Betulinic Acid Induces eNOS Expression via the AMPK-Dependent
KLF2 Signaling Pathway
Gi Ho Lee,§ Jin Song Park,§ Sun Woo Jin, Thi Hoa Pham, Tuyet Ngan Thai, Ji Yeon Kim,
Chae Yeon Kim, Jae Ho Choi, Eun Hee Han, and Hye Gwang Jeong*
Cite This: https://dx.doi.org/10.1021/acs.jafc.0c06250 Read Online
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ABSTRACT: Betulinic acid (BA) is a natural pentacyclic triterpenoid with protective effects against inflammation, metabolic
diseases, and cardiovascular diseases. We have previously shown that BA prevents endothelial dysfunction by increasing nitric oxide
(NO) synthesis through activating endothelial nitric oxide synthase (eNOS) in human endothelial cells. However, the effect of BA
on eNOS expression remains unclear. Thus, the aim of our study was to investigate the intracellular pathways associated with the
effect of BA to regulate eNOS expression in human endothelial cells. BA significantly increased eNOS expression in a time- and
concentration-dependent manner. Additionally, BA upregulated the expression of the transcription factor KLF2, which is known to
regulate eNOS expression. KLF2 silencing in human endothelial cells attenuated the ability of BA to upregulate eNOS. BA also
increased levels of intracellular Ca2+, activating CaMKKβ, CaMKIIα, and AMPK. Inhibition of the TRPC calcium channel abolished
BA-mediated effects on intracellular Ca2+ levels. Moreover, BA increased the phosphorylation levels of ERK5, HDAC5, and MEF2C.
Pretreatment of cells with compound C (AMPK inhibitor), LMK235 (HDAC5 inhibitor), and XMD8-92 (ERK5 inhibitor)
attenuated the BA-induced eNOS expression. Collectively, these findings suggest that BA induces eNOS expression by activating the
HDAC5/ERK5/KLF2 pathway in endothelial cells. The data presented here provide strong evidence supporting the use of BA to
prevent endothelial dysfunction and treat vascular diseases, such as atherosclerosis.
KEYWORDS: betulinic acid, eNOS, KLF2, HDAC5, ERK5, AMPK
■ INTRODUCTION
Endothelial dysfunction can cause various cardiovascular
diseases by dysregulating immune cell adhesion and migration.1 Nitric oxide (NO) produced by endothelial nitric oxide
synthase (eNOS) plays a crucial role in endothelial function,
suppressing platelet adhesion and aggregation, as well as
regulating vascular tone through smooth muscle cell
relaxation.2,3 Expression and activation of eNOS is controlled
at the gene transcription level or by post-translational
modifications, such as phosphorylation.4,5 For instance,
laminar shear stress induces eNOS expression and activity in
the endothelium, modulating vascular homeostasis, enhancing
endothelial cell survival, and exerting antithrombotic, antiadhesive, and anti-inflammatory effects.6,7
Kruppel-like factor 2 (KLF2), a transcription factor, is an
essential regulator of eNOS expression.8 KLF2 belongs to a
subclass of the zinc-finger family that is related to histone
deacetylase 5 (HDAC5) and extracellular signal-regulated
protein kinase 5 (ERK5).9,10 The ERK5/KLF2 signaling
pathway is activated in response to the laminar shear stress,
regulating endothelial function by promoting the expression of
eNOS, thrombomodulin, and other anti-inflammatory mediators.11,12 By suppressing the expression of adhesion molecules,
KLF2 inhibits leukocyte adhesion to the endothelium.13
Moreover, HDAC5 was recently shown to negatively regulate
laminar shear stress-mediated KLF2 signaling.9 Upon
phosphorylation by calcium/calmodulin-dependent kinases,
HDAC5 translocates from the nucleus to the cytosol. Nuclear
export of HDAC5 increases the myocyte enhancer factor 2
(MEF2) and KLF2 transcriptional activity, thereby enhancing
eNOS expression and preventing endothelial dysfunctioninduced inflammation.9,14
Betulinic acid (BA) is a natural compound of pentacyclic
triterpene isolated from Betula. It has been shown to alleviate
inflammation,15 cancer,16 diabetes,17 cardiovascular diseases,18
and platelet activation.19 BA was recently reported to regulate
eNOS expression and vasorelaxation involved in antiatherosclerosis.20,21 Additionally, we have previously shown
that BA increases NO production via eNOS phosphorylation.22 However, the effect of BA on eNOS expression and the
underlying mechanisms remain unclear.
Therefore, in the present study, we assessed the effects of BA
on eNOS expression and the molecular mechanism in human
endothelial cells and found that BA induced eNOS expression
via KLF2 activation. We also explored the signaling pathways
underlying the effect of BA on eNOS expression and identified
AMPK-dependent HDAC5/ERK5 signaling as a key mediator
Received: September 29, 2020
Revised: November 11, 2020
Accepted: November 12, 2020
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© XXXX American Chemical Society A
https://dx.doi.org/10.1021/acs.jafc.0c06250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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of eNOS and KLF2 expression. This is the first study to
demonstrate the role of Ca2+-dependent AMPK/HDAC5/
ERK5 signaling in eNOS expression in response to BA.
■ MATERIALS AND METHODS
Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), and trypsin were purchased from Welgene
(Gyeongsan, South Korea), and fluo-4 acetoxymethyl ester (Fluo-4
AM) was obtained from Invitrogen (Carlsbad, CA). XMD8-92, KN-
62, tetracaine, and SKF96365 hydrochloride were purchased from
Sigma-Aldrich (St. Louis, MO). LMK235 and compound C were
obtained from Tocris (Cookson, Bristol, U.K.), and EDTA was
purchased from GenDEPOT (Barker, TX). Horseradish peroxidaseconjugated anti-rabbit and anti-mouse IgG antibodies, as well as
antibodies against eNOS and p-MEF2C, were purchased from Abcam
(Cambridge, MA); p-AMPK and p-CaMKKβ were purchased from
Cell Signaling Technology (Beverly, MA); KLF2, p-HDAC5, p-ERK5,
p-CaMKIIα, and β-actin were purchased from Santa Cruz
Biotechnology (Dallas, TX). All chemicals were of the highest grade
and commercially available.
Cell Culture. EA.hy926 cells were obtained from the American
Type Culture Collection (Bethesda, MD) and maintained in highglucose DMEM supplemented with 10% FBS at 37 °C in a 5% CO2-
humidified atmosphere. Human umbilical vein endothelial cells
(HUVECs) were purchased from Lonza (Walkersville, MD) and
maintained in EGM-2 Bullet Kit medium containing growth
supplements (Lonza, Walkersville, MD) at 37 °C in a 5% CO2-
humidified incubator. Cells were used between 3 and 7 passages and
treated with BA for experiment when 80−90% confluent. For cell
treatments, BA was dissolved in DMSO. Control cells were treated
with DMSO alone; the final concentration of DMSO did not exceed
0.1%.
KLF2 Short-Interfering RNA (siRNA) Transfection. Doublestranded siRNA oligonucleotides targeting KLF2 and control siRNA
were purchased from Santa Cruz Biotechnology. EA.hy926 and
HUVECs were seeded in 6-well plates and allowed to reach a density
of 70−80%. Subsequently, the cells were transfected with KLF siRNA
or control siRNA (60 nM final concentration) using Lipofectamine
RNAiMAX (Thermo Fisher Scientific, Waltham, MA) according to
the manufacturer’s instructions; transfections were performed at 37
°C for 4 h. After 24 h, the cells were treated with BA (or DMSO
control), and the efficacy of KLF2 silencing was evaluated by western
blot analysis.
Western Blot. After BA treatment, EA.hy926 cells and HUVECs
were lysed and centrifuged at 13,000 rpm for 15 min. Supernatants
were collected, and protein concentrations were measured using a
Pro-Measure protein assay kit (Intron Biotechnology, Seongnam,
Korea). Equal amounts of protein were resolved by 10% SDS-PAGE
and transferred onto nitrocellulose membranes. The membranes were
blocked in 5% skim milk and probed with the indicated primary and
secondary (anti-rabbit or anti-mouse) antibodies. Protein bands were
detected using a Hisol ECL Plus detection kit (BioFact, Daejeon,
Korea). The intensity of the protein bands was processed by Image-J
software, and then the values were normalized to an internal control
(β-actin level and/or Lamin B1).
Real-Time PCR. Total RNA was isolated from EA.hy926 cells
using RNAiso Plus (total RNA extraction reagent; Takara, Shiga,
Japan), and cDNA was synthesized using the BioFact RT Series kit.
PCR amplification was monitored using Bio-Rad CFX Connect RealTime PCR software, version 1.4.1 (Bio-Rad Laboratories, Hercules,
CA). The following primers were used: eNOS forward, 5′-
GAAGGCGACAATCCTGTATGG-3′; eNOS reverse, 5′-
TGTTCGAGGGACACCACGTCAT-3′; KLF2 forward, 5′-ATTCCATGCCATCTGTGCG-3′; KLF2 reverse, 5′-CGTCCCGGCTACATGTGC-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′; and GAPDH reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′.
Measurement of Intracellular Ca2+ Levels. Intracellular Ca2+
levels were measured by Fluo-4 NW staining according to the
manufacturer’s instructions. In brief, cells were cultured in 96-well
black plates (1 × 104 cells/well) and incubated with Fluo-4 NW for
30 min at 37 °C. Subsequently, cells were incubated in the dark for 30
min at room temperature. After the indicated treatments (BA alone or
in combination with inhibitors), the intracellular Ca2+ levels were
measured at 20 s intervals for 15 min using a BioTek Synergy HT
microplate reader (BioTek, Winooski, VT). Fluorescence images were
Figure 1. BA regulates eNOS expression by activating KLF2 signaling in human endothelial cells. (A, B) EA.hy926 cells were treated with 1 μM BA
for 6−24 h or with 0.1−1 μM BA for 24 h, followed by eNOS and KLF2 gene expression analysis by real-time PCR. (C, D) EA.hy926 cells were
treated with 1 μM BA for 6−24 h or with 0.1−1 μM BA for 24 h. eNOS and KLF2 protein levels were determined by immunoblotting. (E)
HUVECs were treated with 0.1−1 μM BA for 24 h, and eNOS and KLF2 protein levels were determined by immunoblotting. (F) EA.hy926 cells
were transfected with 60 nM siRNA of KLF2 siRNA or control siRNA and treated with 1 μM BA for 24 h. eNOS and KLF2 protein levels were
determined by immunoblotting. Data are presented as means ± SD from three independent experiments. *Significantly different from the control
at p < 0.01.
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J. Agric. Food Chem. XXXX, XXX, XXX−XXX
captured under an EVOS fluorescence microscope (Life Technologies, Carlsbad, CA).
Statistical Analysis. Data are presented as means ± standard
deviation (SD) from three independent experiments. One-way
analysis of variance was used to determine significant differences
between treatment groups. The Newman−Keuls test was used to
compare three or more groups. P-values <0.01 were considered
statistically significant.
■ RESULTS
BA Regulates eNOS Expression by Activating KLF2
Signaling in Human Endothelial Cells. In the vascular
Figure 2. HDAC5 and ERK5 are required for KLF2-mediated eNOS expression in human endothelial cells in response to BA. (A, B) EA.hy926
cells were treated with 1 μM BA for 5−60 min or with 0.1−1 μM BA for 15 min. HDAC5, ERK5, and MCF2C phosphorylation levels were
determined by immunoblotting. (C) EA.hy926 cells were treated with 1 μM BA for 0.5−6 h, and nuclear HDAC5 levels were determined by
immunoblotting. (D, E) EA.hy926 cells were pretreated with 10 μM XMD8-92 or 50 nM LMK235 for 30 min and incubated with 1 μM BA for an
additional 24 h. eNOS and KLF2 protein levels were determined by immunoblotting. Data are presented as means ± SD from three independent
experiments. *Significantly different from the control at p < 0.01. #
Significantly different from the BA-treated group at p < 0.01.
Figure 3. BA induces CaMKKβ, CaMKIIα, and AMPK phosphorylation in human endothelial cells by increasing intracellular Ca2+ levels. EA.hy926
cells were treated with 0.1−1 μM BA, and Fluo-4 NW signals were measured at 20 s intervals for 10 min (A) or visualized by fluorescence
microscopy (B). (C, D) EA.hy926 cells were treated with 1 μM BA for 5−60 min or 0.1−1 μM BA for 15 min. CaMKKβ, CaMKIIα, and AMPK
phosphorylation levels were determined by immunoblotting. Data are presented as means ± SD from three independent experiments. *Significantly
different from the control at p < 0.01.
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J. Agric. Food Chem. XXXX, XXX, XXX−XXX
endothelial cells, eNOS expression is activated by KLF2
signaling in response to many stimuli, involving laminar shear
stress.23 However, the effects of BA on eNOS expression are
understudied. Here, we assessed the effect of BA on KLF2
signaling activation and eNOS expression in human
endothelial cells. We treated cells with 1 μM BA or less,
which has been previously shown not to cause significant
cytotoxicity.22 BA treatment on EA.hy926 cells significantly
enhanced eNOS and KLF2 mRNA levels in a time- and
concentration-dependent manner (Figure 1A,B). Consistently,
BA treatment on EA.hy926 cells significantly increased the
protein levels of eNOS and KLF2 in a time- and concentrationdependent manner (Figure 1C,D). Similar findings were
observed in HUVECs (Figure 1E). Importantly, KLF2
silencing in EA.hy926 cells suppressed eNOS induction in
response to BA (Figure 1F). These results suggest that BA
upregulates eNOS in a KLF2-dependent manner in human
endothelial cells.
HDAC5 and ERK5 Are Required for KLF2-Mediated
eNOS Expression in Human Endothelial Cells in
Response to BA. The transcriptional activity of KLF2 is
regulated by HDAC5 and ERK5. Additionally, ERK5-mediated
MEF2C9,10 Therefore, we next evaluated the effect of BA on
the phosphorylation of HDAC5, ERK5, and MEF2C.
Interestingly, BA significantly enhanced the phosphorylation
of HDAC5, ERK5, and MEF2C (Figure 2A,B). Further, we
found that BA-treated endothelial cells had lower nuclear
HDAC5 levels than control cells (Figure 2C). To assess the
relevance of HDAC5 and ERK5 in BA-induced eNOS and
KLF2 expression, we treated cells with LMK235 (HDAC5
inhibitor) and XMD8-92 (ERK5 inhibitor). Interestingly, both
inhibitors attenuated BA-induced eNOS and KLF2 expression
(Figure 2D,E), suggesting that HDAC5 and ERK5 are essential
for BA-induced eNOS upregulation.
BA Induces CaMKKβ, CaMKIIα, and AMPK Phosphorylation in Human Endothelial Cells by Increasing
Intracellular Ca2+ Levels. In the vascular endothelium,
activation of calcium/calmodulin signaling leads to vasodilation via NO production.24 Numerous natural bioactive
compounds have been shown to prevent endothelial
dysfunction by modulating the Ca2+ signaling pathway.25−27
Here, we investigated the effect of BA on the levels of
intracellular Ca2+, as well as the phosphorylation levels of
AMPK, CaMKKβ, and CaMKIIα. BA treatment of EA.hy926
cells increased intracellular Ca2+ influx in a time- and
concentration-dependent manner (Figure 3A,B). In line with
this result, BA significantly increased the phosphorylation of
AMPK, CaMKIIα, and CaMKKβ (Figure 3C,D), suggesting
that BA activates Ca2+-dependent kinases by promoting
intracellular Ca2+ influx.
TRP Channels Are Required for the BA-Induced
Increase in Intracellular Ca2+ Levels and Activation of
CaMKKβ, CaMKIIα, and AMPK in Human Endothelial
Cells. To determine the source of BA-induced intracellular
Ca2+ influx in human endothelial cells, we treated EA.hy926
cells with EDTA and tetracaine (RyR inhibitor). Pretreatment
of cells with EDTA and tetracaine inhibited the increase in
intracellular Ca2+ levels by BA (Figure 4A). Moreover, EDTA
and tetracaine suppressed AMPK, CaMKIIα, and CaMKKβ
phosphorylation in response to BA treatment (Figure 4C).
Several studies have reported that transient receptor potential
(TRP) channels expressed on the endothelial membrane
prevent endothelial dysfunction by regulating intracellular Ca2+
influx.25,28,29 Thus, we used various TRP channel inhibitors to
assess the role of TRP channels in the ability of BA to
modulate intracellular Ca2+ levels and the activity of calciumdependent kinases. Interestingly, pretreatment with the
transient receptor potential cation (TRPC) channel blocker
Figure 4. Role of TRP channels and RyRs in the BA-induced increase in intracellular Ca2+ levels and subsequent phosphorylation of CaMKKβ,
CaMKIIα, and AMPK. (A) Cells were pretreated with 0.5 mM EDTA and 50 μM tetracaine (RyR inhibitor) (B) or 10 μM HC (TRPA blocker),
20 μM SKF (TRPC blocker), and nifedipine (LTCC blocker) for 30 min before treatment with Fluo-4 NW for 30 min at 37 °C. After treatment
with 1 μM BA, Fluo-4 NW signals were measured at 20 s intervals for 10 min. (C) Cells were pretreated with 0.5 mM EDTA and 50 μM tetracaine
for 30 min (D) or 10 μM HC, 20 μM SKF, and 20 μM nifedipine for 30 min, followed by a 30 min treatment with 1 μM BA. CaMKKβ, CaMKIIα,
and AMPK phosphorylation levels were examined by immunoblotting. Data are presented as means ± SD from three independent experiments.
*Significantly different from the control at p < 0.01. #
Significantly different from the BA-treated group at p < 0.01.
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D
SKF attenuated the BA-induced increase in intracellular Ca2+
levels (Figure 4B), as well as suppressed the phosphorylation
of AMPK, CaMKIIα, and CaMKKβ in response to BA (Figure
4D). These findings suggest that in addition to LTCC, TRPC
channels are required for the increase of intracellular Ca2+ and
the activation of AMPK, CaMKIIα, and CaMKKβ in response
to BA.
AMPK Is Required for BA-Induced eNOS Upregulation by Activating HDAC5/ERK5/KLF2 Signaling in
Human Endothelial Cells. In the vascular endothelium,
AMPK plays an important role in various cellular processes,
such as cell proliferation, growth, inflammation, and angiogenesis.30,31 To determine the importance of AMPK in eNOS
expression, we treated EA.hy926 cells with the AMPK inhibitor
compound C prior to BA treatment. Intriguingly, AMPK
inhibition attenuated the ability of BA to upregulate eNOS and
KLF2 in EA.hy926 cells (Figure 5A,B) and HUVECs (Figure
5C). Furthermore, pretreatment with compound C suppressed
the BA-induced phosphorylation of HDAC5, ERK5, and
MEF2C (Figure 5D). We also investigated the roles of AMPK
and CaMKIIα in the nuclear translocation of HDAC5 and
found that pretreatment with compound C and KN-62
(CaMKIIα inhibitor) inhibited the BA-mediated reduction in
nuclear HDAC5 levels (Figure 5E). These data strongly
support that calcium/calmodulin-dependent kinases, such as
AMPK and CaMKIIα, are required for HDAC5, ERK5, and
MEF2C phosphorylation and subsequent eNOS expression in
response to BA treatment.
■ DISCUSSION
NO is produced by eNOS, which is regulated at both the
transcriptional and post-translational levels.4,5 We have
previously reported that BA treatment on human endothelial
cells enhanced NO production via eNOS phosphorylation.22
However, the effect of BA on eNOS expression has remained
hitherto unknown. In this study, BA induced eNOS expression
in human endothelial cells and the BA-mediated upregulation
of eNOS was mediated by AMPK/HDAC5/ERK5 signaling
activation and subsequent KLF2 transcriptional activation.
Numerous transcription factors have been reported to
regulate eNOS expression in vascular endothelial cells. For
instance, eNOS upregulation in response to fluid shear stress is
regulated via the transcription factor KLF2, a zinc-finger
family.9 Deng et al. reported that puerarin, a bioactive
compound isolated from the Chinese herbal medicine Pueraria
lobata, induced eNOS expression by activating the KLF2
signaling pathway in human endothelial cells,32 further
supporting the fact that bioactive compounds derived from
natural products can regulate KLF2 signaling activation and
eNOS expression in vascular endothelial cells. In the present
study, BA upregulated both eNOS and KLF2 in EA.hy926 cells
and HUVECs, which is largely in accordance with a recent
report that grape seed proanthocyanidin extracts increased
eNOS expression via KLF2 pathway activation in HUVECs.33
In addition, we confirmed that KLF2 silencing attenuated the
ability of BA to upregulate eNOS. These results suggest that
KLF2 is essential for eNOS expression in endothelial cells in
response to bioactive compounds, including BA.
Figure 5. AMPK is required for BA-induced eNOS upregulation by activating HDAC5/ERK5/KLF2 signaling in human endothelial cells. (A)
EA.hy926 cells were pretreated with 10 μM compound C (AMPK inhibitor) for 30 min and incubated with 1 μM BA for 24 h. eNOS and KLF2
gene expression levels were analyzed by real-time PCR. (B) EA.hy926 cells were pretreated with 10 μM compound C for 30 min and incubated
with 1 μM BA for 24 h. eNOS and KLF2 protein levels were determined by immunoblotting. (C) HUVECs were pretreated with 10 μM
compound C for 30 min and incubated with 1 μM BA for 24 h. eNOS and KLF2 protein levels were determined by immunoblotting. (D)
EA.hy926 cells were pretreated with 10 μM compound C for 30 min and incubated with 1 μM BA for an additional 30 min. HDAC5, ERK5, and
MEF2C phosphorylation levels were determined by immunoblotting. (E) EA.hy926 cells were pretreated with 10 μM KN-62 or 10 μM compound
C for 30 min and incubated with 1 μM BA for an additional 6 h. Nuclear HDAC5 and cytosol HDAC5 levels were determined by immunoblotting.
Data are presented as means ± SD from three independent experiments. *Significantly different from the control at p < 0.01. #
Significantly different
from the BA-treated group at p < 0.01.
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J. Agric. Food Chem. XXXX, XXX, XXX−XXX
KLF2 activity is tightly regulated by numerous mechanisms,
including MEK5/ERK5/MEF2 and HDAC5 signaling pathways.8,34 Upon phosphorylation by AMPK or CaMKIIα,
HDAC5 translocates from the nucleus to the cytosol; the
reduction in nuclear HDAC5 levels affects the activity of
various transcription factors, including MEF2 and KLF2.9,10,35
Parmar et al. demonstrated that shear stress enhanced KLF2
expression via AMPK-dependent MEK5/ERK5/MEF2 signaling activation.36 Our findings suggest that AMPK is an
upstream regulator of HDAC5 and ERK5, thereby regulating
eNOS expression in response to BA. Notably, BA treatment
increased the phosphorylation levels of HDAC5, ERK5, and
MEF2C and promoted HDAC5 nuclear export in EA.hy926
cells. However, HDAC5 and ERK5 inhibition inhibited the
ability of BA to upregulate eNOS and KLF2 in EA.hy926 cells.
These results indicate that BA enhances KLF2-mediated eNOS
transcription by HDAC5 signaling activation and regulates
KLF2 expression via ERK5/MEF2C signaling activation.
We also found that BA significantly promoted CaMKKβ,
CaMKIIα, and AMPK phosphorylation. AMPK inhibition
using compound C attenuated BA-mediated upregulation of
eNOS and KLF2, as well as inhibited HDAC5 and ERK5
phosphorylation in EA.hy926 cells. Resveratrol has been shown
to induce HDAC5 and ERK5 phosphorylation in an AMPKdependent manner in vascular endothelial cells.37,38 Another
study has shown that HDAC5 phosphorylation by AMPK or
CaMKIIα promoted HDAC5 nuclear export, which affected
the transcriptional activity of KLF2.9,36 Collectively, our results
indicate that in endothelial cells, BA upregulates eNOS and
KLF2 via two distinct AMPK-dependent mechanisms: (i)
AMPK-mediated ERK5 phosphorylation and MEF2C activation, which lead to KLF2 transcriptional activation, and (ii)
AMPK-mediated HDAC5 phosphorylation, which promotes
KLF2 expression.
We previously confirmed that BA prevented endothelial
dysfunction by increasing the intracellular Ca2+ levels via
LTCC and enhancing eNOS activation and NO synthesis in
human endothelial cells.22 TRPs can be activated by both
intracellular and extracellular cues, as well as by physical or
mechanical stimuli. Upon activation, TRPs trigger calcium
signals and membrane depolarization, playing a critical role in
maintaining the barrier integrity.39,40 TRPC is the predominant TRP isoform in human vascular endothelial cells
regulating endothelial permeability.41 In this study, we found
that the block of TRPC and RyR attenuated the BA-mediated
influx in intracellular Ca2+ and phosphorylation of AMPK,
CaMKIIα, and CaMKKβ, consistent with the effect of BA on
Ca2+ release from the endoplasmic reticulum (ER) in beating
rabbit atria.42 TRPs are essential for extracellular Ca2+ influx
and opening of RyR on the ER membrane.43,44 Our findings
indicate that BA induces intracellular Ca2+ influx through both
the TRPC and ER and that the increase in intracellular Ca2+
levels is responsible for the activation of AMPK, CaMKIIα,
and CaMKKβ in response to BA treatment.
In conclusion, our findings suggest that BA increases
intracellular Ca2+ levels in human endothelial cells through
both TRPC-dependent extracellular Ca2+ entry and Ca2+
release from the ER. The enhanced intracellular Ca2+ levels
induce CaMKKβ and CaMKIIα phosphorylation. Upon
activation, CaMKKβ phosphorylates AMPK, which, in turn,
activates HDAC5 and ERK5 by phosphorylation. The AMPKdependent phosphorylation of HDAC5 and ERK5 in human
endothelial cells promotes eNOS expression by the transcription factor KLF2 (Figure 6). These results strongly
support that treatment with the natural compound BA may be
a promising approach to prevent vascular endothelial
dysfunction and treat cardiovascular diseases, including
hypertension, atherosclerosis, and ischemic stroke.
■ AUTHOR INFORMATION
Corresponding Author
Hye Gwang Jeong − College of Pharmacy, Chungnam
National University, Daejeon 34134, Republic of Korea;
orcid.org/0000-0002-8020-8914; Phone: +82-42-821-
5936; Email: [email protected]; Fax: +82-42-823-6566
Authors
Gi Ho Lee − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Jin Song Park − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Sun Woo Jin − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Thi Hoa Pham − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Tuyet Ngan Thai − College of Pharmacy, Chungnam
National University, Daejeon 34134, Republic of Korea
Ji Yeon Kim − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Chae Yeon Kim − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Jae Ho Choi − College of Pharmacy, Chungnam National
University, Daejeon 34134, Republic of Korea
Eun Hee Han − Drug & Disease Target Research Team,
Division of Bioconvergence Analysis, Korea Basic Science
Institute (KBSI), Cheongju 28119, Republic of Korea
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c06250
Author Contributions
G.H.L. and J.S.P. contributed equally to this work.
Figure 6. Proposed molecular mechanism of BA-mediated eNOS
expression through the KLF2 pathway in human endothelial cells. BA
increases the levels of intracellular Ca2+ via the TRPC channel and
then activates phosphorylation of CaMKKβ, CaMKII, and AMPK,
thus leading to an increase in phosphorylation of HDAC5 and ERK5.
Consequently, BA induces eNOS expression by increasing the KLF2
transcriptional activity via both AMPK/HDAC5 and AMPK/ERK5/
MEF2C signaling pathways.
Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article
https://dx.doi.org/10.1021/acs.jafc.0c06250
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by the National Research
Foundation of Korea (NRF) and the grant was funded by
the Korea government (MSIP) (Nos. NRF-
2020R1A2C1007764, NRF-2020R1I1A1A01055488).
■ ABBREVIATIONS
AMPK, AMP-activated protein kinase; BA, betulinic acid;
CaMKII, calmodulin-dependent protein kinase II; CaMKKβ,
calcium/calmodulin-dependent protein kinase kinase 2; eNOS,
endothelial nitric oxide synthase; ERK5, extracellular signalregulated protein kinase 5; HDAC5, histone deacetylase 5;
HUVEC, human umbilical vein endothelial cell; KLF2,
Kruppel-like factor 2; LKLF, lung KLF; MEF2, myocyte
enhancer factor 2; NO, nitric oxide; TRP, transient receptor
potential; TRPC, transient receptor potential cation
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