International Journal of
Molecular Sciences
Article
CK2 Down-Regulation Increases the Expression of
Senescence-Associated Secretory Phenotype Factors through
NF-κB Activation
Junbin Song and Young-Seuk Bae *






Citation: Song, J.; Bae, Y.-S. CK2
Down-Regulation Increases the
Expression of Senescence-Associated
Secretory Phenotype Factors through
NF-κB Activation. Int. J. Mol. Sci.
2021, 22, 406. https://doi.org/
10.3390/ijms22010406
Received: 3 December 2020
Accepted: 30 December 2020
Published: 2 January 2021
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Copyright: © 2021 by the authors. Li-
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This article is an open access article
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tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2021, 22, 406. https://doi.org/10.3390/ijms22010406
School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University,
Daegu 41566, Korea; junbin9292@naver.com
* Correspondence: ysbae@knu.ac.kr
Abstract: Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence
is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the
protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism
aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin
(IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB
(NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation
promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis
and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-
κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP)
expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly
abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b,
miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB
axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide.
Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors
through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and
RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as
anti-inflammatory agents.
Keywords: SASP factors; NF-κB; SIRT1; AKT; protein kinase CK2; miRNA; anti-inflammatory agent
1. Introduction
Cellular senescence is a program of arrested proliferation and altered gene expression
caused by different types of stress. Although senescence has been regarded as an effective
cancer suppression mechanism, it is also involved in some pathological conditions, such
as aging, age-associated diseases, and tumorigenesis [1–3]. One possible mechanism of
this pathological effect is the action of the senescence-associated secretory phenotype
(SASP). SASP is associated with the expression of various cytokines and chemokines,
including interleukin (IL)-1α/β, IL-6, IL-8, growth factors, such as epidermal growth
factor and vascular endothelial growth factor, and matrix metalloproteinases (MMPs),
such as MMP3 and MMP9 [4,5]. SASP affects senescent cells in an autocrine manner and
the microenvironment surrounding senescent cells in a paracrine manner. SASP is; thus,
becoming an attractive pharmacological target for manipulating senescence-mediated
effects [6,7]. However, the molecular mechanism of SASP development remains elusive.
The expression of SASP factors can be modulated by the transcription factor nuclear factor-
κB (NF-κB), which is activated in senescent cells [8]. The most prevalent form of NF-κB
exists as a heterodimer consisting of RelA/p65 and p50. NF-κB is normally retained in the
cytosol by binding to inhibitors of NF-κB (IκB) [9]. Various upstream signaling cascades
can activate the IκB kinase (IKK) complex, which subsequently phosphorylates IκBα. This
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Int. J. Mol. Sci. 2021, 22, 406 2 of 12

leads to IκBα ubiquitination and proteasome-mediated degradation, allowing NF-κB to

enter the nucleus and transcribe its target genes [10]. In addition, NAD+ -dependent histone
deacetylase, SIRT1, inhibits the transcriptional activity of NF-κB by deacetylating RelA/p65
at lysine 310 [11,12].
Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase that catalyzes the
phosphorylation of a large number of proteins. The holoenzyme of CK2 is a heterote-
tramer composed of two catalytic (α and/or α’) subunits and two regulatory β subunits.
The β subunit stimulates the catalytic activity of the α or α’ subunit, thereby mediat-
ing tetramer formation and substrate recognition [13,14]. It has been shown that CK2
down-regulation induces several senescence markers, including senescence-associated
β-galactosidase (SA-β-gal) activity, activation of the p53-p21Cip1/WAF1 axis, and senescence-
associated heterochromatin foci (SAHF) formation [15–17]. CK2 down-regulation stabi-
lizes p53 by accumulating reactive oxygen species (ROS) and inactivating SIRT1 deacety-
lase [18,19]. The activation of the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian
target of rapamycin mTOR) pathway and the inactivation of transcription factors FoxO3a
and Nrf2 are associated with ROS accumulation during CK2 down-regulation-mediated
senescence [20–22]. miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively promote
cellular senescence by inhibiting CK2α [23,24]. In addition, CK2 activity decreased with
advancing age in Caenorhabditis elegans, and kin-10 (the ortholog of CK2β) knockdown led
to a short lifespan phenotype and induced age-related biomarkers in worms [25]. Age-
dependent CK2 down-regulation reduces longevity by associating with ROS generation
via the AGE-1-AKT-1-DAF-16 pathway [23] and SIRT1-FoxO3a pathway [26] in C. elegans.
This study reported for the first time that CK2 down-regulation in human cancer
cells enhances the expression of SASP factors (IL-1β, IL-6, and MMP3) by NF-κB acti-
vation through two pathways: an SIRT1-dependent/AKT-independent pathway and an
SIRT1/AKT-dependent pathway. AKT and SIRT1 are also involved in the increased ex-
pression of zmp-1, -2, and -3 (the orthologs of MMP) mediated by CK2 down-regulation in
nematodes. Moreover, findings established the antisense inhibitors of miR-186, miR-216b,
miR-337-3p, and miR-760 as anti-inflammatory agents.

2. Results
2.1. CK2 Down-Regulation Stimulates the Expression of SASP Factors by Enhancing the Nuclear
Localization of NF-κB in Human Cancer Cells
The role of CK2 on the expression of SASP factors in MCF-7 and HCT116 cells was in-
vestigated. Western blot analysis revealed that treatment with CK2α small interfering RNA
(siRNA) increased the protein levels of SASP factors, including IL-6, IL-1β, and MMP3.
Similarly, treatment with pcDNA-HA-CK2α decreased the expression of these factors
(Supplementary Materials Figure S1). Consistent with previous reports [15,16], CK2α
knockdown increased the levels of p53-p21Cip1/WAF1 protein. Whether CK2 regulates the
mRNA levels of IL-6, IL-1β, and MMP3 was then determined. Reverse transcription-
polymerase chain reaction (RT-PCR) analysis indicated that the mRNA levels of these
factors were increased by CK2α down-regulation and decreased by CK2α overexpression
compared to the control (Figure 1A). Thus, these data suggest that CK2 negatively regulates
the expression of SASP factors at the transcriptional level in human cells. Because NF-κB is
a major transcription factor in expressing SASP factors [6,8], whether CK2 regulates the
protein levels of RelA/p65 and IκB was examined. The RelA/p65 protein levels remained
unchanged after CK2α knockdown or overexpression. However, the protein levels of
IκB were increased by CK2α down-regulation but decreased by CK2α overexpression
compared to the control (Figure 1B). Because NF-κB is normally retained in the cytoplasm
in a complex with IκB [6,8], cytoplasm and nuclei from cells transfected with CK2α siRNA
or pcDNA-HA-CK2α were separated to examine the role of CK2 in the nuclear localization
of RelA/p65. Increased accumulation of RelA/p65 was found in the nuclear extracts com-
pared to the cytosolic extracts of CK2α-silenced cells. In contrast, increased accumulation
of RelA/p65 was observed in the cytosolic extracts compared to the nuclear extracts of
CK2α-overexpressing cells (Figure 1C). Collectively, these results suggest that CK2 down-
 CK2α siRNA or pcDNA-HA-CK2α were separated to examine the role of CK2 in the nu-
clear localization of RelA/p65. Increased accumulation of RelA/p65 was found in the nu-
Int. J. Mol. Sci. 2021, 22, 406 3 of 12
clear extracts compared to the cytosolic extracts of CK2α-silenced cells. In contrast, in-
creased accumulation of RelA/p65 was observed in the cytosolic extracts compared to the
nuclear extracts of CK2α-overexpressing cells (Figure 1C). Collectively, these results sug-
gest that CK2
regulation down-regulation
increases the nuclearincreases
import ofthe nuclear
NF-κB by import of NF-κB by
down-regulating down-regulating
IκB, subsequently
IκB, subsequently promoting
promoting SASP gene expression. SASP gene expression.

Figure 1. CK2 down-regulation stimulates the expression of senescence-associated secretory phenotype (SASP) factors by
Figure 1. CK2
enhancing down-regulation
the nuclear stimulates
localization of NF-kBthe expression
in human of senescence-associated
cancer cells. MCF-7 and HCT116 secretory phenotype
cells were (SASP)
transfected factors
with CK2α by
enhancing the nuclear localization of NF-kB in human cancer cells. MCF-7 and HCT116 cells were transfected with CK2α
siRNA or pcDNA3.1-HA-CK2α for two days. (A) The level of each mRNA was measured by RT-PCR using gene-specific
siRNA or pcDNA3.1-HA-CK2α for two days. (A) The level of each mRNA was measured by RT-PCR using gene-specific
primers (left). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs
primers (left). Representative data from three independent experiments are shown. β-Actin was used as a control. Graphs
represent
represent the
the quantitation
quantitation of each mRNA
of each mRNA relative
relativetotoβ-actin (right).(B)
β-actin(right). (B) The
The level
level of of each
each protein
protein waswas determined
determined by
by im-
immunoblot analysis using specific antibodies (left). Representative data from three independent experiments
munoblot analysis using specific antibodies (left). Representative data from three independent experiments are shown. β- are shown.
β-Actin wasused
Actin was usedasasaacontrol.
control.Graphs
Graphsrepresent
represent the
the quantitation
quantitation of each protein relative
relative to β-actin (right).
toβ-actin (right). (C)
(C)Cytoplasm
Cytoplasm
andnuclei
and nucleiwere
wereisolated
isolatedfrom
fromcells,
cells,and
andboth
both extracts
extracts were
were visualized
visualized byby immunoblotting.
immunoblotting. α-Tubulin
α-Tubulin (cytoplasmic
(cytoplasmicmarker)
marker)
andhistone
and histoneH3H3(nuclear
(nuclearmarker)
marker)were
werequantified
quantifiedas asloading
loadingcontrols
controls(left).
(left).Representative
Representativedata datafrom
fromthree
threeindependent
independent
experiments are
experiments areshown.
shown. Graphs
Graphsrepresent
representthethequantitation
quantitationof ofRelA/p65
RelA/p65 relative
relative toto the
the subcellular
subcellular markers
markers (right).
(right). exp,
exp,
exposure. Data are mean ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***
exposure. Data are mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001. p < 0.001.

2.2. Activation of the AKT-IKK-IκB Pathway Is Associated with the CK2 Down-Regulation-
2.2. Activation of the AKT-IKK-IκB Pathway Is Associated with the CK2
Mediated Nuclear Import of Nuclear
Down-Regulation-Mediated NF-κB Import of NF-κB
BecauseIKK
Because IKKisisknown
knownto tophosphorylate
phosphorylateandandreduce
reducethe
thestability
stabilityofofIκB
IκB[10],
[10],whether
whether
CK2 regulates
CK2 regulates the
the activity
activity of
of IKK
IKKwas
wasexamined
examinedby bymonitoring
monitoringthe thephosphorylation
phosphorylationof of
serine residues 32 and 36 on IκBα. CK2 down-regulation increased the
serine residues 32 and 36 on IκBα. CK2 down-regulation increased the phosphorylation phosphorylation
of these
of these residues
residues on
onIκBα,
IκBα,whereas
whereasCK2α
CK2αoverexpression
overexpression decreased
decreased their phosphorylation
their phosphoryla-
(Figure 2A). Because AKT stimulates IKK activity by phosphorylating
tion (Figure 2A). Because AKT stimulates IKK activity by phosphorylating serine serine 180 on
180IKK
on
[10],[10],
IKK whether CK2
whether CK2regulates
regulatesAKT-mediated
AKT-mediatedIKK IKKphosphorylation
phosphorylation was was investigated by by
monitoring the phosphorylation
monitoring phosphorylation of ofserine
serine180
180on
onIKK.
IKK.Whereas
Whereas CK2
CK2 down-regulation
down-regulation in-
creased IKK
increased phosphorylation,
IKK phosphorylation, CK2α
CK2αoverexpression
overexpressiondecreased its phosphorylation
decreased its phosphorylation
(Figure2A).
(Figure 2A). Furthermore,
Furthermore,treatment
treatmentwith
withthe
the AKT
AKT inhibitor,
inhibitor,triciribine
triciribine(1(1µM),
μM),attenuated
attenuated
the AKT, IKK, and IκBα phosphorylation mediated by CK2 down-regulation
the AKT, IKK, and IκBα phosphorylation mediated by CK2 down-regulation (Figure S2). (Figure S2).
Whether AKT was involved in the CK2 down-regulation-mediated nuclear import of NF-
κB and SASP gene expression was then determined. Treatment with triciribine (1 µM) both
attenuated the nuclear import of RelA/p65 and increased the mRNA levels of SASP factors
induced by CK2 down-regulation (Figure 2B; Figure S3). These results collectively demon-
 Whether AKT was involved in the CK2 down-regulation-mediated nuclear import of NF-
κB and SASP gene expression was then determined. Treatment with triciribine (1 μM)
Int. J. Mol. Sci. 2021, 22, 406 4 of 12
both attenuated the nuclear import of RelA/p65 and increased the mRNA levels of SASP
factors induced by CK2 down-regulation (Figure 2B; Figure S3). These results collectively
demonstrate that the nuclear import of NF-κB and the subsequent expression of SASP
factors are the
strate that mediated
nuclearby the activation
import of NF-κB of thethe
and AKT-IKK-IκB signaling axis
subsequent expression in CK2-down-
of SASP factors are
regulated cells.
mediated by the activation of the AKT-IKK-IκB signaling axis in CK2-down-regulated cells.

Figure 2.
Figure Activation of
2. Activation of the
the AKT-
AKT- IκBIκB kinase
kinase (IKK)-inhibitors
(IKK)-inhibitors of of NF-κB
NF-κB (IκB)
(IκB) pathway
pathway is is associated
associated with
with thethe CK2
CK2 down-
down-
regulation-mediated nuclear
regulation-mediated nuclear import
import of of NF-κB.
NF-κB. (A)(A) MCF-7
MCF-7 and
and HCT116
HCT116 cells
cells were
were transfected
transfected with
with CK2α
CK2α siRNA
siRNA or or
pcDNA3.1-HA-CK2α for
pcDNA3.1-HA-CK2α fortwotwodays.
days.TheThe level
level of of
eacheach protein
protein waswas determined
determined by immunoblot
by immunoblot analysis
analysis usingusing specific
specific anti-
bodies (left).
antibodies Representative
(left). data from
Representative three three
data from independent experiments
independent are shown.
experiments β-Actinβ-Actin
are shown. was usedwasas used
a control.
as a Graphs
control.
represent the quantitation
Graphs represent of p-AKT,
the quantitation IKK, and
of p-AKT, IKK, IκBα
andrelative to β-actin
IκBα relative and that
to β-actin andofthat
p-IKK and p-IκBα
of p-IKK relative
and p-IκBα to the
relative to un-
the
phosphorylated proteins (right). (B) Cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α
unphosphorylated proteins (right). (B) Cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days for two days in the
absence or presence
in the absence of 1 μM
or presence of triciribine (TCN).(TCN).
1 µM triciribine Cytoplasm and nuclei
Cytoplasm were isolated
and nuclei from cells,
were isolated from and
cells,both
and extracts were
both extracts
visualized by immunoblotting. α-Tubulin (cytoplasmic marker) and histone H3 (nuclear marker) were quantified as load-
were visualized by immunoblotting. α-Tubulin (cytoplasmic marker) and histone H3 (nuclear marker) were quantified
ing controls (left). Representative data from three independent experiments are shown. Graphs represent the quantitation
as loading controls (left). Representative data from three independent experiments are shown. Graphs represent the
of RelA/p65 relative to the subcellular markers (right). exp, exposure. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p <
quantitation
0.001. of RelA/p65 relative to the subcellular markers (right). exp, exposure. Data are mean ± SEM. * p < 0.05;
** p < 0.01; *** p < 0.001.
2.3. SIRT1 Attenuates both RelA/p65 Acetylation and Activation of the AKT-IKK-IκB Axis
2.3. SIRT1
Mediated byAttenuates both RelA/p65 Acetylation and Activation of the AKT-IKK-IκB Axis
CK2 Down-Regulation
Mediated by CK2 Down-Regulation
SIRT1 mediates the deacetylation of lysine residue 310 on RelA/p65 [11,12]. There-
fore, SIRT1 mediates
whether the deacetylation
CK2 regulates of lysine residue
SIRT1-mediated 310 on
RelA/p65 RelA/p65 [11,12].
deacetylation Therefore,
was examined.
whether CK2 regulates SIRT1-mediated RelA/p65 deacetylation was examined. Whereas
Whereas CK2 down-regulation increased RelA/p65 acetylation, CK2α overexpression de-
CK2 down-regulation increased RelA/p65 acetylation, CK2α overexpression decreased
creased its acetylation. Furthermore, treatment with the SIRT1 activator, resveratrol (20
its acetylation. Furthermore, treatment with the SIRT1 activator, resveratrol (20 µM), at-
μM), attenuated the increase in RelA/p65 acetylation induced by CK2 down-regulation,
tenuated the increase in RelA/p65 acetylation induced by CK2 down-regulation, whereas
whereas treatment with the SIRT1 inhibitor, nicotine amide (15 mM), restored the de-
treatment with the SIRT1 inhibitor, nicotine amide (15 mM), restored the decrease in
crease in RelA/p65 acetylation induced by CK2 overexpression (Figure 3A). Next, the role
RelA/p65 acetylation induced by CK2 overexpression (Figure 3A). Next, the role of SIRT1
of SIRT1 in IκB activation mediated by CK2 down-regulation was examined. Treatment
in IκB activation mediated by CK2 down-regulation was examined. Treatment with resver-
with resveratrol (20 μM) rescued the CK2 down-regulation-mediated decrease in IκB.
atrol (20 µM) rescued the CK2 down-regulation-mediated decrease in IκB. Treatment with
Treatment with nicotine amide (15 mM) attenuated the CK2 up-regulation-mediated in-
nicotine amide (15 mM) attenuated the CK2 up-regulation-mediated increase in the IκB
crease in the IκB amount (Figure 3A). Furthermore, SIRT1 overexpression or treatment
amount (Figure 3A). Furthermore, SIRT1 overexpression or treatment with resveratrol (20
with resveratrol (20 μM) attenuated both AKT, IKK, and IκBα phosphorylation and IκBα
µM) attenuated both AKT, IKK, and IκBα phosphorylation and IκBα suppression mediated
suppression mediated by(Figure
by CK2 down-regulation CK2 down-regulation (Figure 3B;
3B; Figure S2). Treatment Figure
with S2). Treatment
resveratrol with
(20 µM) attenu-
resveratrol (20 μM) attenuated the increase in SASP gene expression levels induced
ated the increase in SASP gene expression levels induced by CK2 down-regulation (Figure by
S3). Taken together, these data suggest that CK2 down-regulation stimulates SASP gene ex-
pression through both increased RelA/p65 acetylation and activation of the AKT-IKK-IκB
axis mediated by SIRT1 inhibition.
 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 12

CK2 down-regulation (Figure S3). Taken together, these data suggest that CK2 down-reg-
Int. J. Mol. Sci. 2021, 22, 406 5 of 12
ulation stimulates SASP gene expression through both increased RelA/p65 acetylation
and activation of the AKT-IKK-IκB axis mediated by SIRT1 inhibition.

Figure 3. SIRT1 attenuates both RelA/p65 acetylation and activation of the AKT-IKK-IκB axis mediated by CK2 down-
Figure 3. SIRT1 attenuates both RelA/p65 acetylation and activation of the AKT-IKK-IκB axis mediated by CK2 down-
regulation. (A) MCF-7 and HCT116 cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days in
regulation. (A) MCF-7 and HCT116 cells were transfected with CK2α siRNA or pcDNA3.1-HA-CK2α for two days in the
the absence
absence or presence
or presence of 20ofμM
20 resveratrol
µM resveratrol
or 15or
mM 15nicotine
mM nicotine
amide.amide. The
The level of level of eachwas
each protein protein was determined
determined by immunob-by
immunoblot
lot analysis analysis using antibodies
using specific specific antibodies (top). Representative
(top). Representative data fromdata from
three three independent
independent experiments
experiments are shown.
are shown. β-Actin
β-Actin
was usedwasasused as a control.
a control. GraphsGraphs represent
represent the quantitation
the quantitation of acetylated
of acetylated p65 (ac-p65)
p65 (ac-p65) relative
relative to to unacetylated
unacetylated p-65
p-65 (bottom).
RSV, resveratrol;
(bottom). NA, nicotine
RSV, resveratrol; amide. (B)
NA, nicotine Cells(B)
amide. were transfected
Cells with CK2α
were transfected withsiRNA
CK2αand/or
siRNApECE-Flag-SIRT1 for two days.
and/or pECE-Flag-SIRT1 for
Thedays.
two levelThe
of each
level protein was determined
of each protein by immunoblot
was determined analysis
by immunoblot usingusing
analysis specific antibodies
specific (top).
antibodies Representative
(top). data
Representative
from
data three
from independent
three independent experiments
experiments areare
shown.
shown.β-Actin was
β-Actin used
was as as
used a control.
a control.Graphs
Graphsrepresent
representthe
thequantitation
quantitationofofp-
AKT, IKK, and IκBα relative to β-actin and that of p-IKK and p-IκBα relative to unphosphorylated
p-AKT, IKK, and IκBα relative to β-actin and that of p-IKK and p-IκBα relative to unphosphorylated proteins proteins (bottom). Data
(bottom).
are mean ± SEM. * p < 0.05; **p < 0.01; *** p < 0.001.
Data are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

2.4. Triciribine and Resvzeratrol Attenuate the Expression of MMP Orthologs Induced by CK2
2.4. Triciribine and Resvzeratrol Attenuate the Expression of MMP Orthologs Induced by CK2
Down-RegulationininNematodes
Down-Regulation Nematodes

C.C.elegans
eleganshas
hasbeen
beenwidely
widelyusedusedasasaamodel
modelforforexploring
exploringthe themechanisms
mechanismsunderlying
underlying
aging. CK2 down-regulation in nematodes resulted in reduced longevity and onset ofofage-
aging. CK2 down-regulation in nematodes resulted in reduced longevity and onset age-
related biomarkers associated with both AGE-1/PI3K-AKT-1/AKT-DAF-16/FoxO
related biomarkers associated with both AGE-1/PI3K-AKT-1/AKT-DAF-16/FoxO and SIR- and SIR-
2.1/SIRT1-DAF-16/FoxO3a axes
2.1/SIRT1-DAF-16/FoxO3a axes [25,26].
[25,26]. To
To investigate
investigatethe theeffect
effectofofCK2
CK2down-regulation
down-regulation
onthe
on the expression
expression of SASP
of SASP factors
factors in worms,
in worms, the expression
the expression of zmp-1of(the
zmp-1 (the ortholog
ortholog of MMP17of
and MMP21), zmp-2 (the ortholog of MMP21), and zmp-3 (the ortholog of MMP1,ofMMP8,
MMP17 and MMP21), zmp-2 (the ortholog of MMP21), and zmp-3 (the ortholog MMP1,
MMP8, and MMP25) in nematodes treated with kin-10 (the ortholog
and MMP25) in nematodes treated with kin-10 (the ortholog of CK2β) RNA interference of CK2β) RNA inter-
ference (RNAi) was compared to that in nematodes treated
(RNAi) was compared to that in nematodes treated with empty vector control (L4440) with empty vector control
(L4440)
RNAi. RNAi.
kin-10 kin-10 knockdown
knockdown increasedincreased
zmp-1, -2,zmp-1,
and -3-2, and -3 expression
expression in nematodes.
in nematodes. To analyzeTo
analyze
the role ofthe roleand
AKT of AKT
SIRT1 andin SIRT1
zmp-1,in zmp-1,and
zmp-2, zmp-2,
zmp-3 and zmp-3 expression
expression in C.after
in C. elegans elegans after
kin-10
kin-10 knockdown,
knockdown, nematodes nematodes
were furtherweretreated
furtherwith
treated with triciribine
triciribine (25 µM) or (25 μM) or resveratrol
resveratrol (50 µM).
(50 μM). with
Treatment Treatment withortriciribine
triciribine resveratrolorsignificantly
resveratrol abrogated
significantly the abrogated the increased
increased expression of
expression of zmp-1, -2, and -3 induced by kin-10 knockdown (Figure
zmp-1, -2, and -3 induced by kin-10 knockdown (Figure 4). Therefore, these results suggest 4). Therefore, these
results
that AKTsuggest thatare
and SIRT1 AKT and SIRT1
involved in theare involvedexpression
increased in the increased
of zmp-1, expression
zmp-2, and ofzmp-3
zmp-1,
zmp-2, and
mediated byzmp-3 mediated by CK2indown-regulation
CK2 down-regulation nematodes. in nematodes.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 12
Int. J. Mol. Sci. 2021, 22, 406 6 of 12

Figure 4. Effect of triciribine and resveratrol on the expression of matrix metalloproteinases (MMPs)
Figure 4. Effect of triciribine orthologs
and resveratrol
inducedonbytheCK2
expression of matrix in
down-regulation metalloproteinases (MMPs) orthologs
nematodes. Age-synchronized wormsinduced
at the L4 stage
by CK2 down-regulation in nematodes. Age-synchronized worms at the L4 stage were fed on the control or kin-10 RNAi
were fed on the control or kin-10 RNAi plates containing 25 µM triciribine or 50 µM resveratrol for
plates containing 25 μM triciribine or 50 μM resveratrol for three days. Lysates from nematodes were utilized in RT-PCR
three days. Lysates from nematodes were utilized in RT-PCR using specific primers. PCR products
using specific primers. PCR products were resolved on a 1.5% agarose gel. Representative data from three independent
were resolved ondata
experiments are shown (top). Representative a 1.5% agarose
from three gel. Representative
independent data from
experiments three independent
are shown. Act-2 mRNA experiments
served are
as the loading control. Graphs shown (top).the
represent Representative
quantitation ofdata
the from
mRNA three independent
levels of each geneexperiments are shown.
relative to that Act-2 mRNA
of act-2 (bot-
served as the
tom). TCN, triciribine; RSV, resveratrol. loading
Data control.
are mean Graphs
± SEM. *p < represent
0.05; ** p <the quantitation of the mRNA levels of each gene
0.01.
relative to that of act-2 (bottom). TCN, triciribine; RSV, resveratrol. Data are mean ± SEM. * p < 0.05;
2.5.
** p Antisense
< 0.01. Inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 Attenuate SASP
Gene Expression Induced by Lipopolysaccharide (LPS)
2.5. Antisense Inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 Attenuate SASP Gene
It has been
Expression Inducedshown that miR-186, miR-216b,
by Lipopolysaccharide (LPS) miR-337-3p, and miR-760 cooperatively
promote cellular senescence by targeting CK2α, whereas antisense inhibitors of these four
It has been shown that miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively
miRNAs
promote (4 miRs)senescence
cellular suppressedbyCK2 inhibition-mediated
targeting CK2α, whereassenescence by up-regulating
antisense inhibitors of these
CK2α
four miRNAs (4 miRs) suppressed CK2 inhibition-mediated senescence bySASP
[23,24]. Here, the effects of these 4 miRs and antisense inhibitors on gene ex-
up-regulating
pression in human
CK2α [23,24]. Here,cancer cells were
the effects investigated.
of these 4 miRs and Treatment
antisense with the 4 miRs
inhibitors increased
on SASP gene
SASP expression, whereas treatment with the four miR inhibitors (4
expression in human cancer cells were investigated. Treatment with the 4 miRs increased miRi) resulted in de-
creased SASP expression
SASP expression, whereas(Figure 5A). Because
treatment with theLPSfourinduces cellular senescence
miR inhibitors in multiple
(4 miRi) resulted in de-
cell types [27,28], the effect of the 4 miRi on SASP gene expression
creased SASP expression (Figure 5A). Because LPS induces cellular senescence in multiplein cells treated with
LPS was examined. Although treatment with LPS (6 μg/μL) increased SASP
cell types [27,28], the effect of the 4 miRi on SASP gene expression in cells treated with LPS expression
and
wasdecreased
examined.CK2α expression
Although treatmentin human
with LPScancer cells, additional
(6 µg/µL) increased treatment with the
SASP expression 4
and
miRi or CK2α overexpression abrogated the LPS-mediated induction
decreased CK2α expression in human cancer cells, additional treatment with the 4 miRi or of SASP factors
(Figure 5B). Furthermore,
CK2α overexpression treatment
abrogated thewith the 4 miRi induction
LPS-mediated or CK2α overexpression
of SASP factorssuppressed
(Figure 5B).
RelA/p65 acetylation
Furthermore, andwith
treatment IKK theand4IκBα
miRiphosphorylation,
or CK2α overexpression which were mediated
suppressed by LPS
RelA/p65
(Figure 5C). and
acetylation These IKKresults collectively
and IκBα indicate that
phosphorylation, antisense
which inhibitorsby
were mediated ofLPS
miR-186,
(Figure miR-
5C).
216b, miR-337-3p, and miR-760 decrease SASP factors by inhibiting NF-κB
These results collectively indicate that antisense inhibitors of miR-186, miR-216b, miR-337- signaling, sug-
gesting
3p, andthat the 4 miRi
miR-760 together
decrease SASP may act asbyaninhibiting
factors effective anti-inflammatory agent. Finally,
NF-κB signaling, suggesting that
the
theeffect
4 miRioftogether
individual may miR
act inhibitors on the
as an effective LPS-mediated induction
anti-inflammatory of SASP
agent. Finally, thegene
effectex-
of
pression
individualwasmiRinvestigated.
inhibitors Each
on the ofLPS-mediated
the 4 miRi suppressed
inductionthe ofLPS-mediated inductionwas
SASP gene expression of
SASP factors, Each
investigated. but this effect
of the wassuppressed
4 miRi more strongly suppressed byinduction
the LPS-mediated the mixture of the
of SASP four
factors,
miRNA
but thisinhibitors
effect wascompared
more stronglyto individual
suppressed treatment (Figure of
by the mixture S4).
the four miRNA inhibitors
compared to individual treatment (Figure S4).
Int. J. Mol. Sci. 2021, 22, 406 7 of 12
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 12

Figure
Figure 5. Antisense inhibitors
5. Antisense inhibitors of
ofmiR-186,
miR-186,miR-216b,
miR-216b,miR-337-3p,
miR-337-3p,and andmiR-760
miR-760 attenuate
attenuate thethe induction
induction of of senescence-
senescence-as-
associated secretory phenotype (SASP) factors by lipopolysaccharide (LPS).
sociated secretory phenotype (SASP) factors by lipopolysaccharide (LPS). (A) MCF-7 (A) MCF-7 and HCT116 cells were
were transfected
transfected
withthe
with the44miRs
miRs(miR-186,
(miR-186,miR-216b,
miR-216b,miR-337-3p,
miR-337-3p,andand miR-760)
miR-760) oror
thethe 4 miRi.
4 miRi. (B and
(B,C) CellsC) Cells
were were with
treated treatedLPSwith LPS (6
(6 µg/µL)
μg/μL)
in in the presence
the presence or absence
or absence of the 4ofmiRi
the 4ormiRi or pcDNA3.1-HA-CK2α
pcDNA3.1-HA-CK2α for 2The
for 2 days. days. Theoflevel
level eachofprotein
each protein was deter-
was determined
mined
by by immunoblot
immunoblot analysis
analysis using specific
using specific antibodies
antibodies (left). (left). Representative
Representative data from
data from threethree independent
independent experiments
experiments are
are shown. β-Actin was used as a control. Graphs represent the quantitation of p-AKT, IKK, and IκBα
shown. β-Actin was used as a control. Graphs represent the quantitation of p-AKT, IKK, and IκBα relative to β-actin relative to β-actin
and
and that of p-IKK and p-IκBα relative to the unphosphorylated proteins (right). Data are mean ± SEM. *p < 0.05; ** p < 0.01;
that of p-IKK and p-IκBα relative to the unphosphorylated proteins (right). Data are mean ± SEM. * p < 0.05; ** p < 0.01;
*** p < 0.001.
*** p < 0.001.
3. Discussion
3. Discussion
Senescent cells secrete pro-inflammatory proteins known as SASP factors [4–6]. NF-
κB isSenescent
the majorcells secretethat
regulator pro-inflammatory proteins known
induces the appearance of SASPas[8,9].
SASP factors
CK2 [4–6]. NF-κB
down-regulation
is the major regulator that induces the appearance of SASP [8,9]. CK2 down-regulation
was previously reported to induce cellular senescence and organism aging [15–26], but its
was previously
relationship to reported to induce
SASP factors cellular senescence
is unknown. In this study,andweorganism
examined aging
the [15–26], but its
role of CK2 in
relationship to SASP factors is unknown. In this study, we examined the role of CK2 in the
the expression of SASP. The present study shows that CK2 down-regulation induces the
expression of SASP. The present study shows that CK2 down-regulation induces the nuclear
nuclear import of NF-κB by reducing the IκB levels, which promotes the subsequent ex-
import of NF-κB by reducing the IκB levels, which promotes the subsequent expression
pression of SASP factors, such as IL-6, IL-1β, and MMP3 in human cancer cells. In contrast,
of SASP factors, such as IL-6, IL-1β, and MMP3 in human cancer cells. In contrast, CK2
CK2 up-regulation decreases the nuclear import of NF-κB by increasing the IκB levels and
up-regulation decreases the nuclear import of NF-κB by increasing the IκB levels and
reduces the expression of SASP factors, suggesting that CK2 negatively regulates NF-κB
reduces the expression of SASP factors, suggesting that CK2 negatively regulates NF-
signaling (Figure 1; Figure S1). This study demonstrated that CK2 regulates NF-κB signal-
κB signaling (Figure 1; Figure S1). This study demonstrated that CK2 regulates NF-κB
ing through two pathways. First, CK2 regulates NF-κB activity through the AKT-IKK-IκB
signaling through two pathways. First, CK2 regulates NF-κB activity through the AKT-
pathway. CK2 down-regulation induced an increase in nuclear NF-κB and the phosphor-
IKK-IκB pathway. CK2 down-regulation induced an increase in nuclear NF-κB and the
ylation of AKT, IKK, and IκB, which was reversed by treatment with the AKT inhibitor,
phosphorylation of AKT, IKK, and IκB, which was reversed by treatment with the AKT
triciribine (Figure 2; Figure S2). Similarly, treatment with triciribine rescued the CK2
inhibitor, triciribine (Figure 2; Figure S2). Similarly, treatment with triciribine rescued the
down-regulation-mediated
CK2 down-regulation-mediated induction of SASP
induction factors
of SASP in human
factors cellscells
in human (Figure S3) and
(Figure kin-
S3) and
10 knockdown-mediated induction of MMP orthologs in nematodes (Figure
kin-10 knockdown-mediated induction of MMP orthologs in nematodes (Figure 4). Because 4). Because
IKK promotes
IKK promotesthe theubiquitin-dependent
ubiquitin-dependent degradation
degradation of IκBα
of IκBα by phosphorylating
by phosphorylating IκBα IκBα
[10],
[10], this study suggests that CK2 down-regulation induces IκBα degradation
this study suggests that CK2 down-regulation induces IκBα degradation and subsequent and subse-
quent NF-κB
NF-κB nuclearnuclear localization
localization through through the activation
the activation of the AKT-IKK
of the AKT-IKK pathway pathway
(Figure 6).
(Figure 6).
Second, CK2 regulates NF-κB activity through SIRT1. Whereas CK2 down-regulation
increased RelA/p65 acetylation, treatment with the SIRT1 activator, resveratrol, abrogated
the increase in RelA/p65 acetylation induced by CK2 down-regulation. In contrast,
 resveratrol inhibits the PI3K-AKT pathway by SIRT1 activation. Collectively, we conclude
that CK2 functions as a negative regulator of NF-κB in senescent cells. This is not con-
sistent with previous reports that CK2 induces NF-κB activation at multiple levels
through IκB [30], inducible IKK and IKKε [31], IKK2 [32], or RelA/p65 itself [33,34]. Alt-
hough we currently cannot explain this discrepancy, we hypothesize that CK2 may regu-
Int. J. Mol. Sci. 2021, 22, 406
lates NF-κB activity by different mechanisms depending on the state of cell growth (e.g., 8 of 12
proliferation or senescence).

Figure
Figure 6. The working model for the The working model for the CK2
CK26.down-regulation-mediated down-regulation-mediated
expression of SASP factors. CK2expression of SASP factors. CK2
down-regulation
induces SIRT1 inhibition and AKTdown-regulation
up-regulation, which elevate
induces SIRT1the nuclearand
inhibition import
AKTofup-regulation,
RelA/p65 by increasing RelA/p65
which elevate the nuclear import
acetylation and IκB destabilization, respectively. Subsequently, active NF-κB stimulates the transcription of SASP genes,
of RelA/p65 by increasing RelA/p65 acetylation and IκB destabilization, respectively. Subsequently,
including MMP2, IL-6, and IL-1β.active
LPS down-regulates CK2. The antisense inhibitors of miR-186, miR-216b, miR-337-3p,
NF-κB stimulates the transcription of SASP genes, including MMP2, IL-6, and IL-1β. LPS
and miR-760 exhibit anti-inflammatory effects.
down-regulates CK2. The antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760
exhibit anti-inflammatory effects.
These results indicate that antisense inhibitors of miR-186, miR-216b, miR-337-3p,
and miR-760 exhibit
Second, CK2anti-inflammatory effects. through
regulates NF-κB activity These four miRNA
SIRT1. inhibitors
Whereas decreased
CK2 down-regulation
the expression of SASP factors by up-regulating CK2. Furthermore, these
increased RelA/p65 acetylation, treatment with the SIRT1 activator, resveratrol, four miRNA
abrogated
inhibitors successfully
the increase abrogated
in RelA/p65 the LPS-mediated
acetylation induction
induced by CK2 of SASP factors,
down-regulation. RelA/p65
In contrast, whereas
acetylation, and activation decreased
CK2α overexpression of the AKT-IKK axis. Interestingly,
its acetylation, treatment with treatment
the SIRT1with LPS de-
inhibitor, nicotine
creased the level
amide, of CK2αthe
suppressed protein (Figures
decrease 5 and 6).acetylation
in RelA/p65 For many years,
induced CK2bywas
CK2 thought to
up-regulation
be a constitutive,
(Figure 3A).nonregulated
Furthermore,protein kinase [13,14]. Our
SIRT1 overexpression recent reports
or treatment have shownattenuated
with resveratrol that
CK2 isthedownregulated in replicative
AKT-IKK axis-dependent or degradation
IκBα oxidative stress-induced
mediated by CK2senescence in human sug-
down-regulation,
gesting that SIRT1 acts as a negative regulator of AKT-IKK-NF-κB signaling (Figure 3B;
Figure S2). Likewise, resveratrol treatment rescued the CK2 down-regulation-induced
induction of SASP factors in human cells (Figure S3), and kin-10 knockdown-mediated
an increase in the MMP orthologs in nematodes (Figure 4). Taken together, these results
suggest that SIRT1 is involved in CK2 down-regulation-mediated SASP induction through
RelA/p65 deacetylation and AKT activation (Figure 6). This conclusion is consistent with
the results from our previous reports showing that CK2 down-regulation up-regulates
several senescence markers (increased SA-β-gal activity, ROS production, activation of the
p53-p21Cip1/WAF1 axis, and SAHF formation) by activating the PI3K-AKT-mTOR pathway
and inactivating SIRT1 [15–22]. Chai et al. [29] have recently shown that resveratrol inhibits
the PI3K-AKT pathway by SIRT1 activation. Collectively, we conclude that CK2 functions
as a negative regulator of NF-κB in senescent cells. This is not consistent with previous
reports that CK2 induces NF-κB activation at multiple levels through IκB [30], inducible
IKK and IKKε [31], IKK2 [32], or RelA/p65 itself [33,34]. Although we currently cannot ex-
plain this discrepancy, we hypothesize that CK2 may regulates NF-κB activity by different
mechanisms depending on the state of cell growth (e.g., proliferation or senescence).
These results indicate that antisense inhibitors of miR-186, miR-216b, miR-337-3p,
and miR-760 exhibit anti-inflammatory effects. These four miRNA inhibitors decreased
the expression of SASP factors by up-regulating CK2. Furthermore, these four miRNA
inhibitors successfully abrogated the LPS-mediated induction of SASP factors, RelA/p65
acetylation, and activation of the AKT-IKK axis. Interestingly, treatment with LPS decreased
the level of CK2α protein (Figures 5 and 6). For many years, CK2 was thought to be a
Int. J. Mol. Sci. 2021, 22, 406 9 of 12

constitutive, nonregulated protein kinase [13,14]. Our recent reports have shown that CK2
is downregulated in replicative or oxidative stress-induced senescence in human cells
and in nematode aging [15–26]. The present study also indicates that LPS downregulates
CK2 expression. However, how LPS down-regulates CK2α currently cannot be explained.
Among the 4 miRs, it has been reported that miR-186 and miR-216b are associated with
inflammation. miR-186 promotes the secretion of pro-inflammatory cytokines by targeting
cystathionine γ-lyase in THP-1 macrophages [35]. miR-216b down-regulation inhibits
IL-1β-induced chondrocyte injury by up-regulating Smad3 [36]. This study reports, for the
first time, the anti-inflammatory effects of antisense inhibitors of miR-216b and miR-337-3p.
It has been shown that the high mobility group (HMG) B2 protein orchestrates SASP
through the prevention of heterochromatin spreading to exclude SASP gene loci from
senescence-associated heterochromatin [37]. In addition, the CK2-mediated phosphoryla-
tion of the acidic tail of HMGB2 modulates the intranuclear distribution of HMGB2 [38].
A previous study demonstrated that CK2 down-regulation promotes SAHF formation by
enhancing H3K9 trimethylation [17]. Because CK2 down-regulation induces the expression
of SASP factor genes in this study, it is proposed that CK2 down-regulation may allow for
the exclusion of SASP gene loci from SAHF by reducing phosphorylation on the acidic tail
of HMGB2 in senescent cells.
In conclusion, these findings establish CK2 as a key switch to regulate SASP factors.
The results suggest that SIRT1 connects CK2 down-regulation to SASP factors through
NF-κB activation, which is mediated by both RelA/p65 deacetylation and activation of the
AKT-IKK-IκB axis. Because senescence is thought to promote aging-associated diseases
through the resulting inflammatory response, the four inhibitors of miR-186, miR-216b,
miR-337-3p, and miR-760 may provide a strategy for anti-inflammatory intervention.

4. Materials and Methods

4.1. Materials
Antibodies against SIRT1, CK2α, p53-p21Cip1/WAF1 , RelA/p65, IκB, α-tubulin, and β-
actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies
against acetylated RelA/p65 (ac-p65, K310), IKKα/β, phosphorylated IKKα/β (p-IKKα/β,
S180/S181), IL-1β, IL-6, MMP3, and histone H3 were obtained from Abcam (Cambridge,
UK). Antibodies against phosphorylated IκBα (p-IκBα, S32/S36) and phosphorylated
AKT (p-AKT, S473) were obtained from Cell Signaling (Danvers, MA, USA). Resveratrol,
triciribine, nicotine amide, and LPS were obtained from the Sigma Chemical Co. (St. Louis,
MO, USA).

4.2. Cell Culture, RNAi, and DNA Transfection

Human breast cancer MCF-7 cells and human colon cancer HCT116 cells (American
Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s
medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin in a
humidified atmosphere of 95% (v/v) air and 5% (v/v) CO2 at 37 ◦ C. Cells were transfected
with pcDNA3.1-HA-CK2α or pECE-Flag-SIRT1 using Polyfect (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. Mimics for miR-186, miR-216b, miR-337-3p,
and miR-760 and control miRNA were purchased from Genolution, Inc. (Seoul, Korea).
Antisense inhibitors for the 4 miRs were obtained from Panagene, Inc. (Seoul, Korea).
The siRNA for CK2α was 50 -GAUGACUACCAGCUGGUUCdTdT-30 . The siRNA for the
negative control was 50 -GCUCAGAUCAAUACGGAGAdTdT-30 . RNAs were transfected
into cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA) for 48 h.

4.3. Immunoblotting
Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and
then transferred by electrophoresis onto nitrocellulose membranes. The membranes were
blocked with 5% (w/v) non-fat, dry, skim milk dissolved in TBST [20 mM Tris-HCl (pH 7.4),
150 mM NaCl, 0.05% Tween 20] for 2 h and then incubated with specific antibodies diluted
Int. J. Mol. Sci. 2021, 22, 406 10 of 12

in TBST with 1% (w/v) non-fat, dry, skim milk for 1 h. The membranes were washed three
times in TBST and then analyzed with an ECL system (Amersham, UK).

4.4. RT-PCR
Total RNA was extracted from human cancer cells or nematodes. RNA was reverse
transcribed using gene-specific primers and reverse transcriptase (Takara Bio, Inc., Kyoto,
Japan), and the resulting cDNAs were amplified by PCR. The primers used for the assays
are listed in Table S1. PCR products were resolved on a 1.5% agarose gel. The quantitation
of the RT-PCR bands was performed using densitometry. β-Actin RNA levels were used to
standardize the amounts of RNA in each sample.

4.5. Isolation of Nuclear and Cytoplasmic Extracts

The cytoplasmic and nuclear extracts were prepared using an NE-PER Nuclear Cyto-
plasmic Extraction Reagent kit (Pierce, Rockford, IL, USA) according to the manufacturer’s
instructions.

4.6. Culture of Nematodes and RNAi Experiment

The C. elegans N2 strain was acquired from the Caenorhabditis Genetics Center. Nema-
todes were grown at 21 ◦ C on nematode growth medium agar plates with Escherichia coli
strain OP50 as a food source. For some experiments, nematodes were treated with tricirib-
ine or resveratrol. RNAi experiments were performed with E. coli HT115 cells expressing
double-stranded kin-10 RNA, as described previously [25,26].

4.7. Statistical Analysis

Data were analyzed by one-way analysis of variance with the SPSS package program
(IBM, Armonk, NY, USA). The results were considered significant if p < 0.05. Duncan’s
multiple-range test was performed if significant differences between the groups were
identified using α = 0.05.

Supplementary Materials: Supplementary Materials can be found at https://www.mdpi.com/1422

-0067/22/1/406/s1.
Author Contributions: Y.-S.B. conceived and designed the experiments, J.S. performed the experi-
ments, and Y.-S.B. wrote the paper. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning
(NRF-2019R1A2C1005219).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
CK2 protein kinase CK2
HMG high mobility group
IκB inhibitors of NF-κB
IKK IκB kinase
IL interleukin
LPS lipopolysaccharide
mTOR mammalian target of rapamycin
MMP matrix metalloproteinase
NF-κB nuclear factor-κB
RT-PCR reverse transcription-polymerase chain reaction
Int. J. Mol. Sci. 2021, 22, 406 11 of 12

PI3K phosphatidylinositol 3-kinase

ROS reactive oxygen species
SA-β-gal senescence-associated β-galactosidase
SAHF senescence-associated heterochromatin foci
SASP senescence-associated secretory phenotype
siRNA small interfering RNA

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