Identification of Signaling Pathways Mediating Cell Cycle Arrest and
Apoptosis Induced by Porphyromonas gingivalis in Human
Hiroaki Inaba,aMasae Kuboniwa,bHideyuki Sugita,aRichard J. Lamont,cand Atsuo Amanob
Department of Oral Frontier Biology, Center for Frontier Oral Science,aand Department of Preventive Dentistry,bOsaka University Graduate School of Dentistry,
Suita-Osaka, Japan, and Center for Oral Health and Systemic Disease, School of Dentistry, University of Louisville, Louisville, Kentucky, USAc
term delivery of low-birth-weight infants. Porphyromonas gingivalis, a periodontal pathogen, can translocate to gestational tis-
sues following oral-hematogenous spread. We previously reported that P. gingivalis invades extravillous trophoblast cells
The purpose of the present study was to identify signaling pathways mediating cellular impairment caused by P. gingivalis. Fol-
lowing P. gingivalis infection, the expression of Fas was induced and p53 accumulated, responses consistent with response to
was not induced. The inhibition of ATR prevented both G1arrest and apoptosis caused by P. gingivalis in HTR-8 cells. In addi-
associated with Ets1 activation. HTR-8 cells infected with P. gingivalis exhibited activation of Ets1, and knockdown of Ets1 with
siRNA diminished both G1arrest and apoptosis. These results suggest that P. gingivalis activates cellular DNA damage signaling
are various risk factors for preterm delivery of low-birth-weight
infants (PTLBW), many of which involve increased systemic in-
flammation (5, 16). Bacterial infection is one of the major causes
of PTLBW, either ascending from the urogenital tract or occur-
and these infections usually precede the development of preg-
nancy complications. The pathogenesis can result directly from
bacterial invasion of fetoplacental tissue, causing tissue damage
and expulsion of the infected fetus. Alternatively, adverse out-
comes can result from infection-induced disruption of normal
immune and inflammatory status (5, 15, 43).
Various epidemiological studies have shown a link between
periodontal diseases and PTLBW (59). Porphyromonas gingivalis,
a major periodontal pathogen, was detected in the amniotic fluid
of pregnant women with a diagnosis of threatened premature la-
bor (30) as well as in placentas of those with preeclampsia (4). In
addition, P. gingivalis antigens were detected in placental tissues,
including syncytiotrophoblasts, chorionic trophoblasts, decidual
cells, and amniotic epithelial cells, as well as vascular cells, which
were obtained from women with chorioamnionitis at fewer than
gingivalis was also found to invade both maternal and fetal tissues
and result in chorioamnionitis and placentitis. Moreover, P. gin-
els (6, 10, 32). In vitro studies showed that P. gingivalis invaded
placental trophoblasts and induced G1arrest and apoptosis
through pathways involving extracellular signal-regulated kinase
1/2 (ERK1/2) and signaling through cyclins and retinoblastoma
protein (21). In addition, invasion by P. gingivalis induced MEK-
(15) and generally results in low-birth-weight infants. There
p38 mitogen-activated protein kinase (MAPK) pathways and
modulated cytokine expression by trophoblast cell lines (44).
Cell cycle arrest and apoptosis are known to be triggered by
DNA damage (45), following which DNA double-strand breaks
(DSBs) and single-strand breaks (SSBs) induce the activation of
ataxia telangiectasia- and Rad3-related proteins (ATR), as well as
ataxia telangiectasia-mutated kinases (ATM) (12, 45). ATM and
ATR share many biochemical and functional similarities, includ-
ing sequence homology, phosphorylation sites, and downstream
targets (12, 45). The overlap between the target sets includes sub-
strates that promote cell cycle arrest, DNA repair, and apoptosis
via p53. Moreover, most substrates, such as checkpoint kinases
(Chk1 and Chk2) and p53, are phosphorylated by both kinases
(12, 45). However, ATR is essential for the viability of replicating
human and mouse cells, whereas ATM is not. ATM functions
distinctively in response to rare occurrences of DSBs (12). Viral
infection can affect the activation of ATR and/or ATM. ATR acti-
herpes simplex virus and human cytomegalovirus (33, 52). Both
kinases have been reported to be activated in cells infected with
Epstein-Barr virus, human polyomavirus JC virus, and minute
Received 13 March 2012 Returned for modification 12 April 2012
Accepted 30 May 2012
Published ahead of print 11 June 2012
Editor: J. B. Bliska
Address correspondence to Hiroaki Inaba, firstname.lastname@example.org.
Supplemental material for this article may be found at http://iai.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
August 2012 Volume 80 Number 8Infection and Immunityp. 2847–2857iai.asm.org
cobacter pylori can induce DSBs in epithelial and mesenchymal cells,
factors, such as p53-binding protein (53BP1) and mediator of DNA
damage checkpoint protein 1 (MDC1), and histone H2A variant X
The mechanisms by which P. gingivalis induces G1arrest and
apoptosis in trophoblasts are not fully understood. In this study,
cellular DNA damage signaling pathways that act through an
ATR/Chk2/p53-dependent pathway to cause G1arrest and apop-
MATERIALS AND METHODS
Bacterial and cell cultures. The bacterial strains used were P. gingivalis
ATCC 33277 and related mutants, including a long fimbria-null (fimA)
mutant (KDP150) (53) and Rgp- and Kgp-null (rgpA rgpB kgp-deficient)
broth supplemented with yeast extract (1 mg/ml), menadione (1 ?g/ml),
and hemin (5 ?g/ml), as described previously (21). KDP136 was fimbri-
ated as described previously (24). Briefly, the supernatant from fimbria-
null KDP150 culture was filtrated through a 0.2-?m-pore-size filter
(Asahi Glass Co., Ltd., Tokyo, Japan), and membrane vesicle-depleted
supernatants (VDS) containing soluble gingipains were obtained by cen-
trifugation at 100,000 ? g for 50 min. The gingipain-null mutant
(KDP136) was inoculated into fresh culture medium containing 30%
VDS. The HTR-8/SVneo trophoblast cell line was provided by Charles
Graham (Kingston, Ontario, Canada). HTR-8 cells were cultured in
RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with
5% fetal bovine serum at 37°C in 5% CO2.
with phosphate-buffered saline (PBS). For total adhesion and invasion
of the lysate plated and cultured anaerobically for CFU on blood agar
supplemented with hemin and menadione. For invasion assay, extracel-
isms were enumerated. Invasion was calculated from CFU recovered in-
tracellularly as a percentage of total bacteria inoculated.
Western immunoblotting. HTR-8 cells were solubilized in cell lysis
and extraction reagent (Sigma-Aldrich) containing a protease and phos-
phatase inhibitor cocktail (Thermo Scientific, Rockford, IL) and gin-
gipain-specific inhibitors (KYT-1 and KYT-36; Peptide Institute, Osaka,
Japan). Immunoblotting was performed as previously described (21).
Chk1, 1:1,000; anti-phospho-Chk2, 1:1,000; anti-phospho-p53 (Ser15),
1:1,000; anti-phospho-p53 (Ser20), 1:1,000; anti-phospho-MDM2
(Ser166), 1:1,000; anti-phospho-ERK1/2, 1:1,000; anti-ERK1/2, 1:1,000;
anti-p16, 1:1,000; anti-p21, 1:1,000 (Cell Signaling Technology, Beverly,
1:1,000; anti-Chk-2, 1:1,000 (EnoGene, New York, NY); anti-Ets1, 1:500;
anti-Ets2, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-
MDM2, 1:1,000 (NOVUS Biologicals, Littleton, CO). Proteins and phos-
phorylated proteins were detected using the Pierce ECL substrate
(Thermo Scientific). Blots were stripped and probed with anti-?-actin
analysis of bands was performed using ImageJ software.
RNA interference. The following stealth small interfering RNA
(siRNA) duplexes were purchased from Sigma Genosys: human Ets1, 5=-
GGAGAUGGCUGGGAAUUCAAACUUU-3= (22) and 5=-GCUGACCU
CAAUAAGGACA-3= (54); human p53, 5=-GCAUGAACCGGAGGCCC
AUTT-3= (50) and 5=-CUACUUCCUGAAAACAACGTT-3= (49); and
control siRNA, 5=-CAUGUCAUGUGUCACAUCUCTT-3=. The siRNAs
were introduced into HTR-8 cells using Lipofectamine 2000 (Invitrogen,
transfection, the medium was replaced, and cells were incubated for a
further 24 h prior to challenge with P. gingivalis.
Flow cytometry. (i) Cell cycle analysis. Infected or control HTR-8
cells were trypsinized, washed with cold PBS, and then fixed in 70% eth-
anol at ?20°C overnight. Ethanol-fixed cells were washed with PBS and
incubated in 1 ml of 0.1 mg/ml RNase A solution at 37°C for 30 min. The
cells were stained with 50 mg/ml propidium iodide (Sigma-Aldrich). Cell
cycle analysis of 30,000 cells per sample was carried out with excitation at
Fit LT 3.0 (Verity Software, La Jolla, CA).
(ii) Apoptosis. For annexin V staining, cells were harvested and
stained with an annexin V-FITC (fluorescein isothiocyanate) apoptosis
detection kit (BioVision, Palo Alto, CA) according to the manufacturer’s
protocol, and flow-cytometric analysis was performed. Chemicals were
obtained from Sigma-Aldrich. ATM/ATR inhibitor (Calbiochem, Darm-
stadt, Germany), 2.5 ?g/ml in dimethyl sulfoxide (DMSO), was preincu-
bated with HTR-8 cells for 2 h prior to addition of bacteria.
Caspase 3 activity. Caspase 3 activity was measured using a caspase
3/CPP32 colorimetric assay kit (BioVision, Mountain View, CA) accord-
ing to the manufacturer’s instructions on a microplate reader at 405 nm
(Bio-Rad model 680).
Fas (CD95) is a death domain containing a receptor that activates
the extrinsic apoptotic pathway (7). p53 is involved in mediating
key cellular processes, such as DNA repair, cell cycle arrest, senes-
cence, and apoptosis (58). We first examined the effect of P. gin-
givalis on expression of Fas and p53 by HTR-8/SVneo tropho-
blasts (referred to here as HTR-8 cells). Following P. gingivalis
infection at an MOI of 200, Fas expression was induced at 48 h,
and the accumulation of p53 also reached its peak at 48 h (Fig. 1),
while P. gingivalis triggered no induction of Fas expression at
result which has also been observed at an MOI of 200 (21). p53
accumulates in response to DNA damage, and the resulting in-
crease of p53 function induces either cell growth arrest or apop-
tosis (5, 31). Therefore, we next examined the activation status of
DNA damage response proteins up to 48 h after infection in
HTR-8 cells (Fig. 2). ATR and ATM, which activate downstream
signaling molecules such as Chk1, Chk2, and p53, are essential
regulators of DNA damage checkpoints in mammalian cells (45).
Levels of the Ser428-phosphorylated form of ATR and the total
amount of ATR were increased by P. gingivalis from 12 to 48 h
after infection (Fig. 2). On the other hand, phosphorylation of
ATM (Ser1981) and expression of total ATM were not observed.
on Chk2, and Ser15 and Ser20 phosphorylate on p53 (20, 23, 41).
Furthermore, ATR can also activate Chk1, which indirectly phos-
phorylates p53 at Ser20 (63). P. gingivalis was found to induce
Thr68 phosphorylation of Chk2 (Thr68) and p53 (Ser15),
whereas Chk1 (Ser296) and p53 (Ser20) did not show an increase
in phosphorylation levels. MDM2 possesses the E3 ubiquitin li-
gase and binds to the p53 N-terminal transcriptional activation
Inaba et al.
iai.asm.orgInfection and Immunity
domain, leading to degradation of p53 via the 26S proteasome
gingivalis-infected cells. These results indicate that p53 accumu-
lated in response to damage by P. gingivalis via the ATR-Chk2
pathway and by the lack of induction of MDM2.
ATR is associated with P. gingivalis-mediated G1arrest and
apoptosis. DNA damage response is a signal transduction path-
way that coordinates cell cycle transitions and apoptosis (12).
transition and apoptosis responses of HTR-8 cells to P. gingivalis.
P. gingivalis in HTR-8 cells (Fig. 3A). In addition, ATR inhibition
abrogated the induction of apoptosis (Fig. 3B) and caspase 3 ac-
tivity (Fig. 3C). These results suggest that G1arrest and apoptosis
by P. gingivalis in HTR-8 cells are regulated by the ATR-Chk2
Activation of p53 by P. gingivalis controls G1arrest and
apoptosis. To evaluate the effects of p53 activation on cell cycle
arrest and apoptosis caused by P. gingivalis, we silenced p53 ex-
pression using two siRNA duplexes. Expression of p53 was signif-
FIG 1 Fas and p53 expression is upregulated in HTR-8 cells infected with P. gingivalis. HTR-8 cells were infected with P. gingivalis at MOIs of 10, 100, and 200
three independent experiments.
FIG 2 Activation of DNA damage-responsive proteins in HTR-8 cells infected with P. gingivalis. (A) HTR-8 cells were infected with P. gingivalis at an MOI of
200 for the indicated times. Lysates of infected and noninfected cells were immunoblotted with antibodies to DNA damage-responsive proteins. (B) Densito-
metric analysis of blots showing the phosphorylation and total proteins, expressed in arbitrary units. ?-Actin was included as a loading control. Data are
representative of three independent experiments.
Multiple Pathways Activated by P. gingivalis
August 2012 Volume 80 Number 8 iai.asm.org 2849
HTR-8 cells were infected with P. gingivalis and then assayed im-
mediately (time zero) and at 24 and 48 h after bacterial infection.
In cells transfected with the nontarget siRNA, P. gingivalis infec-
tion enhanced the expressions of all signaling molecules tested at
24 and 48 h. Conversely, no induction of p21 or Fas was observed
edly increases Fas expression at the cell surface by transport from
the Golgi complex (7). Thus, Fas induction by P. gingivalis can be
considered due to the p53-dependent response to DNA damage.
In addition, G1arrest that is dependent on p53 requires p21 acti-
p21 was suppressed by p53 knockdown (Fig. 4B and C). More-
over, G1arrest was negligible, and the cell cycle accurately pro-
gressed to S phase in the p53 knockdown cells infected with the
pathogen (Fig. 4D). Additionally, p53 knockdown abrogated the
avoid elicitation of off-targeting, a second siRNA specific to an-
other sequence was provided in the same target mRNA. Figure S1
in the supplemental material shows that cells with alternative p53
siRNA silencing also showed significantly reduced arrest in G1
the apoptotic effect of P. gingivalis. These results suggest that P.
gingivalis diverts p53 signaling events, which causes cell cycle dis-
turbance and ultimately apoptosis.
Activation of Ets1 by P. gingivalis controls G1arrest and
apoptosis. P. gingivalis was previously shown to elevate the levels
of p16 in HTR-8 cells (21), and p53 knockdown did not suppress
p16 induction by the organisms (Fig. 4B). Upregulation of p16 is
mediated by Ets1 and Ets2 proteins through the proximal Ets-
effect of P. gingivalis on Ets1 and Ets2 levels. Infection of HTR-8
cells with P. gingivalis resulted in a significant increase in Ets1
levels compared to noninfected cells, whereas little expression of
Ets2 was observed in infected or control cells (Fig. 5). Ets1 also
induces the expression of apoptotic genes, and p16 mediates cell
cycle arrest at G1(19, 39). In addition, p21 is controlled by the
expression of p16 in hepatoma cells (19). To evaluate the role of
Ets1 activation in P. gingivalis-induced cell cycle arrest and apop-
tosis, we silenced Ets1 expression by using two sets of siRNA du-
plexes (Fig. 6A). Both siRNA constructs reduced expression of
Ets1 at the transcriptional level. Immunoblotting showed that the
cells following P. gingivalis infection. In contrast, the phosphory-
to induce apoptosis and increased the activity of caspase 3 at 48 h
the same effects on cell cycle and apoptosis (see Fig. S2 in the
expresses several virulence factors, including fimbriae as well as
FIG3 Effects of an ATM and ATR inhibitor on G1arrest and apoptosis. HTR-8 cells were infected withP. gingivalis at an MOI of 200 for 48 h. An inhibitor was
added for 2 h prior to infection. (A) DNA content was analyzed by flow cytometry, and cell cycle profiles were obtained with ModFit software. Results are
means ? standard deviations (SD) of cell cycle distribution from three independent experiments with flow cytometry parameters set to exclude cell debris and
(C) Cells were treated with inhibitor were infected with P. gingivalis for 48 h as described for panel B, and caspase 3 activity was measured. Fold changes were
calculated relative to infected cells without inhibitor (t test). * and **, P ? 0.05 and P ? 0.01 (Student’s t test) compared with P. gingivalis-infected cells with
Inaba et al.
iai.asm.org Infection and Immunity
with a nontarget control (siNT). (A) Cells were lysed and immunoblotted with anti-p53 or ?-actin antibodies at 48 h after transfection. (B) siRNA knockdown
cells were infected with P. gingivalis at an MOI of 200 for 24 or 48 h. Expression profiles of signaling molecules were examined by immunoblotting. ?-Actin was
included as a loading control. (C) Densitometric analysis of blots showing the phosphorylation and total proteins, expressed in arbitrary units. Data are
representative of three independent experiments. (D) siRNA knockdown cells were infected with P. gingivalis as described for panel B, and then cell cycle
distribution was determined using flow cytometry. The ratio of cells in each cell cycle phase is expressed relative to that of noninfected controls. (E) siRNA
knockdown cells were infected as described for panel B, followed by staining with annexin V and propidium iodide (PI), and analyzed by flow cytometry. Fold
change was calculated as described for panel C. (F) siRNA knockdown cells were infected with P. gingivalis for 48 h as described for panel D, and then caspase 3
activity was measured. The fold change was calculated as described for panel D. Error bars indicate standard deviations (n ? 3). *, P ? 0.01 (Student’s t test)
compared with the ratio of P. gingivalis-infected NT-siRNA control to NT-siRNA control.
Multiple Pathways Activated by P. gingivalis
August 2012 Volume 80 Number 8iai.asm.org 2851
cysteine proteinases (18). We sought to investigate the role of
are required for adherence to HTR-8 cells (data not shown), we
utilized bacterial culture supernatants containing soluble Rgp to
express mature fimbriae on the surface of Rgp-null mutants as
previously described (24). The Kgp- and Rgp-null mutant
(KDP136) was treated with exogenous gingipains as described in
Materials and Methods, and the treated KDP136 mutant was
tal material). Gingipain activities of treated KDP136 were con-
firmed to be negligible (see Fig. S3B). Furthermore, the adhesion
and invasion abilities did not differ significantly between the wild
type and treated KDP136 (Fig. 7A). Conversely, treated KDP136
failed to cause a progressive increase in G1-phase cells or a corre-
type strain caused rounding and detachment of some cells. In
contrast, treated KDP136 did not induce these changes, though
slightly deformed cell shapes were observed (Fig. 7C). We also
responses were similar between the parental and treated KDP136
strains (Fig. 7D). In addition, treated KDP136 significantly de-
expression and phosphorylation status of ATR, ATM, Chk1,
Chk2, p53, and MDM2 and on levels of Ets1, Fas, p16, and p21 in
HTR-8 cells. Notably, the amount and phosphorylation of
MDM2, which are suppressed by the wild-type strain (Fig. 2),
were increased in response to treated KDP136 (Fig. 7F). For the
to those of the wild type. It is possible that the activation of ATR-
p53 and ERK-Ets1 pathways may be directly affected by loss of
ways, expression of Ets1, p16, Chk2, and p21 mRNAs was deter-
mined using real-time PCR (see Table S1 in the supplemental
material). Wild-type and treated KDP136 elevated levels of Chk2
and p16 mRNA to the same degree, although the increase in Ets1
and p21 mRNA expression following wild-type infection was
higher than that following infection with treated KDP136. These
results suggest that P. gingivalis gingipains may specifically de-
grade MDM2 protein and modulate the activity of multiple sig-
totrophoblasts differentiate into villous intermediate cells, and
these cells are programmed to fuse with the syncytium or into
extravillous migratory cells that transform the maternal vascular
supply. The villous trophoblast bilayer is the primary barrier be-
tween maternal and fetal tissues. The syncytium is a structural
component of maternal-facing layer and a barrier to pathogens
and the maternal cells (2). Extravillous trophoblast (EVT) inva-
fetal growth and well-being (26). HTR-8 cells were established
from first-trimester EVTs (17), while HTR-8/SVneo cells are the
blasts in vitro and cause apoptosis and cell cycle arrest at G1(21).
In addition, P. gingivalis has been detected in the amniotic fluid
(4, 30). A role for P. gingivalis in pregnancy complications is also
maternal and fetal tissues (6, 10, 32). Trophoblasts differentially
regulate the expression of over 2,000 genes following P. gingivalis
infection (44), which may impact many signaling pathways, in-
we examined the effect of P. gingivalis infection on the signaling
pathways which regulate cell cycle and apoptosis. P. gingivalis in-
fection was shown to alter expression profiles of DNA damage
response proteins, which was accompanied by G1cell cycle arrest
and apoptosis. This is the first comprehensive examination of the
signaling pathways of DNA damage response induced by P. gingi-
valis infection. The involvement of p53, Ets1, and ATR in cellular
responses was also shown by siRNA knockdown and chemical
inhibition. Furthermore, gingipain proteolytic activity was found
propose that P. gingivalis initiates the signaling cascade shown in
The Ras-dependent ERK1/2 MAPK pathway plays a central
role in control of cell proliferation, and sustained activation of
ERK1/2 is necessary for G1-to-S-phase progression and positive
regulation of the cell cycle. However, excessive activation of
Downstream of Ras are the effector kinases Raf/MEK/ERK1/2
(19, 61). Moreover, p16 has an antiproliferative function through
activity cycles (19, 61). Hence, activation of this pathway is con-
P. gingivalis. Chk2 was phosphorylated at threonine 68 when cells
were damaged by ionizing radiation (IR), UV irradiation, hy-
activation was shown to be mediated by ATM (20, 37) and ATR
(23, 37, 41). In addition, Chk2 phosphorylation parallels p53
phosphorylation on Ser15 (37). In the present study, DNA dam-
age response induced by P. gingivalis infection led to ATR but not
ATM activation (Fig. 2 and 7F). Moreover, Chk2 was phosphor-
ylated at threonine 68 (Fig. 2 and 7F). Interestingly, ATR-Chk2
gingipain proteases. Accordingly, ATR likely phosphorylates and
HTR-8 cells were infected with P. gingivalis at an MOI of 200 for the indicated
times. Cell lysates were immunoblotted with anti-Ets1 or Ets2 antibodies.
?-Actin was included as a loading control. Data are representative of three
Inaba et al.
iai.asm.orgInfection and Immunity
or with a nontarget control (siNT). (A) Immunoblot with anti-Ets1 or ?-actin antibodies at 48 h after transfection and with ?-actin as a loading control. (B)
siRNA knockdown cells were infected with P. gingivalis at an MOI of 200 for 24 and 48 h. Expression profiles of signaling molecules were examined by
immunoblotting. ?-Actin was included as a loading control. (C) Densitometric analysis of blots of phosphorylated and total proteins, expressed in arbitrary
caspase 3 activity was measured. Fold changes were calculated as described for panel D. Error bars indicate standard deviations (n ? 3). * and **, P ? 0.05 and
P ? 0.01 (Student’s t test) compared with the ratio of P. gingivalis-infected NT-siRNA control to NT-siRNA control.
August 2012 Volume 80 Number 8iai.asm.org 2853
leads to p53 phosphorylation at Ser15. Previous studies have
dent p21 expression (60) and does not require the presence of
ATM kinase (5).
to induce apoptosis mediated by Fas. H. pylori can also induce
characterized. The finding that DNA damage response in HTR-8
cells induced by P. gingivalis increased p53 accumulation and Fas
activation suggests that both proteins are targets of P. gingivalis in
an MOI-dependent manner (Fig. 1). Those findings also suggest
that G1arrest or apoptosis related to p53 and Fas expression may
be MOI dependent in HTR-8 cells infected with P. gingivalis in
vivo. While the levels of P. gingivalis in placental tissue are un-
known, an immunohistochemical study suggested that they can
with siRNA inhibited Fas expression, G1arrest, and apoptosis
(Fig. 4), suggesting that DNA damage response can induce tran-
scriptional upregulation of FAS through p53-dependent mecha-
nisms, as demonstrated with other cell types (14). These findings
suggest that Fas induction is controlled by p53 after P. gingivalis
to a variety of stress signals. Normally, p53 levels are kept low
results in disruption of MDM2-p53 interaction (36, 58). Ser166
phosphorylation of MDM2 has been shown to increase its ubiq-
uitin ligase activity and increase p53 degradation (35). Notably, a
significant decrease in levels of phosphorylated and total MDM2
was observed in P. gingivalis-infected cells, and this decrease was
dependent on the presence of gingipains. Together, these results
contributing to p53 accumulation and apoptosis. This is the first
report of such an MDM2-specific degradation response to patho-
FIG7 HTR-8 cells infected with a P. gingivalis fimbriated gingipain-null mutant strain do not show G1arrest and apoptosis. (A) Antibiotic protection invasion
assay with P. gingivalis ATCC 33277 (wild type) and fimbriated KDP136 (fimbriated gingipain-null mutant strain). HTR-8 cells were infected with P. gingivalis
as percentage of input bacterial cell number. Data are means ? SD of three independent experiments and were analyzed with a t test. (B) Cell cycle distribution
was calculated as described for panel B. The mean percentage ? SD of apoptotic (annexin V?/PI?) cells from three independent experiments was determined
and analyzed with a t test. (E) Fold change in caspase 3 activity. Data are means ? SD from three independent experiments analyzed with a t test. (F) Cells were
infected with P. gingivalis fimbriated KDP136 at an MOI of 200 for the indicated times. Lysates of infected and noninfected cells were subjected to immuno-
blotting to analyze expression profiles of DNA damage-responsive proteins, MDM2 phosphorylated at Ser166, MDM2, Ets1, Fas, p16, and p21. ?-Actin was
included as a loading control. Data are representative of three independent experiments.
Inaba et al.
iai.asm.org Infection and Immunity
response to both the wild-type and treated KDP136 strains. Re-
cent evidence suggests that necrosis can also be programmed,
though in a manner distinguishable from apoptosis (47). In addi-
tion, that report noted that under conditions where caspases are
inhibited or signaling through Fas is otherwise prevented, apop-
totic stimuli can induce necrosis. Our results suggest that caspase
activity and/or Fas induction via the ATR/Chk2/p53 pathway re-
quire gingipains, while induction of p21 by P. gingivalis occurs in
a gingipain-independent manner. The negative regulation of
apoptosis by p21 has been attributed to interaction with pro-
caspase 3, resulting in inhibition of its activity (1). Thus, necrosis
may be induced following infection with treated KDP136. When
important factor in apoptosis induction.
P. gingivalis impacts the cell cycle as well as apoptosis through
the upregulation of p16 and p21 and phosphorylation of ERK1/2
or induce cells to undergo apoptosis (28). In addition, Ets1 con-
trols the regulation of p16 (19, 39). However, the interaction be-
tween p53 and Ets1 in regard to the cell cycle has yet to be inves-
tigated. We found that the levels of p16 and p21 were diminished
by knockdown of p53 and Ets1, respectively, while p16 amounts
were decreased in Ets1-silenced cells following P. gingivalis infec-
tion. Additionally, p21 was diminished by the silencing of p53
and/or Ets1. HTR-8 cells infected with P. gingivalis also exhibited
a sustained activation of Ets1, and knockdown of Ets1 abrogated
both G1arrest and apoptosis. Moreover, the levels of p16, p21,
p53, and Fas were decreased when Ets1 amounts were reduced
(Fig. 4 and 6). This impact of P. gingivalis suggests that p16 and
p21 may be under the control of ERK1/2-Ets1 and p53 pathways.
This is consistent with reports showing that the p53 gene is tran-
scriptionally regulated by Ets1 (3), and a deficiency of Ets1 de-
in response to UV exposure (62). Collectively, these results indi-
cate that P. gingivalis diverts the ERK1/2-Ets1 and p53 signaling
pathways, resulting in both cell cycle arrest and apoptosis.
In conclusion, P. gingivalis infection of HTR-8 cells causes cy-
FIG 7 continued
FIG 8 Proposed schematic model for cell cycle regulation and induction of
progressive Chk2 activation. Chk2 then phosphorylates and activates p53, in-
ducing p21 and the gene expressing proapoptotic Fas. Phosphorylation of
ERK1/2 causes Ets1 activation, which in turn activates p16 and p53.
Multiple Pathways Activated by P. gingivalis
August 2012 Volume 80 Number 8 iai.asm.org 2855
and MAPK activation. We propose that the activation of DNA
trophoblasts, and that the ensuing apoptosis and cell cycle arrest
associated with this periodontal pathogen.
We thank the members of the Amano and Lamont labs for their helpful
discussions. We also thank Mayumi Yoshimori for the technical assis-
This research was supported by a grants-in-aid for Scientific Research
Culture, Sports, Science and Technology and by NIDCR grant DE11111
1. Abbas T, Dutta A. 2009. p21 in cancer: intricate networks and multiple
activities. Nat. Rev. Cancer. 9:400–414.
current research problems. Int. J. Dev. Biol. 54:323–329.
3. Baillat D, Bègue A, Stéhelin D, Aumercier A. 2002. ETS-1 transcription
factor binds cooperatively to the palindromic head to head ETS-binding
sites of the stromelysin-1 promoter by counteracting autoinhibition. J.
Biol. Chem. 277:29386–29398.
4. Barak S, Oettinger-Barak O, Machtei EE, Sprecher H, Ohel G. 2007.
preeclampsia. J. Periodontol. 78:670–676.
5. Behrman RE, Butler AS (ed). 2007. Preterm birth: causes, consequences,
and prevention. National Academies Press, Washington, DC.
6. Bélanger M, et al. 2008. Colonization of maternal and fetal tissues by
Porphyromonas gingivalis is strain-dependent in a rodent animal model.
Am. J. Obstet. Gynecol. 199:e1–e7. doi:10.1016/j.ajog.2007.11.067.
7. Bennett M, et al. 1998. Cell surface trafficking of Fas: a rapid mechanism
of p53-mediated apoptosis. Science 282:290–293.
8. Bhattacharyya A, et al. 2009. Acetylation of apurinic/apyrimidinic endo-
nuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell
apoptosis. Gastroenterology 136:2258–2269.
9. Reference deleted.
10. Boggess KA, Madianos PN, Preisser JS, Moise KJ, Jr, Offenbacher S.
2005. Chronic maternal and fetal Porphyromonas gingivalis exposure dur-
ing pregnancy in rabbits. Am. J. Obstet. Gynecol. 192:554–557.
11. Choudhuri T, Verma SC, Lan K, Murakami M, Robertson ES. 2007. The
ATM/ATR signaling effector Chk2 is targeted by Epstein-Barr virus nu-
clear antigen 3C to release the G2/M cell cycle block. J. Virol. 81:6718–
12. Cimprich KA, Cortez D. 2008. ATR: an essential regulator of genome
integrity. Nat. Rev. Mol. Cell Biol. 9:616–627.
13. Dittmer J. 2003. The biology of the Ets1 proto-oncogene. Mol. Cancer
14. El-Deiry WS. 2001. Insights into cancer therapeutic design based on p53
and TRAIL receptor signaling. Cell Death Differ. 8:1066–1075.
15. Goldenberg RL, Culhane JF, Lams JD, Romero R. 2008. Epidemiology
and causes of preterm birth. Lancet 371:75–84.
16. Goldenberg RL, Goepfert AR, Ramsey PS. 2005. Biochemical markers
for the prediction of preterm birth. Am. J. Obstet. Gynecol. 192:36–46.
17. Graham CH, et al. 1993. Establishment and characterization of first
trimester human trophoblast cells with extended lifespan. Exp. Cell Res.
18. Guo Y, Nquyen KA, Potempa J. 2010. Dichotomy of gingipains action as
virulence factors: from cleaving substrates with the precision of a sur-
geon’s knife to a meat chopper-like brutal degradation of proteins. Peri-
odontol. 2000 54:15–44.
19. Han J, Tsukada Y, Hara E, Kitamura N, Tanaka T. 2005. Hepatocyte
dependent p16INK4aup-regulation, leading to cell cycle arrest at G1in
HepG2 hepatoma cells. J. Biol. Chem. 280:31548–31556.
20. Harper JW, Elledge SJ. 2007. The DNA damage response: ten years after.
Mol. Cell 28:739–745.
21. Inaba H, et al. 2009. Porphyromonas gingivalis invades human tropho-
22. Ito H, et al. 2004. Prostaglandin E2 enhances pancreatic cancer invasive-
ness through an Ets-1-dependent induction of matrix metalloprotei-
nase-2. Cancer Res. 64:7439–7446.
23. Joe Y, et al. 2006. ATR, PML, and CHK2 play a role in arsenic trioxide-
induced apoptosis. J. Biol. Chem. 281:28764–28771.
gingipains in Porphyromonas gingivalis gingipain-null mutant. FEMS Mi-
crobiol. Lett. 273:96–102.
25. Katz J, Chegini N, Shiverick KT, Lamont RJ. 2009. Localization of P.
gingivalis in preterm delivery placenta. J. Dent. Res. 88:575–578.
26. Kaufmann P, Black S, Huppertz B. 2003. Endovascular trophoblast
invasion: implications for the pathogenesis of intrauterine growth retar-
dation and preeclampsia. Biol. Reprod. 69:1–7.
27. Lamont RJ, et al. 1995. Porphyromonas gingivalis invasion of gingival
epithelial cells. Infect. Immun. 63:3878–3885.
28. Lavin MF, Gueven N. 2006. The complexity of p53 stabilization and
activation. Cell Death Differ. 13:941–950.
29. Lee BP, Rushlow WJ, Chakraborty C, Lala PK. 2001. Differential gene
expression in premalignant human trophoblast: role of IGFBP-5. Int. J.
fluid in pregnant women with a diagnosis of threatened premature labor.
J. Periodontol. 78:1249–1255.
31. Li Y, et al. 2011. SIRT2 down-regulation in HeLa can induce p53 accu-
mulation via p38 MAPK activation-dependent p300 decrease, eventually
leading to apoptosis. Genes Cells 6:34–45.
32. Lin D, et al. 2003. Porphyromonas gingivalis infection in pregnant mice
is associated with placental dissemination, an increase in the placental
Th1/Th2 cytokine ratio, and fetal growth restriction. Infect. Immun. 71:
33. Luo MH, Rosenke K, Czornak K, Fortunato EA. 2007. Human cyto-
megalovirus disrupts both ataxia telangiectasia mutated protein (ATM)-
and ATM-Rad3-related kinase-mediated DNA damage responses during
lytic infection. J. Virol. 81:1934–1950.
response that facilitates viral DNA replication and mediates cell death. J.
35. Malmlöf M, Roudier E, Högberg J, Stenius U. 2007. MEK-ERK-
mediated phosphorylation of Mdm2 at Ser-166 in hepatocytes. Mdm2 is
activated in response to inhibited Akt signaling. J. Biol. Chem. 282:2288–
36. Manfredi JJ. 2010. The Mdm2-p53 relationship evolves: Mdm2 swings
both ways as an oncogene and a tumor suppressor. Genes Dev. 24:1580–
37. Matsuoka S, et al. 2000. Ataxia telangiectasia-mutated phosphorylates
Chk2 in vivo and in vitro. Proc. Natl. Acad. Sci. U. S. A. 97:10389–10394.
38. Nakayama K, Yoshimura F, Kadowaki T, Yamamoto K. 1996. Involve-
ment of arginine-specific cysteine proteinase (Arg-gingipain) in fimbria-
tion of Porphyromonas gingivalis. J. Bacteriol. 178:2818–2824.
39. Ohtani N, Yamakoshi K, Takahashi A, Hara E. 2004. The p16INK4a-RB
pathway: molecular link between cellular senescence and tumor suppres-
sion. J. Med. Invest. 51:146–153.
40. Orba Y, et al. 2010. Large T antigen promotes JC virus replication in
G2-arrested cells by inducing ATM- and ATR-mediated G2checkpoint
signaling. J. Biol. Chem. 285:1544–1554.
41. Pabla N, Huang S, Mi QS, Daniel R, Dong Z. 2008. ATR-Chk2 signaling
in p53 activation and DNA damage response during cisplatin-induced
apoptosis. J. Biol. Chem. 283:6572–6583.
42. Paolillo R, Carratelli CR, Rizzo A. 2011. Effect of resveratrol and quer-
cetin in experimental infection by Salmonella enterica serovar Typhimu-
rium. Int. Immunopharmacol. 11:149–156.
43. Pihlstrom BL, Michalowicz BS, Johnson NW. 2005. Periodontal dis-
eases. Lancet 366:1809–1820.
44. Riewe SD, et al. 2010. Human trophoblast responses to Porphyromonas
gingivalis infection. Mol. Oral Microbiol. 25:252–259.
45. Roos WP, Kaina B. 2006. DNA damage-induced cell death by apoptosis.
Trends Mol. Med. 12:440–450.
46. Roshal M, Kim B, Zhu Y, Nghiem P, Planelles V. 2003. Activation of the
ATR-mediated DNA damage response by the HIV-1 viral protein R. J.
Biol. Chem. 278:25879–25886.
47. Rudel T, Kepp O, Kozjak-Pavlovic V. 2010. Interactions between bacte-
Inaba et al.
iai.asm.orgInfection and Immunity
rial pathogens and mitochondrial cell death pathways. Nat. Rev. Micro- Download full-text
48. Sarfaraz S, Afaq F, Adhami VM, Malik A, Mukhtar H. 2006. Cannabi-
noid receptor agonist-induced apoptosis of human prostate cancer cells
cycle arrest. J. Biol. Chem. 281:39480–39491.
49. Scacheri PC, et al. 2004. Short interfering RNAs can induce unexpected
and divergent changes in the levels of untargeted proteins in mammalian
cells. Proc. Natl. Acad. Sci. U. S. A. 101:1892–1897.
50. Scian MJ, et al. 2004. Modulation of gene expression by tumor-derived
p53 mutants. Cancer Res. 64:7447–7454.
51. Shi Y, et al. 1999. Genetic analyses of proteolysis, hemoglobin binding,
and hemagglutination of Porphyromonas gingivalis. Construction of mu-
tants with a combination of rgpA, rgpB, kgp, and hagA. J. Biol. Chem.
52. Shirata N, et al. 2005. Activation of ataxia telangiectasia-mutated DNA
damage checkpoint signal transduction elicited by herpes simplex virus
infection. J. Biol. Chem. 280:30336–30341.
53. Shoji M, et al. 2004. The major structural components of two cell surface
filaments of Porphyromonas gingivalis are matured through lipoprotein
precursors. Mol. Microbiol. 52:1513–1525.
54. Song KS, Lee TJ, Kim K, Chung KC, Yoon JH. 2008. cAMP-responding
element-binding protein and c-Ets1 interact in the regulation of ATP-
dependent MUC5AC gene expression. J. Biol. Chem. 283:26869–26878.
55. Stoicov C, et al. 2005. Major histocompatibility complex class II inhibits
Fas antigen-mediated gastric mucosal cell apoptosis through actin-
dependent inhibition of receptor aggregation. Infect. Immun. 73:6311–
triggers DNA double-strand breaks and a DNA damage response in its
host cells. Proc. Natl. Acad. Sci. U. S. A. 108:14944–14949.
57. Tsai WH, et al. 2008. Streptococcal pyrogenic exotoxin B-induced apop-
tosis in A549 cells is mediated through ?v?3integrin and Fas. Infect. Im-
58. Vazquez A, Bond EE, Levine JA, Bond LG. 2008. The genetics of the p53
pathway, apoptosis and cancer therapy. Nat. Rev. Drug Discov. 7:979–
59. Vettore MV, et al. 2008. The relationship between periodontitis and
preterm low birthweight. J. Dent. Res. 87:73–78.
60. Wang H, et al. 2008. An ATM- and Rad3-related (ATR) signaling path-
way and a phosphorylation-acetylation cascade are involved in activation
Biol. Chem. 283:2564–2574.
61. Xia M, Knezevic D, Vassilev LT. 2011. p21 does not protect cancer cells
from apoptosis induced by nongenotoxic p53 activation. Oncogene 30:
62. Xu D, et al. 2002. Ets1 is required for p53 transcriptional activity in
UV-induced apoptosis in embryonic stem cells. EMBO J. 21:4081–4093.
63. Zhao H, Piwnica-Worms H. 2001. ATR-mediated checkpoint pathways
regulate phosphorylation and activation of human Chk1. Mol. Cell. Biol.
Multiple Pathways Activated by P. gingivalis
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