Báo cáo khoa học: The chromosomal protein HMGN2 mediates lipopolysaccharide-induced expression of b-defensins in A549 cells potx

The chromosomal protein HMGN2 mediates
lipopolysaccharide-induced expression of b-defensins in
A549 cells
Lu-Xia Deng1,2, Gui-Xia Wu1, Yue Cao1, Bo Fan1, Xiang Gao1, Lin Luo3 and Ning Huang1,4
1
2
3
4

Research Unit of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China
Chongqing Lummy Pharmaceutical Co. Ltd, Chongqing, China
Department of Anesthesiology, West China College of Stomatology, Sichuan University, Chengdu, China
State Key Laboratory of Biotherapy, West China Hospital, Sichuan, Chengdu, China

Keywords
high mobility group protein N2; human
b-defensin-2; lipopolysaccharide; p65;
regulation
Correspondence
N. Huang, Research Unit of Infection and
Immunity, West China School of Preclinical
and Forensic Medicine, Sichuan University,
Chengdu, China
Fax ⁄ Tel: +86 28 8506 8208
E-mail:
(Received 13 August 2010, revised 5 April
2011, accepted 11 April 2011)

Human b-defensin-2 (HBD-2) is an antimicrobial peptide produced by the
epithelial cells that plays an important role in innate and adaptive immunity. Here we report that high mobility group protein N2 (HMGN2), a
member of the high mobility group superfamily that affects chromatin

function, modulates the expression of HBD-2 in A549 cells treated by lipopolysaccharide. Mechanistically, HMGN2 prolongs the retention time and
enhances the accumulation of nuclear factor jB p65 in the nucleus, and
promotes the acetylation of p65 through increasing histone acetyltransferase activity and enhancing p65-Ser536 phosphorylation. Additionally, chromatin immunoprecipitation reveals that HMGN2 and p65 synergistically
promote their specific binding to HBD-2 promoter, thereby affecting the
downstream transcription. Taken together, these results suggest that
HMGN2 acts as a positive modulator of nuclear factor jB signalling to
promote lipopolysaccharide-induced b-defensin expression.

doi:10.1111/j.1742-4658.2011.08132.x

Introduction
Antimicrobial peptides are produced by many different
tissues of the body when the innate immune system is
activated by lipopolysaccharide (LPS) or other inflammatory cytokines and chemokines. Defensins are small
cationic antimicrobial peptides which have usually been
categorized into three major families based on the
molecular structure: a-, b-, and h-defensins [1–3]. They
regulate both the innate and adaptive immune
responses, exhibiting broad-spectrum antimicrobial
activity against most Gram-positive and Gram-negative
bacteria, fungi and viruses such as influenza viruses.

b-defensins are largely expressed in epithelial cells
on the surface of respiratory, gastrointestinal and genitourinary tracts and skin [3,4]. Human b-defensin
(HBD) 1 is constitutively expressed in the epithelial
surface of the respiratory and genitourinary tracts [5].
In contrast, the expression of HBD-2, HBD-3 and
HBD-4 is inducible in response to LPS, tumour necrosis factor a (TNF-a), interleukin 1b or Gram-negative
pathogens, underscoring their crucial role in epithelial
host defence under inflammatory conditions [4,6].

Inducible expression of b-defensins by human epithelial

Abbreviations
AA, anacardic acid; ChIP, chromatin immunoprecipitation; Co-IP, co-immunoprecipitation; Glut2, type 2 glucose transporters; HAT, histone
acetyltransferase; HBD, human b-defensin; HDAC, histone deacetylase; HMG, high mobility group; HMGA, HMG-AT-hook family; HMGB,
HMG-box family; HMGN2, high mobility group protein N2; IjBa, NF-jB inhibitor a; LPS, lipopolysaccharide; MAPK, mitogen-activated protein
kinase; NF-jB, nuclear factor jB; PE-H, pEGFPN1-HMGN2; Psi-H, pSilencer-HMGN2-2; siRNA2, small interfering RNA2; TNF-a,
tumour necrosis factor a; TSA, trichostatin.

2152

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L.-X. Deng et al.

HMGN2 mediates expression of b-defensins

cells has been shown to be regulated through several
signalling pathways, such as the nuclear factor jB
(NF-jB) pathway, the p38 mitogen-activated protein
kinase (MAPK) pathway, the c-Jun N-terminal kinase
pathway and the phosphatidylinositol-3-kinase ⁄ AKT
pathway [7–11].
The chromatin-associated high mobility group
(HMG) superfamily includes the HMG-AT-hook
(HMGA) family, HMG-box (HMGB) family and
HMG nucleosome binding (HMGN) family and modulates a wide range of DNA-dependent activities such
as chromatin structure, post-translational modifications, and rates of transcription, replication and
recombination [12–15]. Interestingly, recent studies

have demonstrated that some members of the HMG
family such as HMGA1 and HMGB1 are directly
involved in the regulation of NF-jB activation and its
downstream gene transcription [3,16–18]. It is well
known that NF-jB activation is regulated by
RelA ⁄ p65 acetylation and deacetylation which are
mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), respectively [19,20]. HAT-mediated acetylation of RelA ⁄ p65, particularly at lysine 310
and to a lesser extent at lysine 221, enhances NF-jB ⁄
DNA binding and attenuates its interaction with
NF-jB inhibitor a (IjBa) [21]. Conversely, acetylated
RelA ⁄ p65 is deacetylated by HDACs, leading to the
repression of downstream gene transcription and
IjBa-dependent nuclear export of IjBa ⁄ NF-jB complex [20,22]. The NF-jB activity and the expression of
its downstream gene alter when HAT and HDAC
inhibitors respectively are used [23–26].
HMGN2 has been shown to partially decrease
HDAC activity [27] and also to enhance the H3K14ac
level in chromatin by stimulating HAT activity [13].

Nevertheless, it is unclear whether HMGN2 modulates
NF-jB activity by changing the activity of HATs
and ⁄ or HDACs and thereby regulates the expression
of downstream target HBD-2. Therefore, in the present
study we initially supposed that (a) HMGN2 elevates
the activity of NF-jB, enhances the amount of NF-jB
p65 in the nucleus and regulates the equilibrium
between HATs and HDACs thereby affecting p65 acetylation; and (b) HMGN2 itself or through interaction
with p65 binds to the chromatin in the promoter
region of b-defensin genes to enhance HBD-2 expression. This research aimed to confirm the initial hypothesis through transient transfection and luciferase
experiments, the activity of HAT and HDAC blocking

experiments, chromatin immunoprecipitation (ChIP)
assay and co-immunoprecipitation (Co-IP) etc.

Results
Gene expression profiles after HMGN2
knockdown or ⁄ and LPS stimulation in A549 cells
As the first step to characterizing the function of
HMGN2 in transcriptional regulation in response to
LPS, we prepared knockdown HMGN2 in A549 cells
and found that HMGN2 expression at both mRNA
and protein levels was significantly downregulated in
A549 cells treated by HMGN2-specific small interfering RNA2 (SiRNA-HMGN2-2) (Figs 1A,B and S1).
Next we employed a cDNA microarray to examine the
effect of reduced endogenous HMGN2 level and ⁄ or
LPS treatment on gene expression profiles in A549
cells (Figs S2 and S3). The results showed that
HMGN2 downregulation altered the expression of over
4% of 31 000 genes by twofold or more (Table 1).

A

B
-actin
HMGN2

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS

2

1.0

3

RT-PCR

*

Western blot

0.8
0.6
0.4

*

E
H/P
Psi-

Ps

E-H
H/P

0

i

0.2

Psi-

D

1

-H

Ps
i-H
Ps
i-H
/PE
Ps
i-H
/PE
-H
Ps
i

nk
bla

PE

HMGN2
Histone H3

N

Psi

Ps
i-H
bla
nk
Ma
ke
r
Ps
i-H Psi
/PE
-H
Ps
i-H
/PE

PE
-

H
PE

-actin
HMGN2

B

bla
nk

3

PE

2

PEH

1

HMGN2 RT-PCR western blot

-H

C

N M

HMGN2/ -actin ratio(RT-PCR)
HMGN2/histone H3 western blot

B

PE

Fig. 1. Efficient knockdown of HMGN2 in
A549 cells. Representative RT-PCR (A) or
western blot (B) results showing that
HMGN2 mRNA and protein levels were

reduced by over 80% after siRNA2 treatment. b-actin and histone H3 served as the
loading control for RT-PCR and western blot,
respectively. B, blank group; N, normal
group (without LPS treatment); M, marker.
1–3, different HMGN2 siRNA. (C) Representative RT-PCR and western blot results
showing the expression of HMGN2 mRNA
and protein in the different established stable A549 cells. (D) Values are presented as
mean ± SD for at least five independent
experiments performed in triplicate.
*P < 0.01 versus blank group.

HMGN2
Histone 3

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L.-X. Deng et al.

Table 1. List of genes with changed expression in microarray analysis.
Log 2 ratio
GenBank

CGAP gene
symbol

NM_007315.2
NM_139276.2

STAT1
STAT3

NM_000572.2
NM_000584.2
NM_000600.1
NM_004513.3
NM_000576.2
NM_001315.1
NM_002969.3
NM_002746.1
NM_002749.2
NM_002751.5
NM_002751.5
NM_018661.2
NM_001556.1

IL10
IL8
IL6
IL16
IL-1b
MAPK14
MAPK13
MAPK3
MAPK7
MAPK11
DEFB4 (HBD-2)
DEFB103A(HBD-3)

IKBKB

NM_002468.2
NM_021975.2

MYD88
RELA

NM_000346.2

BVA

CVA

DVA

3.5
5.3

)2.0
)2.4

1.7
2.1

4.1
3.9
4.9
2.9
4.6

3.7
3.4
2.8
3.0
2.1
5.3
4.2
)4.1

)2.3
)2.0
1.9
0.3
)3.7
1.2
)2.1
)1.4
)3.7
)0.1
)3.4
)3.2
3.1

1.5
2.8
6.3
3.4
1.3
5.8
1.2

1.2
)0.3
2.9
2.7
1.8
)1.9

3.4
5.8

1.1
)3.1

4.9
1.2

SOX9

)0.2

)4.2

)4.1

NM_000591.1
NM_002228.3
NM_001569.2
NM_002751.5
NM_014261.1
NM_003266.2

NM_014294.3
NM_005252.2
NM_003225.2

CD14
JUN
IRAK1
TRAF6
TRIF
TLR4
TRAM1
FOS
TFF1

5.3
4.8
4.0
)3.2
2.8
4.9
1.1
3.2
1.2

)1.0
)2.9
)1.4
2.9
)1.4
)2.2

1.4
1.9
3.2

4.1
2.2
2.3
)1.3
1.9
3.6
2.6
5.7
4.8

NM_005080.2
BU739862

XBP1
MED19

3.0
)0.9

1.3
)0.5

4.9
)1.1

NM_003187.3

TAF9

)0.5

)1.1

)1.2

BU739862

LCMR1

0.6

1.6

2.8

BX537584
NM_000125.1
NM_001904.2
NM_001792.2
NM_003508.1
NM_012193.2
NM_004625.3
NM_003392.3
NM_002335.1
NM_002336.1
NM_153426.1

NM_022454.2
NM_052942.2
NM_002093.2

PC4
ESR1
CTNNB1
CDH2
FZD9
FZD4
WNT7A
WNT5A
LRP5
LRP6
PITX2
SOX17
GBP5
GSK3B

3.8
0.5
4.8
4.2
2.8
3.14
2.5
3.3
1.4
1.4
0.4

3.5
0.5
)0.6

3.9
)3.1
3.4
2.6
1.7
2.1
1.3
1.9
1.9
2.3
4.1
)2.4
1.8
2.9

7.0
)2.7
7.2
7.7
5.1
5.1
5.5
4.7
4.2
3.4
4.5

1.8
1.5
1.9

2154

Entrez gene description
Signal transducer and activator of transcription 1, 91 kDa
Signal transducer and activator of transcription 3
(acute-phase response factor)
Interleukin 10
Interleukin 8
Interleukin 6 (interferon, beta 2)
Interleukin 16 (lymphocyte chemoattractant factor)
Interleukin 1, beta
Mitogen-activated protein kinase 14
Mitogen-activated protein kinase 13
Mitogen-activated protein kinase 3
Mitogen-activated protein kinase 7
Mitogen-activated protein kinase 11
Defensin, beta 4
Defensin, beta 103A
Inhibitor of kappa light polypeptide gene enhancer in B-cells,
kinase beta
Myeloid differentiation primary response gene (88)
v-rel reticuloendotheliosis viral oncogene homolog A, nuclear
factor of kappa light polypeptide gene enhancer in B-cells
3, p65
SRY (sex determining region Y) box 9 (campomelic dysplasia,
autosomal sex-reversal)

CD14 antigen
v-jun sarcoma virus 17 oncogene homolog (avian)
Interleukin-1 receptor-associated kinase 1
TNF receptor-associated factor 6
TIR domain containing adaptor inducing interferon-beta
Toll-like receptor 4
Translocation associated membrane protein 1
v-fos FBJ murine osteosarcoma viral oncogene homolog
Trefoil factor 1 (breast cancer, estrogen-inducible sequence
expressed in)
X-box binding protein 1
Mediator of RNA polymerase II transcription, subunit 19
homolog (yeast)
TAF9 RNA polymerase II, TATA box binding protein (TBP)
associated factor, 32kDa
Mediator of RNA polymerase II transcription, subunit 19
homolog (yeast)
Activated RNA polymerase II transcription cofactor 4
Estrogen receptor 1
Catenin (cadherin-associated protein), beta 1, 88kDa
Cadherin 2, type 1, N-cadherin (neuronal)
Frizzled homolog 9 (Drosophila)
Frizzled homolog 4 (Drosophila)
Wingless-type MMTV integration site family, member 7A
Wingless-type MMTV integration site family, member 5A
Low density lipoprotein receptor-related protein 5
Low density lipoprotein receptor-related protein 6
Paired-like homeodomain transcription factor 2
SRY (sex determining region Y) box 17
Guanylate binding protein 5

Glycogen synthase kinase 3 beta

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS


L.-X. Deng et al.

HMGN2 mediates expression of b-defensins

Table 1. (Continued).
Log 2 ratio
GenBank

CGAP gene
symbol

NM_001791.2
BX640852
NM_003387.3
NM_004333.2
NM_002755.2
NM_002745.2
NM_000539.2

BVA

CVA

DVA

Entrez gene description

CDC42
WASL
WASPIP
BRAF
MAP2K1
MAPK1
RHO

4.0
3.7
0.1
3.8
3.6
3.4
4.3

2.5
)2.2
)1.8
)2.3
)1.8
)1.0
1.2

7.1
1.1
)1.1
2.6

2.2
4.6
6.1

NM_005406.1
NM_018890.2

ROCK1
RAC1

0.6
3.1

)2.4
1.6

)2.5
3.7

BQ051103
BM907805
BQ940876

HMGN2
H3F3A
HIST1H1C

)0.3
3.1
)0.3

)8.5
)2.1
)2.1

)7.9
1.8
)2.4

Cell division cycle 42 (GTP binding protein, 25kda)
Wiskott–Aldrich syndrome-like
Wiskott–Aldrich syndrome protein interacting protein
v-raf murine sarcoma viral oncogene homolog B1
Mitogen-activated protein kinase kinase 1
Mitogen-activated protein kinase 1
Rhodopsin (opsin 2, rod pigment) (retinitis pigmentosa 4, autosomal dominant)
Rho-associated, coiled-coil containing protein kinase 1
Ras-related C3 botulinum toxin substrate 1 (rho family, small
GTP binding protein Rac1)
High mobility group nucleosomal binding domain 2
H3 histone, family 3A
Histone 1, H1c

Among these genes, only about 2% were upregulated,
while the rest were downregulated. All these genes have
been annotated in the NCBI Reference Sequence database. In contrast, LPS stimulation obviously activated
several signalling pathways including the NF-jB pathway to induce the expression of cytokines and antimicrobial peptides. About 5% of genes were upregulated
and 3% were downregulated in group C compared with
group A. In addition, 4% of genes were upregulated
and 2% were downregulated in group D compared with

group A (Table 1). Significantly, the expression of
DEFB4 (HBD-2) and DEFB103A (HBD-3) was changed and the ratios of log 2 were increased after LPS
induction and decreased after HMGN2 knockdown.
The microarray analysis of the human genome chip
containing 31 000 genes revealed differential expression
of 3–5% in the four groups (with a false discovery rate
corrected P £ 0.05 and fold change ‡ 1). The changed
gene expression profiles could be categorized into following gene ontology: MAPK, focal adhesion, Toll-like
receptor, epithelial cell, regulation of actin cytoskeleton,
vascular endothelial growth factor, Fc epsilon, Wnt,
cytokine–cytokine receptor interaction, apoptosis, adherens junction, dorso-ventral axis formation, T cell
receptor, insulin and JAK–STAT signalling pathways.
HMGN2 regulates LPS-induced HBD-2 expression
in A549 cells
Since microarray analysis showed that the expression
of HBD-2 was significantly changed after LPS induction and HMGN2 knockdown, next we aimed to confirm these results by RT-PCR and western blot. We
employed a variety of A549 cells with stable transfections of pSilencer-HMGN2-2 (Psi-H), pEGFPN1-

HMGN2 (PE-H), control siRNA (Psi, PE), wild-type
A549 cells (blank), and reintroduction of HMGN2
expression vector or control vector to HMGN2 knockdown A549 cells (Psi-H ⁄ PE-H or Psi-H ⁄ PE, respectively; Figs 1C,D, S4 and S5). These cells were treated
with 0, 20, 40, 60, 80 or 100 lgỈmL)1 LPS for 24 h
and then the RNA and protein were isolated for RTPCR and western blot analysis. The results demonstrated that LPS induced HBD-2 expression in a dosedependent manner which can be abolished by
HMGN2 knockdown. However, reintroduction of
HMGN2 into HMGN2 knockdown cells recovered
LPS-induced expression of HBD-2 (Fig. 2A–D). Taken
together, these data suggest that HMGN2 is crucial
for LPS-induced HBD-2 expression.
To verify that the decreased levels of HBD-2 protein
and transcripts are indeed linked to the expression

levels of HMGN2, the plasmid expressing double-point
mutant HMGN2-S24, 28E was prepared (this protein
enters the nucleus but does not bind to chromatin);
then we examined the levels of HBD-2 expression level
in HMGN2) A549 cells that were stably transformed
with plasmid expressing either the wild-type HMGN2
protein (PEGFPN1-HMGN2) or the double-point S24,
28E HMGN2 protein (PEGFPN1-HMGN2-S24, 28E
deletion mutants were generated by PCR amplification
of the corresponding part of HMGN2 cDNA). In
A549 cells, replenishment of HMGN2 protein upregulated the protein levels of HBD-2, an indication that
the levels of this protein are indeed linked to the cellular levels of HMGN2. In contrast, replenishment of
the S24, 28E HMGN2 double-point mutant, which
does not bind to chromatin, did not change the levels
of HBD-2, suggesting that the interaction of HMGN2
with chromatin regulates HBD-2 expression signifi-

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS

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HMGN2 mediates expression of b-defensins

B

PE-H group
PE group

β-actin

HBD2

B group

6

β-actin
HBD2

Psi-H group

β-actin
HBD2

Psi group

β-actin
HBD2

Psi-H/PE-H group
Psi-H/PE group
0

HBD-2 real time PCR
PE-H
PE group
B group
Psi-H group
Psi group
Psi-H/PE-H group

*
Psi-H/PE group

β-actin
HBD2

Relative expression of HBD-2

A

L.-X. Deng et al.

β-actin
HBD2
β-actin
HBD2

4

*

*
2

*

*
*
*

0

*

20
40
60
80
100
LPS stimulated concentration (µg·mL–1)

20 40 60 80 100

LPS stimulated concentration (µg·mL–1)

C

D

PE-H group

HBD-2 Western blotting

HBD-2
β-actin

PE group

Psi-H group

1.0

HBD2
β-actin

B group

0.8

HBD-2/β-actin ratio

HBD2
β-actin

PE-H group
PE group
B group
Psi-H group
Psi group

HBD2
β-actin

Psi group
HBD2
β-actin
Psi-H/PE-H group
HBD-2
β-actin

Psi-H/PE-H group
Psi-H/PE group
*

0.6

*

*
0.4
0.2

*
*

*

*

*

*

Psi-H/PE group
0

20 40 60 80 100

HBD-2
β-actin

0

20

40
60
80
100
LPS stimulated concentration (µg·mL–1)

LPS stimulated concentration (µg·mL–1)

Cell
Transfected
HMGN2
HBD-2
β-actin

F
HMGN2–A549
(PEGFPN1-HM
GN2)
–

+

HMGN2–A549
(PEGFPN1-HMG
N2-S24,28E)

–

+

Relative expression of HBD-2

E

Transfected–

0.8

Transfected+

0.6
0.4
0.2
0

HMGN2 HMGN2-S24,28E

cantly (Fig. 2E). Overall, the results support an important role of HMGN2 with chromatin regulation in
inducible HBD-2 expression.
HMGN2 regulates NF-jB activity in A549 cells
To reveal potential mechanisms by which HMGN2
regulates LPS-induced HBD-2 expression, we first
examined whether HMGN2 could modulate NF-jB
2156

*

Fig. 2. HMGN2 is crucial for LPS-induced
HBD-2 expression in A549 cells. The cells
were incubated in DMEM with 10% FBS
containing 0, 20, 40, 60, 80 or 100 lgỈmL)1
LPS for 24 h and then HBD-2 mRNA and
protein levels were detected by RT-PCR and
western blot. (A) Representative RT-PCR
results showing the expression of HMGN2
mRNA in the different established stable
A549 cells. b-actin served as the loading
control. (B) Values of RT-PCR are expressed
as relative expression compared with the
blank group and presented as mean ± SD
for at least five independent experiments
performed in triplicate. *P < 0.01 versus
blank group. (C) Representative western
blot results showing the expression of
HMGN2 protein in the different established
stable A549 cells. b-actin served as the loading control. (D) Photodensitometric analysis
of western blot is presented as mean ± SD
for at least five independent experiments
performed in triplicate. *P < 0.01 versus
blank group. (E) Western blot analysis of
stably transfected HMGN2) A549 cells
expressing either HMGN2 or the HMGN2S24, 28E. A549 HMGN2) denotes control,
non-transformed HMGN2) cells. (F) Values
of western blots are expressed as relative
expression compared with b-actin and presented as mean ± SD for at least five independent experiments performed in triplicate.

activity in A549 cells because the promoter region of
HBD-2 contains four NF-jB binding sites [4]. We
observed that LPS led to increased NF-jB levels in the
nucleus and decreased NF-jB levels in the cytoplasm.
However, the LPS-induced change of NF-jB distribution was markedly attenuated by HMGN2 knockdown,
indicating that loss of HMGN2 inhibits LPS-induced
NF-jB accumulation in the nucleus. On the other
hand, gain of HMGN2 due to overexpression repro-

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS


L.-X. Deng et al.

HMGN2 mediates expression of b-defensins

A Cytoplasm

B

Nucleus

C
Cytoplasm

NF-κB Western blotting

PE
B
Psi-H

Psi
Psi-H/
PE-H

0.8

0.6

NF-κB p65 nuleus preotein/whole protein ratio

0.8

*

0

0

0

Psi-H/
PE

20
40
60
80
100
LPS stimulated concentrations (µg·mL–1)

*

*
*

*

0.2

Psi-H/
PE-H

*

Psi-H/PE group

*

Psi

*
*

*

0.2

0.6

0.4

Psi-H

*
*

E

NF-κB western blotting
PE-H group
PE group
B group
Psi-H group
Psi group
Psi-H/PE-H group *

1.0

B

0.4

LPS stimulated concentrations (µg·mL–1)

D

PE

*
*

*

*

A fold induction
(compared to mock infection)

20
40
60
80
100

20
40
60
80
100

Psi-H/
PE

PE-H group
PE group
B group
Psi-H group
Psi group
Psi-H/PE-H group
Psi-H/PE group

1.0

Nucleus

PE-H

0
5
15
30
60
120
180
0
5
15
30
60
120
180

NF-κB p65 nulear preotein/total protein ratio

PE-H

LPS stimulated time (min)

PE-H group
PE group

B group
Psi-H group
Psi group
Psi-H/PE-H group
Psi-H/PE group

40

30

*
20

*
*
10

0
0

5
15
30
60
LPS stimulated time (min)

120

180

PE

-H

PE

B

Ps

i -H

i

E
H
E- H/P
sii -H P
Ps
Ps

/P

Fig. 3. HMGN2 regulates NF-jB activity in A549 cells. (A–D) HMGN2 promotes the nuclear accumulation of p65. At 24 h after transfection,
the cells were incubated in DMEM with 10% FBS containing 0, 20, 40, 60, 80 and 100 lgỈmL)1 LPS. Fresh medium was added 1 h later.
(A) Representative western blot results showing the cytoplasmic and nuclear distribution of p65 in the different established stable A549
cells. (B) Photodensitometric analysis of western blot is presented as mean ± SD for at least five independent experiments performed in
triplicate. *P < 0.01 versus blank group. The cells were incubated in DMEM with 10% FBS containing 100 lgỈmL)1 LPS. Fresh medium was
replaced 0, 5, 15, 30, 60, 120, 180 min later. (C) Representative western blot results showing the cytoplasmic and nuclear distribution of
p65 in the different established stable A549 cells. (D) Photodensitometric analysis of western blot is presented as mean ± SD for at least

five independent experiments performed in triplicate. *P < 0.01 versus blank group. (E) HMGN2 promotes the transcription activity of NF-jB.
The cells were transfected with NF-jB luciferase reporter and treated by LPS. The luciferase activity is presented as mean ± SD for at least
five independent experiments performed in triplicate. *P < 0.01 versus blank group. The luciferase activity in the blank group untreated by
LPS (mock) was utilized as the control value.

ducibly prompted the accumulation of NF-jB in the
nucleus (Fig. 3A–D).
To further confirm that HMGN2 modulates LPSinduced NF-jB activation in A549 cells, A549 cell lines
harbouring a transient NF-jB-dependent luciferase
reporter were utilized. Following LPS (100 lgỈmL)1)
treatment for 4 h, a 20- to 25-fold increase in luciferase
activity from the baseline level was seen in normal
A549 cells (Fig. 3E). Compared with normal A549 cells

(group B), luciferase activity was increased 25% in
HMGN2 overexpressing cells (PE-H) and decreased
50% in HMGN2 knockdown cells (Psi-H). Significantly, restoration of HMGN2 expression in HMGN2
knockdown cells increased luciferase activity to a level
comparable with normal A549 cells (Psi-H ⁄ PE-H)
(Fig. 3E). Taken together, these data prove that
HMGN2 regulates LPS-induced NF-jB activity in
A549 cells.

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HMGN2 mediates expression of b-defensins

L.-X. Deng et al.

(Table 2) that the acetylation of p65-Lys310 was
decreased in HMGN2 knockdown cells with
100 lgỈmL)1 LPS for 5, 15, 30, 60 or 120 min and
increased in HMGN2 overexpressing cells with
100 lgỈmL)1 LPS for 60 and 120 min but the later difference was not statistically significant (Fig. 4A,B),
suggesting that HMGN2 promotes the acetylation of
p65-Lys310 mainly by enhancing HAT activity. Since
p65-Lys310 acetylation depends on p65-Ser536 phosphorylation, we also examined the effect of HMGN2
on p65-Ser536 phosphorylation and found that
HMGN2 could enhance the phosphorylation of p65
on Ser536 as well. Furthermore, western blot analysis
demonstrated that the phosphorylation of p65 on
Ser536 was upregulated in the PE-H group and downregulated in the Psi-H group compared with the blank
group. However, inhibition of HDAC or HAT did not
affect p65 phosphorylation (Fig. 4C,D). These results
demonstrated that HMGN2 promoted p65-Lys310
acetylation via enhancing p65-Ser536 phosphorylation
and increasing HAT activity.

Table 2. Cell grouping in HAT and HDAC activity experiments.
Groups

Descriptions

PE-H group

A549 cells stably transfected with
PEGFPN1-HMGN2

A549 cells stably transfected with
PEGFPN1-HMGN2 and then adding AA
A549 cells induced by LPS
A549 cells stably transfected with
pSilencer-HMGN2-2
A549 cells stably transfected with
pSilencer-HMGN2-2 and then adding TSA

PE-H ⁄ AA group
B group
Psi-H group
Psi-H ⁄ TSA group

HMGN2 modulates the acetylation of p65
The transcription factor NF-jB activity is known to
be regulated by reversible acetylation through HATs
and HDACs. Anacardic acid (AA) inhibits HAT activity while trichostatin (TSA) inhibits HDAC activity.
Therefore, we used these reagents to treat A549 cells.
Western blot analysis for p65-Lys310 acetylation in
AA-treated or TSA-treated cells demonstrated clearly
A

B

Nucleus

Psi-H group
Psi-H/TSA group
B group

Psi-H group

B group

PE-H group

PE-H/AA group

5 15 30 60 120
LPS stimulated time (min)

C

The acetylation level of p65-Lys310

Psi-H/TSA group

a-acetyl-p65
whole cell extract
1.0

PE-H group
PE-H/AA group

0.8

*
*

0.6

*
*

0.4

*

0.2
0
5

15
30
60
LPS stimulated time (min)

PE-H group

PE-H/AA group

5 15 30 60 120
LPS stimulated time (min)

2158

The phosphorylation level of p65-Ser536

B group

120

Psi-H group
Psi-H/TSA group

a-phosph-p65
whole cell extract
Psi-H/TSA

*

*

D

Nucleus
Psi-H group

*

*

B group
PE-H group

1.0

PE-H/AA group

0.8

*

*
*

*

0.6
*

0.4

*
*

*
0.2
0

5

15
30
60
LPS stimulated time (min)

120

Fig. 4. HMGN2 increases the HAT activity

to promote NF-jB activation. The A549 cells
were pre-incubated with 25 lM AA for 4 h
or 100 nM TSA for 18 h and treated with
100 lgỈmL)1 LPS for 5, 15, 30, 60 or
120 min. (A) Representative western blot
results showing the acetylation of p65Lys310 in the indicated A549 cells. (B)
Photodensitometric analysis of western blot
is presented as mean ± SD for at least five
independent experiments performed in
triplicate. *P < 0.01 versus blank group.
P < 0.01 PE-H ⁄ AA versus PE-H group. (C)
Representative western blot results showing the phosphorylation of p65-Ser536 in the
indicated A549 cells. (D) Photodensitometric
analysis of western blot is presented as
mean ± SD for at least five independent
experiments performed in triplicate.
*P < 0.01 versus blank group.

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS


L.-X. Deng et al.

HMGN2 mediates expression of b-defensins

The ratio of chromosome immunoprecipitation
8.0

A

B

M

1

HMGN1/INPUT
HMGN2/INPUT
HMGN2-/HMGN1/INPUT

2

Fig. 5. HMGN2 is enriched in HBD-2 promoter chromatin in A549 cells. (A) Enrichment of each DNA sequence in the HMGN2
or HMGN1 immunoprecipitate relative to
the input DNA is normalized and plotted as
the position of the PCR primer pair within
the HBD-2 gene locus. Each point is an
averaged value from three independent
experiments. (B) The electrophoretogram
chromosome ultrasonication. M, maker; 1,
the whole chromosome; 2, chromosome
analysis following ultrasonication.

Fold enrichment

6.0

P1

4.0

2.0

0.0

–10

–8

–6

–4

–2

0

2

4

Ex1

8 7 6 5 4 3 2 1 0 9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8
P1 P1 P1 P1 P1 P1 P1 P1 P1 P P P P P P P P P P G G G G G G G G

HMGN2 binds to HBD-2 promoter upon LPS
stimulation
Members of the HMGN family have been reported to
affect the chromatin structure to alter the recruitment

of transcription factors to promoter [28]. To explore
the mechanism by which HMGN2 affects LPS-induced
expression of HBD-2, we performed ChIP analysis of
the 13 kb region of HBD-2 gene using antibodies to
HMGN1 or HMGN2 and demonstrated a twofold to
fourfold enrichment of HMGN2 over a )3 kb region
5¢ to the start of transcription, suggesting that the
region for P1 amplification contains the enrichment
region for HMGN2 and HBD-2 chromatins (Fig. 5).
More importantly, in the HBD-2 chromatin derived
from the HMGN2) A549 cells, the level of HMGN1
was enriched in several regions, especially those spanning primer sets P13–P6 and P5–P2 (Fig. 5). The
increased level of HMGN1 in the HBD-2 chromatin of
the HMGN2) A549 cells suggested that HMGN1 and
HMGN2 had functional redundancy among them.
Furthermore, to address the possibility that
HMGN2 promotes the interaction of NF-jB p65 with
HBD-2 promoter, we produced knockdown HMGN2
and performed ChIP analysis to show that depletion
of HMGN2 affected the chromatin binding of NF-jB
(Figs 6A and S6), indicating that HMGN2 enhances
the interaction between p65 and HBD-2 promoter.
Thus we hypothesized that HMGN2 and p65 may preassemble into a regulatory complex on HBD-2 promoter. However, Co-IP experiments demonstrated that
p65 and HMGN2 did not assemble into a complex in
the nuclear compartment because HMGN2 antibody

efficiently precipitated HMGN2 from nuclear extracts
of A549 cells but the precipitates did not contain p65
(Fig. 6B). Likewise, anti-p65 immunoprecipitated p65
efficiently and the precipitates did not contain

HMGN2 (Fig. 6B). Next, we examined whether
HMGN2 and p65 share the same binding sites on
HBD-2 promoter. ChIP analysis indicated that the
antibodies to HMGN2 and p65 efficiently immunoprecipitated chromatin containing the respective proteins
(Fig. 6C), but the chromatin with HMGN2 enrichment
does not contain p65 (Fig. 6C, upper left panel) and
the chromatin with p65 enrichment does not contain
HMGN2 (Fig. 6C, lower right panel). Based on these
results we conclude that HMGN2 could not interact
with p65 in nuclear extracts and they did not share the
same chromatin binding site.
In contrast, results of reciprocal sequential ChIP
analysis revealed that chromatin that was sequentially
immunoprecipitated with HMGN2 and p65 antibody
was enriched in the DNA of HBD-2 promoter (P1)
while the HBD-2 promoter was enriched in chromatin
that was sequentially immunoprecipitated with antip65 and anti-HMGN2 (Fig. 6D,E), suggesting that
HMGN2 and p65 mutually promote their binding to
the promoter of HBD-2.

Discussion
The mechanism underlying the regulation of the
expression of antimicrobial peptides including b-defensins has not been elaborated at the transcriptional
level. Our studies indicate that chromatin binding protein HMGN2 mediates the LPS-induced expression of

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS

2159

HMGN2 mediates expression of b-defensins

HBD-2 P1

1.0
Input
Control IgG
α-p65
+
α-HMGN2 –

0.5

–
+

+
–

–
+

0
a-HMGN2

B

a-NF-κB p65

C

gG

WB:

IP:

IP:

65

α-P

ut

ol l

inp

ntr

Co

10%

lgG

IP:

N2

MG

α-H

inp

Con

10%

trol

ut

IP:

No
lgG
Co
ntr
ol l
gG
α-P
65
1%
inp
ut
5%
inp

ut

:

HMGN2–

Normal

lgG
ntr
o
α-H l lgG
MG
N2
1%
inp
ut
5%
inp
ut

IP

Psi
Psi-H

No

Fold enrichment at P1

1.5

Co

A

L.-X. Deng et al.

p65
HMGN2

D
1stChIP
Control IgG
α-HMGN2

E

1stChIP
Control IgG α-p65

Fold enrichment at P1

Input
control IgG
α-p65
α-HMGN2

+
–

ro
ll
gG
HM
Co GN
2
nt
ro
ll
g
αHM G
G
N2

HBD-2 in A549 cells. Our microarray analysis indicated that depletion of HMGN2 protein altered the
expression level of over 4% of genes by twofold or
more in A549 cells. Significantly, the HBD level in the
LPS group was fivefold higher than that in the control
group, while the HBD-2 level in the HMGN2 knockdown group was threefold less than in the LPS group.
The results of the microarray are reliable because they
agree well with the expression of genes known to be
modulated by HMGN2, such as N-cadherin, Sryrelated HMG-box gene 9 (Sox9), pituitary homeobox
2, heat shock proteins and type 2 glucose transporters
(Glut2) [4,29–35]. Pathway analysis of the microarray
results showed that HMGN2 modulates the Toll–
NF-jB pathway upon LPS stimulation because the
expression of RELA (p65), IKBKB (IjB) and myeloid
differentiation primary response gene 88 (MyD88),
which were the main members on the Toll–NF-jB

pathway, was changed in HMGN2 knockdown plus

2160

–
+

1st: α-p65
2nd: α-HMGN2

α-

2nd ChIP

nt

Co
nt
ro
lI
gG
α
Co -p6
5
nt
ro
ll
gG
αp6
5

2nd ChIP

Co

Fold enrichment at P1

HBD-2 P1

+
–

–
+

α-HMGN2
α-p65

1st: α-HMGN2
2nd: α-p65

Fig. 6. HMGN2 and p65 independently bind
HBD-2 promoter in A549 cells. (A) Depletion
of HMGN2 reduced the binding of p65 to
HBD-2 promoter. (B) Co-IP assay showing
that HMGN2 and p65 did not form a complex in the nucleus. IP, antibody used for
immunoprecipitation; WB, antibody used for
western blot. (C) ChIP assay showing that
HMGN2 and p65 were not co-localized in
the chromatin. (D) HMGN2 and p65 were

co-localized in the promoter region of HBD-2
chromatin, with the IgG as a negative control. (E) ChIP assay showing that HMGN2 or
p65 bound to HBD-2 chromatin at P1.

LPS groups compared with the LPS group. The
changes of gene expressions observed in the microarray
were identified by RT-PCR and western blotting.
Finally, the results detected in the microarray were
consistent with those achieved through RT-PCR and
western blotting, including the change of HBD-2
expression. Compared with A549 cells stimulated by
LPS, HBD-2 expression was 50% less in HMGN2
knockdown cells and was over 30% higher in HMGN2
overexpressing cells. In addition, reintroduction of
HMGN2 re-expression led to the recovery of HBD-2
expression by over 70% in HMGN2 knockdown cells.
Overall, these findings prove that HMGN2 plays an
essential role in LPS-induced HBD-2 expression in
A549 cells.
Next we aimed to elucidate the molecular mechanism by which HMGN2 regulates HBD-2 expression.
HBD-2 promoter contains several binding sites for
transcription factors including NF-jB, NF-IL-6 and

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS


L.-X. Deng et al.

AP-1 [36–38]. Previous studies found that the upregulation of HBD-2 promoter activity was mainly dependent on NF-jB in A549 cells, and LPS is known to
induce the activation of NF-jB. Therefore, we proposed that HMGN may mediate LPS-induced HBD-2

expression through the NF-jB signalling pathway. To
examine this possibility we first performed western blot
analysis to show that the accumulation of p65 protein
in the nucleus, indicative of NF-jB activation, was
increased in HMGN2 overexpresssing A549 cells but
decreased in HMGN2 knockdown cells. Next, we
employed NF-jB luciferase reporter assay to quantify
NF-jB activation [39]. The NF-jB-induced luciferase
activity was significantly diminished in HMGN2
knockdown cells and increased in HMGN2 overexpressing cells. Based on these data we could conclude
that HMGN2 is crucial for LPS-induced NF-jB activation.
NF-jB activation is known to be reciprocally regulated by RelA ⁄ p65 acetylation and deacetylation mediated by HATs and HDACs. HDACs and HATs are
enzymes that influence transcription by selectively deacetylating or acetylating the e-amino groups of lysine
located near the amino termini of core histone proteins. Acetylation of p65 at lysines 218, 221 and 310
by HATs including the general transcriptional coactivators CBP and P300 would impair the association
between IjBa and p65, thus enhancing the binding
affinity of p65 for DNA [40]. The effect of HATs is
compromised by HDACs that deacetylate p65 and
thus promote the interaction between p65 and IjB.
HDACs are categorized into two classes: class I
HDAC 1, 2, 3, 8 and 11, and class II HDAC 4, 5, 6,
7, 9 and 10. Previous studies reported that the association of NF-jB with HDAC1 and HDAC2 co-repressor
proteins functions to repress the expression of NF-jB
regulated genes [19,20,22].
Interestingly, several studies suggested the relationship between HMGN proteins and the activity of
HATs and HDACs. An in vitro assay showed that
HMGN1 and HMGN2 partially inhibit the endogenous mouse HDAC activity [27]. HMGN1 enhances
the acetylation level of lysine 14 in the tail of H3,
while HMGN1 and HMGN2 increase the acetylation
through enhancing the activity of HATs [13]. AA is

an HAT inhibitor and inhibits the nuclear translocation and acetylation of p65, repressing TNF-induced
NF-jB dependent reporter gene expression [25]. In
contrast, TSA, an HDAC inhibitor, enhances p65 acetylation induced by Gram-negative bacteria and transforming growth factor b1 [41]. In addition, HDAC
inhibitor SFN led to a time- and dose-dependent
upregulation of HBD-2 mRNA and protein expression

HMGN2 mediates expression of b-defensins

in Caco-2, HT-29 and SW480 cells [42]. This is paralleled by changes in the acetylation of distinct core proteins, H4 and HMGN2, resulting in the induction of
LL-37, a member of the antimicrobial peptide that
protects the urinary tract against invasive bacterial
infection [43].
In the current study, AA (HAT inhibitor) was
selected to test whether blocking HATs diminishes the
acetylation level of the p65-Lys310 subunit in the PE-H
transfected A549 cells. If HMGN2 promotes the acetylation of p65 through increasing HAT activity, AA pretreatment would reduce the acetylation level of p65Lys310 in the nucleus in the PE-H ⁄ AA group compared
with the PE-H group. On the other hand, TSA (HDAC
inhibitor) was used to examine the acetylation level of
p65-Lys310 in the Psi-H transfected A549 cells. If
HMGN2 promotes the acetylation of p65 through
inhibiting HDAC activity, the TSA pretreatment would
augment the acetylation level of p65-Lys310 in the
nucleus in the Psi-H ⁄ TSA group compared with the
Psi-H group. Because p65-Lys310 acetylation was only
allowed when p65-Ser536 was phosphorylated, the p65
global phosphorylation status on Ser536 was also tested
by western blotting in five groups.
The results demonstrated that the amount of p65Lys310 acetylation in the nucleus of A549 cells in the
PE-H group was low compared with that in the presence of AA (Fig. 4A,B). Adding AA to the HMGN2
overexpressing cells helped to reduce the acetylation

level of p65-Lys310 in the nuclei to the normal level,
while adding TSA to the HMGN2 knockdown cells
did not bring the acetylation level of p65-Lys310 in the
nucleus to normal; the difference was not statistically
significant. Results of western blotting for p65-Lys310
acetylation in AA-treated or TSA-treated cells indicated that HMGN2 increased acetylation of p65Lys310 mainly by enhancing the activity of HATs.
Since p65-Lys310 acetylation depends on p65-Ser536
phosphorylation, we also examined the effect of
HMGN2 on p65-Ser536 phosphorylation. Subsequently, thePE-H plus AA group or the Psi-H plus
TSA group did not modify p65-Ser536 phosphorylation compared with the PE-H or Psi-H group respectively, the difference not being statistically significant.
However, western blot analysis demonstrated that the
phosphorylation of p65 on Ser536 was upregulated in
the PE-H group and downregulated in the Psi-H group
compared with the blank group. Collectively, these
results indicated that HMGN2 promotes p65-Lys310
acetylation mainly via increasing HAT activity and
enhancing p65-Ser536 phosphorylation.
Lastly, we performed ChIP analysis to demonstrate
that HMGN2 enhances HBD-2 transcription directly by

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2161


HMGN2 mediates expression of b-defensins

L.-X. Deng et al.

binding to the promoter region of this gene. HMGN1

also binds to HBD-2 chromatin but not within the
enrichment region in A549 cells. However, in the HBD2 chromatin derived from the HMGN2) A549 cells, the
level of HMGN1 was enriched in several regions, especially those spanning primer sets P13–P6 and P5–P2.
Moreover, a previous study had reported that it is likely
that homeostatic mechanisms, perhaps involving
HMGN2, compensate for the loss of the HMGN1 protein based on structural similarity among them. Indeed,
the ChIP analyses in that study indicated an increase of
HMGN2 in the Sox9 chromatin obtained from
HMGN1) ⁄ ) mice, suggesting functional redundancy
among these proteins [31]. Moreover, the downregulation of HMGN2 decreased the binding of p65 to HBD2 promoter significantly. To address the potential relationship between HMGN2 and p65 in the regulation of
the HBD-2 promoter region, we did reciprocal sequential precipitation experiments and observed that
HMGN2 and p65 specifically target the HBD-2 promoter. Co-IP and ChIP experiments demonstrate that
HMGN2 does not interact with NF-jB directly and the
two proteins do not form a complex in the nucleus and
or share the same binding sites throughout the entire
chromatin. These findings are consistent with previous
studies reporting the relationship between HMGB1 and
glucocorticoid receptor in glucocorticoid receptor binding sites and HMGN3 and PDX1 (a Glut2 transcription
factor). In particular, HMGN3 and PDX1 mutually
reinforce their specific binding to the Glut2 chromatin
without interaction with each other or sharing the same
binding sites in Glut2 chromatin [28,44]. Based on these
data, we propose that HMGN2 and p65 mutually reinforce their specific binding to the chromatin in the promoter of the HMGN2 gene, thereby regulating
HMGN2 protein levels in A549 cells.
We noticed that the expression of HBD-2 was
decreased by only 40–50% when endogenous
HMGN2 was knocked down. The reason is that the
efficiency of RNA interference and the signalling
pathways that induce b-defensin expression upon LPS
stimulation are not limited to NF-jB. In addition,

other members of the HMGN family that are structurally similar to HMGN2 may play a complementary role. Some people have confirmed that depletion
of HMG-14 and HMG-17 is associated with a delay
in early embryonic development from the two- to
four-cell stage, but depleting either one does not
delay the development [45]. In summary, we identified
a novel mechanism by which HMGN2 modulates the
expression of HBD-2 in A549 cells. HMGN2 prolongs the retention time and enhances the accumulation of NF-jB p65 in the nucleus, and promotes the
2162

acetylation of p65 through increasing HAT activity.
Additionally, chromatin immunoprecipitation revealed
that HMGN2 and p65 synergistically promote their
specific binding to the HBD-2 promoter, thereby
affecting downstream transcription. Taken together,
these results suggest that HMGN2 acts as a positive
modulator of NF-kB signalling to promote LPSinduced b-defensin expression.

Materials and methods
Materials
The antibodies were p65 (Santa Cruz Biotechnology, Santa
Cruz, CA, USA), p65 (for ChIP; Abcum, Cambridge, UK),
acetyl-p65 (Lys310) (Cell Signaling, Danvers, MA, USA),
b-actin (Santa Cruz), histone H3 (Abcum) and HBD-2
(Santa Cruz). The affinity pure HMGN2 antibodies were
prepared in our laboratory. Escherichia coli LPS was from
Sigma (St Louis, MO, USA).

Cell culture
Human lung adenocarcinoma A549 cells were obtained
from the School of Pharmaceutical Science, Sun Yat-Sen

University (Guangzhou, China). These cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a
humidified incubator containing 5% CO2.

siRNA transfection
Three HMGN2-specific siRNAs (1, 2 and 3) (SiRNA-HMG
N2-1: sense, 5¢-CUA AUA GAA UGU CUC CAA ATT-3¢;
antisense, 5¢-UUU GGA GAC AUU CUA UUA GTG-3¢;
SiRNA-HMGN2-2: sense, 5¢-AGU CAG GGU CGG CUU
GUG ATT-3¢; antisense, 5¢-UCA CAA GCC GAC CCU
GAC UTT-3¢; SiRNA-HMGN2-3: sense, 5¢-UAA UAG
AAU GUC UCC AAA GTT-3¢; antisense, 5¢-CUU UGG
AGA CAU UCU AUU AGT-3¢) and two non-specific control siRNAs (positive siRNA sense, 5¢-GAC TTC ATAA
GGCG CATGC-3¢; antisense, 5¢- GCATGCGCCTTAT
GAAGTC-3¢; negative siRNA sense, 5¢-AUU GUA UGC
GAU CGC AGA CTT-3¢; antisense, 5¢-GUC UGC GAU
CGC AUA CAA UGA-3¢) were designed and synthesized by
RiboBio (Guangzhou, China). The positive control siRNA
was targeted to the b-actin gene, while the negative control
did not match any genes of known function in GenBank.
A549 cells were placed in six-well plates (Costar, CA, USA)
at a density of 2 · 105 cells per well. After 24 h, cells were
grown to 40–50% confluence and transfected by HMGN2specfic siRNA or control siRNAs using LipfectamineÔ
2000 (Invitrogen, Carlsbad, CA, USA) according to the

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS


L.-X. Deng et al.

HMGN2 mediates expression of b-defensins

Table 3. A549 cell groups in microarray analysis.

Table 4. A549 cell groups in this study.

Groups

Description

Groups

Description

Blank group (group A)
HMGN2 knockdown
group (group B)
LPS group (group C)
HMGN2 knockdown
plus LPS group
(group D)

A549 cells untreated
A549 cells transfected with
HMGN2-specific siRNA2
A549 cell treated by 100 lgỈmL)1 LPS
A549 cell transfected with
HMGN2-specific siRNA2 for 48 h and
then treated by 100 lgỈmL)1 LPS for
24 h

PE-H group

A549 cells stably transfected with
PEGFPN1-HMGN2
A549 cells stably transfected with
PEGFPN1
A549 cells treated by LPS
A549 cells stably transfected with
pSilencer-HMGN2-2
A549 cells stably transfected with
pSilencer
A549 cells stably transfected with
pSilencer-HMGN2-2 and transfected with PEGFPN1-HMGN2
A549 cells stably transfected with
pSilencer-HMGN2-2 and transfected with PEGFPN1

PE group
B group
Psi-H group
Psi group
Psi-H ⁄ PE-H group

manufacturer’s instructions. The medium was replaced 6 h
after transfection and the cells were cultured for 24 h before
the efficiency of HMGN2 knockdown was determined.

Psi-H ⁄ PE group

cDNA microarray and data analysis

A549 cells were placed into six-well plates at a density of
2 · 105 cells per well. The cells were divided into four
groups in triplicate (Table 3). The microarray was performed at Shanghai Biochip according to instructions from
SBC GeneChips (shbiochip, Shanghai). Total RNA was
isolated from A549 cells using Trizol (Invitrogen) following
the manufacturer’s instructions. About 10–20 lg RNA was
converted to cDNA using an oligo (dT) T7 primer, and the
double-stranded cDNA was labelled with Cy3 (as the reference sample) and Cy5 (as the treatment sample), respectively. The double-stranded cDNA was purified and used as
a template in subsequent transcription reactions. The
labelled cDNA probes for the four groups were mixed with
hybridization buffer. The samples were hybridized competitively under coverslips to the microarray slides at 50 °C for
16 h in a dark humidity chamber and washed as follows:
2 · NaCl ⁄ Cit for 5 min, 0.5 · NaCl ⁄ Cit for 5 min, and
30% ethanol ⁄ water for 30 s at 42 °C. Hybridization experiments were performed in duplicate using cDNA derived
from the four different groups.
Images were scanned and analysed by the Agilent
GeneChip Scanner. The original signals of the images
were normalized using the reference spots on the slide
and the fluorescence signals were balanced and amended
using the genespring software. Three biological replicates
(independent RNA isolations), each with a technical replicate (dye swap), were performed for each condition.
Genes with a Cy5 ⁄ Cy3 signal ratio ‡ 2.0 were considered
upregulated, whereas those with a ratio £ 0.5 were downregulated.

Plasmid constructs
Short hairpin (sh) RNA constructs were prepared using pSilencer 4.0-CMV (Ambion, Austin, TX, USA) as the vector.
The HMGN2-shRNA plasmids were named pSilencerHMGN2-1 and pSilencer-HMGN2-2, respectively (HM

GN2-shRNA-1: sense, 5¢-GATCCTCTGCGAGGTTGTC
TGCTATTCAAGAGATAGCAGACAACCTCGCAGAT

CA-3¢; antisense, 5¢-AGCTTGATCTGCGAGGTTGTCTG
CTATCTCTTGA ATAGCAGACAACCTCGCAGAG-3¢;
HMGN2-shRNA-2: sense, 5¢-GATCCA AATGGAGATGC
CAAAACATTCAAGAGATGTTTTGGCATCTCCATTT
TCA-3¢;antisense, 5¢-AGCTTGAAAATGGAGATGCCAA
AACATCTCTTGAATGTTTTGGCATCTCCATTTG-3¢).
Additional shRNAs were used as positive and negative controls (positive shRNA: sense, 5¢-GATCCCGTATATGATA
CCAACAGTAATTC AAGAGATTACTGTTGGTATCA
TATACGTTTTCA-3¢; antisense, 5¢-AGCTTT GAAAACG
TATATGATACCAACAGTAATCTCTTGAATTACTGT
TGGTATCATATACGGG-3¢; negative shRNA: sense, 5¢-G
ATCCGACTTCATAAGGCGACTGCT TCAAGACGGC
ATGCGCCTTATGAAGTCTTTTTTGTCGACA-3¢; antisense, 5¢-AGCTTAGTTCGACAAAAAAGACTTCTTCAT
AAGGCGCATGCCGTCTTGAAGCACGCCTTATGAA
GT-3¢). The complete HMGN2 cDNA was amplified from
the total RNA of A549 cells by RT-PCR with the primers
5¢-CACCATGGCGGAAGGCGGAGCGGC-3¢ and 5¢-GT
TAGTGTGCATACAGTTTT-3¢, and cloned into PEGFP
N1 vector (Invitrogen). The positive clone was verified by
DNA sequencing and named as PEGFPN1-HMGN2.

Establishment of stable cell lines
A549 cells were seeded in 12-well plates at a density of
2 · 105 cells per well. Following overnight incubation in
DMEM containing 10% FBS at 37 °C in 5% CO2, pSilencer-HMGN2-1, pSilencer-HMGN2-2, PEGFPN1-HMGN2
or the control vector was transfected into A549 cells using
LipofectamineÔ 2000 (Invitrogen) as per the manufacturer’s instructions. Resistant colonies were selected by
G418 at 800 lgỈmL)1 based on the killing curve patterns
for A549 cells. Single clones were picked and tested for

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HMGN2 mediates expression of b-defensins

L.-X. Deng et al.

Table 5. Primers for HMGN2 and HBD-2.
Primers

Sequences

HMGN2 sense
HMGN2 antisense
HBD-2 sense
HBD-2 antisense

5¢-CGATG CCC AAG AGA AAG G-3¢
5¢-GCAA CTT GGC ATC TCC AGC A-3¢
5¢-CCAGCCATCAGCCATGAGGGT-3¢
5¢-GGAGCCCTTTCTGAATCCGCA-3¢

co-transfected with 200 ng reporter plasmid pGL3-3 · jBLuc and pRL-TK (Promega) using Lipfectomine 2000 (Invitrogen). Then the cells were untreated or treated with
LPS (100 lgỈmL)1) for 4 h before the cell lysate was collected by passive lysis for luciferase activity assay. The
experiments were done in triplicate for each group.

ChIP assay and Co-IP
HMGN2 expression by RT-PCR and western blot. The stable A549 cell lines are listed in Table 4.

RNA isolation, RT-PCR and real time PCR
Total RNA was isolated from A549 cells in the seven groups
using Trizol (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized from 1 lg RNA with high quality (260 versus 280 nm: 1.8–2.0). The resulting cDNA was
analysed by SYBR Green PCR kit or normal RT-PCR kit
according to the manufacturers’ protocols. 5 lL cDNA was
amplified in a 25 lL reaction mix containing 22.5 lL SYBR
Green supermix (Bio-Rad, CA, USA) and 1 lL each primer
(Table 5). The amplification conditions were initial denaturation at 95 °C for 15 min, 35 cycles of denaturation at 95 °C
for 15 s, annealing at 58 °C for 15 s and elongation at 72 °C
for 30 s. To determine HBD-2 mRNA level, the experiment
was repeated three times under each experimental condition
and the comparative threshold cycle method was employed
with human b-actin as the internal control. Analysis of relative
gene expression data using real-time quantitative PCR and the
)2DDCT method [46].

Western blot
Whole cell lysate was extracted from A549 cells using Protein Extraction Reagent (Pierce, Rockford, IL, USA).
Alternatively, the cytoplasmic and nuclear extracts were
extracted from A549 cells using NE-PER nuclear and cytoplasmic extraction reagents (Pierce) following the manufacturer’s instructions. The protein concentration of the
extract was determined using the BCA protein assay
(Pierce). The proteins were separated with SDS ⁄ PAGE and
transferred to poly(vinylidene difluoride) membranes. The
membranes were incubated with HBD-2 (1 : 1000, Santa
Cruz), HMGN2 (1 : 500, Santa Cruz), NF-jB p65
(1 : 1000, Santa Cruz), histone H3 (1 : 1000, Santa Cruz)
or b-actin (1 : 1000, Santa Cruz) antibody followed by
incubation with the horseradish peroxidase conjugated antigoat IgG serum (Santa Cruz). Finally the membranes were
developed using ECL Plus reagent (Pierce).

Luciferase assay
Stable A549 cells were placed in 24-well plates at a density
of 2.5 · 105 cells per well. After 24 h the cells were

2164

ChIP assay was performed using ChIP IT express kit
(Active Motif, Carlsbad, CA, USA). Briefly, A549 cells
were treated with 100 lgỈmL)1 LPS for 1 h, fixed with
1% formaldehyde for 10 min at room temperature, sonicated to  500 bp soluble DNA fragments and immunoprecipitated with specific antibodies. Antibodies for ChIP
experiments included affinity pure HMGN2 or p65 antibody, or IgG (Upstate Biotechnology, Lake Placid, NY,
USA) as the negative control, and the primers used for
ChIP are given in Table S1. Immunoprecipitated DNA
was analysed and the relative enrichment value was calculated as described previously [19]. Co-IP was conducted
using the nuclear complex Co-IP kit (Active Motif).
Nuclear extracts of A549 cells were prepared and precipitated with affinity pure HMGN2 or p65 antibody, and
the co-precipitated proteins were detected by western
blot.

Acknowledgements
The research was supported by grants from the
National Natural Science Foundation of China (No.
30671963, No. 30470763 and CMB 98681).We thank
Dr Bustin (NIH) for advice on the experiments and Dr
Yingqun Wang for revising the manuscript.

Conflict of interest
The authors declare that there is no conflict of interest.

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Supporting information
The following supplementary material is available:
Fig. S1. HMGN2 expression is not affected by LPS
stimulation in A549 cells.
Fig. S2. The quantity of RNA in four groups was
detected by NanoDrop ND-1000.
Fig. S3. (A) The boxplot of the microarray. (B) Part
of the gene map changed significantly in four groups.
The red colour indicated upregulated genes with a
detection ratio ‡ 2, while the green colour indicated
downregulated genes with a detection ratio £ 0.5. (C)
The diagrammatic sketch of the microarray.
Fig. S4. (A) The fragment of HMGN2 cDNA detected
by RT-PCR and HMGN2 protein detected by western
blotting: B, blank group; 1, pSilencer-HMGN2-1; 2,
pSilencer-HMGN2-2. (B) Representative RT-PCR and
western blotting images are shown. b-actin served as
the loading control for PCR, while histone H3 served
as the loading control for western blotting.
Fig. S5. HMGN2 has no effect on the proliferation of
A549 cells.
Fig. S6. ChIP assay demonstrated that HMGN2 and
HMGN1 in normal A549 cells and HMGN1 in HMGN2
knockdown A549 cells bind to HBD-2 chromatin.

Fig. S7. Model for the regulation of transcriptional
activity by HMGN2 to HBD-2 expression.
Table S1. The primers used for ChIP.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.

FEBS Journal 278 (2011) 2152–2166 ª 2011 The Authors Journal compilation ª 2011 FEBS