close

Вход

Забыли?

вход по аккаунту

?

Reduced p53 in peripheral blood mononuclear cells from patients with rheumatoid arthritis is associated with loss of radiation-induced apoptosis.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 52, No. 4, April 2005, pp 1047–1057
DOI 10.1002/art.20931
© 2005, American College of Rheumatology
Reduced p53 in Peripheral Blood Mononuclear Cells From
Patients With Rheumatoid Arthritis Is Associated With
Loss of Radiation-Induced Apoptosis
Kevin Maas, Matthew Westfall, Jennifer Pietenpol, Nancy J. Olsen, and Thomas Aune
apoptosis in patients with RA. We hypothesize that this
liability may contribute to autoimmunity.
Objective. Patients with autoimmune disorders
exhibit highly reproducible gene expression profiles in
their peripheral blood mononuclear cells. This profile
includes, at least in part, a collection of underexpressed
genes that encode proteins that inhibit cell cycle progression and stimulate apoptosis. We aimed to determine whether this gene expression profile confers functional liability on lymphocytes from patients with
rheumatoid arthritis (RA).
Methods. Viability studies in response to a panel
of proapoptotic stimuli revealed that T lymphocytes
from patients with RA were resistant to gamma
radiation–induced apoptosis, a process known to be
dependent on p53. To assess p53 function in RA peripheral blood mononuclear cells, baseline levels of p53
protein and TP53 transcript were measured in patients
with RA and controls. The cellular p53 response to
gamma radiation was also assessed by immunoblotting.
Results. Lymphocytes from patients with RA had
lower baseline levels of TP53 messenger RNA (mRNA)
and p53 protein than did those from control subjects
and were deficient in their ability to increase p53 after
exposure to gamma radiation. A subgroup of patients
with RA had a second biochemical defect characterized
by expression of very low baseline levels of checkpoint
kinase 2 mRNA and protein.
Conclusion. We conclude that defects in the expression of TP53 mRNA and, in a subgroup, defects in
expression of CHK2 mRNA, lead to severe defects in
Autoimmune diseases, which are characterized
by the site(s) of immune-mediated destruction, affect
3–5% of the population. Autoimmune disorders range
from those associated with organ-specific attacks, as in
multiple sclerosis (MS) and type I diabetes (insulindependent diabetes mellitus [IDDM]), to those with
more systemic manifestations, as in rheumatoid arthritis
(RA) and systemic lupus erythematosus (SLE). Despite
the prevalence of these disorders, the mechanisms underlying their pathogenesis remain poorly understood
(1). There is also significant clinical heterogeneity within
patient groups, with variation in disease activity, time of
onset, and response to therapy (2). Because of this
clinical heterogeneity, finding common underlying defects in human autoimmune disease has proven difficult (3).
There are numerous ways to induce clinical manifestations of autoimmunity in animal models (4). This
raises the following question: are there also numerous
underlying mechanisms that cause human autoimmune
disease? Recent studies examining gene expression profiles in peripheral blood mononuclear cells (PBMCs)
revealed identifiable similarities among patients with
autoimmune disease. For example, interferon-␣ (IFN␣)–
and IFN␤-responsive gene expression signatures are
present in PBMCs from both children (5) and adults (6)
with severe lupus, supporting a potential role of these
cytokines in SLE pathogenesis. IFN signatures are
linked to disease activity.
Common genes encoding proteins that regulate
cell cycle progression and apoptosis are differentially
expressed in many autoimmune disorders (7–9). In
particular, our previous study revealed a common gene
expression profile among patients with RA, SLE,
IDDM, or MS (8). This expression signature was ob-
Supported by grants from the NIH (T32-GM-07347, RR00095, DK-58765, AI-44924, AI-53984, and CA-070856).
Kevin Maas, BS, Matthew Westfall, BS, PhD, Jennifer Pietenpol, BS, PhD, Nancy J. Olsen, MS, MD, Thomas Aune, BS, PhD:
Vanderbilt University, Nashville, Tennessee.
Address correspondence and reprint requests to Thomas
Aune, BS, PhD, Vanderbilt University Medical Center, MCN T3219,
1161 21st Avenue South, Nashville, TN 37232. E-mail: thomas.
aune@vanderbilt.edu.
Submitted for publication April 6, 2004; accepted in revised
form December 13, 2004.
1047
1048
MAAS ET AL
served in all of the patient populations studied and
perfectly discriminated autoimmune patients from controls. In contrast to the expression patterns described in
the lupus studies cited above, this expression pattern
does not correlate with disease severity and is present in
unaffected first-degree relatives, suggesting that there is
a heritable component.
To gain insight into the significance of these
altered gene expression profiles, we examined lymphocytes obtained from patients with RA for defects in
apoptosis. We observed a marked resistance to gamma
radiation–induced apoptosis in patients with RA. The
tumor suppressor protein p53 plays a key role in the
gamma radiation–induced apoptotic response by T cell
receptor ␣/␤ T cells and B lymphocytes in the peripheral
blood of both murine models and humans (10,11).
PBMCs obtained from patients with RA had lower
baseline levels of TP53 messenger RNA (mRNA) and
p53 protein and were deficient in their ability to increase
p53 after exposure to gamma radiation. PBMCs from
approximately half of the RA patients had a second
defect, which was markedly reduced expression of
checkpoint kinase 2 (CHK2) mRNA and protein. Chk2
is one of the upstream regulators of p53.
We conclude that lymphocytes from patients with
RA have at least 2 prominent defects in the p53 damageresponse pathway. One of these defects is underexpression of TP53 mRNA and p53 protein (which was
observed in all RA patients), and the second is underexpression of CHK2 mRNA and Chk2 protein (which
was observed in ⬃50% of RA patients). We hypothesize
that these defects represent a cellular liability that may
contribute to development of autoimmune disease.
PATIENTS AND METHODS
Patient populations. The study group comprised 43
control subjects who had no current chronic or acute infection
and no family history of autoimmunity, and 52 patients meeting the American College of Rheumatology (ACR; formerly,
the American Rheumatism Association) clinical criteria for
RA (12). In both the control and patient populations, the
approximate female-to-male ratio was 3:1. The age ranges
(22–55 years) and racial distributions in both groups were
similar. Human subject studies were approved by the Committee for the Protection of Human Subjects of the Vanderbilt
University Institutional Review Board.
Sample processing and PBMC isolation. PBMCs were
isolated from heparinized blood by centrifugation on a Histopaque gradient (Sigma, St. Louis, MO). Isolated PBMCs
were washed twice in HEPES buffered saline.
Analysis of cell viability by flow cytometry. PBMCs
were suspended at 1 ⫻ 106 cells/ml in complete media (RPMI
1640 medium, 10% fetal calf serum, glutamine, and penicillin/
streptomycin). Pilot experiments with control PBMCs were
used to establish optimal doses and time points for each
apoptosis-inducing agent. Based on results from these pilot
experiments, cells were left untreated or were treated with
different apoptosis-inducing agents, as follows: 10-Gy gamma
radiation, 5 ␮M dexamethasone, 1 ␮M staurosporine, or 100
J/m2 ultraviolet (UV) radiation. At appropriate time points,
cells were harvested and washed with fluorescence-activated
cell sorting (FACS) buffer (10% bovine serum albumin in
phosphate buffered saline [PBS] with 0.2% sodium azide) and
incubated with fluorescein isothiocyanate– and phycoerythrinlabeled antibodies against CD3, CD4, CD14, CD19, and
CD45RO (Becton Dickinson, San Jose, CA). Cells were
washed and suspended in 500 ␮l FACS buffer supplemented
with 2 ␮l of 0.5 ␮M 7-aminoactinomycin D (7-AAD; Molecular
Probes, Eugene, OR) as a viability marker prior to flow
cytometric analysis. Samples were analyzed for 1 minute at
high flow rate. Cells positive for 7-AAD were excluded from
analysis, and the total numbers of remaining lymphocytes were
tabulated to determine viability.
RNA isolation and quantitative polymerase chain
reaction (PCR). TRI Reagent (Molecular Research Center,
Cincinnati, OH) was used to isolate total RNA from PBMCs.
Five micrograms of total RNA was reverse transcribed with
SuperScript II reverse transcriptase (Gibco BRL Life Technologies, Rockville, MD) to prepare complementary DNA
(cDNA). Complementary DNA was also prepared from the
HCT116 cell line in order to construct relative standard curves.
TP53- and GAPDH-specific primers were used to amplify
cDNA samples with SYBR Green PCR Master Mix (Applied
Biosystems, Foster City, CA). Fluorescence was monitored
using an ABI PRISM 7000 detector (Applied Biosystems).
Relative quantities of TP53 and GAPDH transcripts in control
and patient samples were calculated using a standard curve
derived from the HCT116 cell line.
Western blot analysis. Whole cell lysates were prepared in 1⫻ PBS, 1% Nonidet P40, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulfate, plus a protease inhibitor cocktail
(Sigma). For each sample, equal amounts of total protein were
electrophoresed and transferred to Immobilon-P membranes
(Millipore, Bedford, MA). Membranes were blocked in a 5%
nonfat milk, 0.1% Tween 20 in PBS, and probed with combinations of the following primary antibodies diluted in 1%
nonfat milk with PBS plus 0.1% Tween 20: p53, Chk2, ␤-actin
(Santa Cruz Biotechnology, Santa Cruz, CA), p21 (Oncogene
Research Products, San Diego, CA), p45 up-regulated modulator of apoptosis (PUMA; Abcam, Cambridge, MA),
poly(ADP-ribose) polymerase 1 (PARP-1; Cell Signaling
Technology, Beverly, MA), and proliferating cell nuclear antigen (PCNA; Calbiochem, San Diego, CA). Membranes were
washed 3 times with PBS–Tween (PBST), and probed with
goat anti-mouse horseradish peroxidase–conjugated secondary
antibodies (Santa Cruz Biotechnology) in 1% nonfat milk in
PBST. Membranes were washed 3 times for 20 minutes with
PBST. The ECL Plus Chemiluminescent Immunodetection Kit
(Applied Biosystems) was used to visualize bands.
Western blot analysis of luminescence intensity. Multiple exposures of films were captured using the Fluor-S MAX
imaging system (Bio-Rad, Hercules, CA). Both background
chemiluminescence and chemiluminescence intensities for individual bands were measured. Background chemilumines-
TP53 AND APOPTOSIS DEFECTS IN RA
1049
Figure 1. Defects in gamma radiation–mediated apoptosis in rheumatoid arthritis (RA) peripheral blood mononuclear cells
(PBMCs). A, Cell viability as assessed by flow cytometry of PBMCs from control (ctrl) subjects (n ⫽ 5) and RA patients (n ⫽
6) 1–3 days after gamma radiation (10 Gy). B, PBMCs from control subjects (n ⫽ 10) or RA patients (n ⫽ 12) were treated
with different apoptosis-inducing agents. Relative cell viability for whole PBMCs and for CD4⫹, CD8⫹, and CD19⫹
lymphocytes was determined 3 days (dexamethasone, ultraviolet [UV] radiation, and gamma radiation) or 1 day (staurosporine)
after treatment, by flow cytometry. C, Scatter plot of CD4⫹ T lymphocyte viability in controls (n ⫽ 6) and RA patients (n ⫽
12) 3 days after exposure to 10-Gy gamma radiation. D, Viability of CD3⫹,CD45ROhigh and CD3⫹,CD45ROlow T lymphocytes
in controls (n ⫽ 6) and RA patients (n ⫽ 12) 3 days after exposure to 10-Gy gamma radiation. Bars show the mean ⫾ SD.
cence was subtracted from all band intensities. Images of bands
that were overexposed (as detected in the Fluor-S MAX
software suite) were excluded from analysis. Intensities of
samples were normalized relative to baseline HCT116 levels in
order to make interblot comparisons and compensate for
differences in exposure time.
Reverse transcription–PCR (RT-PCR) and semiquantitative PCR analysis. Complementary DNA prepared from
PBMC RNA from control subjects or patients with RA was
used for Chk2 and GAPDH amplifications. Thirty cycles were
used for initial RT-PCR amplification of samples. Semiquantitative PCR was performed on control and RA samples. PCRs
were carried out as described above, with the exception that
cDNA was serially diluted, and 35 cycles were used for
amplification.
Statistical analysis. Results are expressed as the
mean ⫾ SD. Statistically significant differences between
groups were determined by Student’s t-test. P values less than
0.05 were considered significant.
RESULTS
Defects in gamma radiation–induced apoptosis
in RA PBMCs. A unique gene expression profile in
PBMCs obtained from patients with autoimmune disease suggests that these cells may exhibit defects in
apoptosis (7–9). For example, one of the most underexpressed genes in RA PBMCs is TP53, the gene that
encodes the tumor suppressor protein p53. To test the
hypothesis that p53 function is compromised in PBMCs
obtained from patients with RA, PBMCs from 6 patients
with RA and from 5 control subjects were exposed to
10-Gy gamma radiation (a known p53-dependent proapoptotic stimulus), and cellular viability was determined over a 3-day time course. RA PBMCs exhibited
resistance to gamma radiation–induced cell death com-
1050
pared with control PBMCs (Figure 1A) (P ⬍ 0.005 for
all time points).
To determine whether this defect in apoptosis
reflected a generalized defect or a selective defect in
p53-dependent apoptosis, we performed additional studies using a panel of additional agents that induce apoptosis, as follows: dexamethasone, staurosporine, UV
radiation, and gamma radiation. PBMCs from 10 control
subjects and from 12 patients with RA were treated with
these agents and analyzed by flow cytometry for viability
of whole PBMCs and CD4⫹, CD8⫹, and CD19⫹
lymphocytes. Each apoptosis-inducing agent caused apoptosis in both PBMCs and lymphocytes (Figure 1B).
We found no difference in the level of apoptosis between RA and control PBMCs or lymphocytes after
treatment with dexamethasone and staurosporine.
Treatment with UV radiation revealed no significant
difference in overall PBMC or T lymphocyte viability;
however, B lymphocytes from control subjects were
more resistant to UV-mediated apoptosis compared
with those from RA patients.
Consistent with our initial observations, both
PBMCs and lymphocytes from patients with RA were
markedly resistant to gamma radiation–induced apoptosis relative to controls. This defect in apoptosis was most
pronounced in CD4⫹ and CD8⫹ T lymphocytes (P ⬍
0.002 and P ⬍ 0.001, respectively) and insignificant in
CD19⫹ lymphocytes (Figure 1B). A scatter plot of
CD4⫹ T cell viability after exposure to gamma radiation
in different individuals demonstrated a highly homogeneous gamma radiation–induced apoptosis response in
the control population, while there was substantial variability among lymphocytes from patients with RA (Figure 1C). Quantitative differences were observed in the
response to gamma radiation by CD45ROlow and
CD45ROhigh T cells. CD45ROlow T cells exhibited
greater cell death compared with CD45ROhigh cells
(Figure 1D). Both populations of RA lymphocytes were
more resistant to apoptosis induced by gamma radiation
than were control lymphocytes. These results revealed a
significant defect in gamma radiation–induced apoptosis
in T lymphocytes from patients with RA.
Underexpression of TP53 mRNA and p53 protein
in RA PBMCs. Because gamma radiation–induced apoptosis is dependent on functional p53 (10,13), low baseline levels of TP53 mRNA and p53 protein could
contribute to defective apoptosis in RA. Results from
previous microarray studies (8) revealed that TP53
transcript levels were lower in RA patients than in
controls. We included additional microarray data from
12 previously unanalyzed RA samples, in order to
MAAS ET AL
Figure 2. Baseline TP53 transcript and p53 protein levels in RA
PBMCs. A, Scatter plot displaying microarray results for TP53 transcript levels in control PBMCs (n ⫽ 9), PBMCs from patients with
early RA (mean ⫾ SD disease duration 1 ⫾ 0.2 years; n ⫽ 16), and
PBMCs from patients with established RA (mean ⫾ SD disease
duration 10 ⫾ 1 years; n ⫽ 9). B, Normalized TP53 transcript levels in
control (n ⫽ 4) and RA PBMCs (n ⫽ 5) as determined by quantitative
polymerase chain reaction (PCR). C, Representative immunoblot
comparing baseline p53 levels in PBMCs from a control cell line
(HCT116), control subjects (C1–3), and RA patients (RA1–3). ␤-actin
was included as an internal loading control. D, Normalized luminescence data for all individuals (control, n ⫽ 9; RA, n ⫽ 10) expressed
in relative units of p53 protein. Bars in B and D show the mean and SD.
See Figure 1 for other definitions.
compare TP53 expression levels with our previously
reported findings in control subjects and RA patients.
TP53 mRNA levels in control subjects were relatively
heterogeneous but were uniformly higher than the levels
in patients with RA (Figure 2A). Real-time RT-PCR
analysis confirmed that TP53 message levels were lower
in patients with RA than in controls (Figure 2B). These
TP53 AND APOPTOSIS DEFECTS IN RA
results revealed that TP53 mRNA was consistently underexpressed in the RA patient population relative to
the control population.
To further characterize p53 in the RA patient
population, we used Western blotting techniques to
measure baseline p53 levels. Whole cell extracts were
prepared from PBMCs obtained from 10 previously
unanalyzed control subjects and from 10 patients with
RA. Luminescence intensities were measured using a
Fluor-S MAX imaging system. A representative blot
using samples from human cell line HCT116 (as a
positive control), 3 control subjects, and 3 patients with
RA clearly demonstrated a marked reduction in p53
protein levels in RA PBMCs in the resting state (Figure
2C). Compiled luminescence data revealed that baseline
p53 levels were significantly lower (⬎10-fold) in RA
PBMCs compared with those in control PBMCs (P ⬍
0.001) (Figure 2D).
Levels of p53 after exposure to gamma radiation.
In addition to measuring basal levels of p53 in the RA
patient population, we examined p53 protein levels after
exposure to gamma radiation. Under normal circumstances, p53 levels are relatively low in the resting state
due to rapid turnover through the ubiquitin–proteasome
pathway (14). In response to DNA-damaging agents
(i.e., gamma radiation), the N-terminal domain of p53 is
phosphorylated, blocking protein turnover (13,15). As a
result, levels of p53 increase markedly after exposure to
gamma radiation. Protein p53 acts as a transcription
factor to induce damage-response target genes such as
CDKN1A (p21) (16), GADD45A (17), NOXA1 (18), and
BBC3 (PUMA) (19).
We measured increases in steady-state levels of
p53 and p21 induction in response to gamma radiation in
previously unanalyzed samples. PBMCs from control
subjects (n ⫽ 12) and RA patients (n ⫽ 12) were
challenged with exposure to 10-Gy gamma radiation or
remained untreated. Twenty-four hours after challenge,
whole cell extracts were prepared and immunoblotted
for p53, p21, and ␤-actin. The high intensity of ␤-actin
on the immunoblots made obtaining reproducible exposures between blots difficult, and thus it served as a
control for equal protein loading within a blot rather
than as a loading control among blots. Instead, the
HCT116 cell line was included as a positive control for
p53 responsiveness to gamma irradiation (20) and to
serve as an indicator for exposure time and normalization in later quantitative analyses. Representative immunoblots for control subjects and RA patients are
presented in Figure 3.
As expected, levels of p53 in PBMCs from con-
1051
Figure 3. Defects in p53 protein stabilization after exposure to
gamma radiation. Representative immunoblots of peripheral blood
mononuclear cells (PBMCs) cultured for 24 hours (control, n ⫽ 12;
rheumatoid arthritis [RA], n ⫽ 12) and either left untreated (⫺) or
treated with 10-Gy gamma irradiation (⫹). Samples were blotted for
p53 and p21, with ␤-actin indicating equal protein loading within a
blot. Based on steady-state p53 protein levels with and without
radiation, PBMCs from patients with rheumatoid arthritis (RA) were
segregated into gamma radiation nonresponders (n ⫽ 5) or gamma
radiation partial responders (n ⫽ 5).
trol subjects (n ⫽ 5) increased markedly in response to
gamma irradiation (Figure 3A). Increased levels of p21
confirmed downstream p53 transcriptional activity in
response to gamma irradiation. The p53-dependent response to gamma irradiation in PBMCs from patients
with RA (n ⫽ 10) differed from that in controls. The
most striking difference was that approximately half of
the patients failed to increase p53 steady-state levels
following exposure to gamma radiation (Figure 3).
Based on these results, RA patients were organized into
2 distinct groups. PBMCs from RA patients who were
1052
gamma radiation nonresponders (GNRs) failed to increase p53 levels in response to irradiation (Figure 3B).
PBMCs from RA patients who were gamma radiation
partial responders (GPRs) displayed a modest increase
in p53 levels, albeit to lower levels than those observed
in controls (Figure 3C). In addition to their failure to
increase levels of p53, PBMCs from GNRs did not
increase p21 levels after exposure to gamma radiation
(Figure 3B). In contrast, PBMCs from GPRs had increased p53 and p21 levels in response to gamma
irradiation, albeit to lower levels than those in controls
(Figure 3C).
Two distinct defects in the p53 damage response
in RA patients. In order to quantitate the relative
protein levels for all of the control, RA GNR, and RA
GPR samples examined in the gamma radiation experiment, we calculated luminescence intensities from the
immunoblots (Figure 4) (control subjects, n ⫽ 10; RA
GNRs, n ⫽ 7; RA GPRs, n ⫽ 5) using the Fluor-S MAX
imaging system. Luminescence intensities were normalized using the untreated HCT116 cell line, present on all
blots, in order to make comparisons among blots. Initially, we examined protein levels for the untreated and
irradiated conditions in control, GPR, and GNR groups.
To accomplish this, we normalized luminescence intensities using the untreated HCT116 cell line present on all
blots to make comparisons among blots. These results
revealed that p53 levels in untreated and gammairradiated cells from both the RA GNR and GPR groups
were significantly lower than those in controls (P ⬍
0.001 for both groups) (Figure 4A). We believe that
these lower p53 levels arise from lower TP53 transcript
levels in the RA patient population.
We also calculated the gamma radiation–induced
fold induction of p53 and p21 for each individual in the
control, GPR, and GNR groups. Fold induction was
calculated as the ratio of the luminescence intensity of
protein bands between the gamma-irradiated group and
the untreated group. Fold induction was averaged for all
individuals within the control, GPR, and GNR groups.
Both controls and RA GPRs demonstrated increased
induction of p53, in comparable magnitude, in response
to gamma irradiation (Figure 4B). This implies that,
despite low baseline p53 levels, the signaling events
needed to increase p53 levels after exposure to gamma
radiation are intact in GPRs. In contrast, the GNR
group showed undetectable induction of p53 after exposure to gamma radiation, suggesting that additional
defects are present in the p53-dependent damageresponse pathway in this group of RA patients. To a
MAAS ET AL
Figure 4. Quantitative analysis of response to irradiation in controls,
patients with rheumatoid arthritis (RA) who were gamma radiation
partial responders (GPRs), and RA gamma radiation nonresponders
(GNRs). Normalized luminescence intensities were compiled to permit quantitative comparisons among the groups described in Figure 3.
A, Untreated (Unt) and postradiation (IR) p53 levels were determined
in controls (n ⫽ 10), RA GPRs (n ⫽ 5), and RA GNRs (n ⫽ 7). B,
Average fold induction of protein in response to exposure to gamma
radiation was calculated for p53, p21, and ␤-actin among the different
groups. Fold induction is defined as the ratio of gamma-irradiated
luminescence intensity versus the untreated luminescence intensity.
Bars show the mean and SD. MDM2 ⫽ mouse double minute 2.
large degree, induction of p21 after gamma irradiation
mirrored the p53 results for the control, GPR, and GNR
groups.
Defective induction of effectors of apoptosis in
RA PBMCs. Protein p53 that accumulates after exposure to gamma radiation acts as a transcription factor to
induce proapoptotic target genes (21,22). Because we
observed defects in gamma radiation–induced apoptosis,
we investigated whether this was accompanied by defective induction of proapoptotic target genes. This was
accomplished by immunoblotting representative untreated and gamma-irradiated extracts from previously
analyzed controls (n ⫽ 2), RA GPRs (n ⫽ 2), and RA
TP53 AND APOPTOSIS DEFECTS IN RA
Figure 5. Defective induction of p53 downstream effectors in peripheral blood mononuclear cells from patients with RA. Whole cell
extracts from representative controls (n ⫽ 2), GPRs (n ⫽ 2), and
GNRs (n ⫽ 2). Untreated (⫺) and gamma-irradiated (⫹) samples
were immunoblotted 24 hours after challenge. Lysates were probed for
poly(ADP-ribose) polymerase 1 (PARP-1), proliferating cell nuclear
antigen (PCNA), p45 up-regulated modulator of apoptosis (PUMA),
and ␤-actin. See Figure 4 for other definitions.
GNRs (n ⫽ 2). Extracts were analyzed for p53 levels to
confirm their partial responder/nonresponder status.
Extracts were also probed for PUMA (19,23), PARP-1
cleavage, a biochemical marker for apoptosis (24),
PCNA as a measure of cellular proliferative status, and
␤-actin as a control. PCNA and ␤-actin levels were
relatively consistent among samples and thus also served
as protein-loading controls (Figure 5).
PBMCs from RA GNRs and, to a more limited
extent, those from RA GPRs demonstrated marked differences when compared with controls. Control PBMCs exhibited increased p53 levels in response to gamma irradiation, with corresponding increases in PUMA and
PARP-1 cleavage compared with untreated samples
(Figure 5). In contrast, PBMCs from RA GNRs exhibited negligible increases in p53 levels and PUMA after
exposure to gamma radiation. RA GNRs had lower
levels of both full-length and cleavage forms of PARP-1
and did not display increased PARP-1 cleavage after
exposure to gamma radiation. The RA GPRs exhibited
variability in p53 levels and downstream effector function in response to gamma radiation. One patient had
low p53 levels as well as negligible PUMA induction and
PARP-1 cleavage. The other RA patient had a somewhat higher response. These results provide further
evidence that downstream p53 apoptotic effector function is compromised in PBMCs from patients with RA.
1053
Correlation of decreased Chk2 expression with
RA GNR status. Our quantitative analysis of relative
protein levels and protein induction revealed that PBMCs
obtained from patients with RA contain lower levels of
p53 both before and after exposure to gamma radiation.
However, the GNR group appeared to have additional
defects in the p53-dependent damage response that
prevented them from increasing p53 levels after irradiation. To explore potential causes for this additional
defect, we examined Chk2, an upstream kinase that can
phosphorylate p53 (25) and prevent its ubiquitinmediated degradation (26). Extracts from previously
identified RA GNRs (n ⫽ 5), RA GPRs (n ⫽ 5), and
control subjects (n ⫽ 5) were reanalyzed for Chk2. All
control PBMCs contained comparable levels of Chk2
(Figure 6A). Levels of Chk2 in control PBMCs decreased after gamma radiation. RA GNRs had uniformly low levels of Chk2 (Figure 6B), while PBMCs
from RA GPRs contained levels of Chk2 equivalent to
those of control PBMCs (Figure 6C).
To determine whether low Chk2 levels in patients
with RA correlated with low transcript levels of CHK2,
we analyzed PBMC RNA derived from previously unexamined RA patients and controls (Figure 6D). A portion
of the RA patients had CHK2 transcript levels similar to
those of controls, and a portion had very low CHK2
transcript levels. Semiquantitative RT-PCR analysis was
used to more accurately determine relative levels of
transcript in a representative control sample and RA
sample with very low CHK2 transcript abundance. This
confirmed that CHK2 transcript levels were substantially
lower in a subset of RA patients compared with controls
(Figure 6D). We believe that underexpression of CHK2
mRNA may account, at least in part, for the heterogeneity in the response of RA PBMCs to gamma radiation.
Differential expression of many p53-regulated
genes in PBMCs from patients with RA. The abovementioned studies focused on p53 protein, TP53 transcript levels, and a few well-characterized downstream
transcriptional targets of p53. We also wanted to determine whether additional known p53-regulated genes
were differentially expressed in RA compared with
control PBMCs. To do so, we compared results from our
microarray analysis of differential gene expression between control and RA PBMCs (8) to microarray analysis
of differential gene expression between cell lines that
have or do not have functional p53 (27,28). Genes
identified in these studies include both known direct
transcriptional targets of p53 (p21, PUMA) as well as
genes that may be direct transcriptional targets of p53 or
may be differentially expressed as a result of secondary
1054
effects of the presence or absence of p53. We examined
our microarray data to determine expression levels of
genes (identified in cell line studies) that have altered
MAAS ET AL
Table 1.
Expression levels of p53-regulated genes*
Fold reduction
or increase
Gene title
p53-inducible genes
Transglutaminase
E-cadherin
CDKN1B, p27
Caspase 6
Myosin 1B
Epoxide hydrolase
RAD52 homolog
Ubiquinone
APAF1
GADD45A
PIG11
c-fos
Endoglin
BTG family, 2
Tyrosinase
p53-repressible genes
Adenosine deaminase
DAG kinase
Fibronectin 1
IL-8
EGF receptor
Cyclin A2
Stathmin
NF-IL3
RAD51 homolog
Carboxypeptidase
CDC28 PK2
COP9
Cyclin E1
CDC6 homolog
Galectin 3
SGK
⫺11.1
⫺6.3
⫺5.0
⫺5.3
⫺5.6
⫺8.3
⫺4.8
⫺4.5
⫺4.3
⫺3.2
⫺2.6
⫺1.8
⫺2.5
⫺2.1
⫺2.1
3.8
4.1
4.8
5.4
3.6
3.4
2.9
3.1
2.9
3.5
2.4
1.9
2.5
1.9
2.5
2.1
* Values for p53-inducible genes are the fold reduction; values for
p53-repressible genes are the fold increase. Data were compiled from
previous microarray experiments (8). Genes that were p53 responsive
(26,27) were selected from our microarray data.
Figure 6. Low levels of Chk2 protein and CHK2 transcript in peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA). Whole cell extracts from the same patient lysates
(untreated [⫺], gamma-irradiated [⫹]) described in Figure 4 were
immunoblotted for Chk2 protein. A–C, Immunoblots of controls (n ⫽
5), RA gamma radiation nonresponders (GNRs) (n ⫽ 4), and RA
gamma radiation partial responders (GPRs) (n ⫽ 5). D, Reverse
transcription–polymerase chain reaction (PCR) performed on cDNA
from total RNA in previously unexamined control (n ⫽ 3) and RA
(n ⫽ 6) PBMCs. Top panel shows amplification results after 30 cycles.
Thirty-five–cycle semiquantitative PCR was performed on serial dilutions of cDNA. Results for control (C8) and RA PBMCs with low
CHK2 transcript levels (RA16) are shown in the paired figures in the
lower panel. HCT ⫽ HCT116 cell line.
expression profiles in the presence or absence of p53
(Table 1). All genes that were overexpressed in p53positive cell lines were underexpressed in RA PBMCs.
Conversely, all genes that were underexpressed in p53positive cell lines were overexpressed in RA PBMCs.
These results further support our hypothesis that defects
in p53 expression and induction in PBMCs from patients
with RA may account for a significant portion of the
unique gene expression profile observed in these affected individuals.
DISCUSSION
In this study, we found uniform defects in the p53
damage-response pathway in PBMCs from patients with
RA. T lymphocytes from patients with RA were significantly more resistant to gamma radiation–induced cell
death than were control T lymphocytes. In contrast,
TP53 AND APOPTOSIS DEFECTS IN RA
lymphocytes from RA patients did not display defects in
p53-independent modes of apoptosis. Both p53 protein
and TP53 mRNA baseline levels were substantially
reduced in RA PBMCs, providing a possible explanation
for defects in gamma radiation–induced apoptosis. Levels of p21 (a cyclin-dependent kinase inhibitor) were
also substantially lower in RA patients. Furthermore,
half of the patients with RA failed to exhibit increased
steady-state levels of p53 after exposure to gamma
radiation. PBMCs from these RA GNRs also contained
negligible levels of Chk2, an upstream kinase that phosphorylates p53 (25) and may prevent ubiquitin-mediated
degradation of p53 (29) after DNA damage.
Our previous work revealed the presence of a
gene expression signature in patients with a range of
autoimmune disorders (8). Many underexpressed genes
present in this signature encode proteins necessary for
apoptosis. One of the most consistently underexpressed
genes is TP53, a central mediator of cellular responses to
stress that induces cell cycle arrest or apoptosis (30,31).
This series of biochemical studies support our initial
observations of significantly reduced expression of TP53
mRNA in autoimmunity and demonstrate that lymphocytes from patients with RA have a defective p53mediated damage-response pathway.
Defects in lymphocyte apoptosis are hypothesized to contribute to development of autoimmunity.
Some of the best support for this theory comes from
observations of lpr or gld mutations (mutations in Fas or
Fas ligand [FasL], respectively) on the MRL murine
background (32). These mice develop autoantibodies
and succumb to fatal glomerulonephritis. With the exception of the rare autoimmune lymphoproliferative
syndrome (33), efforts to identify defects in Fas or FasL
in more common human autoimmune disorders have
been relatively unsuccessful (34). Our results clearly
demonstrate that there are uniform defects in apoptosis
in PBMCs from patients with RA, but that these defects
are present in the p53 damage-response pathway.
Other investigations have addressed the role of
p53 in RA. The majority of these studies have focused
on the synovium. It has been proposed that high levels of
oxidative stress in rheumatoid synovium may cause
somatic mutations in the TP53 gene (35). Presumably,
mutations in synovial p53 may allow pathologic proliferation of synovial cells that may lead to joint destruction
and other clinical manifestations of RA. Alternatively, it
has been proposed that the cytokine, macrophage migratory inhibitory factor (MIF), may cause decreased
cellular p53 levels (36,37). MIF lowers endogenous p53
levels both in vivo and in vitro, and high levels of MIF
could contribute to synovial proliferation and pannus
1055
formation. Although these results specifically address
the impact of MIF on the synovium, serum levels of MIF
are also elevated in RA patients compared with controls
(38). Elevated MIF levels may contribute to the underexpression of p53 in PBMCs from RA patients. However, our results clearly show that T lymphocytes, representing 80% of our PBMC preparations, are defective
in p53-mediated apoptosis, and T lymphocytes are not
known to respond to MIF.
There is also evidence that p53 maintains tolerance in lymphocytes by regulating cell cycle progression.
Human T lymphocytes from peripheral blood or intestinal lamina propria show an inverse relationship between p53 levels and the rate of progression through the
cell cycle (39). Cell cycle delays mediated by elevated
levels of p53 in lamina propria T lymphocytes may be a
mechanism that maintains tolerance against environmental antigens. Preliminary studies by Leech et al,
using an antigen-induced arthritis model on a p53⫺/⫺
background, revealed that T lymphocytes proliferate
more readily and produce more IFN␥ in the absence of
p53 (40). Similar results in models of collagen-induced
arthritis (41) suggest that inflammatory responses may
be exacerbated in the absence of p53.
Lymphocytes from healthy individuals with the
HLA–DRB1*04 allele (42) and patients with RA (43)
show signs of inappropriate aging as measured by telomeric shortening. T lymphocytes from RA patients also
proliferate less readily (compared with controls) in
response to stimulation with anti-CD3 or recall antigens.
Similar observations have been made in p53⫺/⫺ mice
(44). These studies revealed no defects in lymphocyte
development. Rather, lymphocytes from p53⫺/⫺ mice
exhibited signs of accelerated aging and nonresponsiveness to T cell receptor (TCR) stimuli. Recent studies
also demonstrated that p53 is a necessary element of
cellular senescence (45–47). Therefore, we speculate
that persistent underexpression of TP53 mRNA and p53
protein, and perhaps CHK2 mRNA and Chk2 protein,
may explain the accelerated aging and lack of lymphocyte responsiveness to TCR signals observed in patients
with RA.
We observed that approximately half of the patients with RA failed to demonstrate increased steadystate levels of p53 in response to exposure to gamma
radiation, and that this correlated with depressed levels
of Chk2. CHK2 mRNA levels are almost absent in this
subset of RA patients. Chk2 is believed to be an
upstream regulator of p53 stability in response to certain
types of DNA damage (15,29), although there is now
debate in the field about its absolute necessity for p53
stabilization (48,49). Studies in Chk2⫺/⫺ mice reveal no
1056
MAAS ET AL
abnormalities in lymphocyte development; however,
nothing more is known about the role of Chk2 in the
immune system.
Defects in downstream p53 target genes may also
play a role in promoting systemic autoimmunity. Microarray results and the results of our studies presented
here demonstrate that many p53 downstream gene
targets are dysregulated in a manner consistent with p53
dysfunction. For example, p21, a downstream cyclindependent kinase inhibitor and transcriptional target of
p53, is also underexpressed in patients with RA. Although controversial (50), studies have documented
increased autoantibody production, glomerulonephritis,
and mortality in p21⫺/⫺ female mice (51). T lymphocytes
from these mice are hyperproliferative when cultured
with interleukin-2 after activation compared with wildtype littermates. Additional studies examining
GADD45A, another downstream target of p53 effector
function, link this gene to systemic autoimmunity and
abnormalities in T lymphocyte function (52).
GADD45A⫺/⫺, p21⫺/⫺ mice exhibit aggressive autoimmunity comparable with that observed in MRL/lpr mice.
These observations, combined with our results in
the RA patient population, indicate that defects in the
expression of molecules in the DNA damage-response
pathway might play a role in autoimmune pathogenesis.
These proteins may also represent new targets for
therapeutic approaches. It might be possible to design
therapies to either correct defects in the p53 damage
response pathway or inhibit downstream effectors that
are normally inhibited by damage-response proteins
such as p53, p21, or GADD45A.
ACKNOWLEDGMENTS
We thank Drs. Theodore Pincus, Howard Fuchs, Victor Byrd, Tuulikki Sokka, and their patients for access to their
clinics and for providing blood samples. We would also like to
thank Bo Yelverton, Sukumar Narasimhulu, and Xuan Li for
technical assistance with experiments.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
REFERENCES
21.
1. Marrack P, Kappler J, Kotzin BL. Autoimmune disease: why and
where it occurs. Nat Med 2001;7:899–905.
2. Moxley G, Cohen HJ. Genetic studies, clinical heterogeneity, and
disease outcome studies in rheumatoid arthritis. Rheum Dis Clin
North Am 2002;28:39–58.
3. Gregersen PK. Genetics of rheumatoid arthritis: confronting
complexity. Arthritis Res 1999;1:37–44.
4. Wakeland EK, Liu K, Graham RR, Behrens TW. Delineating the
genetic basis of systemic lupus erythematosus. Immunity 2001;15:
397–408.
5. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau
22.
23.
24.
25.
J, et al. Interferon and granulopoiesis signatures in systemic lupus
erythematosus blood. J Exp Med 2003;197:711–23.
Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann
WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc
Natl Acad Sci U S A 2003;100:2610–5.
Bomprezzi R, Ringner M, Kim S, Bittner ML, Khan J, Chen Y, et
al. Gene expression profile in multiple sclerosis patients and
healthy controls: identifying pathways relevant to disease. Hum
Mol Genet 2003;12:2191–9.
Maas K, Chan S, Parker J, Slater A, Moore J, Olsen N, et al.
Cutting edge: molecular portrait of human autoimmune disease.
J Immunol 2002;169:5–9.
Ramanathan M, Weinstock-Guttman B, Nguyen LT, Badgett D,
Miller C, Patrick K, et al. In vivo gene expression revealed by
cDNA arrays: the pattern in relapsing-remitting multiple sclerosis
patients compared with normal subjects. J Neuroimmunol 2001;
116:213–9.
Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC,
Hooper, ML, et al. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 1993;362:849–52.
Seki H, Kanegane H, Iwai K, Konno A, Ohta K, Yachie A, et al.
Ionizing radiation induces apoptotic cell death in human TcR
␥/␦⫹ T and natural killer cells without detectable p53 protein. Eur
J Immunol 1994;24:2914–7.
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,
Cooper NS, et al. The American Rheumatism Association 1987
revised criteria for the classification of rheumatoid arthritis.
Arthritis Rheum 1988;31:315–24.
Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW.
Participation of p53 protein in the cellular response to DNA
damage. Cancer Res 1991;51:6304–11.
Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid
degradation of p53. Nature 1997;387:296–9.
Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida
H, et al. DNA damage-induced activation of p53 by the checkpoint
kinase Chk2. Science 2000;287:1824–7.
El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R,
Trent JM, et al. WAF1, a potential mediator of p53 tumor
suppression. Cell 1993;75:817–25.
Kastan MB, Zhan Q, el-Deiry WS, Carrier F, Jacks T, Walsh WV,
et al. A mammalian cell cycle checkpoint pathway utilizing p53 and
GADD45 is defective in ataxia-telangiectasia. Cell 1992;71:
587–97.
Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T,
et al. Noxa, a BH3-only member of the Bcl-2 family and candidate
mediator of p53-induced apoptosis. Science 2000;288:1053–8.
Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA
induces the rapid apoptosis of colorectal cancer cells. Mol Cell
2001;7:673–82.
Kennedy AS, Harrison GH, Mansfield CM, Zhou XJ, Xu JF,
Balcer-Kubiczek EK. Survival of colorectal cancer cell lines
treated with paclitaxel, radiation, and 5-FU: effect of TP53 or
hMLH1 deficiency. Int J Cancer 2000;90:175–85.
Wu X, Deng Y. Bax and BH3-domain-only proteins in p53mediated apoptosis. Front Biosci 2002;7:d151–6.
Yu J, Zhang L. No PUMA, no death: implications for p53dependent apoptosis. Cancer Cell 2003;4:248–9.
Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is
induced by p53. Mol Cell 2001;7:683–94.
Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw
WC. Cleavage of poly(ADP-ribose) polymerase by a proteinase
with properties like ICE. Nature 1994;371:346–7.
Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human
homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev
2000;14:289–300.
TP53 AND APOPTOSIS DEFECTS IN RA
26. Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD. Phosphorylation of Ser-20 mediates stabilization of human p53 in response
to DNA damage. Proc Natl Acad Sci U S A 1999;96:13777–82.
27. Zhao R, Gish K, Murphy M, Yin Y, Notterman D, Hoffman WH,
et al. Analysis of p53-regulated gene expression patterns using
oligonucleotide arrays. Genes Dev 2000;14:981–93.
28. Kannan K, Amariglio N, Rechavi G, Jakob-Hirsch J, Kela I,
Kaminski N, et al. DNA microarrays identification of primary and
secondary target genes regulated by p53. Oncogene 2001;20:
2225–34.
29. Chehab NH, Malikzay A, Appel M, Halazonetis TD. Chk2/hCds1
functions as a DNA damage checkpoint in G(1) by stabilizing p53.
Genes Dev 2000;14:278–88.
30. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB. Wild-type p53
is a cell cycle checkpoint determinant following irradiation. Proc
Natl Acad Sci U S A 1992;89:7491–5.
31. Stewart ZA, Pietenpol JA. p53 signaling and cell cycle checkpoints. Chem Res Toxicol 2001;14:243–63.
32. Nagata S, Suda T. Fas and Fas ligand: lpr and gld mutations.
Immunol Today 1995;16:39–43.
33. Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin
AY, et al. Dominant interfering Fas gene mutations impair
apoptosis in a human autoimmune lymphoproliferative syndrome.
Cell 1995;81:935–46.
34. McNally J, Yoo DH, Drappa J, Chu JL, Yagita H, Friedman SM,
et al. Fas ligand expression and function in systemic lupus erythematosus. J Immunol 1997;159:4628–36.
35. Yamanishi Y, Boyle DL, Rosengren S, Green DR, Zvaifler NJ,
Firestein GS. Regional analysis of p53 mutations in rheumatoid
arthritis synovium. Proc Natl Acad Sci U S A 2002;99:10025–30.
36. Hudson JD, Shoaibi MA, Maestro R, Carnero A, Hannon GJ,
Beach DH. A proinflammatory cytokine inhibits p53 tumor suppressor activity. J Exp Med 1999;190:1375–82.
37. Leech M, Lacey D, Xue JR, Santos L, Hutchinson P, Wolvetang E,
et al. Regulation of p53 by macrophage migration inhibitory factor
in inflammatory arthritis. Arthritis Rheum 2003;48:1881–9.
38. Leech M, Metz C, Hall P, Hutchinson P, Giantis K, Smith M, et al.
Macrophage migration inhibitory factor in rheumatoid arthritis:
evidence of proinflammatory function and regulation by glucocorticoids. Arthritis Rheum 1999;42:1601–8.
39. Sturm A, Itoh J, Jacobberger JW, Fiocchi C. p53 negatively
regulates intestinal immunity by delaying mucosal T cell cycling.
J Clin Invest 2002;109:1481–92.
1057
40. Leech M, Xue JR, Poulos G, Hall P, Morand E. p53 modulates the
systemic immune response and arthritis severity in antigeninduced arthritis [abstract]. Arthritis Rheum 2003; 48 Suppl
9:S348.
41. Yamanishi Y, Boyle DL, Pinkoski MJ, Mahboubi A, Lin T, Han Z,
et al. Regulation of joint destruction and inflammation by p53 in
collagen-induced arthritis. Am J Pathol 2002;160:123–30.
42. Schonland SO, Lopez C, Widmann T, Zimmer J, Bryl E, Goronzy
JJ, et al. Premature telomeric loss in rheumatoid arthritis is
genetically determined and involves both myeloid and lymphoid
cell lineages. Proc Natl Acad Sci U S A 2003;100:13471–6.
43. Koetz K, Bryl E, Spickschen K, O’Fallon WM, Goronzy JJ,
Weyand CM. T cell homeostasis in patients with rheumatoid
arthritis. Proc Natl Acad Sci U S A 2000;97:9203–8.
44. Ohkusu-Tsukada K, Tsukada T, Isobe K. Accelerated development and aging of the immune system in p53-deficient mice.
J Immunol 1999;163:1966–72.
45. Sharpless NE, DePinho RA. p53: good cop/bad cop. Cell 2002;
110:9–12.
46. Campisi J. Cancer and ageing: rival demons? Nat Rev Cancer
2003;3:339–49.
47. D’Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H,
Carr P, von Zglinicki T, et al. A DNA damage checkpoint response
in telomere-initiated senescence. Nature 2003;426:194–8.
48. Ahn J, Urist M, Prives C. Questioning the role of checkpoint
kinase 2 in the p53 DNA damage response. J Biol Chem 2003;
278:20480–9.
49. Keramaris E, Hirao A, Slack RS, Mak TW, Park DS. Ataxia
telangiectasia-mutated protein can regulate p53 and neuronal
death independent of Chk2 in response to DNA damage. J Biol
Chem 2003;278:37782–9.
50. Lawson BR, Kono DH, Theofilopoulos AN. Deletion of p21
(WAF-1/Cip1) does not induce systemic autoimmunity in female
BXSB mice. J Immunol 2002;168:5928–32.
51. Balomenos D, Martin-Caballero J, Garcia MI, Prieto I, Flores JM,
Serrano M, et al. The cell cycle inhibitor p21 controls T-cell
proliferation and sex-linked lupus development. Nat Med 2000;6:
171–6.
52. Salvador JM, Hollander MC, Nguyen AT, Kopp JB, Barisoni L,
Moore JK, et al. Mice lacking the p53-effector gene Gadd45a
develop a lupus-like syndrome. Immunity 2002;16:499–508.
Документ
Категория
Без категории
Просмотров
1
Размер файла
226 Кб
Теги
loss, associates, periphery, patients, apoptosis, induced, p53, reduced, blood, cells, radiation, arthritis, mononuclear, rheumatoid
1/--страниц
Пожаловаться на содержимое документа