Calyculin A reveals serine/threonine phosphatase protein phosphatase 1 as a regulatory nodal point in canonical signal transducer and activator of transcription 3 signaling of human microvascular endothelial cells.
ABSTRACT Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflammation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3 dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1) inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells (HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity.
- SourceAvailable from: Cornelia Wilson[Show abstract] [Hide abstract]
ABSTRACT: Tau phosphorylation is regulated by a balance between tau kinase and phosphatase activities. Disruption of this equilibrium was suggested to be at the origin of abnormal tau phosphorylation and thereby that might contributes to tau aggregation. Thus, understanding the regulation modes of tau dephosphorylation is of high interest in determining the possible causes at the origin of the formation of tau aggregates and to elaborate protection strategies to cope with these lesions in AD. Among the possible and relatively specific interventions that reverse tau phosphorylation is the stimulation of certain tau phosphatases. Here, we reviewed tau protein phosphatases, their physiological roles and regulation, their involvement in tau phosphorylation and the relevance to AD. We also reviewed the most common compounds acting on each tau phosphatase including PP2A.Ageing research reviews 07/2012; 12(1):39-49. · 7.63 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Multiple lines of evidence suggest that the transcription factor STAT3 is linked to a protective and reparative response in the heart. Thus, increasing duration or intensity of STAT3 activation ought to minimize damage and improve heart function under conditions of stress. Two recent studies using genetic mouse models, however, report findings that appear to refute this proposition. Unfortunately, studies often approach the question of the role of STAT3 in the heart from the perspective that all STAT3 signaling is equivalent, particularly when it comes to signaling by IL-6 type cytokines, which share the gp130 signaling protein. Moreover, STAT3 activation is typically equated with phosphorylation of a critical tyrosine residue. Yet, STAT3 transcriptional behavior is subject to modulation by serine phosphorylation, acetylation, and redox status of the cell. Unphosphorylated STAT3 is implicated in gene induction as well. Thus, how STAT3 is activated and also what other signaling events are occurring at the same time is likely to impact on the outcome ultimately linked to STAT3. Notably STAT3 may serve as a scaffold protein allowing it to interact with other singling pathways. In this context, canonical gp130 cytokine signaling may function to integrate STAT3 signaling with a protective PI3K/AKT signaling network via mutual involvement of JAK tyrosine kinases. Differences in the extent of integration may occur between those cytokines that signal through gp130 homodimers and those through heterodimers of gp130 with a receptor α chain. Signal integration may have importance not only for deciding the particular gene profile linked to STAT3, but for the newly described mitochondrial stabilization role of STAT3 as well. In addition, disruption of integrated gp130-related STAT3 signaling may occur under conditions of oxidative stress, which negatively impacts on JAK catalytic activity. For these reasons, understanding the importance of STAT3 signaling to heart function requires a greater appreciation of the plasticity of this transcription factor in the context in which it is investigated.JAK-STAT. 04/2012; 1(2):101-10.
- [Show abstract] [Hide abstract]
ABSTRACT: We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.PLoS ONE 08/2012; 7(8):e43313. · 3.53 Impact Factor
Calyculin A Reveals Serine/Threonine Phosphatase Protein
Phosphatase 1 as a Regulatory Nodal Point in Canonical
Signal Transducer and Activator of Transcription 3 Signaling
of Human Microvascular Endothelial Cells
Carlos Zgheib,1Fouad A. Zouein,1Rony Chidiac,1Mazen Kurdi,1,2and George W. Booz1
Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflam-
mation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but
their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal
transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate
immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3
dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting
p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1)
inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells
(HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without
affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid
and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced
gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing
the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus
resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a
protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity.
rosis, systolic/diastolic heart failure, metabolic syndrome,
diabetes, and hypertension (Lo ´pez Farre ´ and Casado 2001;
Yung and others 2006; Coccheri 2007; Ganne and Winer
2008; Dawood and Schlaich 2009; Lakshmi and others 2009).
Inflammation is often initiated by stimuli, such as the inter-
leukin 6 (IL-6) type cytokines, acting on endothelial cells to
enhance reactive oxygen species (ROS) generation, as well as
leukocyte chemotaxis and adherence (Nian and others 2004;
Hou and others 2008; Brasier 2010). The IL-6 type cytokines
include IL-6, IL-11, leukemia inhibitory factor (LIF), cardio-
trophin 1, oncostatin M, ciliary neurotrophic factor, and
cardiotrophin-like cytokine (Kurdi and Booz 2007). On
binding to their cell surface receptors, these cytokines acti-
vate several intracellular signaling events, notably the Janus
kinase 1 (JAK1)-signal transducer and activator of tran-
ascular inflammation occurs in coronary heart dis-
ease, myocardial infarction, arteriosclerosis, atheroscle-
scription 3 (STAT3) pathway. STAT3 is a transcription factor
that is activated by phosphorylation of tyrosine residue 705
(Y705). After phosphorylation, STAT3 forms homodimers or
heterodimers with other STAT family members that bind
specific promoters to induce target gene expression (Kurdi
and Booz 2007). STAT3 is also phosphorylated by various
kinases on serine residue 727 (S727) within the C-terminus
transcription activation domain. Previous reports have
shown that S727 phosphorylation is required for maximal
transcriptional activity and DNA binding of STAT3, as well
as STAT3 homodimerization (Zhang and others 1995; Kurdi
and Booz 2007).
Others have reported that treatment of ALK+TCL cells,
glioblastoma multiforme cells, 293T cells, human antigen-
specific CD4+T cell lines, and cutaneous T cell lymphoma
lines with the PP1/PP2A inhibitor calyculin A caused a
marked increase in STAT3 S727 phosphorylation (Woetmann
and others 1999; Zhang and others 2002a; Ghosh and others
2005). In this study, we tested the hypothesis that by
1Department of Pharmacology and Toxicology, Center for Excellence in Cardiovascular-Renal Research, School of Medicine, The Uni-
versity of Mississippi Medical Center, Jackson, Mississippi.
2Department of Chemistry and Biochemistry, Faculty of Sciences, Lebanese University, Hadath, Lebanon.
JOURNAL OF INTERFERON & CYTOKINE RESEARCH
Volume 32, Number 2, 2012
ª Mary Ann Liebert, Inc.
simultaneously increasing nuclear STAT3 S727 and Y705
phosphorylation with calyculin A and LIF, we could enhance
STAT3-related gene expression in human microvascular en-
dothelial cells (HMEC). Unexpectedly, we observed contrary
findings that reveal a novel point of control for STAT3-
mediated gene response which has significance for under-
standing the inflammatory process.
Materials and Methods
Tissue culture reagents were from Invitrogen. Fetal bovine
serum (FBS, SH30070.03) was from Thermo Scientific. Oka-
daic acid, xanthine, and protease inhibitor cocktail for use
with mammalian cell and tissue extracts were from Sigma-
Aldrich. Antibodies for STAT3, STAT3 pY705, histone H4,
and LSD1 were from Cell Signaling Technology. The antibody
against pS727 STAT3 was from Millipore. Fostriecin and an-
tibodies for Ac-histone H4 K5, histone H1, p300, phospho-
p300 S89, and GAPDH were from Santa Cruz Biotechnology.
RIPA-based kinase extraction buffer and activated vanadate
were from Boston Bioproducts. Calyculin A was from Santa
Cruz Biotechnology, and Sigma-Aldrich. Xanthine oxidase
from buttermilk was obtained from EMD Chemicals. Binding
of nuclear extracts to a STAT3 consensus oligonucleotide was
measured using the TransAM STAT3 kit from Active Motif.
Nuclear extraction kits were from Active Motif (STAT3 oli-
gonucleotide binding) and Thermo Scientific (Westerns). RNA
was extracted with the RNAqueous kit from Ambion.
HMEC-1 were obtained from the Centers for Disease
Control and Prevention. Cells were cultured in MCDB 131
medium with 15% FBS, 10ng/mL epidermal growth factor,
10mM glutamine, 1mg/mL hydrocortisone, and antibiotic-
antimycotic. For experiments, cells were grown to near
confluency on 60 or 100mm diameter culture dishes. Twelve
to 15h beforehand, growth medium was replaced with me-
dium containing 0.5% FBS.
Whole-cell lysates were prepared by scraping cells into ice-
cold RIPA-based buffer containing 100mM vanadate and pro-
tease inhibitor cocktail and were cleared by centrifugation at
100,000 g for 20min at 4?C. Equal amounts of protein in
Laemmli’s-sodium dodecyl sulfate (SDS) reducing buffer were
separated by SDS-PAGE. Separated proteins were blotted onto
nitrocellulose membranes, and the immunoreactive bands were
quantified using the Li-COR Odyssey infrared imaging system.
Real-time polymerase chain reaction
cDNA was prepared using SuperScript VILO cDNA syn-
thesis Kit from Invitrogen. Gene amplification was per-
formed using TaqMan Gene Expression Master Mix and
TaqMan Gene expression assays from Applied Biosystems.
TaqMan Gene Expression primers used were suppressor of
cytokine signaling 3 (SOCS3) (Hs02330328_s1), chemokine
(C-C motif) ligand 2 (CCL2) (Hs00234140_m1), and GAPDH
(Hs99999905_m1). Real-time polymerase chain reaction was
carried out in a BioRad iQ5 multicolor machine. Normal-
ization of gene expression was carried out with housekeep-
ing gene GAPDH. Data were analyzed using the iQTM 5
optical system software provided by Biorad.
Results are expressed as mean–SEM for n number of
independent experiments. The data in Figs. 2 and 8 were
analyzed by 2-way analysis of variance (ANOVA) and
paired t-test, respectively. In all other cases, statistical sig-
nificance was assessed by one-way ANOVA followed by an
appropriate post hoc test. P£0.05 was taken as significant.
LIF-induced STAT3 phosphorylation
in endothelial cells
Initial studies were performed to determine the appro-
priate dosing regimen with calyculin A to assess the impact
of enhanced S727 phosphorylation on LIF-induced STAT3
activation. Treatment of HMEC-1 with LIF (2ng/mL) pro-
duced a time-dependent increase in phosphorylation levels
of both Y705 and S727 STAT3 with the latter being less
intense of the 2 (Fig. 1A). Pretreatment with calyculin A
enhanced basal levels of STAT3 S727 phosphorylation, but
LIF-induced Y705 phosphorylation was markedly attenuated
under this condition (Fig. 1B). We had previously made a
similar observation using cardiac myocytes, and provided
evidence that S727 STAT3 phosphorylation serves to recruit
a tyrosine phosphatase (Booz and others 2003). However,
ment with calyculin A (CA) on LIF-induced STAT3 signal-
ing. HMEC-1 were (A) treated with 2ng/mL LIF for various
times, (B) pretreated for 60min with 100nM CA and then
treated with 2ng/mL LIF, or (C) treated with 2ng/mL LIF
for various times along with 100nM CA. Proteins were
extracted, and levels of pY705 STAT3, pS727 STAT3, and
STAT3 were evaluated by Western blotting. Blots shown
are representative of (A) 5, (B) 2, and (C) 3 independent
experiments. STAT3, signal transducer and activator of
transcription 3; HMEC, human microvascular endothelial
cells; LIF, leukemia inhibitory factor; DMSO, dimethyl
Differential effect of pretreatment versus cotreat-
88ZGHEIB ET AL.
when cells were co-treated with LIF and calyculin A, we
were able to produce an increase in STAT3 S727 phosphor-
ylation without any reduction in LIF-induced Y705 phos-
phorylation (Fig. 1C). For that reason, cotreatment with
calyculin A and LIF was performed in subsequent experi-
ments. These findings are quantified in Fig. 2.
Calyculin A inhibits the activity of both PP1 and PP2A
(Ishihara and others 1989; Resjo ¨ and others 1999). To address
which of these PP was involved in STAT3 S727 dephosphor-
ylation, we used okadaic acid, which inhibits PP2A activity
but has little effect on PP1 activity (Resjo ¨ and others 1999). As
seen from Fig. 3A, treatment with okadaic acid (1mM) failed to
further increase STAT3 S727 phosphorylation when cotreated
with LIF. We also tested the effect of fostriecin on STAT3 S727
phosphorylation. Fostriecin is a selective inhibitor of PP2A
(IC50=3.2nM) with little effect on PP1 (IC50=131mM) (Walsh
and others 1997) and was reported to inhibit PP2A in human
endothelial cells (Grethe and Po ¨rn-Ares 2006). As seen from
Fig. 3B and C, cotreatment with fostriecin had no effect on
STAT3 Y705 or S727 phosphorylation.
Increased STAT3 S727 phosphorylation
with calyculin A cotreatment did not enhance
To assess the effect of enhancing STAT3 S727 phosphor-
ylation on LIF-induced gene expression, we selected 2 genes
that are activated by LIF in HMEC-1cells and which contain
a STAF_HOXF_STAT framework in their promoters (un-
published observation), namely, SOCS3 and CCL2, also
cotreatment with CA on LIF-
induced STAT3 signaling. Re-
described in Fig. 1 were quan-
tified using the LiCor Odyssey
system. Values shown are the
mean of 2 independent experi-
ments (Pretreatment) or mean –
analyzed by 2-way analysis of
variance with CA treatment and
STAT3, time was significant as
source of variation (P£0.001),
whereas cotreatment (CA) and
were not, thus indicating that
CA cotreatment did not affect
LIF-induced STAT3 Y705 phos-
phorylation. For pS STAT3, co-
cotreatment-time interaction as
sources of variation were sig-
nificant at P£0.01, P£0.01, and P£0.05, respectively, thus indicating that cayculin A cotreatment resulted in greater
STAT3 S727 phosphorylation in response to LIF stimulation in a time-dependent manner.
enhance STAT3 S727 phosphorylation. (A)
HMEC-1 were treated with 2ng/mL LIF for
various times along with 1mM okadaic acid
(OA) or vehicle (0.04% v/v DMSO). Proteins
were extracted, and levels of pY705 STAT3,
pS727 STAT3, and STAT3 were evaluated by
Western blotting. Results shown are repre-
sentative of 2 independent experiments. (B)
HMEC-1 were treated with vehicle or 2ng/
mL LIF in the presence or absence of 1mM
fostriecin for 2h. Proteins were extracted,
and levels of pY705 STAT3, pS727 STAT3,
and STAT3 were evaluated by Western
blotting. (C) Levels of pS727 STAT3 and
pY705 STAT3 were normalized to total
STAT3 using the LiCor Odyssey system.
Values are mean–SEM for 3 independent
experiments. ***P£0.001 versus control or
fostriecin. PP2A, protein phosphatase 2A.
PP2A inhibitor cotreatment did not
PP1 AND STAT3 GENE EXPRESSION 89
known as monocyte chemotactic protein-1 (MCP-1). As seen
from Fig. 4, contrary to our expectations, enhancing STAT3
S727 phosphorylation by cotreatment with calyculin A did
not enhance LIF-induced expression of these genes, but ra-
ther a marked inhibition was observed.
Increased S727-STAT3 phosphorylation did not
impact STAT3 nuclear localization
To address the possibility that STAT3 S727 phosphoryla-
tion caused nuclear export of STAT3 as reported by others
(Woetmann and others 1999), we isolated nuclei after LIF
and/or calyculin A treatment. Notably, calyculin A co-
treatment resulted in a marked increase in the proportion of
nuclear STAT3 that was phosphorylated on S727 without
changing the proportion which was phosphorylated on Y705
in response to LIF stimulation (Fig. 5A, B). In addition, as
Fig. 5C shows, there was no significant difference between
LIF and LIF-calyculin A treatments in nuclear STAT3 levels.
By itself, calyculin A did not significantly affect nuclear
STAT3 levels. Nuclear STAT3 levels assessed at 60min did
show a trend toward being higher with LIF treatment, but
this did not reach statistical significance.
PP1 inhibition decreased STAT3 DNA
Since calyculin A did not have any significant effect on
STAT3 nuclear levels, we considered the possibility that the
decrease in LIF-induced SOCS3 and CCL2 gene expression
was due to impaired DNA binding of STAT3. To address
that possibility, nuclear extracts were assessed for their
ability to bind an oligonucleotide containing STAT3 con-
sensus binding sites. Figure 6 shows that calyculin A treat-
ment alone produced a significant decrease in STAT3 nuclear
binding. As expected, LIF treatment enhanced STAT3 bind-
ing. Binding resulting from co-treatment, although markedly
enhanced compared with calyculin A treatment alone, was
no different from vehicle control. These results indicate that
calyculin A causes changes in STAT3 that attenuate DNA
Calyculin A causes p300 inhibitory phosphorylation
The first function identified for the phosphorylation of the
S727 residue in STAT3 was the recruitment of transcriptional
co-activating proteins p300/CBP (Schuringa and others 2001;
Ray and others 2002). We, thus, asked whether the complete
block by calyculin A of LIF-induced gene expression could
be due, in part, to an inhibitory action on p300/CBP. As
expression. HMEC-1 were treated for 1h with nothing (Con-
trol), 0.04% v/v DMSO (vehicle control), 100nM CA, 2ng/mL
LIF, or LIF with DMSO or with CA. RNA was extracted, re-
verse transcribed, and analyzed by real-time polymerase chain
reaction for SOCS3 and CCL2 expression. Results were nor-
malized to GAPDH and expressed as the fold increase over
control levels. Values are mean–SEM for 4 independent ex-
periments. ***P£0.001 versus LIF or LIF + DMSO.
CA inhibited LIF-induced SOCS3 and CCL2 gene
levels. Cells were treated with vehicle, 100nM
CA, 2ng/mL LIF (with vehicle), or LIF + CA.
(A) After 1h, nuclear extracts were prepared
and analyzed by Western blotting for STAT3
pS727, STAT3 pY705, STAT3, and LSD1 or
histone H1 (not shown) as a loading control.
Results were quantified using the LiCor
Odyssey system and for each experiment
normalized to the control. (B) pS727 STAT3
and pY705 to total STAT3 ratios. For pS
STAT3/STAT3: *P£0.05 versus control or
LIF, **P<0.01 versus control or LIF. For pY
STAT3/STAT3: *P<0.05 versus control or
CA, ***P<0.001 versus control or CA. (C)
Nuclear STAT3 levels were normalized to
histone H1 or LSD1 levels as a loading con-
trol. Values are mean–SEM of 9 independent
CA did not affect nuclear STAT3
90ZGHEIB ET AL.
Fig. 7A and B show, treatment of HMEC-1 with calyculin A
caused a marked increase in the phosphorylation of p300/
CBP on S89, which others have reported to be associated
with inhibition of acetyltransferase activity (Yuan and
Gambee 2000). Fostriecin (1mM) by itself or with LIF had no
effect on p300 S89 phosphorylation or degradation (data not
shown). Moreover, as can be seen from Fig. 7A and C, ca-
lyculin A caused a remarkable decrease in the protein levels
of p300/CBP. To further establish that calyculin A was tar-
geting p300/CBP, we looked at the effect of this inhibitor on
histone H4 acetylation at K5, as p300/CBP preferentially
acetylates Histone H4 at this site and at K8 (Schiltz and
others 1999). As Fig. 7D shows, calyculin A markedly re-
duced the fraction of histone H4 acetylated on K5.
Oxidative stress increases STAT3 and p300
We next sought to investigate whether phosphorylation of
STAT3 S727 and p300 S89 that is associated with PP1 inhi-
bition might have pathological or physiological relevance.
Others have reported that PP1 is inhibited by oxidative stress
(O’Loghlen and others 2003). We, thus, asked whether long-
term exposure to oxidative stress would affect STAT3 and
p300 phosphorylation. HMEC-1 were incubated with xan-
thine oxidase and xanthine to generate ROS. As Fig. 8 shows,
exposure of HMEC-1 to oxidative stress produced a modest
but significant increase in both STAT3 S727 and p300 p89
phosphorylation. Notable oxidative stress produced a
marked increase in STAT3 Y705 phosphorylation, which is in
keeping with the well-established observation that protein
tyrosine phosphatases are inhibited in cellular systems by
ROS (Winterbourn and Hampton 2008).
We report here that the PP1 inhibitor, calyculin A, caused
a marked increase in phosphorylation of STAT3 on S727,
which had been previously linked to enhanced gene ex-
pression by STAT3 at certain promoters (Zhang and others
1995; Schuringa and others 2001; Ray and others 2002; Kurdi
and Booz 2007). Okadaic acid and fostriecin, which exhibit
almost an exclusively inhibitory effect on PP2A, did not
cause a similar increase in STAT3 S727 phosphorylation, thus
showing that PP1 or PP1-related protein is the phosphatase
responsible for STAT3 S727 dephosphorylation in HMEC-1.
Although several studies have implicated PP2A in depho-
sphorylating STAT3 S727 (Liang and others 1999; Woetmann
nuclear extracts to a STAT3 consensus binding sequence.
Cells were treated for 1h with 0.04% DMSO (control), 100
nM CA, 2ng/mL LIF with DMSO, or LIF with 100nM CA.
Nuclear extracts (20mg protein) were assayed for binding to
a STAT3 consensus binding site using the TransAM STAT3
kit from Active Motif. Values are mean–SEM of 4 inde-
pendent experiments. Values labeled with the same letter are
CA cotreatment inhibited LIF-induced binding of
p300/CBP on S89 and p300/CBP deg-
radation. (A) Cells were treated for 1h
with 0.04% DMSO (control), 100nM
CA, 2ng/mL LIF with DMSO, or LIF
with 100nM CA. Cell extracts were an-
alyzed by Western immunoblotting for
p300 pS89, p300, and GAPDH (loading
control). Results were quantified using
the LiCor Odyssey system and show
that (B) CA caused p300 phosphoryla-
tion on p89 and (C) p300 degradation
regardless of the presence or absence of
LIF. (D) Cell extracts were analyzed by
Western immunoblotting for Ac-histone
H4 K5 and histone H4. Values are
mean–SEM of 3 independent experi-
ments. ***P<0.001 versus control or LIF,
*P<0.05 versus control.
CA causes phosphorylation of
PP1 AND STAT3 GENE EXPRESSION91
and other 1999; Zhang and others 2002a; Togi and others
2009), a recent study found evidence that PP1 and not PP2A
is responsible for STAT3 S727 dephosphorylation in a variety
of human tumor cell lines (Haridas and others 2009).
The role of STAT3 S727 that lies within the transcription
activation domain is complicated and best described as
myriad. Evidence has been reported supporting the conclu-
sion that phosphorylation of this residue regulates STAT3
subcellular distribution (Woetmann and others 1999), tyro-
sine phosphorylation (possibly through recruitment of a
phosphatase) (Zhang and others 2002a; Booz and others
2003), cofactor recruitment (e.g., histone deacetylase p300
and Sp1) (Schuringa and others 2001; Yang and others 2005),
maximal transcriptional activity (Zhang and others 1995),
receptor binding (Zhang and others 2002b), and homo-
dimerization (Zhang and others 1995).
Based on what has been reported, one might expect that
simultaneous Y705 and S727 phosphorylation would en-
hance STAT3-related gene expression. However, we ob-
served just the opposite, which was due in part to the
inhibition and degradation of p300/CBP seen with calyculin
A. Most likely, these actions occurred indirectly as the result
of enhanced (stress) kinase signaling that would occur with
inhibition of the repressive phosphatase actions of PP1 on
these signaling pathways. Factors that control the stability of
p300/CBP are poorly understood. In cardiac myocytes, the
anticancer agent doxorubicin was found to cause p300 hy-
perphosphorylation and degradation via the p38 mitogen-
activated protein kinases (Poizat and others 2005). Protein
kinase Cd, AMP-activated protein kinase, and salt inducible
kinase 2 were reported to phosphorylate p300 on S89,
thereby attenuating its ability to function as an acetylase
(Yang and others 2001; Yuan and others 2002; Bricambert
and others 2010). Whether p300/CBP is a direct target of PP1
is a question for future investigation.
Inhibition of DNA binding also partially explains the ef-
fect of calyculin A on STAT3-related gene expression. We
observed that treatment with calyculin A alone strongly re-
duced binding of nuclear extracts to a STAT3 consensus
binding site (Fig. 6), although nuclear levels of STAT3 were
not affected (Fig. 5C). Others reported that calyculin A re-
duced binding of STAT3 from cutaneous T lymphoma cells
to various STAT3 binding elements (Woetmann and others
1999). The explanation suggested was that increased serine
phosphorylation of STAT3 caused decreased STAT3 tyrosine
phosphorylation and consequently reduced STAT3 DNA
binding. However, we did not observe a marked decrease in
STAT3 phosphorylated on Y705 in response to calyculin A
(Fig. 5B). An alternative explanation may be increased
phosphorylation of STAT3 at 2 other sites, namely S691 or
T714, which are known to be affected by DNA damage or
the cell cycle, respectively. In fact, calyculin A was reported
to cause increased threonine phosphorylation of STAT3
(Woetmann and others 1999). Although S691 and T714
phosphorylation likely have physiological relevance, to the
best of our knowledge, the effect of phosphorylation of
these sites on STAT3 DNA binding has not been reported
and warrants investigation. In any case, the results shown in
Fig. 6 indicate that the positive effect of STAT3 Y705 phos-
phorylation on DNA binding can offset any potential dimi-
nution in DNA binding produced by phosphorylation at
LIF-induced STAT3 nuclear levels at 60min tended to be
higher in the absence of calyculin A (Fig. 5C). However,
the increase did not reach statistical significance. Although
STAT3 was originally identified as a latent cytoplasmic
transcription factor that translocates to the nucleus on cyto-
kine-induced Y705 phosphorylation, STAT3 is now known
to constitutively shuttle between the cytoplasm and nu-
cleus independent of its phosphorylation state (Sehgal 2008).
Thus, subcellular distribution is unlikely to explain our
findings on impaired LIF-induced SOCS3 and CCL2 gene
expression on cotreatment with calyculin A (Fig. 4). Both
SOCS3 and CCL2 contain a STAF_HOXF_STAT framework
within their promoters, and their induction is commonly
associated with an inflammatory state. SOCS3 acts as a
classic negative inhibitor of JAK-STAT signaling, whereas
CCL2 is important for the recruitments of lymphocytes to
sites of tissue injury and inflammation. Our results raise the
possibility that expression of CCL2 and SOCS3 may be
tamped or kept under control with increasing stress by at-
tenuation of PP1 activity.
Although we demonstrated that oxidative stress mim-
icked the effects of calyculin A on STAT3 and p300 phos-
phorylation, other stress stimuli may be involved as well.
Notably, PP1 inhibition by calyculin A in lung microvascular
cells has been linked to disruption of barrier function, thus
suggesting a role for this phosphatase in cell architecture
and p300 phosphorylation. HMEC-1cells
were incubated for 6h in the absence or
presence of 20U/L xanthine oxidase and
500mM xanthine. Cell extracts were prepared
and analyzed for STAT3 Y705 and S727
phosphorylation and p300 S89 phosphory-
lation. (A) Representative blots from 3 inde-
pendent experiments. (B) The fraction of
phosphorylated to total protein (pY705/
STAT3, pS727/STAT3, and pS89/p300) was
determined using the LiCor Odyssey detec-
tion system and for each experiment nor-
mean–SEM of 3 independent experiments.
***P<0.001 and *P<0.05 versus control.
Oxidative stress increases STAT3
92ZGHEIB ET AL.
(Kelly and others 1998). Consistent with that possibility, we
observed that HMEC-1 seemed to adopt a more rounded
appearance in response to treatment with calyculin A (un-
published observation). The regulatory mechanism de-
scribed here may come into play in attenuating certain
aspects of IL-6 type cytokine inflammatory signaling when
other stresses are present in the cell.
In conclusion, our findings highlight the pivotal role of a
serine threonine phosphatase that is most likely PP1 in con-
trolling the nuclear actions of STAT3 by balancing opposing
signaling processes (Fig. 9). On one hand, PP1 exerts positive
actions on the transcriptional activity of STAT3 by preventing
p300/CBP phosphorylation and degradation, and potentially
opposing phosphorylation of STAT3 at 1 or 2 inhibitory sites.
On the other hand, PP1 serves to attenuate STAT3 transcrip-
tional activity by dephosphorylating the site responsible for
p300/CBP recruitment. p300/CBP enhances STAT3 tran-
scriptional activity by allowing for the acetylation of histone
at certain promoters containing STAT3 responsive elements
(Ray and others 2002; Ray and others 2008). Understanding
how these opposing signaling events are coordinated and
affected by stress could lead to novel therapeutic strategies
to control the impact of inflammation on microvascular
The authors gratefully acknowledge the technical assis-
tance of Mr. Hani Jamal Alturkmani with some of the ex-
periments. This work was supported by grants from the
National Heart, Lung, and Blood Institute (5R01HL088101-03
and 3R01HL088101-02S1) to GWB and from the Lebanese
University, the Lebanese National Council for Scientific Re-
search (CNRS # 05-10-09), and the COMSTECH-TWAS (09-
122 RG/PHA/AF/AC_C) to M.K.
Author Disclosure Statement
No competing financial interests exist.
Booz GW, Day JN, Baker KM. 2003. Angiotensin II effects on
STAT3 phosphorylation in cardiomyocytes: evidence for Erk-
dependent Tyr705 dephosphorylation. Basic Res Cardiol
Brasier AR. 2010. The nuclear factor-kB-interleukin-6 signalling
pathway mediating vascular inflammation. Cardiovasc Res
Bricambert J, Miranda J, Benhamed F, Girard J, Postic C, Dentin
R. 2010. Salt-inducible kinase 2 links transcriptional coacti-
vator p300 phosphorylation to the prevention of ChREBP-
dependent hepatic steatosis in mice. J Clin Invest 120:
Coccheri S. 2007. Approaches to prevention of cardiovascu-
lar complications and events in diabetes mellitus. Drugs 67:
Dawood T, Schlaich MP. 2009. Mediators of target organ dam-
age in hypertension: focus on obesity associated factors and
inflammation. Minerva Cardioangiol 57:687–704.
Ganne S, Winer N. 2008. Vascular compliance in the cardiome-
tabolic syndrome. J Cardiometab Syndr 3:35–39.
Ghosh MK, Sharma P, Harbor PC, Rahaman SO, Haque SJ. 2005.
PI3K-AKT pathway negatively controls EGFR-dependent
DNA-binding activity of Stat3 in glioblastoma multiforme
cells. Oncogene 24:7290–7300.
Grethe S, Po ¨rn-Ares MI. 2006. p38 MAPK regulates phosphor-
ylation of Bad via PP2A-dependent suppression of the MEK1/
2-ERK1/2 survival pathway in TNF-a induced endothelial
apoptosis. Cell Signal 18:531–540.
Haridas V, Nishimura G, Xu ZX, Connolly F, Hanausek M,
Walaszek Z, Zoltaszek R, Gutterman JU. 2009. Avicin D: a
protein reactive plant isoprenoid dephosphorylates Stat 3 by
regulating both kinase and phosphatase activities. PLoS One
Hou T, Tieu BC, Ray S, Recinos Iii A, Cui R, Tilton RG, Brasier
AR. 2008. Roles of IL-6-gp130 signaling in vascular inflam-
mation. Curr Cardiol Rev 4:179–192.
Ishihara H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato
Y, Fusetani N, Watabe S, Hashimoto K, Uemura D, Hart-
shorne DJ. 1989. Calyculin A and okadaic acid: inhibitors of
protein phosphatase activity. Biochem Biophys Res Commun
Kelly JJ, Moore TM, Babal P, Diwan AH, Stevens T, Thompson
WJ. 1998. Pulmonary microvascular and macrovascular en-
dothelial cells: differential regulation of Ca2+and permeabil-
ity. Am J Physiol 274:L810–L819.
Kurdi M, Booz GW. 2007. Can the protective actions of JAK-
STAT in the heart be exploited therapeutically? Parsing the
regulation of interleukin-6-type cytokine signaling. J Cardio-
vasc Pharmacol 50:126–141.
Lakshmi SV, Padmaja G, Kuppusamy P, Kutala VK. 2009. Oxi-
dative stress in cardiovascular disease. Indian J Biochem
Liang H, Venema VJ, Wang X, Ju H, Venema RC, Marrero MB.
1999. Regulation of angiotensin II-induced phosphorylation of
STAT3 in vascular smooth muscle cells. J Biol Chem 274:
trolling the actions of STAT3 on gene expression in HMEC-1
by exerting both positive and negative actions. STAT3 tran-
scriptional activation by the IL-6 type cytokines is due to
enhanced phosphorylation of S727 and Y705. PP1 attenuates
STAT3 transcriptional activity by dephosphorylating the site
(S727) responsible for p300/CBP recruitment, but potentiates
STAT3 DNA binding by dephosphorylating other sites (S691
and T714) in the C-terminus of STAT3. PP1 also favors
STAT3-mediated transcription by opposing phosphorylation
and subsequent inhibition and degradation of p300/CBP.
Under conditions of PP1 inhibition (occurring perhaps with
oxidative stress), increased STAT3 DNA binding due to S727
phosphorylation would be offset by p300 phosphoryla-
tion and degradation due to stress kinase activation. In the
absence of an additional (stress) signal, IL-6 type cytokine-
induced STAT3 activation occurs in the absence of p300 S89
phosphorylation. IL, interleukin.
Scheme illustrating the pivotal role of PP1 in con-
PP1 AND STAT3 GENE EXPRESSION 93
Lo ´pez Farre ´ A, Casado S. 2001. Heart failure, redox alterations,
and endothelial dysfunction. Hypertension 38:1400–1405.
Nian M, Lee P, Khaper N, Liu P. 2004. Inflammatory cytokines
and postmyocardial infarction remodeling. Circ Res 94:
O’Loghlen A, Pe ´rez-Morgado MI, Salinas M, Martı ´n ME. 2003.
Reversible inhibition of the protein phosphatase 1 by hydro-
gen peroxide. Potential regulation of eIF2a phosphorylation in
differentiated PC12cells. Arch Biochem Biophys 417:194–202.
Poizat C, Puri PL, Bai Y, Kedes L. 2005. Phosphorylation-
dependent degradation of p300 by doxorubicin-activated p38
mitogen-activated protein kinase in cardiac cells. Mol Cell Biol
Ray S, Lee C, Hou T, Boldogh I, Brasier AR. 2008. Requirement
of histone deacetylase1 (HDAC1) in signal transducer and
activator of transcription 3 (STAT3) nucleocytoplasmic dis-
tribution. Nucleic Acids 36:4510–4520.
Ray S, Sherman CT, Lu M, Brasier AR. 2002. Angiotensinogen
gene expression is dependent on signal transducer and acti-
vator of transcription 3-mediated p300/cAMP response ele-
ment binding protein-binding protein coactivator recruitment
and histone acetyltransferase activity. Mol Endocrinol 16:
Resjo ¨ S, Oknianska A, Zolnierowicz S, Manganiello V, Deger-
man E. 1999. Phosphorylation and activation of phosphodi-
esterase type 3B (PDE3B) in adipocytes in response to serine/
threonine phosphatase inhibitors: deactivation of PDE3B
in vitro by protein phosphatase type 2A. Biochem J 341:
Schiltz RL, Mizzen CA, Vassilev A, Cook RG, Allis CD, Naka-
tani Y. 1999. Overlapping but distinct patterns of histone
acetylation by the human coactivators p300 and PCAF within
nucleosomal substrates. J Biol Chem 274:1189–1192.
Schuringa JJ, Schepers H, Vellenga E, Kruijer W. 2001. Ser727-
dependent transcriptional activation by association of p300
with STAT3 upon IL-6 stimulation. FEBS Lett 495:71–76.
Sehgal PB. 2008. Paradigm shifts in the cell biology of STAT
signaling. Semin Cell Dev Biol 19:329–340.
Togi S, Kamitani S, Kawakami S, Ikeda O, Muromoto R, Nanbo
A, Matsuda T. 2009. HDAC3 influences phosphorylation of
STAT3 at serine 727 by interacting with PP2A. Biochem Bio-
phys Res Commun 379:616–620.
Walsh AH, Cheng A, Honkanen RE. 1997. Fostriecin, an anti-
tumor antibiotic with inhibitory activity against serine/thre-
onine protein phosphatases types 1 (PP1) and 2A (PP2A), is
highly selective for PP2A. FEBS Lett 416:230–234.
Winterbourn CC, Hampton MB. 2008. Thiol chemistry and
specificity in redox signaling. Free Radic Biol Med 45:549–561.
Woetmann A, Nielsen M, Christensen ST, Brockdorff J, Kaltoft
K, Engel AM, Skov S, Brender C, Geisler C, Svejgaard A,
Rygaard J, Leick V, Odum N. 1999. Inhibition of protein
phosphatase 2A induces serine/threonine phosphorylation,
subcellular redistribution, and functional inhibition of STAT3.
Proc Natl Acad Sci USA 96:10620–10625.
Yang W, Hong YH, Shen XQ, Frankowski C, Camp HS, Leff T.
2001. Regulation of transcription by AMP-activated protein
kinase: phosphorylation of p300 blocks its interaction with
nuclear receptors. J Biol Chem 276:38341–38344.
Yang XP, Irani K, Mattagajasingh S, Dipaula A, Khanday F,
Ozaki M, Fox-Talbot K, Baldwin WM 3rd, Becker LC. 2005.
Signal transducer and activator of transcription 3a and spec-
ificity protein 1 interact to upregulate intercellular adhesion
molecule-1 in ischemic-reperfused myocardium and vascular
endothelium. Arterioscler Thromb Vasc Biol 25:1395–1400.
Yuan LW, Gambee JE. 2000. Phosphorylation of p300 at serine 89
by protein kinase C. J Biol Chem 275:40946–40951.
Yuan LW, Soh JW, Weinstein IB. 2002. Inhibition of histone
acetyltransferase function of p300 by PKCd. Biochim Biophys
Yung LM, Leung FP, Yao X, Chen ZY, Huang Y. 2006. Reactive
oxygen species in vascular wall. Cardiovasc Hematol Disord
Drug Targets 6:1–19.
Zhang Q, Raghunath PN, Xue L, Majewski M, Carpentieri DF,
Odum N, Morris S, Skorski T, Wasik MA. 2002a. Multilevel
dysregulation of STAT3 activation in anaplastic lymphoma
Zhang T, Seow KT, Ong CT, Cao X. 2002b. Interdomain inter-
action of Stat3 regulates its Src homology 2 domain-mediated
receptor binding activity. J Biol Chem 277:17556–17563.
Zhang X, Blenis J, Li HC, Schindler C, Chen-Kiang S. 1995.
Requirement of serine phosphorylation for formation of
STAT-promoter complexes. Science 267:1990–1994.
Address correspondence to:
Dr. George W. Booz
Department of Pharmacology and Toxicology
Center for Excellence in Cardiovascular-Renal Research
School of Medicine
The University of Mississippi Medical Center
2500 North State St.
Jackson, MS 39216-4505
Received 30 June 2011/Accepted 20 September 2011
94ZGHEIB ET AL.