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Respiratory Research
GPS2 ameliorates cigarette smoking-induced
pulmonary vascular remodeling bymodulating
theras-Raf-ERK axis
Ting Hu1,2†, Chaohui Mu1,2†, Yanmiao Li1,2, Wanming Hao2,3, Xinjuan Yu2,3, Yixuan Wang2,3, Wei Han2,3* and
Qinghai Li2,3*
Abstract
Background Mitogen-activated protein kinase (MAPK)signaling-mediated smoking-associated pulmonary vascular
remodeling (PVR) plays an important role in the pathogenesis of group 3 pulmonary hypertension (PH). And G protein
pathway suppressor 2 (GPS2) could suppress G-protein signaling such as Ras and MAPK, but its role in cigarette smok-
ing -induced PVR (CS-PVR) is unclear.
Methods An in vivo model of smoke-exposed rats was constructed to assess the role of GPS2 in smoking-induced
PH and PVR. In vitro, the effects of GPS2 overexpression and silencing on the function of human pulmonary arterial
smooth cells (HPASMCs) and the underlying mechanisms were explored.
Results GPS2 expression was downregulated in rat pulmonary arteries (PAs) and HPASMCs after CS exposure. More
importantly, CS-exposed rats with GPS2 overexpression had lower right ventricular systolic pressure (RVSP), right
ventricular hypertrophy index (RVHI), and wall thickness (WT%) than those without. And enhanced proliferation
and migration of HPASMCs induced by cigarette smoking extract (CSE) can be evidently inhibited by overexpressed
GPS2. Besides, GPS2siRNA significantly enhanced the proliferation, and migration of HPASMCs as well as activated Ras
and Raf/ERK signaling, while these effects were inhibited by zoledronic acid (ZOL). In addition, GPS2 promoter meth-
ylation level in rat PAs and HPASMCs was increased after CS exposure, and 5-aza-2-deoxycytidine (5-aza) inhibited
CSE-induced GPS2 hypermethylation and downregulation in vitro.
Conclusions GPS2 overexpression could improve the CS-PVR, suggesting that GPS2 might serve as a novel therapeu-
tic target for PH-COPD in the future.
Keywords Pulmonary hypertension, Cigarette, GPS2, MAPK, DNA methylation
†Ting Hu and Chaohui Mu contributed equally to this work.
*Correspondence:
Wei Han
hanw@uor.edu.cn
Qinghai Li
wflqh88@163.com
Full list of author information is available at the end of the article
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Page 2 of 16
Huetal. Respiratory Research (2024) 25:210
Graphical Abstract
Introduction
Pulmonary hypertension (PH) is defined as a mean pul-
monary arterial pressure (mPAP) >20 mmHg at rest [1].
It is accompanied by complex etiologies and high mor-
tality [1]. PH is classified into 5 clinical subgroups, and
the group 3 PH (PH due to chronic lung disease (CLD),
PH-CLD is a common type [2]. As the most common
CLD, chronic obstructive pulmonary disease (COPD) is
the 3rd most common cause of death in the world, and
its major complication is PH associated with COPD (PH-
COPD), which is closely related to acute exacerbation or
poor prognosis of COPD patients [2]. Cigarette smok-
ing (CS), a shared risk factor for COPD and PH-COPD
[3], could induce airway injury and pulmonary vascular
remodeling (PVR) [4, 5]. And advanced COPD patients
with severe hypoxemia (about 50–70%) are frequently
complicated with mild-moderate PH, while a small part
of COPD patients (1–3%) with minor airway damage also
have severe PH (mPAP >45mmHg) [1]. Due to the com-
plex pathophysiology of PH-COPD, there is currently
no effective drugs but conservative oxygen therapy for
PH-COPD.
In general, PVR is the major pathological change of
PH-COPD, which is characterized by intimal damage as
well as the thickening of the media and adventitia [6, 7].
And the phenotypic changes of pulmonary artery smooth
muscle cells (PASMCs) including increased prolifera-
tion, enhanced migration and reduced apoptosis, are the
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Page 3 of 16
Huetal. Respiratory Research (2024) 25:210
pathologic basis of CS-induced medial thickening and
PVR [8]. Perivascular inflammation, oxidative stress,
senescence, and glycolipid metabolism have been reported
to be involved in the occurrence and development of CS-
induced PVR (CS-PVR) [9, 10], but the pathogenesis of
CS-PVR is still uncertain.
Chronic inflammation caused by CS is a common cause
of airway injury and PVR, in which mitogen-activated
protein kinase (MAPK) signaling plays a key role [9, 11].
But the specific role of MAPK signaling in CS-PVR has
not been fully elucidated. e extracellular signal-regu-
lated kinase (ERK) cascade is one of the classical MAPK
signals, consisting of a series of key protein kinases (Ras,
Raf, MEK, and ERK). As an important regulatory path-
way for modulating smooth muscle function, the ERK
pathway has been found to be associated with the occur-
rence and progression of PH of various types [12–14].
Zhang etal. found that Raf1 expression was significantly
elevated in the serum of patients with idiopathic pulmo-
nary arterial hypertension (IPAH) compared with healthy
subjects, suggesting that Raf1 might be a potential bio-
marker of this disease [15]. And inhibition of the Ras/
ERK signaling has been shown to improve PVR induced
by silymarin in rats [16]. In addition, ERK1/2 pathway
activation has been reported to closely linked to CS-PVR
and PH development [17, 18]. However, the roles of Ras
and Raf1 in CS-PVR have not been fully elucidated and
whether they regulate the ERK pathway in CS-PVR is still
not clear.
It is well known that ERK1/2 MAPK signaling, which
is closely related to CS-PVR, is mainly transduced and
regulated by the G protein family [19]. As a repressor of
G protein signaling, the G protein pathway suppressor
2 (GPS2) is widely expressed in various mammalian tis-
sues and is involved in many physiological and pathologi-
cal processes, including metabolism [20–22], immune
responses [23], erythroid differentiation [24], and brain
development [25]. In addition, GPS2 expression is
reduced in a variety of tumors (such as osteosarcoma and
melanoma) and adipose tissues, indicating that GPS2 is
closely related to tumor cell proliferation and lipid-asso-
ciated inflammation [26, 27]. Importantly, Zhuang etal.
found that GPS2 overexpression upregulated potassium
channel protein expression in human embryonic kidney
cells by inhibiting ERK1/222. Given that PH is a chronic
inflammatory proliferative disease and GPS2 has been
reported to play a regulatory role in ERK signaling, we
hypothesize that GPS2 may be involved in CS-PVR by
regulating the ERK1/2 pathway. However, relevant stud-
ies have not been reported yet.
In addition, DNA methylation, as the most common
epigenetic modification, is involved in the development
of various diseases such as COPD and lung cancer by
affecting multigene expression through methylation
modification of the genome [28–31]. Currently, most
of the studies on DNA methylation in the field of PH
have focused on pulmonary arterial hypertension (PAH)
[32–36]. However, in 2019, our team was the first to pro-
pose CS-PVR, and we revealed that cigarette smoking
enhanced RASEF methylation in pulmonary arteries of
rats and further induced PVR [37]. And in this study, we
will explore whether GPS2 expression is also regulated by
DNA methylation after CS exposure.
is study aims to investigate the role of GPS2 in CS-
PVR and PH, as well as the possible molecular mecha-
nisms. Our study will further reveal the pathogenesis of
CS-PVR, which may provide a new target for the preven-
tion and treatment of PH-COPD.
Methods
Animal models
Adult male SD rats were obtained from Jinan Pengyue
Laboratory Animal Breeding Company. Rats were ran-
domly divided into a “smoking group” and an “air group”.
e “smoking group” was exposed to cigarette smoke
from 10 cigarettes (Hong Jin Long, 1.2 mg nicotine,
15mg tar per cigarette, Wuhan, China) in a plexiglass
ventilated box for 1h each time, two times per day and
5 days per week for 3 months, as previously noted [37].
e “air group” rats were housed in clean air during the
same period. After receiving the approval from the Eth-
ics Committee of Qingdao Municipal Hospital, our study
was carried out in accordance with the regulations of the
Chinese Animal Ethics Committee.
Preparation ofcigarette smoke extract (CSE)
CSE was acquired by combusting Kentucky Research
Cigarettes (CODE 3R4F, Class A cigarettes, University
of Kentucky, USA). e method for CSE preparation
was carried out following previously reported proce-
dures [6, 37] with a few modifications. In brief, CSE
was freshly prepared by bubbling the smoke from one
research cigarette into 25ml culture medium, at a burn-
ing rate of one cigarette every 5min. A 0.22 μm filter
was used to filter the extract, which was considered to
be CSE of 100% concentration.
Infection withGPS2 overexpressing adeno‑associated virus
GPS2 overexpressing adeno-associated virus type 1
(AAV1-GPS2-GFP) and adeno-associated virus type
1-negative control (AAV1-NC-GFP) were both pur-
chased from Hanheng Biotechnology Company. We con-
structed a smoking-exposed rat model, as described in a
previous study [37]. SD rats were divided into the “smok-
ing group” and the “air group”. After 3 months of cigarette
exposure, SD rats in the “smoking group” were randomly
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Huetal. Respiratory Research (2024) 25:210
subdivided into the intervention group and the control
group. AAV1-GPS2-GFP (2*1010vg/rat) was injected into
rats in the intervention group via airway, and thus this
group is called “Smoking + AAV1.GPS2” group. And rats
in the control group and the “air group” were injected
with equivalent doses of AAV1-NC-GFP, which were
combined as “AAV1.NC” group. And the control group is
termed “Smoking + AAV1.NC” group”. After virus infec-
tion, the previous exposure (cigarette smoking or air)
was continued for 6 weeks, followed by a hemodynamic
assessment. Upon the completion of the treatment, rats
were killed and samples were retained. Isolation of rat
PAs was performed as previously described [38]. In brief,
under the stereo microscope, the isolation of the rat pul-
monary arteries was performed. We started to separate
the pulmonary arterial trunk which is connected to the
right ventricle. We separated the left side into a branch of
left pulmonary arteries until the pulmonary hilum. And
we separated the right side until the hilum and continu-
ously isolated the arteries downward into the right upper
lobe pulmonary artery, the right interlobar pulmonary
artery, and the right lower lobe pulmonary artery. e
isolated pulmonary arteries were used in subsequent
experiments.
Culture andtransfection ofHPASMCs
Human pulmonary artery smooth muscle cells
(HPASMCs) were purchased from Proximity Life Sci-
ences (China). e cells were cultured in DMEM solu-
tion containing 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin and placed in a constant tem-
perature incubator at 37°C with 5% CO2. HPASMCs
were infected with Ad.GPS2 (adenovirus for GPS2
overexpression) and NC.Ad (the control adenovirus)
(MOI = 200); and we also transfected HPASMCs with
GPS2 siRNA (siGPS2; 50 nM) using Lipofectamine 2000
(Invitrogen, USA) for silencing GPS2. And SiGPS2 target
sequences were as follows: the first 5′-CAG CCA GCT
TAT AGT CCT A − 3′, the second 5′-GGA GAA GCT TTT
GGC TCT A − 3′.
Hemodynamic measurements
Right ventricular systolic pressure (RVSP) was measured
by a PowerLab system (AD Instruments, Australia) as a
previous study has described [39]. Briefly, animals were
anesthetized with intraperitoneal sodium pentobarbital
(80 mg/kg), followed by tracheal intubation and inva-
sive ventilation. e right ventricular wall was punctured
using a 26G needle (Sigma-Aldrich, St. Louis, MO) con-
nected to a pressure transducer. Data recording was initi-
ated after a stable tracking waveform was obtained, and
data were analyzed using Lab Chart software (AD Instru-
ments, Australia).
Sample processing andassessment ofright ventricular
hypertrophy
Following the hemodynamic analysis, rat blood samples
were collected, and the hearts were dissected. en, the
hearts were flushed with saline to remove residual blood;
the atria and valves were excised; and the left and right
ventricles were preserved. Finally, the right ventricle (RV)
was separated from the left ventricle (LV) and interven-
tricular septum (S). RV hypertrophy was assessed by the
weight ratio of RV to (LV + S), which was known as Ful-
ton’s index.
Assessment ofPVR
HE staining was performed for the transverse and longi-
tudinal sections of the rat left lung, and then we assessed
the wall thickness of all small pulmonary arteries on the
sections that matched the outer diameter (50–150μm).
Compared to pulmonary veins, pulmonary arteries are
less dilated, accompanying the airway. ey have higher
smooth muscle content and thicker walls. ese features
make them easier to identify. e images were analyzed
using Image Pro Plus 6.0 (Media Cybernetics, MD). e
wall thickness (WT%) index was calculated to assess PVR
(WT% = [(external diameter − internal diameter)/exter-
nal diameter] × 100%), as a previous study described [40].
Apoptosis assay
After being processed using the methods mentioned
above, stimulated HPASMCs were collected. Annexin
V-FITC and PI were added to the cell suspensions after
incubation for 15min according to the instructions of
the Apoptosis Detection Kit (Elabsience, China). And the
level of apoptosis was detected by flow cytometry.
Migration assay
A transwell migration experiment was conducted using
24-well transwell plates containing chambers with an
8-µm pore size (Transwell, Corning, USA). Migrated
HPASMCs were fixed with 4% paraformaldehyde and
stained with 0.1% crystal violet, according to the manu-
facturer’s instructions. Quantification was carried out by
counting the number of cells in ten randomly selected
fields viewed with a light microscope (Olympus, Tokyo,
Japan).
Immunouorescence andimmunohistochemical staining
Immunofluorescence and immunohistochemical stain-
ing were conducted as previously reported [37, 39, 41]
with minor changes. And primary antibodies were used
as follows : a-SMA (Abcam, Cambridge, UK) and pho-
tographed using an orthogonal fluorescence microscope
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Huetal. Respiratory Research (2024) 25:210
(Olympus, Tokyo, Japan); GPS2 (ProteinTech, Wuhan,
China) and photographed using an ordinary light micro-
scope (Olympus, Tokyo, Japan).
qRT‑PCR
Total RNA was extracted from rat pulmonary arter-
ies or HPASMCs by applying Trizol reagent, reverse
transcribed, and then subjected to RT-PCR using cor-
responding kits according to the manufacturer’s instruc-
tions (Takara, Japan). And the relative value of mRNA
(with GAPDH or β-actin as internal reference) was calcu-
lated by the 2^(-ΔΔCt) method.
e primers used were as follows: human β-actin, 5′- AGA
AAA TCT GGC ACC ACA CCT-3′ (forward) and 5′- GAT
AGC ACA GCC TGG ATA GCA-3′ (reverse); rat β-actin, 5′-
CGT AAA GAC CTC TAT GCC AACA-3′ (forward) and 5′-
CGG ACT CAT CGT ACT CCT GCT − 3′ (reverse); human
GPS2, 5′- CAG CAG AGC CTG ACT GTT CA − 3′ (forward)
and 5′- AAG CAC TTG GGG TCC AAA CA − 3′ (reverse); rat
GPS2, 5′- AAC TGC AGC AGA AGC TTT CA − 3′ (forward)
and 5′- GCA GCT GAT GTC AGA GTG GT − 3′ (reverse). e
ratio for the mRNA of interest was normalized by β-actin.
Western blot analysis
Total protein was extracted from rat pulmonary arter-
ies or HPASMCs and its concentration was measured
by BCA kit (Elabsience, China). Primary antibodies we
used were as follows: GPS2 (1:1000), ERK1/2 (1:3500),
p-ERK1/2 (r202/Tyr204) (1:3500) (Wuhan Sanying,
China), Raf1 (1:1000), p-Raf1 (ser259) (1:1000) (CST,
USA), proliferating cell nuclear antigen (PCNA) (1:3000),
matrix metalloproteinase 9 (MMP9) (1:1000), TP53
(1:1000) (Abcam, UK), and the relevant secondary anti-
bodies. Finally, ECL luminescent solution was applied
to develop the color; a fully automated chemilumines-
cence gel-imaging analyzing system (Beijing Sage Sci-
ence and Technology Co., Ltd.) was used for exposure
and photography; and Image J software was utilized for
analysis. Quantitative results are expressed as gray value
ratios of target proteins to GAPDH or β-Actin, except for
the signaling pathway proteins, such as “Active-Ras/Ras,
p-Raf/Raf, and p-ERK/ERK” which were presented as a
ratio of each phosphorylated protein to their respective
total proteins.
Ras activity assay
Ras Pull-Down Activation Assay Biochem Kit (Cat#:
16,117, ermo Scientific, USA) was applied to rat pul-
monary arteries and treated HPASMCs to assess the
level of activated Ras. Lastly, ECL luminescent solu-
tion was used to develop the color; a fully automated
chemiluminescent gel imaging analysis system (Sage
Science and Technology, China.) was used for exposure
and photography; and Image J software was utilized for
analysis.
DNA methylation sequencing oftarget regions
DNA was extracted from rat pulmonary arteries or
HPASMCs under the combined stimuli (CSE and 5-aza)
using the Genomic DNA Extraction Kit (Tiangen Biochem-
ical Technology, China). e extracted DNA was amplified
according to the corresponding methylation primers. Sub-
sequently, using Acegen Targeted Methyl Panel Custom-
ized System (Acegen Technology, China), DNA sequencing
was performed to assess the methylation level of CpGs in
its GPS2 promoter. e following methylation primers
were used: rat GPS2 5′-TTT YGT TTT TGT TTT TGG TTA
TTG TGA ATT TTA AAG -3′ (forward), 5′-CTA TAC AAA
AAA CCC ACA ACT TCC TC-3′ (reverse); human GPS2
5′-TTG TAT GTT TTT TGT TTY GAG GGG ATAA-3′ (for-
ward) 5′-CCR TTA TAA TAA AAT TTA AAC RTC CTA TTC
CAC-3′ (reverse).
Statistical analysis
GraphPad Prism 8.0 was used for statistical analyses. All
quantitative data were expressed as “mean ± standard
deviation”. Comparisons between the two groups were
performed using Student’s t tests. For multiple com-
parisons, normally distributed data were analyzed using
one-way analysis of variance followed by Newman–
Keuls tests. A P-value < 0.05 was considered statistically
significant.
Results
A rat model ofCS‑induced PH
Compared with the “air group”, the “smoking group”
showed significant increases in RVSP (46.84 ± 5.76
mmHg vs. 22.33 ± 6.42 mmHg), right ventricular hyper-
trophy index (RVHI) and WT%, as well as inflammatory
cell infiltration and lung tissue damage (Fig.1A-E), indi-
cating that the PH model was successfully constructed in
the CS-exposed rats.
CS reduced GPS2 expression inrat PAs andHPASMCs
In vivo, the “smoking group” showed significantly
decreased GPS2 expression in medial layer of rat PAs
compared with the “air group” (Fig. 2A-D). In vitro,
2% CSE significantly promoted the proliferation of
HPASMCs (Figure S1). However, GPS2 expression was
significantly reduced in the HPASMCs stimulated by 2%
CSE for 24–48h, which was consistent with the changes
invivo (Fig.2E-G).
GPS2 overexpression alleviated CS‑induced PH inrats
To investigate the effects of GPS2 on CS-PVR and PH in
rats, we performed hemodynamic and histopathological
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Huetal. Respiratory Research (2024) 25:210
assessments of cardiopulmonary tissues by a single tran-
stracheal injection of AAV1.GPS2 in the PH rats after
chronic exposure to CS for 3months and continued the
exposure for 1.5 months.
We found that the “Smoking + AAV1.GPS2 group”
had significant increases in the GPS2 expression (Figure
S2A-D), as well as decreases in RVSP (23.14 ± 0.99 mmHg
vs. 42.03 ± 6.14 mmHg) (Fig.3A, B), RVHI (Fig.3C) and
WT% (Fig. 3D-E) in the rat PAs, compared with the
“Smoking + AAV1.NC” group. Besides, GPS2 overexpres-
sion attenuated inflammatory cell infiltration and the
lesions of lung tissue in CS-exposed rats (Fig.3D). is
finding indicated that high GPS2 expression alleviated
PVR and PH in CS-exposed rats.
GPS2 overexpression inhibited CSE‑induced proliferation
andmigration ofHPASMCs
To investigate the effect of GPS2 on CSE-induced bio-
logical behaviors of HPASMCs, we applied GPS2.Ad
in combination with CSE to stimulate HPASMCs and
assessed their functional changes after stimulation. qRT-
PCR assays showed that GPS2 was successfully overex-
pressed in HPASMCs (Fig.4A).
As for proliferation, the cell confluency and cell viabil-
ity of HPASMCs, as well as the fraction of EDU-positive
cells were significantly increased after 48h of CSE stim-
ulation (Fig. 4B-D), while GPS2.Ad inhibited the CSE-
induced HPASMC proliferation. Moreover, Annexin V/
PI flow assay revealed that GPS2.Ad increased HPASMC
apoptosis (Fig. 4E), while Transwell assay showed that
GPS2.Ad also inhibited CSE-induced migration of
HPASMCs (Fig.4F). Next, we evaluated the expression of
proteins associated with cell proliferation, apoptosis and
migration, and found that CSE increased the expression
of PCNA and MMP9 and downregulated TP53 expres-
sion in HPASMCs, which could be inhibited by GPS2.Ad
(Figure S3).
GPS2 overexpression inhibited activation oftheRas/Raf/
ERK pathway
As mentioned before, we found that GPS2 overexpres-
sion in rat pulmonary vessels alleviated CS-induced
PVR and PH. To elucidate the possible molecular
mechanisms, we performed western blot analysis of
potential downstream signaling pathways in rat PAs
and HPASMCs. Invivo, rats in the “Smoking + AAV1.
Fig. 1 Hemodynamic and histopathological changes in rats. A, B Power Lab system for detection of RVSP in rats; (C) right ventricular hypertrophy
index in rats; (D, E) HE staining showing wall thickness of small pulmonary arteries in rats (400X). ( n = 6, ** P < 0.01, *** P < 0.001; bar = 50 μm)
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Huetal. Respiratory Research (2024) 25:210
GPS2” group showed higher levels of GPS2 pro-
tein and significantly lower expression of p-Raf1 and
p-ERK1/2 than those in the “Smoking + AAV1.NC”
group (Fig. 5A, B). In vitro, we assessed the expres-
sion of ERK1/2 MAPK signaling proteins in HPASMCs
after being infected with GPS2.Ad and stimulated
with 2% CSE. And we found reduced expression of
GPS2 protein and activated Ras, Raf1 and ERK1/2
signaling in HPASMCs after being stimulated by CSE,
which could be inhibited by GPS2.Ad (Fig.5C-F). This
finding suggested that GPS2 overexpression might
improve CS-PVR by regulating Ras/Raf1/ERK signal-
ing in HPASMCs.
Fig. 2 Figure 2 CS exposure affects GPS2 expression in pulmonary artery smooth muscle. A Immunohistochemical staining of GPS2 in rat lung
tissues (bar=100 μm, arrows indicate positively stained cells in rat PAs); (B) qRT-PCR were used to detect GPS2 expression in rat PAs (n=6, *P < 0.05);
(C, D) Quantification of GPS2+ cells in the intimal and mesial layers of PAs (bar= 100 μm,n=6, **P < 0.01). E Western blot were used to detect GPS2
expression in rat PAs (n=6, *P < 0.05)； (F, G) qRT-PCR and Western blot were used to detect GPS2 expression in HPASMCs after CSE stimulation;
(n=3, *P < 0.05, **P < 0.01)
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Huetal. Respiratory Research (2024) 25:210
siGPS2‑induced proliferation andmigration ofHPASMCs
viaRas/Raf/ERK pathway
To further explore the connection between the inhibitory
role of GRPS2 in CS-induced PVR and the Ras/Raf/ERK
pathway, we combined zoledronic acid (ZOL) (an inhibi-
tor of Ras signaling) and siGPS2 to stimulate HPASMCs,
and then monitored the changes in the biological behav-
iors of stimulated HPASMCs and the activation of Ras/
Raf/ERK pathway in these cells.
Our results were as follows. (a) ZOL is not toxic to
HPASMCs at a concentration of 100µM or less (Figure
S4B), and ZOL at a concentration of 40µM significantly
inhibited the activation of Raf1 and ERK1/2 (Figure S4C);
(b) siGPS2 promoted cell confluency (Figure S4D), cell
viability and the proportion of “EDU-positive cells” in
HPASMCs, whereas ZOL inhibited the pro-proliferative
effect of siGPS2; (c) siGPS2 increased the migration of
HPASMCs, while ZOL inhibited the pro-migratory effect
of siGPS2 (Fig. 6A-C); (d) siGPS2 decreased HPASMC
apoptosis, whereas ZOL inhibited the anti-apoptotic
effect of siGPS2 (Fig. 6D); (e) siGPS2 increased the
expression of PCNA and MMP9, as well as decreased
TP53 expression, all of which could be reversed by ZOL
(FigureS5); (f) siGPS2 attenuated the expression of GPS2
proteins and promoted the activation of Ras, Raf1 and
ERK1/2; whereas ZOL inhibited the activation of Ras,
Raf1 and ERK1/2 caused by siGPS2 (Fig.6I-L).
e above results indicated that ZOL (a Ras inhibitor)
could inhibit siGPS2-induced proliferation, migration,
resistance to apoptosis of HPASMCs, as well as the acti-
vation of Ras, Raf1 and ERK1/2 proteins by siGPS2 in the
cells.
CS enhanced GPS2 promoter methylation inrat PAs
andHPASMCs
To explore whether DNA methylation is involved in
the regulation of GPS2 expression by CS, we conducted
methylation sequencing and found that GPS2 promoter
methylation was significantly increased in the PAs of
CS-induced PH rats (Fig.7A). To better understand the
effects of DNA methylation on GPS2 expression, we
conducted DNA methylation sequence, PCR and WB
in vitro. As the result, CSE decreased the cell conflu-
ency and increased DNA methylation. But after 5-aza-
2-deoxycytidine (5-Aza-CdR), a DNA methyltransferase
inhibitor, was added in culture medium of HPASMCs,
Fig. 3 Effects of GPS2 overexpression on hemodynamics and cardiopulmonary structure in CS-exposed rats. A, B Hemodynamic conditions of rats
in the three groups; (C) RVHI of rats; (D, E) HE staining to assess the thickness of pulmonary arterioles vessel wall in rats (400×). (Air+NC.AAV1 group
(n=8), Smoking+NC.AAV1 group (n=8), Smoking+GPS2.AAV1 group (n=8), ***P< 0.001; bar= 50 μm)
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Huetal. Respiratory Research (2024) 25:210
DNMT1 expression and GPS2 promoter methylation
were decreased and GPS2 expression was increased (F7
B-F).
(A) Targeted bisulfite sequencing (TBS) was used to
evaluate the methylation level of GPS2 promoter in rat
PAs (n = 4); (B, C) the cell confluency of HPASMCs and
DNMT1 mRNA expression; (D, E) qRT-PCR and West-
ern blot were used to assess the expression of GPS2
mRNA and protein in HPASMCs after the combined
stimulation by CSE and 5-aza; (F) TBS measured the
methylation level of GPS2 promoter in HPASMCs after
the combined stimulation by CSE and 5-aza. (n = 3,
*P < 0.05, **P < 0.01, ***P < 0.001)
Discussion
As a common complication of COPD, COPD-associated
pulmonary hypertension (PH-COPD) seriously under-
mines the quality of life and prognosis of COPD patients.
Unfortunately, there are currently no effective therapeu-
tic drugs for PH-COPD. Our study is the first to explore
the role of GPS2 in CS-PVR and its mechanism using
in vivo and in vitro experiments, which might provide
a scientific basis for the development of new drugs for
PH-COPD. is study yielded several important findings.
(1) CS exposure downregulated GPS2 expression in rat
PAs and HPASMCs; (2) GPS2 overexpression alleviated
smoking-induced PVR and PH in rats, as well as inhib-
ited the proliferation and migration of HPASMCs; (3)
ZOL reversed siGPS2-induced proliferation and migra-
tion of HPASMCs, as well as the activation of Ras/Raf1/
ERK1/2 signaling; and (4) CS exposure increased GPS2
promoter methylation in rat PAs and HPASMCs.
Smoking is a major risk factor for PH-COPD. CS-PVR
is an important pathological alteration in PH-COPD,
in which the phenotypic switch of pulmonary arterial
smooth muscle cells (PASMCs) (excessive proliferation
and migration) is the pathological basis of CS-PVR [37,
42, 43]. Experimental evidence from animals has shown
that CS-PVR often precedes the occurrence of emphy-
sema [44, 45]. Besides, the historical “vascular hypoth-
esis” for the cause of emphysema has been proposed [46].
Consistently, our study found that RVSP, WT% and RVHI
were significantly increased in rats exposed to CS for 3
months without significant alveolar structure changes,
indicating that CS-PVR precedes the occurrence of
emphysema which usually requires more than 6-month
Fig. 4 GPS2 overexpression alleviated CSE-induced behavioral changes in HPASMCs. A GPS2 expression in GPS2.Ad-infected HPASMCs; (B‑D)
CCK8 and EDU to detect the proliferative ability of HPASMCs; (E) Flow assay to detect the apoptosis of HPASMCs; (F) Transwell assay to detect
the migratory ability of HPASMCs. (n=3, *P < 0.05, ***P < 0.001)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 16
Huetal. Respiratory Research (2024) 25:210
Fig. 5 Activation of Ras/Raf1/ERK signaling in PAs of CS-exposed rats and CSE-stimulated HPASMCs. A, B Western blot to detect Raf1and
ERK1/2 activation levels in the PAs of rats. (n=8, *P < 0.05 vs Air+NC.AAV1, #P< 0.05 vs Smoking+NC.AAV1); (C, D) Ras pull-down activation assay
to determine activated Ras level (n=3, *P < 0.05, **P < 0.01); (E, F) Levels of GPS2 protein and the Ras/MAPK signaling pathway proteins (Ras, Raf1
and ERK1/2) (n=3, *P < 0.05, **P < 0.01 vs "NC.Ad ";#P < 0.05, ##P < 0.01 vs "NC.Ad+CSE")
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Page 11 of 16
Huetal. Respiratory Research (2024) 25:210
Fig. 6 Effects of ZOL on the function of HPASMCs and the activation of Ras/Raf/ERK pathway in the cells. A, B CCK8 and EDU to detect
the proliferative ability of HPASMCs; (C) Transwell assay to detect the migration ability of HPASMCs; (D) Flow cytometry to detect apoptosis
of HPASMCs; (E) Ras pull-down activation assay to determine activated Ras level; (F) GPS2 protein level and activation levels of Raf1 and ERK1/2.
(n=3, *P < 0.05, **P < 0.01, ***P < 0.001 vs “siNC”; #P < 0.05, ###P < 0.001，##P < 0.01, ####P < 0.0001 vs“siGPS2”)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 16
Huetal. Respiratory Research (2024) 25:210
CS-exposure. In addition, Marija Gredic etal. found that
myeloid-cell-specific deletion of inducible nitric oxide
synthase (iNOS) prevented smoke-induced PH in mice
but did not affect hypoxia-induced PH [47]. All the above
evidence suggests that the development of CS-PVR is not
dependent on emphysema-associated hypoxemia, which
may explain the pathophysiological features of cases with
mild COPD and severe PH.
G protein-coupled receptors (GPCRs) can participate
in the contraction and remodeling of the pulmonary
Fig. 7 Effect of CS on GPS2 promoter methylation. ATargeted bisulfite sequencing (TBS) was used to evaluate the methylation level of GPS2
promoter in rat PAs (n=4); (B, C)the cell confluency of HPASMCs and DNMT1 mRNA expression; (D, E) qRT-PCR and Western blot were used to assess
the expression of GPS2 mRNA and protein in HPASMCs after the combined stimulation by CSE and 5-aza; (F) TBS measured the methylation level
of GPS2 promoter in HPASMCs after the combined stimulation by CSE and 5-aza. (n=3, *P<0.05, **P <0.01, ***P <0.001)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 13 of 16
Huetal. Respiratory Research (2024) 25:210
arteries by responding to various extracellular stimuli
(including hormones, neurotransmitters, calcium ions,
sugars/lipids/proteins/amino acids), and regulating vir-
ous signaling pathways such as Ca2+, endothelin, and
prostacyclin [48]. As a classical G protein signaling
inhibitor, GPS2 has been found to be involved in lipid
inflammation and tumorigenesis [49–51]. And PH, a
chronic inflammatory proliferative disease of the pul-
monary vasculature, might be associated with GPS2.
But the role of GPS2 in PH is less investigated. ere-
fore, our study investigated the role of GPS2 in a CS-
induced PH rat model and CSE-stimulated HPASMCs.
We found that CS exposure reduced GPS2 expres-
sion in rat PAs and HPASMCs and that GPS2 over-
expression mitigated the proliferative phenotype of
HPASMCs as well as CS-PVR and CS-induced PH in
rats, which suggested that CS exposure might induce
PVR by downregulating GPS2. ese effects of GPS2
on CS-PVR/CS-induced PH might be related to the
antiproliferative properties of GPS2 which has been
extensively studied in the field of tumors. It has been
reported that GPS2 deletion activates AKT signal-
ing to promote proliferation of triple-negative breast
cancer cells (MDA-MB-231) [51], and it inhibits the
proliferation of gastric cancer cells by increasing the
ubiquitylation of the epidermal growth factor receptor
(EGFR) [52]. Now the involvement of GPS2 in CS-PVR/
CS-induced PH is well established, but the underlying
mechanisms remain unclear.
GPS2 was first found in yeast cells and it could inhibit
Ras activation [53]. e Ras/Raf/ERK (MAPK) signaling
is a key pathway which regulates the inflammation and
proliferation in a variety of inflammatory proliferative
disorders [54, 55]. Our preliminary study and previous
studies from others have found that Raf/ERK signaling
is closely associated with the development of IPAH and
CS-induced PH [56, 57]. However, whether GPS2 par-
ticipates in CS-PVR by regulating the Ras/Raf/ERK axis
has not been investigated. To explore this, we examined
ERK cascade signaling and assessed the effects of the Ras
inhibitor-ZOL on the function and related signaling of
HPASMCs. We found that CS exposure promoted Raf/
ERK1/2 activation in rat PAs and HPASMCs, and that
ZOL inhibited siGPS2-induced Ras/Raf/ERK activation
as well as the proliferation and migration of HPASMCs.
Notably, ZOL is widely used in the clinical treatment
of osteoporosis and osteolytic bone disease by inhibit-
ing osteoclast function and survival. Consistent with
our study, Hassan’s team reported that ZOL inhibited
the proliferation and migration of human aortic smooth
muscle cells (HASMCs) [58]. Our finding that ERK cas-
cade signaling played an important role in PVR was also
supported by many previous studies [47, 48].
Numerous studies have confirmed that ERK1/2 MAPK
activation signaling is closely related to the remod-
eling phenotype (enhanced proliferation and migration
and apoptosis resistance) and the expression of related
markers (e.g., PCNA, MMP9, p53, etc.) in pulmonary
vascular smooth muscle cells. And the present study
demonstrated that GPS2 modulated Ras/Raf/ERK signal-
ing to indirectly affect the expression of p53 and MMP9.
In addition, it has been reported that GPS2 can consti-
tute a transcriptional repressor complex directly involved
in the transcription of p5359, 60, but there is no relevant
study on the direct regulation of the transcription of
genes such as MMP9 and PCNA.
DNA methylation is the most common epigenetic
modification. Environmental exposures, such as chronic
cigarette smoking, often alter DNA methylation and
participate in gene expression regulation and disease
development [61, 62]. To investigate the mechanisms of
GPS2 expression in CS-exposed rat PAs and HPASMCs,
we evaluated the methylation level of the GPS2 pro-
moter. And we found that CS increased the methylation
level of the GPS2 promoter in rat PAs and HPASMCs,
while 5-aza (a DNA methylation inhibitor) inhibited CS-
induced GPS2 hypermethylation in HPASMCs, which
suggested the involvement of “DNA methylation” in the
regulation of GPS2 expression by CS. Similarly, our team
previously reported that CS enhanced RASEF methyla-
tion in rat PAs, contributing to CS-PVR [37]. In addition,
aberrant methylation of superoxide dismutase 2 (SOD2)
and bone morphogenetic protein receptor 2 (BMPR2)
has been found to be involved in the development of PAH
[32, 63]. However, unlike the specific treatments taken
for other epigenetic abnormalities such as miRNAs, there
currently is a lack of DNA methylation therapeutic strat-
egies for PH due to a paucity of gene-specific methylation
editing technologies [64, 65].
is study explored the role of GPS2 in CS-PVR
and its possible mechanisms using invivo and invitro
experiments, but it also had some limitations. First,
this study focused on the antiproliferative properties
of GPS2 in CS-PVR. In addition to pulmonary vascular
cell dysfunction, lung inflammation and hypoxia asso-
ciated with interstitial lung lesions can also trigger PH
[66, 67]. In our study, we observed that CS-exposed rats
with overexpressed GPS2 exhibited attenuated intersti-
tial lung lesions, suggesting that GPS2 might play a role
in pulmonary hypertension by modulating interstitial
lung lesions. However, our study did not conduct an
animal experiment to verify this finding. Second, e
lungs of smoke-exposed rats exhibited extensive infil-
tration of inflammatory cells, e.g. macrophages and
neutrophils [68]. And macrophage activation is closely
related to PH [69]. Besides, previous studies have
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Page 14 of 16
Huetal. Respiratory Research (2024) 25:210
shown that GPS2 expression is associated with mac-
rophage activation [70, 71]. erefore, it is reasonable
to hypothesize that apart from its direct influence on
small arteries, GPS2 might also alleviates CS-PH via its
interaction with lung macrophages or other immune
cells. However, this hypothesis requires further verifi-
cation. ird, the present study showed that ZOL inhib-
ited Ras/Raf/ERK signaling, as well as suppressed the
proliferation and migration of HPASMCs, but its ability
to improve CS-PVR and PH needs to be further veri-
fied by invivo animal experiments. Fouth, many stud-
ies have found that CS exposure can trigger pulmonary
vascular endothelial dysfunction, which is involved in
the development of pulmonary hypertension [72, 73].
And the immunohistochemistry in our study suggests
that GPS2 is expressed not only in the smooth muscle
of pulmonary arteries, but also in the endothelium of
the pulmonary vasculature, which suggests that GPS2
may play a role in maintaining endothelial cellular
homeostasis. After quantifying GPS2 expression in the
pulmonary vascular endothelium, we found no signifi-
cant difference between the “air group” and the “CS-
exposed group”. However, we did not further verify this
finding in PAECs.
In summary, our study showed that CS enhanced
GPS2 methylation and downregulated its expression,
while high GPS2 expression inhibited Ras/Raf1/ERK1/2
activation as well as attenuated CS-PVR and PH. e
present study provides new insights into the pathogen-
esis of PH-COPD and offers a novel target for the drug
development in PH-COPD.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12931- 024- 02831-0.
Supplementary Material 1.
Supplementary Material 2.
Authors’ contributions
T.H., C.M., W.H., and Q.L. conceived and planned the experiments. T.H., C.M., Y.L.
and Y.W. carried out the animal experiments., T.H., C.M., Y.L., X.Y. and W.H. con-
tributed to cell experiments. W.H and Q.L. supervised the study. T.H. and Q.L.
took the lead in writing the manuscript. All authors provided critical feedback
and helped shape the research, analysis and manuscript. Q.L. was responsible
for the overall content as the guarantor.
Funding
This work was supported by the National Natural Science Foundation of China
(NO. 81900048), Medicine and Health science and technology develop-
ment program of Shandong Province (NO. 202103020630) and Science and
technology development plan program of Shinan district of Qingdao (NO.
2022-4-002-YY).
Availability of data and materials
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
All animal experiments were approved by the Ethics Committee of Qingdao
Municipal Hospital.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1 Department of Respiratory and Critical Care Medicine, Qingdao Municipal
Hospital, Qingdao University, Qingdao, China. 2 Qingdao Key Lab of Common
Diseases, Qingdao Municipal Hospital, University of Health and Rehabilita-
tion Sciences, 5 Donghai Middle Road, Qingdao 266071, China. 3 Depart-
ment of Respiratory and Critical Care Medicine, Qingdao Municipal Hospital,
University of Health and Rehabilitation Sciences, 5 Donghai Middle Road,
Qingdao 266071, China.
Received: 10 January 2024 Accepted: 1 May 2024
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