phosphorylation and inhibit cardiomyocyte hypertrophy Wenqiang Liao, Shuyi Wang, Chide Han and Youyi Zhang Institute of Vascular Medicine, Peking University Third Hospital and Key Labora
Trang 1phosphorylation and inhibit cardiomyocyte hypertrophy Wenqiang Liao, Shuyi Wang, Chide Han and Youyi Zhang
Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, PR China
14-3-3 Proteins were first discovered in 1967 as acidic
proteins found abundantly in the brain 14-3-3
Pro-teins comprise a family of highly conserved proPro-teins
having a molecular mass of 30 kDa and an
isoelec-tric point of around 5 [1] The proteins of this family
are distributed ubiquitously and have been found in all
eukaryotic organisms, ranging from yeast to mammals
Many organisms contain multiple isoforms: at least
seven isoforms (b, c, e, f, g, h⁄ s and r) exist in
mam-mals and two to 12 isoforms in yeast, fungi, and
plants In all organisms, 14-3-3 proteins form
homo-or heterodimeric structures 14-3-3 Proteins have been
shown to bind with over 200 cellular proteins It is
possible that these interactions, like many of those
shown previously, occur through the conserved amphipathic groove of 14-3-3 [1–3] 14-3-3 Proteins specifically recognize phosphoserine⁄ threonine-contain-ing sequence motifs on target proteins, such as RSXpSXP, RXSX (S⁄ T) XP or RX (Y ⁄ F) XpSXP In addition, they can bind to unphosphorylated motifs: GHSL and WLDLE [4–6]
14-3-3 Proteins have been shown to interact with an array of partners, ranging from enzymes to structural proteins Often, these proteins are important in vital cellular processes including cell cycle control and apop-tosis Through its interaction, 14-3-3 either regulate the catalytic activity of its bound enzymes, determine the subcellular localization of target proteins, or both
Keywords
14-3-3 proteins; cardiomyocyte; hypertrophy;
NFAT; PKB ⁄ GSK3b
Correspondence
Y Zhang, Institute of Vascular Medicine,
Peking University Third Hospital and Key
Laboratory of Molecular Cardiovascular
Science, Ministry of Education, Beijing
100083, PR China
Fax: +86 10 82802306
E-mail: zhangyy@bjmu.edu.cn
(Received 28 September 2004, revised 28
January 2005, accepted 14 February 2005)
doi:10.1111/j.1742-4658.2005.04614.x
14-3-3 Proteins are dimeric phophoserine-binding molecules that participate
in important cellular processes such as cell proliferation, cell-cycle control and the stress response In this work, we report that several isoforms of 14-3-3s are expressed in neonatal rat cardiomyocytes To understand their function, we utilized a general 14-3-3 peptide inhibitor, R18, to disrupt 14-3-3 functions in cardiomyocytes Cardiomyocytes infected with adeno-virus-expressing YFP-R18 (AdR18) exhibited markedly increased protein synthesis and atrial natriuretic peptide production and potentiated the responses to norepinephrine stimulation This response was blocked by the pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor Consistent with a role of PI3K in the R18 effect, R18 induced phospho-rylation of a protein cloned from the vakt oncogene of retrovirus AKT8 (Akt – also called protein kinase B, PKB) at Ser473 and glycogen synthase 3b (GSK3b) at Ser9, but not extracellular signal-regulated kinase 1⁄ 2 (ERK1⁄ 2) AdR18-induced PKB and GSK3b phosphorylation was com-pletely blocked by LY294002 In addition, a member of the nuclear factor
of activated T cells (NFAT) family, NFAT3, was converted into faster mobility forms and translocated into the nucleus upon the treatment of AdR18 These results suggest that 14-3-3s inhibits cardiomyocytes hyper-trophy through regulation of the PI3K⁄ PKB ⁄ GSK3b and NFAT pathway
Abbreviations
a 1 -AR, a 1 -adrenergic receptor; AdR18, adenovirus expressing R18 peptide; ANP, atrial natriuretic peptide; PI3K, phosphoinositide 3-kinase; GSK3b, glycogen synthase 3b; ERK1⁄ 2, extracellular signal-regulated kinase 1 ⁄ 2; MOI, multiplicity of infection; NE, norepinephrine; NFAT, nuclear factor of activated T cells; LY, LY294002; PD, PD98059; TDT, terminal deoxynucleotidyl transferase.
Trang 2[1,7] For example, 14-3-3 inhibits ASK1 (apoptosis
signal regulating kinase-1) activity by binding to
speci-fic residues surrounding Ser967 [8,9] This interaction
also controls the subcellular distribution of ASK1
[10,11] The binding of 14-3-3 with PI3K, PKC and
Raf can either inhibit or enhance the activities of these
enzymes [12,13] 14-3-3 Proteins associate with cdc25c,
FKHRL1, HDAC5⁄ 7, NFATc, p27 and PKUa,
pre-venting their entry into the nucleus [14,15] 14-3-3
Pro-teins can also modulate protein–protein interactions
For example, 14-3-3 interacts with the
apoptosis-promoting protein BAD, preventing BAD from binding
to and inhibiting the antiapoptotic function of Bcl-XL
[16,17]
Although many 14-3-3 binding partners have been
identified, the physiological functions of 14-3-3 remain
elusive in many biological systems This is especially
true in the cardiovascular system One method to
determine the importance of 14-3-3 is to use ligand
binding-defective 14-3-3 mutants Examples of these
include dominant-negative forms of 14–3-3f and g
with the point mutation K49E and the double
muta-tion R56A and R60A [8,18] It is hypothesized that
these mutants produce a dominant negative effect by
dimerizing with endogenous 14-3-3 monomers, thereby
inhibiting the function of these proteins However, this
inhibition is partial and only disrupts a certain
iso-form-mediated processes Additionally, there are
tech-nical limitations related to the use of stable cell lines,
which place restrictions on its applicability to many
14-3-3-mediated processes
R18 is a 20-mer peptide that was isolated from
a phage display screen [19] With the core motif
WLDLE, it was found to globally inhibit
14-3-3–lig-and interactions in a specific 14-3-3–lig-and isoform-independent
manner [4,20,21] In this study, we utilized the
adeno-virus-expressing YFP-R18 (AdR18) and found that
14-3-3 can inhibit cardiomyocyte hypertrophy and
negatively modulate a1-adrenergic receptor (a1
-AR)-mediated hypertrophy The phosphoinositide 3-kinase
(PI3K)⁄ protein kinase B (PKB) ⁄ glycogen synthase 3b
(GSK3b) and nuclear factor of activated T cells
(NFAT) pathway most likely contributes to this
pro-cess
Results
Different isoforms of 14-3-3 proteins are
expressed in cardiomyocytes
To determine which isoforms of 14-3-3 exist in
cardio-myocytes, northern blot analysis was performed using
specific probes for the b, c, e, f and h⁄ s isoforms of
14-3-3 Figure 1A shows that the mRNAs for the b, c,
e and f isoforms were detected in isolated cardiomyo-cytes, but no signal of h⁄ s isoform was observed (data not shown) Furthermore, western blot analysis using antibodies specific for 14-3-3b, c, e and f confirms the expression of these isoforms in cardiomyocytes (Fig 1A)
In adult rats treated with osmotic mini-pumps for continuous norepinephrine (NE) infusion, the expres-sion of 14-3-3f protein in the heart tissue was increased one day after the NE infusion (data not shown) To identify whether the expression of 14-3-3
in isolated cardiomyocytes could be affected by activa-tion of the a1-adrenergic receptor (a1-AR), cells were treated with 10 lm NE in the presence of 10 lm pro-pranolol for indicated times Through western blot analysis, we did not find any difference in the expres-sion of 14–3-3f, b, c and e (Fig 1B)
R18 significantly potentiates NE-induced protein synthesis
To investigate the role of 14-3-3 in a1-AR induced hypertrophy of cardiomyocytes, adenovirus expressing R18 peptide (AdR18), a specific and
isoform-independ-0 1 3 6 12 24 48 time (h)
14-3-3β ζ ε γ
14-3-3 14-3-3 14-3-3
B
14-3-3 mRNA
14-3-3 protein 18sRNA
A
β γ
ε ζ
Fig 1 (A) Different isoforms of 14-3-3 s were expressed in cardio-myocytes 14-3-3f, e, c and b isoforms expressed in isolated neo-natal cardiomyocytes Cardiomyocytes cultured in 10 cm plates were serum-free for 24 h, then total RNA was extracted for northern blot analysis The whole cell lysate was harvested for western blot analysis The results were representative of four inde-pendent experiments No signal was detected for the 14-3-3h ⁄ s isoform (B) No effects of the treatment of NE on the expression
of each 14-3-3 isoform Cardiomyocytes were deprived of serum for 24 h, and incubated with propranolol (10 l M ) for 30 min, then stimulated with NE (10 l M ) for the times indicated before lysis and analysis by western blotting The experiment was repeated three times with the same result.
Trang 3ent inhibitory peptide of 14-3-3, was used A [3
H]leu-cine incorporation assay was performed to measure
protein synthesis; an important parameter of
cardio-myocyte hypertrophy Incorporation of [3H]leucine
into cardiomyocytes was increased either by treatment
with NE (expressed as the fold of control, compared
with the control, the NE treated group 1.56 ± 0.31,
P< 0.05, n¼ 5) or by infection with AdR18 (the
AdR18 group 1.51 ± 0.23 vs the Ad group
1.14 ± 0.11, P < 0.05, n¼ 5) (Fig 2A)
Infection of AdR18 in cardiomyocytes significantly
potentiated the NE-induced hypertrophy (Fig 2B)
Compared with the Ad control group, the protein
syn-thesis of the Ad + NE treatment group (expressed as
the fold of Ad control, 1.51 ± 0.31, P < 0.05, n¼ 4)
increased about 50%, and the AdR18-infected group
(1.45 ± 0.26, n¼ 4) increased about 45% However,
for the AdR18 + NE treatment group (2.41 ± 0.38,
n¼ 4), the protein synthesis increased about 150%,
which is much more than the total increment induced
by the NE or R18 treatment These results indicate
that R18 potentiated NE-induced protein synthesis in
cardiomyocytes
PI3K is critical for R18-induced protein synthesis
We next examined which signaling molecule was
responsible for the effect of 14-3-3 on protein synthesis
and on NE-induced protein synthesis in cardiomyo-cytes For these experiments, the extracellular signal-regulated kinase 1⁄ 2 (ERK1 ⁄ 2) inhibitor, PD98059 (PD), and the PI3K inhibitor, LY294002 (LY), were used Figure 2C,D shows that the R18-induced protein synthesis was blocked significantly by LY (10 lm; expressed as fold of control, the AdR18 + LY group
vs the AdR18 group, P < 0.05, n¼ 3), whereas the NE-induced protein synthesis was blocked by PD (10 lm; the NE + PD group vs the NE group,
P < 0.01, n¼ 3) The treatment with PD decreased the protein synthesis of the AdR18 + NE group [the AdR18 + NE + PD group 1.31 ± 0.09 (n¼ 3) vs the AdR18 + NE group 2.56 ± 0.47 (n¼ 5), P < 0.05] to the level of the AdR18 treatment alone (the AdR18 group 1.38 ± 0.23, n¼ 5) (Fig 2E) Further-more, the protein synthesis was markedly reduced by the LY treatment (the AdR18 + NE + LY group 0.62 ± 0.07 vs the AdR18 + NE group, n¼ 3,
P < 0.01)
R18 induces ANP expression in cardiomyocytes
in a PI3K-dependent manner One of the characteristic phenotypic changes of cardio-myocyte hypertrophy is the enhanced expression of the embryonic gene atrial natriuretic peptide (ANP) A detectable level of ANP (40.3 ± 3.2 ng mL)1, n¼ 3)
0
1
2
protein synthesis fo
*
*
A
0 1 2 3
Ad Ad +NE AdR18 AdR18+NE
protein synthesis fo
*
*
B
0 1 2
Ad AdR18 AdR18+LY AdR18+PD
protein synthesis fo
*
C
0
1
2
CON NE NE+PD NE+LY
protein synthesis fo
**
0 1 2 3
Ad AdR18 AdR18
+NE AdR18 +NE +LY
AdR18 +NE +PD
protein synthesis fo
** *
Fig 2 R18 induced protein synthesis and potentiated the NE-induced protein synthesis in cardiomyocytes in a PI3K dependent manner Car-diomyocytes cultured in 24-well plates were infected with or without AdR18 or Ad at a MOI of 10 After starvation for 24 h and treatment with propranolol (10 l M ) as well as a different inhibitor for 30 min, cells were stimulated with NE (10 l M ) for 48 h and 1 lCiÆmL)1[ 3 H]Leu was added 6 h before analysis by [3H]Leu incorporation assay (A and B) R18 induced the protein synthesis (n ¼ 5) and also potentiated the NE-induced protein synthesis (C–E) PI3K was required for the R18-induced protein synthesis, and ERK1 ⁄ 2 for the NE-induced protein syn-thesis Cardiomyocytes were treated with or without propranolol (10 l M ) plus LY294002 (LY, 10 l M ) or PD98059 (PD, 10 l M ), respectively, for 30 min and then stimulated with NE (10 l M ) for 48 h, the protein synthesis was measured (n ¼ 3) The values shown are means ± SD and expressed as the fold of control, *P < 0.05; **P < 0.01.
Trang 4was found in the culture medium of untreated myocytes.
The ANP production was increased approximately
threefold upon the treatment of NE (10 lm) in the
pres-ence of propranolol (10 lm) for 40 h (expressed as the
fold of control, 116.8 ± 6.3 ngÆmL)1 vs the control
group, P < 0.05, n¼ 3), and about fourfold in the
medium of AdR18-infected myocytes (165.6 ± 35.4
ngÆmL)1, P < 0.01 vs the Ad control group, n¼ 3),
indicating an enhanced production of ANP in these cells
(Fig 3A) Figure 3B shows that compared with the Ad
control group, the level of ANP in the AdR18 + NE
group was increased appromimately fivefold (204.8 ±
23.3 ngÆmL)1, n¼ 3), fourfold in the AdR18 group and
threefold in the Ad + NE group Compared with the
Ad + NE group, the level of ANP in the AdR18 + NE
group was increased about 1.5-fold (P < 0.05, n¼ 3),
indicating that R18 enhanced the NE-induced ANP
production
Furthermore, we determined whether PI3K was
required for R18 enhancement of ANP production; as
in protein synthesis As shown in Fig 3C–E, treatment
with LY (10 lm), but not with PD (10 lm), markedly
blocked the R18-induced ANP expression (33.6 ±
0.3 ngÆmL)1, vs the AdR18 or the AdR18 + NE
group, respectively, P < 0.05, n¼ 3), whereas the
NE-induced ANP production was blocked by PD, not
by LY (the NE + PD group 45.37 ± 12.46 ngÆmL)1
vs the NE group, P < 0.01, n¼ 3)
PKB and GSK3b phosphorylation are induced
by R18 and blocked by PI3K inhibitor Glycogen synthase kinase-3 beta (GSK3b), a down-stream signaling molecule of the PI3K⁄ PKB pathway, and ERK1⁄ 2 play very important roles in the regula-tion of hypertrophic response Phosphorylated ERK1⁄ 2 (the active form of the enzyme) positively regulates the hypertrophic response, while dephosphorylated GSK-3b (the active form of the enzyme) negatively regulates the hypertrophic response [22–25] This caused us to speculate whether these signaling mole-cules are involved in the AdR18-induced cardiomyocyte hypertrophy The effect of R18 on ERK1⁄ 2, PKB and GSK3b phosphorylation is shown in Fig 4 Activation
of cardiac a1-AR significantly increased the ERK1⁄ 2 phosphorylation compared with the control group (Fig 4A) The infection of AdR18 in cardiomyocytes had no effect on the ERK1⁄ 2 phosphorylation treated either with or without NE However, the infection of AdR18 in cardiomyocytes markedly induced the PKB and GSK3b phosphorylation Compared with the Ad control, AdR18 induced about twofold increase on the GSK3b phosphorylation (n¼ 3, P < 0.05), and activation of a1-AR with NE (in the presence of 10 lm propranolol to block beta-ARs) also induced about a twofold increase (Fig 4B), but the PKB phospho-rylation was not induced by the treatment of NE
0
2
4
6
CON NE Ad AdR18
CON NE NE+PD NE+LY
*
**
A
0 2 4 6
Ad AdR18
Ad AdR18 AdR18
AdR18+NE Ad+NE
*
B
0 2 4 6
Ad AdR18 AdR18+LY AdR18+PD
**
C
0
2
D
0 2 4 6
+NE AdR18 +NE +LY
AdR18 +NE +PD
**
E
Fig 3 R18 induced ANP expression and enhanced the NE-induced ANP expression in cardiomyocytes in a PI3K-dependent manner Cardio-myocytes cultured in 24-well plates were infected with or without AdR18 or Ad at an MOI of 10 After starvation for 24 h and treatment with propranolol (10 l M ) and different inhibitors for 30 min, cells were stimulated with NE (10 l M ) for 40 h The culture medium was collec-ted for ANP assay using an ELISA kit (A and B) R18 induced ANP production, and also enhanced the NE-induced ANP production (C–E) PI3K was responsible for the role of R18, and ERK1 ⁄ 2 responsible for the NE-induced ANP production Cardiomyocytes were treated with
or without propranolol (10 l M ) plus LY294002 (LY, 10 l M ) or PD98059 (PD, 10 l M ) for 30 min and stimulated with NE (10 l M ) for 40 h Then, the ANP production was measured The values shown were means ± SD and expressed as the fold of control The level of ANP in control was 40.25 ± 3.23 ngÆmL)1, *P < 0.05; **P < 0.01 (n ¼ 3).
Trang 5(Fig 4A) The AdR18-induced PKB and GSK3b
phosphorylation was completely blocked by the PI3K
inhibitor LY (10 lm, n¼ 3, P < 0.05, compared with
the AdR18 treatment only), but the NE-induced
GSK3b phosphorylation was not blocked by LY Taken together, these results indicate that the regu-lation of GSK3b phosphoryregu-lation is involved in R18-induced cardiomyocyte hypertrophy
R18 converts NFAT3 into faster mobility forms and induces its nuclear translocation
NFAT3, a member of the nuclear factor of activated
T cells (NFAT) family, plays a pivotal role in cardio-myocyte hypertrophy [26] It is phosphorylated by activated GSK3b (dephosphorylated form) As the infection of AdR18 induced GSK3b phosphorylation – and thus inactivation – in isolated cardiomyocytes, we hypothesized that AdR18 expression may result in the dephosphorylation of NFAT3, inducing faster gel mobility Figure 5 shows that AdR18 indeed converts NFAT3 into the faster mobility forms and this effect
is abolished by cyclosporin A (400 nm), an inhibitor of calcineurin Next, we examined the cellular localization
of NFAT3 by immunofluorescence analysis As shown
in Fig 6A, NFAT3 was present predominantly in the nucleus upon the treatment of AdR18, but was mainly found in the cytoplasm of the control and Ad group
To confirm the above results, cytoplasmic and nuclear extracts were prepared for western blot analysis with
an anti-NFAT3 Ig Clearly, the nuclear fraction of NFAT3 was increased upon the treatment with AdR18 (Fig 6B) Together, these results indicate that the localization of NFAT3 can be regulated by the treat-ment with AdR18
Discussion
14-3-3 proteins are a family of regulatory molecules that are found ubiquitously in eukaryotes Through interaction with target proteins, 14-3-3 proteins partici-pate in regulation of cell cycle, intracellular signal transduction, cytoskeletal structure and apoptosis In
A
ERK1/2
Ad AdR18
Phospho-ERK1/2
NE
Phospho-PKB PKB
LY
Phospho-PKB PKB
B
0
1
2
3
4
+NE
AdR18 AdR18 +NE
*
*
0
1
2
3
4
con AdR18 AdR18
+LY
NE NE +LY
*
Fig 4 R18 induced PKB and GSK3b phosphorylation, which was
blocked by PI3K inhibitor, LY294002 (A) Cardiomyocytes were
infected with or without AdR18 or Ad and 24 h later, the cells were
serum-starved for 24 h prior to treatment with propranolol (10 l M )
as well as LY294002 (LY, 10 l M ) for 30 min, and then treated with
or without NE (10 l M ) for 10 min Phosphorylated PKB, GSK3b and
ERK1 ⁄ 2 were detected by western blot with antibodies to
phos-pho-Ser473 PKB, phospho-Ser9 GSK-3b and phospho-ERK1 ⁄ 2 The
same membranes were stripped and re-probed with general
GSK-3b and ERK1 ⁄ 2 antibody (B) The data is means ± SD and
expressed as the fold of control, *P < 0.05 (n ¼ 3).
CON Ad AdR18 AdR18
+CysA
NFAT3
Fig 5 R18 converted NFAT3 into the faster mobility forms Neona-tal cardiac myocytes were infected with or without Ad or AdR18 in the presence or absence of cyclosporin A (Cys A, 400 n M ) for
30 min The whole cell lysate from these cells was subject to west-ern blot (6% gel) with anti-NFATc3 Ig The experiment was repea-ted three times with the same result.
Trang 6the present investigation, we have evaluated the role of
14-3-3 in cardiomyocyte hypertrophy by using an
adenovirus vector expressing the YFP-R18 fusion
pep-tide (AdR18) to inhibit 14-3-3 interactions Compared
with dominant-negative forms of 14-3-3s, the use of a
global inhibitor of 14-3-3 provides a more complete
view of the role of these proteins [20,21]
While some isoforms of 14-3-3 proteins were found
in the whole rat heart by using northern blot and
west-ern blot analysis previously [27], our results
demon-strate that 14-3-3c, e, b and f isoforms are expressed
in cultured neonatal rat cardiomyocytes R18 markedly
increased protein synthesis and ANP production and
also potentiated the a1-AR-mediated protein synthesis
and ANP production These were decreased by PI3K
inhibition, but not by ERK1⁄ 2 inhibition In addition,
R18 induced both PKB and GSK3b phosphorylation,
which was blocked completely by LY294002, whereas
NE only induced GSK3b phosphorylation, which was
not blocked by LY294002 Lisa et al have reported
that the a1-AR-induced GSK3b phosphorylation is
mediated by PKC, but not by PI3K [25] Further, we
found that NFAT3, a member of the nuclear factor
of activated T cells family, was converted into the
dephosphorylated, faster mobility forms, and
translo-cated into the nucleus upon AdR18 treatment These
results indicate that the PI3K⁄ PKB ⁄ GSK3b and
NFAT pathway is probably involved in the hyper-trophic response induced by R18
Using the R18 peptide as an inhibitor of 14-3-3, pre-vious work has shown that the R18 peptide negatively regulates early Xenopus development and induces apoptosis under some apoptotic stimulation [20,21] Using dominant-negative (DN)-14-3-3 transgenic mice
as model, Muslin et al found that transgenic mice, after transverse aortic constriction, developed signifi-cant cardiac hypertrophy and left ventricular dilation, and the survival of these mice decreased markedly [18] Until now, the effect of 14-3-3 on cardiomyocyte hypertrophy has not been reported
In this study, R18 treatment increased markedly protein synthesis and ANP production in cardio-myocytes, which was blocked by LY294002 but not
by PD98059 In addition, R18 potentiated the NE-induced protein synthesis and enhanced the NE-induced ANP production The effects of R18 on NE-induced hypertrophy were not caused by inhibiting 14-3-3 expression, because 14-3-3 protein levels were not altered upon the stimulation with NE To our sur-prise, the protein synthesis in the AdR18 + NE group was blocked by either LY294002 or PD98059 but the ANP production in this group was blocked only by LY294002 and not by PD98059 The reason for this difference was attributed to the probability that only a
Ad
CON
AdR18
A
NFAT3
cytoplasm
CON Ad AdR18 CON Ad AdR18
nucleus
B
Fig 6 R18 induces NFAT3 nuclear localiza-tion (A) Cardiomyocytes grown on glass coverslips were infected with or without AdR18 at an MOI of 10 and then starved for
24 h After fixation, the cellular localization
of NFAT3 was detected using an antibody against rabbit NFAT3 After washing in NaCl ⁄ P i , samples were incubated with Cy5-conjugated goat anti-(rabbit IgG) Ig (red) plus Hoechst 33342 (blue) and examined by con-focal microscopy NFAT3 was predominantly localized in the nucleus upon the treatment
of AdR18, whereas, in the CON and Ad group, NFAT3 was mainly localized in the cytoplasm Scale Bar, 16 lm (B) Cardio-myocytes were infected with Ad and AdR18, respectively, and then starved for
24 h Cytoplasmic and nuclear protein extra-ction were prepared and subject to western blot analysis using anti-NFAT3 Ig The experi-ment was repeated two times with the same result.
Trang 7portion of the signaling molecules and transcription
factors modulated by 14-3-3 proteins were shared by
the processes of protein synthesis and ANP
produc-tion In addition, we found that the effect of NE on
ANP production was not blocked by LY alone, but
the combination of NE and AdR18 is inhibited by LY
to a level even greater than that of NE alone On the
other hand, PD could inhibit the effect of NE alone,
but could not affect the combination of NE and
AdR18 on the ANP production (Fig 3D,E) These
results suggest that cross talk may occur between NE
and R18 in regulation of ANP expression The
mech-anism of this cross talk remains to be clarified
One of the established roles of 14-3-3 proteins is to
inhibit apoptosis The disruption of 14-3-3 interactions
has been shown to lower the apoptotic threshold of
cells Interestingly, we found that R18 induced
cardio-myocyte hypertrophy, and the phosphorylation of
GSK3b on Ser9 was involved in this hypertrophic
response Similarly, a previous study has revealed that
the activation of the Fas receptor, another molecule
related to apoptosis, could induce cardiomyocyte
hypertrophy, which also was dependent on the
inacti-vation of GSK3b by Ser9 phosphorylation [28]
GSK3b is an established target of the PI3K⁄ PKB
signaling pathway, where PKB phosphorylates and
thereby inactivates GSK3b Phosphorylation and
inacti-vation of GSK3b, a negative regulator of cardiomyocyte
hypertrophy, has been identified to be necessary and
suf-ficient for the hypertrophy induced by hypertrophic
stimuli [25,29] GSK3b phosphorylated various cellular
substrates, including glycogen synthase, cyclin D1,
c-Jun, and NFAT Phosphorylation of cellular
sub-strates by GSK3b either directly suppressed enzyme
activities or changed subcellular localizations [30]
NFAT3 plays a crucial role in cardiomyocyte
hypertro-phy NFAT phosphorylation by GSK3b leads to NFAT
interaction with 14-3-3 proteins, causing the
redistribu-tion of NFAT from the nucleus to the cytoplasm This
results in the subsequent inhibition of NFAT-mediated
transcription [26,31] In our study, we found that R18
could convert NFAT3 into the faster mobility forms
(unphosphorylated NFAT) Cyclosporin A, an inhibitor
of calcineurin, abolished this effect of AdR18 on
NFAT3 In addition, R18 could induce the nuclear
localization of NFAT3 Therefore, the R18-induced
hypertrophy is probably caused by one or all of the
fol-lowing mechanisms: (a) R18 removes the negative
con-straint of GSK3b on NFAT; (b) R18 disrupts the
NFAT)14-3-3 interaction and inhibits the protective
role of 14-3-3 on phosphorylated NFAT; (c) R18
pre-vents NFAT translocation from nucleus to cytoplasm
Figure 7 shows a working model depicting the effects of
14-3-3 on these molecules In this model, NFAT is a pivotal molecule and R18 disrupts the balance between the unphosphorylated and phosphorylated forms of NFAT However, it is probable that R18 induced cardio-myocyte hypertrophy involves disruption of 14-3-3 inter-action with other binding proteins such as PI3K As R18 inhibits 14-3-3 proteins in an isoform-independent manner, the role of each isoform of 14-3-3 in cardio-myocyte hypertrophy remains to be elucidated
In summary, our findings establish that several iso-forms of 14-3-3 proteins (c, e, b and f) are expressed
in rat cardiomyocytes We have also shown that 14-3-3 inhibits cardiomyocyte hypertrophic responses and negatively regulates the a1-AR-induced hypertrophy, in which the PI3K⁄ PKB ⁄ GSK3b and NFAT pathway is likely involved The regulation of GSK3b phosphoryla-tion and the compartmentaphosphoryla-tion of NFAT by 14-3-3 probably contributes to this process
Experimental procedures
Materials The ERK1⁄ 2 inhibitor (PD98059), PI3K inhibitor (LY294002), propranolol and norepinephrine (NE) were
Hypertrophic stimuli
1 -AR NE
Cytoplasm
NFAT Nucleus
PKC
Hypertrophy
14-3-3
calcineurin
Cys A
PKB
-ser-9-P GSK3
PI3K
P NFAT
14-3 P NFAT GSK3
Hypertrophic stimuli
1 -AR
NE α1-AR NE
Cytoplasm
NFAT Nucleus
PKC
Hypertrophy
14-3-3
calcineurin
Cys A
PKB
-ser-9-P
Active Inactive
GSK3β
PI3K
P NFAT P NFAT
14-3
14-3 P NFAT P NFAT GSK3 β
Fig 7 A working model depicts 14-3-3 proteins inhibiting the cardio-myocyte hypertrophy Upon stimulation, PKB is phosphorylated via activated PI3K and GSK3b is phosphorylated via both PKC and PI3K, leading to GSK3b inhibition The active, dephosphorylated GSK3b phosphorylates NFAT and counteractes the effect of calcineurin on NFAT 14-3-3 Proteins inhibit PI3K and activate GSK3b, keeping NFAT in cytoplasm by binding to phosphorylated NFAT R18 induces cardiomyocyte hypertrophy in part by removal of the modulatory effect of 14-3-3 on PI3K, GSK3b, and NFAT, leading to transcrip-tional activation of NFAT in nucleus NE, norepinephrine; Cys A, cyclosporin A; PI3K, phosphoinositide 3-kinase; NFAT, nuclear factor
of activated T cells PKB, protein kinase B.
Trang 8purchased from Sigma Chemical Co (St Louis, MO, USA).
Terminal deoxynucleotidyl transferase (TDT) was from
Invitrogen Corporation (Carlsbad, CA, USA) [3H]Leucine
was from Amersham Biosciences (Little Chalfont, Bucks,
UK) Other reagents were obtained from commercial
sup-pliers
Isolation and culture of neonatal ventricular
myocytes
Procedures with experimental animals followed the National
Institute of Environmental Health Sciences Animal and
Use Committee guidelines Primary cultures of
cardio-myocytes were prepared from the ventricles of 1-day-old
Sprague–Dawley rats (from the experiment animal
depart-ment of the Medical Science Center, Peking University,
Beijing, China) by enzymatic digestion in 0.1% trypsin,
0.03% collagenase II as described previously [32] Neonatal
rats were put into a glass beaker containing a cotton mass
wetted with ethyl ether After anaesthesia and decapitation,
hearts were taken out immediately and put into ice-cold
NaCl/Pi, and then cut into pieces Cells in suspension were
collected after several rounds of digestion of heart pieces,
then divided into several 100-mm culture dishes and
incuba-ted for 1 h The suspension containing unattached
cardio-myocytes was then collected and seeded at a density of
1.5· 105cellsÆcm)2 in culture media (Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum, 0.1 mm
5-bromodeoxyuridine, 50 lgÆmL)1 penicillin and 50 lgÆ
mL)1streptomycin) After incubation at 37C in humid air
with 5% (v⁄ v) CO2for 24 h, the cardiomyocytes were then
deprived of serum and incubated for another 24 h before
treatment Cells were preincubated with 10 lm propranolol
to block b-adrenergic receptors with or without different
inhibitors for 30 min before stimulation with 10 lm NE
Recombinant adenovirus vectors
The plasmid containing R18 peptide, pAAV-EYFP-R18 was
made by subcloning the 100 bp NheI ⁄ XhoI fragment of
pSCM-136 plasmid into the 4.5 kbp XbaI ⁄ XhoI fragment of
pAAV-MCS (Stratgene, Heidelberg, Germany), and the
pAAV-EYFP was made by subcloning the 800 bp NheI ⁄
Hin-dIII fragment of pEYFP-C1 into the 4.5 kbp XbaI ⁄ HindIII
fragment of pAAV-MCS The adenovirus shuttle constructs
pAdTrack-EYFP-R18 and pAdTrack-EYFP were made by
subcloning the BamHI⁄ XhoI fragments of pAAV-EYFP-R18
and pAAV-EYFP, respectively, into
pAdTrack-cytomegalo-virus (CMV) digested with BglII⁄ XhoI Recombinant
adeno-viruses expressing EYFP-R18 (AdR18) or EYFP (Ad, as a
control) were constructed using a method described
previ-ously [33] Briefly, shuttle construct was linearized with PmeI
and electroporated into Escherichia coli BJ5183 (ATCC,
Manassas, VA, USA) together with the adenoviral backbone
plasmid pAdEasy-1 Homologous recombinants were selected
and were identified by restriction analysis Finally, the PacI-linearized recombinant was transfected into HEK293A (ATCC) packaging cells The adenoviruses produced were used to infect additional HEK293A cells, and a high titer adenovirus stock was made following several rounds of amplification All recombinant adenoviruses were tested for transgene expression in cardiac myocytes by reverse transcriptase-polymerase chain reaction and western blot Cardiomyocytes were infected with AdR18 or Ad at a multi-plicity of infection (MOI) of 10 for 24 h and then subjected
to experiments after deprived of serum for 24 h
Northern blot analysis The total cellular RNA was extracted from cardiomyocytes using a total RNA isolation system kit (Promega Corp., Madison, WI USA) The total RNA (15 lg) was separated
on a horizontal 1.0% agarose⁄ 2.2 m formaldehyde gel and transferred onto a nylon membrane (Millipore Corp., Bill-erica, MA, USA) The membrane was then hybridized with probe at 42C overnight, washed and autoradiographed [34] The synthesized 45-mer oligonucleotide probes were labeled using terminal deoxynucleotidyl transferase with [32P]dATP[aP] The sequences of probes are as follows: 14-3-3f probe: 5¢-TGAGTGTAGTCTGTGTGGGTACTG TAAGGCTTGGAGCACTTGTGA-3¢; 14-3-3h probe: 5¢-TC CTCTAGCAAGGAAGCCCATTCATGTGTATGGGGTC AACTGTTT-3¢; 14-3-3b probe: 5¢-GTCTATTGAGCTCT GTGATCTGTTTGGTGTCACTGTATCCTCCAC-3¢; 14-3-3c probe: 5¢-CAGGTGGACTGGCAGCGCACGCTGATGC TACTACTGCAGTCTTTA-3¢; 14-3-3e probe: 5¢-ACCTAA AGCTGGGACCAGTAAAATCCACAGAAATTCACTCT TGCC-3¢; 18sRNA probe: 5¢-ACGGATTCTGATCGTCTT CGAACC-3¢
Western blot analysis Cells seeded on 30-mm plates were washed once with ice-cold NaCl⁄ Piat the appropriate time after treatment, and lysed in 0.15 mL lysis buffer [20 mm Tris⁄ HCl, pH 7.4,
100 mm NaCl, 10 mm sodium pyrophosphate, 5 mm EDTA, 50 mm NaF, 1 mm sodium vandate, 0.1% (w⁄ v) SDS, 10% (w⁄ v) glycerol, 1% (v ⁄ v) Triton X-100, 1% (w⁄ v) sodium deoxycholate] containing 1 lm leupeptin, 0.1 lm aprotinin, 1 mm phenylmethanesulfonyl fluoride and
1 lm pepstatin Protein concentration was calculated using the BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) Protein was loaded onto a 10% SDS⁄ polyacrylamide gel and electrophoretically transferred
to nitrocellulose membranes (Pall Corporation, East Hill,
NY, USA), analyzed with antibodies according to the supplier’s protocol, and visualized with peroxidase and
an enhanced-chemiluminescence system (ECL kit, Pierce Biotechnology, Inc.) The following antibodies were used
in this study: anti-14-3-3b, anti-14-3-3c, anti-14-3-3e,
Trang 9anti-14-3-3f, anti-eIF-5, anti-GSK3b and anti-NFATc3
(1 : 1000 dilution, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA), and anti-ERK1⁄ 2 (1 : 2000 dilution,
Upstate Biotechnology, Charlottesville, VA, USA),
anti-PKB, anti-(Ser473-phospho-PKB),
anti-(Ser9-phospho-GSK3b), anti-(Thr202⁄ Tyr204-phospho-ERK1 ⁄ 2) Igs (1 : 1000
dilution, Cell Signaling Technology, Inc., Beverly, MA, USA)
Immunofluorescence and confocal microscopic
assay
Cardiomyocytes grown on glass coverslips in six-well dishes
were infected with or without AdR18 for about 24 h and
then starved for 24 h After washing with 37C NaCl ⁄ Pi,
cells were fixed with 4% paraformaldehyde, permeabilized
with 0.2% Triton X-100, and incubated with an
anti-NFAT3 Ig (1 : 250) at 4C overnight, and Cy5-conjugated
AffiniPure Goat anti-(rabbit IgG) Ig (Jackson
Immuno-Research, West Grove, PA, USA) (1 : 500) at 37C for 1 h
Cells were counterstained with 5 lgÆmL)1 Hoechst 33342
(Sigma-Aldrich) to visualize the nucleus Microscopic images
were acquired using a Leica Confocal Microscope
Cytoplasmic and nuclear protein extract
preparation
Cardiomyocytes cultured in 10-cm plates were infected with
Ad and AdR18, respectively, at an MOI of 10, and then
starved for 24 h Cytoplasmic and nuclear protein
extrac-tions were prepared according to the instrucextrac-tions of
NE-PER Nuclear and Cytoplasmic Extraction Reagents kit
(Pierce Biotechnology, Inc.) Briefly, cells were washed
twice with ice-cold NaCl⁄ Pi, recovered by scraping, then
pelleted and resuspended in 0.2 mL ice-cold CER I
contain-ing protease inhibitors (Halt Protease Inhibitor Cocktail
Kit, Pierce Biotechnology) Cells were broken by vortexing
vigorously and then adding 11 lL ice-cold CER II and
vortexed vigorously again After centrifugation (5 min at
16 000 g, 4 C), the supernatant (cytoplasmic extract) was
collected and the insoluble fraction containing nuclei was
resuspended in 0.1 mL ice-cold NER containing protease
inhibitors After four rounds of vortexing (15 s) and
incu-bating on ice (10 min), then centrifuging for 10 min at
16 000 g, 4 C, the supernatant (nuclear extract) fraction
was collected After protein quantification, cytoplasmic and
nuclear proteins (30 lg) were electrophoresed on an 8%
SDS⁄ polyacrylamide gel, transferred to nitrocellulose, and
immunoblotted as described above
Atrial natriuretic peptide (ANP) enzyme-linked
immunosorbent assay
Cardiomyocytes cultured in 24-well plates were
preincu-bated with different inhibitors for 30 min and treated with
NE in serum-free medium for 40 h The supernatants were collected for the ANP assay using an ELISA kit (Phoenix Pharmaceuticals Inc., Belmont, CA, USA) following the manufacturer’s instruction
Protein synthesis assay ([3H]leucine incorporation)
Cardiomyocytes cultured in 24-well plates were serum deprived for 24 h, pretreated with or without a variety of inhibitory agents, and then incubated for 48 h with NE in serum-free medium [3H]leucine (1 lCiÆmL)1) was added 6 h before the harvest At the end of the incubation, the plates were quickly washed twice with ice-cold NaCl⁄ Pi, kept for
30 min with ice-cold 10% (v⁄ v) trichloroacetic acid at 4 C, and washed with NaCl⁄ Pi Precipitates were solubilized in 0.1 m NaOH with gentle shaking at 37C for 1 h The radioactivity incorporated into trichloroacetic acid-precipita-ble materials was determined by liquid scintillation spectro-metry (Beckman Coulter Inc., Fullerton, CA, USA)
Statistical analysis All data represent the mean ± SD of at least three inde-pendent experiments The analysis of variance (anova) was performed for the comparison of three or more groups and the post-test comparison was performed by the method of Tukey A value of P < 0.05 was accepted as significant
Acknowledgements
The authors wish to thank Prof Haian Fu (Pharmacol-ogy Department at Emory University, USA) for the generous gift of pSCM136 and pEYFP-C1 plasmids and Lisa M Cockrell (Emory University) for critical reading
of the manuscript This work was supported by grants from the foundation of national key basic research and development project (G2000056906) and national nat-ural science foundation (30270540, 30200321)
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