Anti-apoptotic activity of Chlamydia trachomatis Pgp3 protein involves activation of
the ERK1/2 pathway mediated by up-regulation of DJ-1 protein

Fangzhen Luo1#, Mingyi Shu1#, Silu Gong1#

1
Institute of Pathogenic Biology, Hengyang Medical College, Hunan Provincial Key
Laboratory for Special Pathogens Prevention and Control, Hunan Province Cooperative
Innovation Center for Molecular Target New Drug Study, University of South China,
Hengyang 421001, P.R. China
#These authors contributed equally to this work
*Corresponding author
Zhongyu Li,
Institute of Pathogenic Biology, Hengyang Medical College, University of South China, No.
28, Changsheng West Road, Hengyang 421001, Hunan, China.
Tel: 86-734 8282 907
Fax: 86-734 8282 907
Email: [email protected]
Abstract
Chlamydia trachomatis has evolved strategies to prevent host cell apoptosis to evade the host
immune defense. However, the precise mechanisms of anti-apoptotic activity of C.
trachomatis still need to be clarified. Pgp3, one of eight plasmid proteins of C. trachomatis,
has been identified to be closely associated with chlamydial virulence. In this study, we
attempted to explore the effects and the mechanisms of Pgp3 protein on apoptosis in HeLa
cells, the results showed that Pgp3 increased Bcl-2/Bax ratio and prevented caspase-3
activation to suppress apoptosis induced by TNF-α and cycloheximide (CHX) through
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ERK1/2 pathway activation. Down-regulation of DJ-1 with siRNA-DJ-1(si-DJ-1) reduced
ERK1/2 phosphorylation and elevated apoptotic rate significantly in Pgp3-HeLa cells.
However, inhibition of ERK1/2 signal pathway with ERK inhibitor PD98059 had little effect
on DJ-1 expression. These findings confirm that plasmid protein Pgp3 contributes to
apoptosis resistance through ERK1/2 signal pathway mediated by up-regulation of DJ-1
expression. Therefore, the present study provided novel insights into the role of Pgp3 in
apoptosis and suggested that manipulation of the host apoptosis response could be a new
approach for the prevention and treatment of C. trachomatis infection.
Keywords: Chlamydia trachomatis; Pgp3 protein; Apoptosis; ERK1/2 pathway; DJ-1
protein.
Introduction
Chlamydia trachomatis (C. trachomatis), which is an obligate intracellular bacterial pathogen
with a biphasic life cycle, is responsible for both ocular and sexually transmitted diseases
(Amza et al. 2019; Pinto et al. 2017; Rowhani-Rahbar et al. 2006; Wang et al. 2016).
Trachoma, caused by serovars A-C, is the leading cause of infectious blindness in the world;
Infections with genital serovars D-K can lead to urethritis, cervicitis and prostatitis; The L
serovars (L1-L3) are associated with lymphogranuloma venereum disease (Boutin et al. 2018;
Totten et al. 2015). Furthermore, genital infection of C. trachomatis can also increase
the susceptibility to HPV infection and subsequent cervical cancer (Mancini et al. 2018; Shew
et al. 2006), and enhance the risk of HIV infection (Buckner et al. 2016; Peterman et al.
2015). However, the mechanism of chlamydial pathogenesis is not fully elucidated.
Apoptosis is a major host defense mechanism against microbial infection (Hu et al. 2017; Li
et al. 2019; Wang et al. 2018). As an intracellular bacterium, C. trachomatis is able to inhibit
cell apoptosis to escape from the host cell immune response and complete their obligate
intracellular growth cycle in the host cells (Fischer et al. 2004a; Waguia Kontchou et al.
2016). It has been demonstrated that the anti-apoptotic mechanisms in infected cells are
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involved in activation of Raf/MEK/ERK and PI3K/AKT signal pathways by promoting the
expression of the anti-apoptotic proteins (Du et al. 2011; Rajalingam et al. 2008), and
degradation of pro-apoptotic BH3-only proteins (Fischer et al. 2004b; Ying et al. 2005). Fan
et al found that C. trachomatis inhibited host cell apoptosis by blocking mitochondrial
cytochrome c release and caspase activation (Fan et al. 1998). Some other studies (Gonzalez
et al. 2014; Kun et al. 2013) have also reported that Chlamydia-infected host cells are
resistant to apoptosis by inducing Bag-1 via the MAPK/ERK survival pathway or MDM2-p53
axis activation. Zou et al and Lei et al showed C. trachomatis inhibits apoptosis through
activation of PI3K-AKT-mediated MDM2-p53 axis and up-regulation of HMGB1 expression
induced by chlamydial secreted protein Pgp3 (Lei et al. 2017; Zou, et al. 2019).
Pgp3 (pORF5) is the only secreted protein encoded by the cryptic plasmid and distributed
mostly in the cytosol of infected cells (Li et al. 2008), which has been identified to be closely
related to chlamydial virulence (Hou et al. 2015; Hou et al. 2019; Liu et al. 2014; Zhang et al.
2019). Recent studies have shown that Pgp3 protein induced alterations in the host cell
proteome (Zou et al. 2018) and protected mitochondrial function by inducing mitophagy and
inhibiting apoptosis (Lei et al. 2017). Pgp3 also plays important roles in ascending infection
from female lower genital tract to the upper genital tract, resulting in induction of oviduct
pathology (Liu et al. 2014) and the production of TNF-α, IL-1β and IL-8 via ERK/MAPK and
p38/MAPK signal pathways (Zhou et al. 2013).
DJ-1, also known as PARK7, is a molecular chaperone, transcriptional regulator and
mitochondrial function regulator, which plays roles in oxidative stress (Jo et al. 2017;
Martin-Nieto et al. 2019; Oh et al. 2018: 211-7), pathogenesis of cancer (Kawate, et al. 2017;
Zhou, et al. 2018), and inflammation (Choi et al. 2019; Kim et al. 2014). Cells with
overexpression of DJ-1 were resistant to apoptosis by enhancing ERK-dependent mitophagy
(Gao et al. 2012), and attenuated effects of curcumin on colorectal cancer cell proliferation
and apoptosis (Shang et al. 2019), while down-regulation of DJ-1 with DJ-1 siRNA made
cells vulnerable to apoptosis through inhibiting AKT phosphorylation (Wang et al. 2016). It
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has been reported that DJ-1 is translocated to the mitochondria in response to oxidative stress
increasing the interaction between DJ-1 and Bcl-xL and stabilizing Bcl-X(L) protein level
(Ren et al. 2011). Bcl-xL, a member of the Bcl-2 family, acts as an anti-apoptotic protein by
preventing the release of mitochondrial cytochrome c and oligomerization of pro-apoptotic
Bax and Bak to inhibit apoptosis activation (Yang et al. 2015). Fan showed that anti-apoptotic
activity of DJ-1 on apoptosis was associated with its ability of decreasing Bax level through
inhibiting p53-Bax-caspase pathway (Fan et al. 2008).
Here, we investigated the roles of DJ-1 and ERK1/2 signal pathway in the regulation of
apoptosis in Pgp3-transfected HeLa cells. Our results showed that C. trachomatis
plasmid-encoded Pgp3 inhibited apoptosis via ERK1/2 signal pathway by up-regulating DJ-1
expression. In contrast, targeted knockdown of DJ-1 or utilization of ERK1/2-specific
chemical inhibitor both triggered apoptosis. Down-regulation of DJ-1 with
siRNA-DJ-1(si-DJ-1) reduced ERK1/2 phosphorylation and elevated apoptotic rate
significantly. Consequently, we summarized that ERK1/2 pathway activation mediated by
DJ-1 protein was required for Pgp3 anti-apoptotic activity.
1 Materials and methods
Cell culture and treatment
Pgp3 stable transfected HeLa cell line (Pgp3-HeLa) and control HeLa cell line (Ctrl-HeLa,
which was transfected with control vector ), prepared as previously described (Zou et al.
2018), were cultured in MEM (Hyclone, South Logan, UT, USA) supplemented with 10%
(vol/vol) fetal bovine serum (GIBCO, Grand Island, NY, USA) at 37℃, 5% CO2 incubator.
For the induction of apoptosis, Pgp3-HeLa cells and Ctrl-HeLa cells were treated for 6 h with
TNF-α (20 ng/ml; PeproTech, Rocky Hill, NJ, USA) and 2 µg/ml cycloheximide (CHX;
Sigma-Aldrich, St. Louis, MO, USA). For some experiments, Pgp3-HeLa cells were
pretreated with the ERK inhibitor PD98059 (30 µM; Sigma-Aldrich) for 30 min prior to
treatment with TNF-α and CHX.
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RNA interference and gene transfection
The transfection of siRNA-DJ-1(si-DJ-1) was used to down-regulate the expression of DJ-1.
siRNAs were designed by GenePharma (Shanghai, China). The siRNA sequences targeting
against DJ-1 were 5’-GGGCGCACAGAATTTATCTTT-3’ (sense) and
5’-AGATAAACACCAGATCCTCTT-3’ (antisense), non-target siRNA negative control
(sense 5-TTCTCCGAACGTGTCACGTTT-3 and antisense 5-ACGTGACACGTTCG
GAGAATT-3) were also synthesized to serve as a control. Pgp3-HeLa cells were seeded in
6-well plates for 24 h and then transfected with si-DJ-1 or control siRNA (NC-DJ-1) using
Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s protocol. After transfection for 24 h, the cells were treated with TNF-α/CHX
for 6 h, the protein levels of DJ-1, p-ERK, total ERK were verified by Western blot analysis.
Hoechst staining
After treated with TNF-α and CHX as described above, cells were fixed with 4%
paraformaldehyde for 15 min, followed by staining with Hoechst 33342 (Sigma-Aldrich) for
30 min at room temperature. After washing with PBS for three times, the condensed or
fragmented apoptotic nuclei were observed under fluorescence microscope, five fields per
well were randomly selected for apoptotic cell counting, and the apoptotic rate was calculated
as the ratio of number of apoptotic cells to total cells.
TUNEL assay
Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, after washing
with PBS for three times, cells were permeabilized with 0.1% Triton X-100 in 0.1%
sodium citrate buffer for 5 min, then subjected to the TUNEL assay using in situ cell death
detection kit (Roche, Basel, Switzerland), according to the manufacturer’s instruction. The
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stained cells were counted under fluorescence microscope in at least five randomly
selected fields. Nuclei were simultaneously counted by counterstaining with Hoechst 33342.
Flow cytometry analysis
Apoptotic cells were measured with Annexin V-PE/7AAD apoptosis detection kit (BD
Biosciences, San Jose, CA, USA). Briefly, cells were washed with PBS and resuspended in
100 µl 1×binding buffer, followed by addition of 5 µl Annexin V-PE and 5 µl 7-AAD. After
incubating the cells at room temperature in the dark for 15 min, the stained cells were
analyzed by flow cytometry.
Reverse transcription and quantitative Real-time PCR
For detection of target gene amplification products, total RNAs were isolated with Trizol
reagent (Invitrogen), and reversely transcribed to cDNA as a template for PCR amplification
with RevertAid™First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA,
USA) as described by the manufacturer. Quantitative real-time PCR reactions were performed
using the SYBR Green (TIANGEN, Beijing, China) in a final volume of 20 µl, including 2 μl
of cDNA template, 10 µl of 2 × SuperReal PreMix Plus, 0.6 µl of both upstream and
downstream primers (10 µM). The amplification conditions were 95℃ for 10 min, followed
by 40 cycles of denaturation at 95℃ for 10 s, annealing at 65℃ for 20 s and elongation at
72°C for 30 s. The primer sequences were shown in Table 1. The relative mRNA expression
levels of target gene were calculated using 2−∆∆Ct method, and normalized to GAPDH.
Western blot analysis
Cells were solubilized with 2% sodium dodecyl sulfate sample buffer, then loaded onto 12%
SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membranes. The
membranes were incubated in blocking buffer containing 5% nonfat milk, and then primary
antibodies were applied, including rabbit antibodies against Bax (Cell Signaling Technology,
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Danvers, MA, USA), Bcl-2, t-ERK1/2, phospho-ERK1/2, DJ-1, cleaved caspase-3 p17 and
procaspase-3 p32 (all from Cell Signaling Technology). After washing, the membranes were
incubated with horseradish peroxidase-conjugated secondary antibody (Biosharp, Hefei,
China) for 1 h at room temperature. and visualized with an ECL detection reagents (Pierce,
Rockford, IL, USA).
Statistical analysis
Statistical analysis was assessed using the SPSS 19.0 software (Chicago, IL, USA).
Comparisons between groups were evaluated by one-way analysis of variance (ANOVA)
followed by Student’s t test. All data from three independent experiments were presented as
mean ± standard deviations (SD). P<0.05 was considered to be statistically significant.
2 Result
Pgp3 plasmid protein of C. trachomatis contributes to apoptosis resistance
Host cells infected with C. trachomatis are profoundly resistant to diverse apoptotic stimuli
(Du et al. 2011; Fan et al. 1998; Fischer et al. 2004b; Rajalingam et al. 2008; Ying et al.
2005). To investigate whether Pgp3 was involved in chlamydial anti-apoptotic activity,
Pgp3-HeLa cells and Ctrl-HeLa cells were treated with TNF-α/CHX for 6 h, and examined
for apoptosis by Hoechst 33342 staining. As shown in Figure 1A, the apoptotic cells exhibited
an apoptotic-like abnormal morphology, characterized by marked chromatin condensation and
fragmentation. The nuclei of Pgp3-HeLa cells mostly exhibited dispersed and weak
fluorescence. Apoptotic bodies were more common in Ctrl-HeLa cells when compared to
Pgp3-HeLa cells (P<0.01). As illustrated in Figure 1B, after treated with TNF-α/CHX, about
11.5% of Pgp3-HeLa cells showed a typical cell apoptosis as revealed by typical chromatin
condensation which was significantly decreased compared with that of Ctrl-HeLa cells
(42.6%, P<0.01). These above results strongly indicated that Pgp3 inhibited apoptosis.
Since Bcl-2 family members have been shown to play a pivotal role in chlamydial
anti-apoptotic activity, we evaluated whether Pgp3 protein regulated the apoptosis-related
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proteins Bcl-2 and Bax expression. Upon induction of apoptosis, the mRNA level of Bcl-2
was increased, whereas Bax mRNA was decreased significantly in the Pgp3-HeLa cells, when
compared with those of Ctrl-HeLa cells (Figure 2A, P<0.01). In parallel with the
expression of mRNA of Bcl-2 and Bax, Bcl-2 protein expression was increased by 4.8 times,
and Bax protein expression was reduced by 84.3% in Pgp3-HeLa cells as compared with that
in Ctrl-HeLa cells (Figure 2B and 2C, P<0.01), thereby leading to an increase of the
Bcl-2/Bax ratio. Taken together, our results indicated that an increase in Bcl-2/Bax ratio was
involved in Pgp3-induced anti-apoptotic activity.
Caspase-3, an apoptotic biomarker, is an important effector protease that is thought to be
essential for apoptosis. Activation of caspase-3 results in irreversible biochemical,
physiologic and morphological changes in cells. Therefore, we hypothesized that Pgp3
anti-apoptotic activity may function by inhibition of caspase-3 activation. Western blot
analysis was used to reveal the expression of the cleaved caspase-3 in Pgp3-HeLa cells and
Ctrl-HeLa cells after treated with TNF-α/CHX for 6 h. As indicated in Figure 2D and 2E, the
cleaved caspase-3 (p17) and procaspase-3 (p32) in Pgp3-HeLa cells were decreased by 72.9%
(P<0.01) and 46.4% (P<0.05) respectively as compared with those in Ctrl-HeLa cells,
confirming that Pgp3 protein inhibited caspase-3 activation.
Pgp3 protein anti-apoptotic activity involves activation of the ERK1/2 pathway
ERK signal pathway has been proved to play an important role in chlamydial anti-apoptotic
activity (Du et al. 2011; Kun et al. 2013), therefore we first detected whether ERK1/2
pathway was activated in Pgp3-HeLa cells after treated with TNF-α/CHX. As shown in figure
3A and 3B, ERK1/2 phosphorylation was stronger in Pgp3-HeLa cells than that in Ctrl-HeLa
cells (P<0.01). As expected, ERK inhibitor PD98059 effectively inhibited ERK1/2
phosphorylation in Pgp3-HeLa cells, the level of ERK1/2 phosphorylation was decreased by
53.6% (P<0.01), but in Ctrl-HeLa cells, PD98059 had no effect on ERK1/2 phosphorylation
(P>0.05). These results indicated that Pgp3 activated ERK1/2 signal pathway.
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To evaluate the role of ERK1/2 pathway in Pgp3-induced anti-apoptotic activity, Pgp3-HeLa
cells were pretreated with PD98059 for 30 min prior to treatment with TNF-α/CHX. The
results of Hoechst staining and TUNEL assay showed that the apoptotic rates were increased
by 28.5% and 42.1% respectively after treated with PD98059, compared with the control
group (Figure 3C and 3D, P<0.01). The cell apoptosis was confirmed by flow cytometry. As
shown in figure 3E and 3F, PD98059 induced an obvious increase in cell apoptosis, the
apoptotic rate were about 13.4% and 26.0% respectively in Pgp3-HeLa cells in the presence
or absence of PD98059, a statistical difference was observed between the two groups
(P<0.01), indicating that inhibition of ERK1/2 signal pathway counteracted apoptosis
resistance induced by Pgp3 protein. These results suggested that ERK activation was required
for Pgp3 anti-apoptotic activity.
Next, we wanted to test whether Pgp3 selectively regulated the expression of Bcl-2 and Bax
and caspase-3 activation to inhibit apoptosis through ERK1/2 signal pathway. The results
showed that both Bcl-2 mRNA and protein expression levels were significantly decreased in
Pgp3-HeLa cells after treated with PD98059 compared with control group (Figure 3G, 3H and
3I, P<0.01). In contrast, Bax was up-regulated in comparison with the control cells (Figure
3G, 3H and 3I, P<0.01). The alteration of Bcl-2 and Bax levels resulted in a
decrease in Bcl-2/Bax ratio. Similar to Bax, the cleaved caspase-3 p17 was significantly
increased in Pgp3-HeLa cells after treated with PD98059 (Figure 3H and 3I, P<0.01),
conforming that ERK1/2 pathway was involved in regulation of Bcl-2/Bax ratio and
activation of caspase-3.
Pgp3 promotes the expression of DJ-1 protein
The isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with
nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis was
applied to identify and quantify the differentially expressed proteins in the Pgp3-Hela and the
Ctrl-HeLa cells as described by Zou et al (Zou et al. 2018). A total of 314 proteins with fold
change ratios < 0.8 or > 1.25 were successfully identified. DJ-1 was one of up-regulated
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proteins, the MS/MS mass spectrometry and relative quantitative information of DJ-1 were
shown in figure 4A and 4B. Quantitative real time-PCR and Western blot analysis were
further performed to validate the mRNA and protein expression levels of DJ-1. As it was
shown in figure 4C, 4D and 4E, DJ-1 mRNA expression was significantly increased (P<0.01)
and the protein expression was also up-regulated (P<0.01) in Pgp3-Hela cells when compared
with Ctrl-HeLa cells, the results were consistent with the proteomic data.
DJ-1 is essential for Pgp3 anti-apoptotic activity
To further detect whether DJ-1 was participated in anti-apoptotic activity induced by Pgp3,
siRNA targeting DJ-1 was designed and validated by Western blot (Figure 5A and 5B), the
results showed that DJ-1 protein expression was decreased by about 42.6% in the Pgp3-HeLa
cells transfected with si-DJ-1 compared with that in control cells transfected with NC-DJ-1
(P<0.05), suggesting that DJ-1-siRNA had a stronger suppressive effect at protein level. The
apoptosis rate of Pgp3-HeLa cells transfected with si-DJ-1 significantly increased by about
30.8% and 36.9% than that of the control cells (P<0.01), according to Hoechst staining and
TUNEL staining respectively after apoptosis induction with TNF-α/CHX (Figure 5C and 5D).
By flow cytometry analysis, we found that down-regulation of DJ-1 resulted in the increased
apoptosis in Pgp3-HeLa cells (Figure 5E and 5F). The apoptotic rate of Pgp3-HeLa cells
transfected with si-DJ-1 increased by 17.1% compared with control group (P<0.05).
ERK1/2 pathway activation is dependent on up-regulation of DJ-1 expression induced
by Pgp3 protein
We further addressed whether ERK1/2 pathway was involved in Pgp3anti-apoptotic activity
through up-regulation of DJ-1 protein. Western blot was used to determine ERK1/2
phosphorylation in Pgp3-HeLa cells transfected with si-DJ-1 or NC-DJ-1. As shown in figure
6A and 6B, ERK1/2 phosphorylation in Pgp3-HeLa cells transfected with si-DJ-1 was
decreased by about 50% when compared with that of the same cells transfected with negative
control (P<0.05). However, treatment with ERK1/2 inhibitor PD98059 did not significantly
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alter DJ-1 expression in Pgp3-HeLa cells (Figure 6C and 6D, P>0.05). These observations
demonstrated that ERK1/2 pathway activation was required for DJ-1 expression.
Discussion
Like other obligate intracellular pathogens, C. trachomatis has evolved strategies to prevent
host cell apoptosis to complete the obligate intracellular growth cycle (Fischer et al. 2004a;
Waguia Kontchou, et al. 2016). Although antiapoptotic effects of C. trachomatis have been
described previously (Du et al. 2011; Fan et al. 1998; Fischer et al. 2004b; Rajalingam et al.
2008; Ying et al. 2005), the precise mechanisms on how C. trachomatis prevents apoptosis,
and which protein(s) is involved in chlamydial anti-apoptotic activity remain unclear. In this
study, we have demonstrated that Pgp3 protein is profoundly resistant to
TNF-α/CHX-induced apoptosis through ERK1/2 signal pathway activation mediated by
up-regulation of DJ-1 protein.
Apoptosis is induced mainly through two cellular pathways: the extrinsic death-receptor
pathway and the intrinsic mitochondrial pathway (Fan et al. 2008; Fulda et al. 2006). The two
pathways are functionally interconnected to some extent (Chou et al. 2015; Jin et al 2005).
The Bcl-2 family proteins and caspase-3 are important regulatory proteins in cell apoptosis.
Members of Bcl-2 family can regulate the mitochondrial outer membrane permeability (Kale
et al. 2018) and control cell apoptosis by activating the caspase-3 mediated pathway (Sun et
al. 2012). Bcl-2 family can be divided into anti-apoptotic proteins (such as Bcl-2, Bcl-xL,
etc.) and pro-apoptotic proteins (such as Bax, Bak, etc.) two different groups, therefore, the
ratio of anti-apoptotic to pro-apoptotic proteins is involved in determination of the cellular
fate. Our results firstly showed that the apoptotic rate of Pgp3-HeLa cells was significantly
decreased when compared with that of Ctrl-HeLa cells after apoptosis induction with
TNF-α/CHX, indicating that Pgp3 had anti-apoptotic activity and inhibited apoptosis strongly.
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Further research found that Pgp3 up-regulated Bcl-2 expression and down-regulated Bax
expression and caspase-3 activation, this observation suggested that apoptosis resistance
mediated by Pgp3 protein in HeLa cells was closely associated with up-regulation of
Bcl-2/Bax ratio and inhibition of caspase-3 activation.
ERK pathway is an important signal pathway for the host immune response, and regulates
many important cellular functions including cell survival and proliferation (Howie et al. 2008;
Nozawa et al. 2019; Wang et al. 2019). Accumulating evidence has demonstrated that ERK
signal pathway is not only essential for chlamydial acquisition of host glycerophospholipids
(Su et al. 2004), which benefits in chlamydial growth in the infected host cells, but also
important for anti-apoptotic activity to obtain advantages in persistent chlamydial infection
(Du et al. 2011; Kun et al. 2013). Our previous studies have shown that ERK signal pathway
activated by Pgp3 was involved in the production of pro-inflammatory cytokines (Zhou et al.
2013), we therefore supposed that ERK pathway was also participated in Pgp3 anti-apoptotic
activity. This hypothesis was subsequently validated by the experimental data. Firstly, we
found that after treated with TNF-α/CHX, ERK1/2 phosphorylation was stronger in
Pgp3-HeLa cells than that in Ctrl-HeLa cells, and ERK inhibitor PD98059 effectively
inhibited ERK1/2 phosphorylation induced by Pgp3 protein, which suggesting that ERK
signal pathway was activated in Pgp3-HeLa cells. Secondly, inhibition of the ERK1/2 signal
pathway with an ERK inhibitor reduced the ratio of Bcl-2/Bax by down-regulation of Bcl-2
expression and up-regulation of Bax expression at both mRNA and protein levels, PD98059
also prevents effectively activation of caspase-3 in Pgp3-HeLa cells. Finally, the
apoptotic rate significantly increased in Pgp3-HeLa cells after inhibition of ERK signal
pathway activation with PD98059, when compared with that of control group.
In our previous study, we found that Pgp3 increased PI3K-AKT activation leading to
apoptosis resistance via an up-regulation of the Bcl-2/Bax ratio and p53 degradation,
anti-apoptotic activity of Pgp3 protein was closely related to the activation of
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PI3K-AKT-mediated MDM2-p53 axis (Zou, et al. 2019). It is well-known that both
PI3K-AKT and ERK signal pathways play important role in controlling cell proliferation,
survival and apoptosis. These pathways are regulated by positive and negative feedback and
crosstalk mechanisms (Guan et al. 2015; Butler et al. 2017; Ersahin et al. 2015). Increasing
evidence demonstrates that the PI3K-AKT and ERK signal pathways may interact to promote
the growth and inhibit apoptosis (Cui et al. 2015; Lee et al. 2009; Jiang et al. 2017; Semaan et
al. 2016). Apoptosis is a complex and multi-step process, a large number of genes, including
members of the Bcl-2 family, Caspases family, and signal pathways
are involved in this process (Gonzalez et al. 2014; Kun et al. 2013; Du et al. 2011;
Rajalingam et al. 2008). In this regard, our future studies should focus on determining the
relationship between ERK and PI3K/AKT pathways following activation with Pgp3. This
would contribute to further clarifying the mechanism underlying the mechanism of
anti-apoptosis by Pgp3 and its potential as a therapeutic intervention.
DJ-1, as a multi-functional protein, is involved in various cellular processes, and mutations in
its gene are associated with many diseases. Studies have shown that DJ-1 affects cell viability
by up-regulating the phosphorylation of ERK1/2 (Gu et al. 2009), and protects dopaminergic
neurons against rotenone-induced apoptosis by enhancing ERK-dependent mitophagy (Gao et
al. 2012). DJ-1 binds to p53, resulting in suppression of DUSP1 expression, thereby
regulating the activation level of ERK (Kato et al. 2013). Moreover, DJ-1 decreases Bax
expression through repression of p53 transcriptional activity (Fan et al. 2008). In order to
determine the correlation among DJ-1, ERK and Pgp3 anti-apoptotic activity, we first tested
the expression of DJ-1 in Pgp3-Hela cells by quantitative real time-PCR and Western blot
analysis, the results showed that Pgp3 significantly enhanced DJ-1 expression. Further study
showed that down-regulation of DJ-1with si-DJ-1 reduced ERK1/2 phosphorylation,
furthermore, dramatically promoted apoptosis of Pgp3-HeLa cells, these results are consistent
with those of previous studies (Gao et al. 2012; Jo et al. 2017). However, inhibition of
the ERK1/2 signal pathway had little effect on DJ-1 expression. These observations indicated
that DJ-1 was involved in Pgp3 anti-apoptotic activity through ERK1/2 signal pathway, which
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may be associated with the escape of C. trachomatis from host immune systems and complete
their obligate intracellular growth cycle in the host cells.
In conclusion, we have demonstrated that Pgp3 plasmid protein of C. trachomatis can prevent
host cells apoptosis by regulating expression levels of Bax and Bcl-2 and activation of
caspase-3. This anti-apoptotic activity involves ERK activation via up-regulation of DJ-1
protein, combined with our previous research (Zou, et al. 2019), the anti-apoptotic activity of
Pgp3 protein was depicted in Figure 7. Our findings not only help define the basis of
chlamydial pathogenesis, but also provide new information on understanding the mechanisms
of chlamydia anti-apoptotic activity. A therapeutic interference with Pgp3 anti-apoptotic
activity could perhaps facilitate elimination of the bacteria and might prevent and control the
risk of C. trachomatis infection.
Conflicts of Interest
All authors declare no financial competing interests.
All authors declare no non-financial competing interests.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
81772210 and 31470277), Construct Program of the Key Discipline in Hunan Province (No.
2011-76), Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control
(No. 2014-5), and Hunan Province Cooperative innovation Center for Molecular Target New
Drug Study (No. 2016-429).
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Graphical Abstract The Chlamydia trachomatis Pgp3 plasmid protein plays an important
role in apoptosis inhibition and the anti-apoptotic activity of Pgp3 protein involves ERK
activation through up-regulation of DJ-1 protein
Figure 1. Pgp3 protein inhibits apoptosis induced by TNF-α/CHX
Pgp3-HeLa cells and Ctrl-HeLa cells were treated with TNF-α/CHX for 6 h. The cells were then
stained with Hoechst 33342 and viewed under a fluorescence microscope. (A) Apoptotic nuclei
appeared dense and fragmented (marked with arrows). (B) Histogram for apoptotic rates assayed
by Hoechst staining. Data represented from three independent experiments. *P<0.01.
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Figure 2. Pgp3 protein regulates apoptosis related factors
(A) Expression levels of Bcl-2 and Bax were analyzed by quantitative real time-PCR. The relative
expression level was normalized to the level of GAPDH. Data from three independent
experiments were analyzed and presented as the mean ± SD. *P<0.01. (B) Expression of Bcl-2
and Bax was analyzed by Western blot, GAPDH served as a protein loading control. (C) The
histogram for expression levels of Bcl-2 and Bax assayed by Western blot. Y-axis represented the
fold change of relative density. *P<0.01. (D) The protein levels of cleaved caspase-3 p17 and
procaspase-3 p32 were detected by Western blot. (E) The histogram for expression levels of
cleaved caspase-3 p17 and procaspase-3 p32. *P<0.01, **P<0.05.
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Figure 3. Pgp3 protein anti-apoptotic activity involves activation of the ERK1/2 pathway
Pgp3-HeLa cells and Ctrl-HeLa cells were treated with TNF-α/CHX for 6 h prior to treatment
with or without ERK inhibitor PD98059. (A) Activation of ERK1/2 signal pathway was detected
by Western blot. (B) The histogram for ERK1/2 phosphorylation level assayed by Western blot,
Y-axis represented the fold change of relative density. *P<0.01, NS P>0.05. (C) Apoptotic cells
were identified by using Hoechst 33342 and TUNEL staining. (D) Histogram for apoptotic rates
assayed by Hoechst and TUNEL staining. Data represented from three independent experiments.
*P<0.01. (E) Apoptotic cells were determined by Annexin V-PE/7-AAD flow cytometry. (F)
Histogram represented apoptotic rates assayed by flow cytometry analysis. *P<0.01. (G) The
transcription levels of Bcl-2 and Bax were measured by real time-PCR. The relative mRNA level
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was normalized to GAPDH. Data represented from three independent experiments. *P<0.01. (H)
Protein expression levels of Bcl-2, Bax and cleaved caspase-3 p17 in Pgp3-HeLa cells were
analyzed by Western blot. GAPDH served as a protein control. (I) The histogram for relative
protein expression levels of Bcl-2, Bax and cleaved caspase 3 p17 assayed by Western blot, Y-axis
represented the fold change of relative density. *P<0.01.
Figure 4. DJ-1 expression was up-regulated in Pgp3-Hela cells
Pgp3-HeLa cells and Ctrl-HeLa cells were harvested, and lysed with the lysis buffer. The iTRAQ
labeling was performed, and peptides derived from Pgp3-HeLa cells were labeled with iTRAQ tag
113 or 114, while peptides derived from Ctrl -HeLa cells were labeled with tags 115 or 116. After
strong cation exchange fractionation, peptide mass fingerprinting (A) and iTRAQ relative
quantitation (B) of DJ-1 protein were performed with nano liquid chromatography-tandem MS
(nanoLC-MS/MS) and ProteinPilot software. DJ-1 expression was further validated by
quantitative real time-PCR (C) and Western blot analysis (D, E), the results showed that DJ-1
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expression was increased in Pgp3-Hela cells than that in Ctrl-HeLa cells at both mRNA and
protein levels. *P<0.01.
Figure 5. Role of DJ-1 in anti-apoptotic activity induced by Pgp3
(A) Effects of si-DJ-1 or NC-DJ-1 on DJ-1 expression in Pgp3-HeLa cells. The expression of DJ-1
was detected in Pgp3-HeLa cells transfected with si-DJ-1 or a scrambled negative control (NC) by
Western blot. (B) Histogram for interference effect of si-DJ-1, Y-axis represented the fold change
of relative density. **P<0.05. (C) Effects of DJ-1 on apoptosis in Pgp3-HeLa cells treated with
TNF-α/CHX using Hoechst 33342 and TUNEL staining. (D) The histogram represented the
percentage of positive cells by Hoechst 33342 and TUNEL staining. *P<0.01. (E) Apoptotic cells
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were detected by flow cytometry analysis. (F) The histogram represented the percentage of
apoptotic cells. Data were shown from three independent experiments. **P<0.05.
Figure 6. DJ-1 promoted ERK1/2 phosphorylation in Pgp3-HeLa cells
Pgp3-HeLa cells were transfected with si-DJ-1 and NC-DJ-1 respectively for 24 h or incubated
with ERK inhibitor PD98059 for 30 min prior to treatment with TNF-α/CHX. ERK1/2
phosphorylation (A) and DJ-1 protein expression (C) were detected by Western blot analysis. The
histogram represented ERK1/2 phosphorylation levels (B) and DJ-1 expression levels (D) assayed
by Western blot. Y-axis represented the fold change of relative density. **P<0.05, NS P>0.05.
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Figure 7. Schematic diagram Cycloheximide of Pgp3-induced apoptosis inhibition through activation of
ERK signal pathway and PI3K-AKT-mediated MDM2-p53 axis
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