c). Because DNA damage occurs under
these conditions, FOS expression leads to proliferation of cells that
contain DNA damage. Taken together, these data indicate that Gs–
PKA-dependent signalling, which leads to the generation of reactive
oxygen species
27
, and ARRB1-dependent p53 degradation, which
results in impaired DNA checkpoint and repair mechanisms
28
,
synergistically lead to accumulation of DNA damage and consequently
may have effects on genomic integrity.
DNA damage may promote rearrangements in chromosomes. To
quantify the occurrence of catecholamine-induced rearrangements, we
analysed inter-chromosomal rearrangements between Tcrg (the T-cell-
receptor-clocus) and Tcrb (the T-cell-receptor-blocus) in thymocytes
(see Methods and Supplementary Fig. 6d). Both wild-type and Arrb1
2/2
mice were infused for four weeks with either saline or isoproterenol, and
genomic DNA was isolated from the thymus. Consistent with the accu-
mulation of DNA damage (Figs 1a, 3f), isoproterenol infusion leads to
an increase in these Tcr rearrangements in wild-type mice; however,
the effects are no longer observed in Arrb1
2/2
mice (Fig. 4e and
Supplementary Fig. 6e). This indicates that catecholamine-stress-
hormone-dependent accumulation of DNA damage promotes re-
arrangements in chromosomes.
Because chronic isoproterenol stimulation affects DNA damage and
chromosomal rearrangements, we examined whether this cascade also
affects genome integrity in the testes, in which paternal stress may
affect the offspring’s genome. Using the isoproterenol-infusion model
of chronic stress
7,13
, we observed that isoproterenol stimulation leads
a
b
RFP-Arrb1 RFP
Iso: Arrb1–/–
DNA γ-H2AX MergedRFP
c
3
7
g
d
12 4
865
h
0
0.4
0.8
1.2
p53 levels (arbitrary units)
Arrb1–/–
WT
Mouse testes
Saline
Iso
WB:p53
WB:β-tubulin
P = 0.0014
DNA γ-H2AX MergedRFP
Ns
pcDNA
+RFP
p53
+RFP
Iso
p53
+RFPArrb1
5678
9101112
13 14 15 16
17 18 20
p53-null H1299 cells
Thymus
01.0–1.0
110
115
120
125
130
01.0–1.0
qD1 qD2.2
WT
Testes
110
115
120
125
130
qD1 qD2.2
Arrb1–/–
(Mb)
log2 ratio
–0.2 0.2 0.6–1.0
GainLoss
Nucleus
β2ARs
P
U
U
Prolonged
lowering
of p53
ARRB1
p53
P
PI3K/AKT
p53
ARRB1
Accumulation of
DNA damage MDM2
MDM2
Iso-induced
chr4
*
Ns
1234
f
Gs/PKA
WB:p53
WB:ARRB1
WB:γ-H2AX
p53
–++
pcDNA
+–+
H-89 ––++
WB:β-tubulin
p53-null H1299 cells
Iso + + + +
12 3 4
1
2
3
4
NsIso
DNA
γ-H2AX
Arrb1–/–
WT
Saline
Iso
Tcr trans-
rearrangement
Total
Arrb1–/– WT
Tcr trans-
rearrangement
Total
e
Iso Iso
19
Figure 4
|
Chronic catecholamine stimulation leads to accumulation of
DNA damage by an ARRB1- and p53-dependent mechanism.
a, Isoproterenol stimulation leads to formation of c-H2AX foci in wild-type but
not in Arrb1
2/2
MEFs. Wild-type and Arrb1
2/2
MEFs were chronically
stimulated with 10 mM isoproterenol every 12 h for 3 days. Cells were
immunostained and examined by confocal microscopy. Scale bar, 50mm.
b, Rescuing ARRB1 expression in Arrb1
2/2
MEFs restores isoproterenol-
induced c-H2AX foci. Two days after transfection, cells were stimulated and
examined as described in a. RFP, red fluorescent protein. Scale bar, 10mm.
c, ARRB1-dependent regulation of p53 levels mediates the accumulation of
isoproterenol-induced DNA damage. Two days after the transfection, cells
were stimulated as in a. Transfected cells, indicated with arrowheads, were
visualized with RFP. Scale bar, 10mm. d, Isoproterenol-induced accumulation
of DNA damage is synergistically suppressed by PKA inhibition and by
rescuing p53 expression. e, Isoproterenol stimulated, ARRB1-dependent
accumulation of DNA damage promotes rearrangements in chromosomes.
Total indicates PCR amplification of a non-specific locus (see Methods).
f, Isoproterenol infusion leads to decreased p53 levels in the testes from wild-
type, but not Arrb1
2/2
, mice. Wild-type and Arrb1
2/2
mice (n55 for each
condition) were treated as in Fig. 1a. All bars represent mean6s.e.m.
g, Isoproterenol-infused mice develop chromosomal rearrangements in an
ARRB1-dependent manner. Wild-type and Arrb1
2/2
mice were infused as in
Fig. 1a. Genomic DNA from each organ was examined in an array-CGH. The
data represent log
2
ratio plots (isoproterenol/saline) of genomic content in
chromosome 4 (chr4, 105–130 Mb), comparing isoproterenol-infused mice
with saline-infused mice of a same genotype. *, significance threshold of
1.0 310
27
(rank segmentation algorithm). The direction of isoproterenol-
induced chromosomal gain (green arrow) or loss (red arrow) is indicated.
Yellow highlights represent sites of isoproterenol-induced rearrangements.
h, Schematic diagram of b
2
-adrenoreceptor (b
2
AR)-dependent regulation of
DNA damage in response to prolonged secretion of catecholamines during
chronic stress.
RESEARCH LETTER
4 | NATURE | VOL 000 | 00 MONTH 2011
Macmillan Publishers Limited. All rights reserved
©2011
to a lowering of p53 levels in the testes. The effects are abolished in
Arrb1
2/2
mice (Fig. 4f). To examine these phenomena in a genome-
wide context, we conducted an array-comparative genomic hybridiza-
tion (array-CGH). In the same model of chronic stress
7,13
, genomic
DNA was isolated from the testes and thymus, which allowed us to
eliminate any changes due to meiotic recombination by considering
only rearrangements that occurred in both organs (see study design in
Supplementary Fig. 6f). These studies show that the only such
rearrangement occurring upon isoproterenol-infusion results in a
duplication of more than 1 megabase (Mb) in regions 4qD2.2 and
4qD1 in wild-type mice; however, these events are not observed in
the testes from Arrb1
2/2
mice (Fig. 4g and Supplementary Fig. 6f).
Quantitative PCR (qPCR) of the testicular genome from each mouse
also confirms isoproterenol-induced duplication at 4qD2.2 in an
ARRB1-dependent manner (Supplementary Fig. 6g). Taken together,
these data support the hypothesis that b
2
-adrenoreceptor- and
ARRB1-dependent signalling regulates catecholamine-induced degra-
dation of p53, thus leading to the accumulation of DNA damage in
both somatic and germline cells (Fig. 4h).
The stress response is conserved in mammals, and is probably
required for survival. However, psychosocial stress in humans is not
time-limited, because aspects of this type of stress response can be
sustained over months or even years. This may lead to prolonged
secretion of stress hormones and consequent adverse effects for the
individual. Indeed, clinicalstudies have shown marked risk-reductions
for prostate cancer, lung adenocarcinoma and Alzheimer’s disease
associated with chronic b-blocker (b-adrenoreceptor-antagonist)
therapy
4,29,30
. It also seems plausible that such hormonal influences
on DNA damage may not be limited to the b
2
-adrenoreceptors.
METHODS SUMMARY
Experimental procedures. Each experiment was repeatedat least three times with
comparable results, unless indicated otherwise.
Cell culture conditions and treatments. Isoproterenol was prepared fresh for
each experiment by dissolving bitartrate salt (Sigma) immediately before stimu-
lation. To study chronic b-adrenergic effects, U2OS cells and MEFs were cultured
until confluent,then stimulated with 10 mM isoproterenol for 24 h unlessotherwise
indicated. To study c-H2AX formation, cells were cultured until 40–50% con-
fluent, then stimulated with 10mM isoproterenol every 12h for 3days. To study
phosphorylation of MDM2 at Ser 166 in MEFs, cells were serum-starved for 4 h,
then stimulated with 10 mM isoproterenol for 1 h. H-89 (10 mM), leptomycin B
(10 nM), ICI 118,551 (10 mM), wortmannin (100 nM), 5-(2-benzothiazolyl)-3-
ethyl-2-(2-(methylphenylamino)ethenyl)-1-phenyl-1H-benzimidazolium iodide
(AKTi, 1 mM) or LY294002 (10 mM, Sigma) were addedto the media 30 min before
stimulation with isoproterenol.
Isoproterenol infusion. Mice were subcutaneously implanted with ALZET
osmotic pumps to administer saline or isoproterenol (30mg kg
21
d
21
) continu-
ously, dissolved in saline, for 28 days (mini-osmotic pump model 2004), following
the manufacturer’s procedure. After administration, animals were killed and the
indicated organs were dissected out. All animals used in these studies were adult
male mice of 8–12weeks of age. Animals were handled according to approved
protocols and animal welfare regulations of the Institutional Review Board at
Duke University Medical Center.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 16 July 2010; accepted 18 July 2011.
Published online 21 August 2011.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
AcknowledgementsR.J.L. is a Howard Hughes Medical Instituteinvestigator. This work
was supportedby HL16037 and HL70631 (R.J.L.).We thank D. Addison and Q. Lennon
for secretarialassistance; S. H. Snyder and M. A. Koldobskiy for providing p53 deletion
constructs; B. K. Kobilka and H. A. Rockman for providing Adrb2
2/2
mice; M. C. Hung
for providing the MDM2-S166D plasmid; A. K. Shukla, A. Kahsai, J. Kim, J. Sun,
S. M. DeWire and N. Odajima for discussion and comments.
Author Contributions M.R.H. and R.J.L. designed experiments, directed the study and
wrote the paper. M.R.H. performed most of the experiments, analysed the data and
prepared the figures. R.J.L. supervised the study and provided financial support. J.J.K.
performed some experiments, helped to analyse the data and helped to write the
paper.E.J.W., S.R., K.X., S.K.S.and S.A. helped to analysethe data. E.J.W.,S.R., R.T.S., B.W.,
C.M.L. and S.A. performed some experiments. A.J.T. and S.G.G. performed the
array-CGH and helped to analyse the data. W.G. and D.R.D. helped to characterize the
functionality of p53 and to analyse the data.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to R.J.L. (lefko001@receptor-biol.duke.edu).
LETTER RESEARCH
00 MONTH 2011 | VOL 000 | NATURE | 5
Macmillan Publishers Limited. All rights reserved
©2011
METHODS
Reagents. Unless otherwise noted, chemicals were purchased from Sigma.
Antibodies. Antibodies used were as follows, indicated WB for western blotting,
IP for immunoprecipitation and CM for confocal microscopy. PARP-1 (WB,
1:500 dilution, Alexis Biochemicals). FOXO3a (WB, 1:500), MDM2 phosphory-
lated at Ser 166 (WB, 1:1,000), FOS (WB, 1:500), PUMA (WB, 1:500), p21 (WB,
1:500), all from CellSignaling. Mouse p53 (FL-393: IP, 2 mg;WB, 1:200; CM, 1:50),
human p53 (DO-1: WB, 1:5,000), MDM2 (SMP14: IP, 1 mg), ARRB1 (K-16: IP,
1mg), ubiquitin (P4D1: WB, 1:200), human b
2
-adrenoreceptor (H-20: WB,
1:3,000), mouse b
2
-adrenoreceptor (M-20: WB, 1:200), anti-goat IgG–HRP
(WB, 1:10,000), all from Santa Cruz. ARRB1 (10: WB, 1:200; CM, 1:50; BD
Biosciences). LDH (WB, 1:300; Calbiochem). Mdm2 (HDM2-323: WB, 1:200),
b-tubulin I (SAP.4G5: WB, 1:10,000), both from Sigma. Histone H2B (WB,
1:5,000), histone H2B phosphorylated on Ser14 (WB, 1:5,000), c-H2AX (WB,
1:1,000; CM, 1:100), all from Millipore. 53BP1 (WB, 1:1,000; Novus). p21 (WB,
1:200; Rockland). Anti-mouse IgG–HRP (WB, 1:10,000), anti-rabbit IgG–HRP
(WB, 1:10,000), both from GE Healthcare. Rabbit polyclonal ARRB1 antibody
(A1CT: WB, 1:20,000) was generated as previously described
31
.
Primers. Tcrg a1: 59-ACCATACACTGGTACCGGCA-39,Tcrg b1: 59-ACCCC
TACCCATATTTTCTTAG-39,Tcrb a2: 59-TCTACTCCAAACTACTCCAG-39,
Tcrb b2: 59-CCTCCAAGCGAGGAGATGTGAA-39, non-specific locus (chr 7)
forward: 59-AGGCCTGGCTAGGCTTTTGGAATCTTTC-39, non-specific locus
(chr 7) reverse: 59-TGCCAGTGCTGGTGCGTGTGCACGGCTGT-39, qPCR
chr4 qD2.2 forward: 59-TGGTGCTGGCACAACTGGCA-39, qPCR chr4 qD2.2
reverse: 59-TGACGGTGTCTTTTGCCTTACAGAAGC-39, qPCR control (chr1)
forward: 59-CCTCCCATCAACGTTCAGGAGCC-39, qPCR control (chr1)
reverse: 59-ACTGCTTCTGCTCCAAACCCTGC-39,p21 promoter forward:
59-CCAGAGGATACCTTGCAAGGC-39,p21 promoter reverse: 59-TCTCTGT
CTCCATTCATGCTCCTCC-39.
Peptides. The synthesis of ARRB1-bindingpeptide (ARRB1-BP: V
2
Rpp) has been
described elsewhere. The sequence of the peptide, with phosphorylation sites
underlined, is: ARGRTPPSLGPQDESCTTASSSLAKDTSS (ref. 32).
Plasmids. The MDM2-S166D plasmid
17
and p53 deletion constructs
33
and were
gifts from M. C. Hung and S. H. Snyder, respectively. Plasmids encoding shRNAs
against p53 (psiRNA-mp53 and psiRNA-hp53), and the control psiRNA-LucGL3,
were purchased from InvivoGen.
Experimental procedures. Each experiment was repeatedat least three times with
comparable results, unless indicated otherwise.
Immunoblotting. SDS polyacrylamide gel electrophoresis (SDS–PAGE) was per-
formed on 1.0-mm-thick NuPAGE 4–12% Bis-Tris gels (Invitrogen) and sepa-
rated proteins were transferred to nitrocellulose membranes by semi-dry transfer,
using trans-blot transfer medium (Bio-Rad). Blots were blocked with blocking
buffer (5% skimmed milk in PBS with 0.02% Tween-20) before incubation at
4uC overnight with primary antibodies, diluted in blocking buffer as described
above. Blots were washed threetimes for 5 min each in PBS with 0.02% Tween-20,
and then incubated with secondary antibodies in blocking buffer. Blots were
washed three times for 5min each in PBS with 0.02% Tween-20, and developed
by SuperSignal West Pico/Femto solution (Pierce). Each protein band of interest
on the immunoblot was quantified by densitometryusing the GeneTools program
(SynGene).
Co-immunoprecipitation. Cells were lysed in a lysis buffer(50 mM Tris (pH 7.4),
150 mM NaCl, 0.1% CHAPS buffer, 0.1 mg ml
21
BSA, 1 mM PMSF and 1 mM
EDTA, with Halt protease and phosphatase inhibitor cocktail (Pierce)), and
homogenized by passing through a 28-gauge needle 20 times. Crude lysates were
cleared of insoluble debris by centrifugation at 14,000g. Extra lysis buffer was
added to 100–500 mg of cell lysate to bring samples to a total volume of 1 ml.
Immunoprecipitating antibody (1–2 mg) was added and incubated on a rotator
at 4 uC overnight. On the following day, 25ml (50% slurry) of the appropriate
TrueBlot IP beads (eBioscience) was added and incubated on a rotator at 4 uC
for 1 h. The beads were washed five times with the lysis buffer and quenched with
30 ml of SDS sample buffer (32). For detection of p53 ubiquitination (Fig. 3b),
10 mM N-ethylmaleimide and 20 mM MG132 were added to the lysis buffer. Co-
immunoprecipitation after cell fraction was conducted as previously described
34
.
Briefly, cells were lysedin RIPA A buffer (0.3% Triton X-100, 50 mM Tris (pH 7.4)
and 1 mM EDTA), with rotationat 4 uCfor 30 min. Cell lysates were centrifugedat
14,000gfor 10min and the supernatant was used as the cytosolic fraction. The
nuclear fraction was extracted from the pellet with RIPA B buffer (1% Triton
X-100, 1% SDS, 50 mM Tris (pH7.4), 500mM NaCl and 1 mM EDTA),
affinity-precipitated with the indicated antibodies, and subjected to SDS–PAGE.
Subcellular fractionation. U2OS cells from a 10-cm plate were resuspended in
300 ml of buffer B (0.25M sucrose, 10 mM Tris (pH 7.4), 10 mM MgCl
2
,10mM
KCl, 1 mM DTT and protease inhibitorcocktail without EDTA) and homogenized
with 150 strokes in a 1-ml dounce tissue grinder (Wheaton) using a tight pestle on
ice. After centrifugation at 750gfor 10 min, the supernatant was isolated to
separate the cytosolic fraction. The pellet was washed twice with buffer B, and
resuspended in 100–200 ml of buffer B. The suspension was analysed as the nuclear
fraction. Cytosolicfractions and nuclei were also prepared by using Nuclei EZ prep
nuclei isolation kit (Sigma), following the manufacturer’s protocol, and com-
parable results were obtained. To detect the effects of PI3K inhibition by
LY294002 on isoproterenol-induced p53 nuclear export, U2OS cells were pre-
incubated with 10 mM LY294002 for 30min, and then stimulated with 10mM
isoproterenol for 1h. To detect the effects of nuclear export on decreased nuclear
p53, U2OS cells werepre-incubated with 10 nM leptomycin B for 30 min,and then
stimulated with 10mM isoproterenol for 1h. To prepare a total-cell extract, cell
pellets were lysed in a lysis buffer (50mM Tris (pH7.4), 150 mM NaCl, 0.1%
CHAPS, 0.1 mg ml
21
BSA, 1 mM PMSF and 1 mM EDTA, with Halt protease
and phosphatase inhibitorcocktail (Pierce)) and homogenized by passing through
a 28-gauge needle 20 times. Crude lysates were cleared of insoluble debris by
centrifugation at 20,000g.
Cell culture conditions and treatments. Wild-type MEFs(passage number ,72),
Arrb1
2/2
MEFs (passage number ,76) and Arrb2
2/2
MEFs (passage number
,49) were prepared according to the 3T3 protocol
35,36
. Established MEF cultures
and RAW264.7 cells were maintained in Dulbecco’s modified Eagle medium
(DMEM)with 10% FBS and 2 mM L-glutamineat 37 uCwitha5%CO
2
atmosphere
in a humidified incubator. U2OS,HEK-293 and NCI-H1299 cells were maintained
in modified Eagle medium (MEM) with 10% FBS and 2 mM L-glutamine, withthe
same conditions as above. U2OS and NCI-H1299 cells were transfected with
FuGENE6 transfection reagent (Roche) following the manufacturer’s protocol.
For RNA interference for ARRB1,the vector system shRNA was usedas previously
described
37,38
. Briefly, U2OScells in 10-cm plates were transfected with either 10 mg
controlshRNA plasmid (59-ACGTGACACGTTCGGAGAATTGATATCCGTTC
TCCGAACGTGTCACGTTT-39)or10mgARRB1 shRNA plasmid (59-ATTCT
CCGCGCAGAAGGCTTT GATATCCG AGCCTTCTGCGCGGAGAATTT-39),
and incubated for 72 h. HEK-293 cells and MEFs were transfected with lipofecta-
mine 2000 (Invitrogen) following the manufacturer’s protocol. Briefly, 2 mgofDNA
was dissolved in 35 ml of serum- and antibiotic-free medium per well, in a 6-well
plate. Lipofectamine 2000 (10 ml) was mixedwith 25 ml of serum-and antibiotic-free
medium, and incubated for 5 min. The prepared DNA and lipofectamine 2000
solutions were mixed and the mixture was incubated for 20 min at 18–23 uC.
During the incubation, normal cell-culture medium was replaced with serum-
and antibiotic-free medium. The transfection mixture was added to the cells in
serum-free culture, and incubated overnight. On the following day, the medium
was replaced with normal serum- and antibiotic-containing growth medium, and
the cells were incubated for 48–72h before testing.
Isoproterenol, epinephrine and norepinephrine were prepared fresh for each
experiment by dissolving the bitartrate salts (Sigma) immediately before stimulation.
To study chronic b-adrenergic effects, U2OS cells and MEFs were cultured until
confluent, then stimulated with 10 mM isoproterenol for 24 h, unless otherwise indi-
cated. To study c-H2AX formation, cells were cultured until 40–50% confluent, then
stimulated with 10 mM isoproterenol every 12 h for3 days. H-89 (10 mM), leptomycin
B (10 nM), ICI 118,551 (10 mM), wortmannin (100 nM), 5-(2-benzothiazolyl)-3-
ethyl-2-(2-(methylphenylamino)ethenyl)-1-phenyl-1H-benzimidazolium iodide
(AKTi, 1 mM) or LY294002 (10 mM, Sigma) were addedto the media 30 min before
stimulationwith isoproterenol. To study the effectsof isoproterenol stimulation on
cell proliferation after DNA damage, cells were ultraviolet-irradiated (50 Jper m
2
)
and incubated for 6 h, followedby stimulation with 10 mM isoproterenol every12 h
for 3 days. Cell lysates were examined by immunoblotting for FOS,an indicator of
cell survival and proliferation
39
. To study phosphorylation of MDM2 at Ser 166 in
MEFs, cells were serum-starved for 4h, thenstimulated with 10 mM isoproterenol
for 1 h. To study this phosphorylation event in U2OS cells, cells were serum-
starved for 36h, then stimulated with 10mM isoproterenol for 10 min.
In vitro
ubiquitination assay. 10 nM His-p53 (ProteinOne) was mixed with
200 ng E1 (BostonBiochem), 200 ng UbcH5b (BostonBiochem), 5mg ubiquitin
(BostonBiochem) and 25 nM MDM2 in 20 ml of reaction mixture (40mM Tris
(pH 7.6), 2 mM ATP-Mg
21
, 1 mM dithiothreitol and 5 mM MgCl
2
). Purified
recombinant ARRB1 (0, 50 or 500nM) was added to the reaction mixture in
the presence or absence of 300nM ARRB1-BP. The sample was incubated for
60 min at 30 uC, resolved by SDS–PAGE and analysed by immunoblotting with
anti-p53 antibody (DO-1).
Isoproterenol infusion. Wild-type (C57BL/6), Arrb1 knockout (Arrb1
2/2
)
40
or
b
2
-adrenoreceptor knockout (Adrb2
2/2
)
41
mice were subcutaneously implanted
with ALZET osmotic pumps to administer saline or isoproterenol (30mg kg
21
d
21
) continuously, dissolved in saline, for 28days (mini-osmotic pump model
2004), following the manufacturer’s procedure. After administration, animals
were killed and the indicated organs were dissected out. For protein preparation,
dissected organ tissues were lysed and sonicated in RIPA buffer (50mM Tris
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©2011
(pH 7.4), 500mM NaCl, 1% SDS, 1% Triton X-100 and 1mM EDTA, with Halt
protease and phosphatase inhibitor cocktail). Genomic DNA was prepared from
dissected organ tissues by DNeasy blood & tissue kit (Qiagen), following the
manufacturer’s protocol. All animals used in these studies were adult male mice
of 8–12 weeks of age. All mouse strains were backcrossed to the C57BL/6
background for $10 generations. Animals were handled according to approved
protocols and animal welfare regulations of the Institutional Review Board at
Duke University Medical Center.
Quantitative real-time PCR (qPCR). qPCR was performed with Power SYBR
Green PCR Master Mix (Applied Biosystems) and StepOne Real-time PCR system
(Applied Biosystems) following the manufacturer’s protocol. To validate the
array-CGH analysis, relative genomic content (copy number) was determined
with the comparative C
T
(DDC
T
) method
42
.
GST pulldown assay. Wild-type rat Arrb1 or human MDM2 were subcloned into
the pGEX4T1 vector and prepared according to the manufacturer’s recommenda-
tions (Amersham Biosciences). The GST tag was cleaved with thrombin protease
(Hematologic Technologies Inc.). p53 (1nM) was co-incubated overnight with
10 nM of GST–ARRB1 or 10nM of GST at 4uC in 1 ml binding buffer (50mM
Tris (pH 7.4), 150 mM NaCl, 0.1 mg ml
21
BSA and 10 mM D-myo-inositol
1,2,3,4,5,6-hexakisphosphate), and 20 ml of 50% glutathione–sepharose was then
added to the mixture. The mixture was further incubated at 4 uC for 1 h with
rotation. The beads were washed once with 1ml binding buffer, separated by
SDS–PAGE and analysed by immunoblotting.
Immunofluorescence experiments. Immunofluoresence using confocal micro-
scopy was carried out as previously described
43
. For detection of c-H2AX foci, we
captured images of more than 20 fields per preparation, which were randomly
chosen in a blind manner. Cells positive for c-H2AX foci in each field were tallied
and added together to determine the percentage. The total number of cells was
counted with 49,6-diamidino-2-phenylindole (DAPI) nuclear staining. For rescue
experiments using the expression of RFP–ARRB1 (or RFP as a control) in MEFs,
RFP-positive cells were counted for c-H2AX foci. For p53 rescue experiments in
NCI-H1299 cells, RFP–Arrb1 (or RFP) and p53 were co-transfected in a 1:3 ratio.
Detection of interchromosomal rearrangements between
Tcrg
and
Tcrb
.The
trans-rearrangement between Tcrg and Tcrb loci were detected by nested PCR,
using first the ‘a’ set of primers and then the ‘b’ set of primers, as previously
described
44
(Supplementary Fig. 6d). The number of rearrangements is expressed
as the reciprocal of the highest dilution of DNA yielding an amplified product (for
example, the number of trans-rearrangements per 1.5 310
5
cells (1 mgofDNA)is
1,000, yielding an amplifiable fragment at a 1:1,000 dilution (1ng of DNA))
44
.
DNA preparation. DNA was prepared from the testes and thymus by using the
DNeasy blood & tissue kit (Qiagen) following the manufacturer’s protocol. To
enrich sperm from excised testis grafts, the testis was minced and the epithelial
tissue, containing leydig and sertoli cells, was removed.
Array-comparative genomic hybridization (Array-CGH). A tiling-path CGH
array for the genome analysis in mouse (UCSC Build mm9) was designed and
constructed by NimbleGen Systems (NimbleGen). The resulting array contained
720,000 probes with a median probe spacing of 3,537 base pairs. Probes were
synthesized using an isothermal format (melting temperature 76 uC), and varied
in length from 50 to 75base pairs. Genomic DNA from five mice for each experi-
mental condition was pooled and examined. Genomic DNAs (1 mg) from test
(isoproterenol-treated) and reference (saline-treated) mice were differentially
labelled with 59-Cy3 and 59-Cy5 random nonamers (TriLink Biotechnologies),
respectively, and hybridized to the oligoarray for 72 h using the MAUI hybridiza-
tion station (BioMicro Systems Inc.). Image-capture of the hybridized arrays for
fluorescent intensity extraction was performed using a Genepix 4100A scanner
(Molecular Dynamics) and normalized using Nimblescan v2.5 microarray soft-
ware (Nimblegen) before importing into Nexus Copy-Number (BioDiscovery) for
analysis.
Array-CGH analysis. BioDiscovery’s rank segmentation algorithm, whichis similar
to circular binary segmentation
45
, was used to identify genomic rearrangements. The
significance threshold was set as 1.0 310
27
. The calling algorithm used cluster values
and defined log
2
thresholds of 60.2. We applied a cutoff of ten oligomer clones
showing the same trend in copy-number change to define chromosomal rearrange-
ments. Black lines in the plot indicate a ‘cluster value’, which is the median log-ratio
value of all the probes in that region. Isoproterenol-induced rearrangements in the
testes were determined, and identical rearrangements were detected in the thymus
(see study design in Supplementary Fig. 6f).
Radioligand binding experiments. For ICI 118,551 and CGP 20712A affinity
measurements, subtype-selective ligand affinities were determined from competi-
tion radioligand binding experiments, conducted according to previousworks
46,47
.
Briefly, 25 mg of cell membranes, prepared via differential centrifugation, were
resuspended in assay buffer (50mM Tris-HCl (pH 7.4), 12.5 mM MgCl
2
,2mM
EDTA and 1 mM ascorbic acid) containing 60pM [
125
I]cyanopindolol (NEX189,
2,200 Ci mmol
21
) and concentrations of ICI 118,551 or CGP 20712A ranging
from 1 mM to 1 pM. Nonspecific binding was determined in the presence of
10 mM propranolol. After incubation at 25uC for 90 min, membranes were
collected and washed via vacuum filtration (Brandel) and the bound radioactivity
was quantified using a Packard Cobra gamma counter(Perkin Elmer). Equilibrium
inhibition constant (K
i
) values were calculated from nonlinear regression analysis
(Graphpad) using the method in ref. 48.
p53 reporter assay. U2OS cells were transfectedwith the p53-luc reporter plasmid
(Stratagene) in the presence of serum, using FuGENE6 transfection reagent
(Roche). Three hours after the transfection, media were changed to serum-free
media containing 100mM ascorbic acid and appropriate concentrations of iso-
proterenol. Cells wereincubated for 24 h and lysed in 31 passive lysisbuffer (PLB,
Promega). The firefly luciferase reporter was analysed with addition of luciferase
assay reagent II (Promega).
Chromatin immunoprecipitation assay (ChIP) and Re-ChIP. ChIP was per-
formed as previously described
49
. In brief, both wild-type and Arrb1
2/2
MEFs
were incubated with 50 mM etoposide for 20 h. After incubation, cells were treated
with 2 mM disuccinimidyl glutarate (Pierce) to crosslink protein complexes, then
treated with formaldehydeto link protein to DNA covalently. Cells were lysed and
the nucleoprotein complexes were sonicated. DNA–protein complexes enriched
by the initial immunoprecipitation withanti-ARRB1 (K-16) antibody were eluted
from beads with elution buffer (1% SDS, 0.1M NaHCO
3
), and further immuno-
precipitated with anti-p53 (FL-393) antibody for Re-ChIP. The retrieved com-
plexes were then analysed by PCR amplification of p53-binding elements in the
p21 promoter.
Statistics. Unless otherwise noted, Pvalues were calculated with Student’s t-test
(two-tailed). Analysis of variance was performed with Prism (GraphPad).
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©2011
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The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor (β2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.
Article
A theoretical analysis has been made of the relationship between the inhibition constant (KI) of a substance and the (I50) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between KI and I50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I50 values in agreement with the admonitions previously described in the literature. The analysis described shows KI does not equal I50 when competitive inhibition kinetics apply; however, KI is equal to I50 under conditions of either noncompetitive or uncompetitive kinetics.