Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular Endothelial Cells

Article (PDF Available)inMolecular and Cellular Biology 26(17):6333-46 · October 2006with26 Reads
DOI: 10.1128/MCB.00207-06 · Source: PubMed
Abstract
Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.
MOLECULAR AND CELLULAR BIOLOGY, Sept. 2006, p. 6333–6346 Vol. 26, No. 17
0270-7306/06/$08.000 doi:10.1128/MCB.00207-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction
of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular
Endothelial Cells
William A. Sands, Hayley D. Woolson, Gillian R. Milne, Claire Rutherford, and Timothy M. Palmer*
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and
Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
Received 6 February 2006/Returned for modification 28 March 2006/Accepted 8 June 2006
Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs)
by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors
inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling
complex (soluble IL-6R/IL-6). This was associated with the induction of suppressor of cytokine signaling 3
(SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor
complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts
abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was
without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require
cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative
cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small
interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3
induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and
sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/
SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP
may mediate its potent anti-inflammatory effects.
Vascular endothelial cells (ECs) represent a major cellular
target for many pro- and anti-inflammatory cytokines. Some of
the most important are class I cytokines, including the adipo-
cytokine leptin and “interleukin 6 (IL-6) family” members such
as IL-11, oncostatin M, and prototypical member IL-6 (19).
Activated vascular ECs, smooth muscle cells, monocytes, and
macrophages are each capable of producing IL-6, and accu-
mulation of IL-6 has been noted within arterial lesions in
several models of atherosclerosis (42, 54). Responsiveness to
IL-6 requires two components: an IL-6 -receptor (IL-6R)
which binds the cytokine with high affinity, and gp130, which
cannot bind IL-6 directly but can interact with IL-6-bound
IL-6R to transduce an intracellular signal (19). While the
endothelium is unresponsive to IL-6 alone due to a lack of
endogenous IL-6R, soluble IL-6R (sIL-6R) derived from
hepatocytes or shed from monocytes and macrophages re-
cruited to sites of vascular injury can bind IL-6 to form a
sIL-6R/IL-6 so-called trans-signaling” complex that can in-
teract with and activate gp130 homodimers (30). This has been
reported to trigger the initiation of angiogenesis, the up-regu-
lation of adhesion molecules, such as E-selectin, ICAM-1 and
VCAM-1, and the accumulation of chemokines such as mono-
cyte chemoattractant protein-1 (MCP-1/CCL2) (19, 22, 37). In
contrast, adipocyte-derived leptin can directly bind and acti-
vate any of several splice variants of the leptin receptor Ob-R,
although ECs appear to express only the full-length Ob-Rb
isoform (58).
Signaling from both gp130 and Ob-Rb is initiated upon
receptor clustering and subsequent activation of receptor-as-
sociated “Janus kinases” (JAKs), which catalyze the phosphor-
ylation of each receptor on specific Tyr residues within their
cytoplasmic domains. This triggers recruitment of members of
the “signal transducers and activators of transcription” (STAT)
family and their subsequent phosphorylation by JAKs. Upon
Tyr phosphorylation, STATs dissociate from the receptors to
form either homo- or heterodimeric complexes that translo-
cate to the nucleus to initiate target gene transcription upon
binding specific DNA elements and recruiting transcriptional
coactivators. The protein Tyr phosphatase SH2 domain-con-
taining protein tyrosine phosphatase 2” (SHP-2) also binds to
specific phospho-Tyr residues within gp130 and Ob-Rb,
thereby mediating the recruitment of Grb2-Sos complexes that
initiate the Ras/Raf/mitogen-activated protein kinase-ERK ki-
nase (MEK)/extracellular signal-related kinase (ERK) signal-
ing cascade (19, 58).
Signaling from cytokine receptors is subject to strict negative
regulation via several mechanisms designed to prevent inap-
propriately sustained activation of downstream responses (55).
For example, the transcriptional activity of phosphorylated
STAT proteins can be inhibited via SUMOylation by PIAS
proteins (“protein inhibitors of activated STATs”) or by tar-
geting for proteasomal degradation via ubiquitylation by E3
ligases, such as SLIM (36, 52). Also, in addition to initiating
the ERK pathway, SHP-2 is capable of inhibiting downstream
signaling by dephosphorylating Tyr phosphorylated cytokine
receptors, JAKs, and STATs (19, 55). However, one of the
* Corresponding author. Mailing address: 425 Davidson Bldg., Mo-
lecular Pharmacology Group, Division of Biochemistry and Molecular
Biology, Institute of Biomedical and Life Sciences, University of Glas-
gow, Glasgow G12 8QQ, United Kingdom. Phone: 44 141 330 4626.
Fax: 44 141 330 4620. E-mail: T.Palmer@bio.gla.ac.uk.
6333
most important inhibitory mechanisms identified is the induc-
tion of suppressor of cytokine signaling” (SOCS) proteins.
These constitute a family of eight related proteins (CIS and
SOCS-1 to -7), of which SOCS-1 and SOCS-3 have been char-
acterized most intensively, that function as end points in a
classical negative feedback loop whereby activation of STATs
triggers the induction of SOCS proteins, which then bind and
terminate signaling from activated cytokine receptors (2, 27).
A role for SOCS-3 in specifically terminating gp130 and Ob-Rb
signaling has been demonstrated by several observations, in-
cluding the unrestricted agonist-stimulated activation of
STAT3 seen in macrophages, hepatocytes, and neurons from
cell-specific conditional SOCS-3-deficient mice (8, 32, 56).
SOCS-3 terminates signaling by binding to JAK-phosphory-
lated receptors via its SH2 domain and interacting with and
inhibiting adjacent JAKs via its “kinase inhibitory region,”
thereby preventing the recruitment and Tyr phosphorylation of
STATs (41). SOCS-3 can also potentially competitively block
receptor recruitment of SHP-2 to Tyr759 of gp130, thus inhib-
iting ERK activation (43). In addition, the C-terminal “SOCS
box” domain can target SH2 domain-bound partners for
proteasomal degradation by directing interaction with an
elongin B/C-Cul5-Rbx1 complex to form an E3 ubiquitin
ligase (23, 38).
SOCS-1 and SOCS-3 can also be induced by noncytokine
stimuli, thus providing a mechanism by which otherwise distinct
signaling pathways can negatively control cytokine responsive-
ness. These include the chemokine IL-8 and bacterially derived
chemoattractant formylmethionyl-leucyl-phenylalanine follow-
ing activation of their cognate G protein-coupled receptors
(50), and lipopolysaccharide (LPS) via Toll-like receptor 4
(51). One recently identified signal for SOCS induction is el-
evation of intracellular cyclic AMP (cAMP) (16, 34). The abil-
ity of this prototypical second messenger to suppress activation
of the NF-B pathway at several levels in many cell types has
been the most intensively studied aspect of its anti-inflamma-
tory effects (31). However, the induction of SOCS-1 and SOCS-3
observed in leukocytes and FRTL-5 thyroid cells suggests a po-
tential mechanism by which cAMP could block pro-inflammatory
signaling from multiple JAK-STAT-mobilizing cytokine receptors
(16, 34). Classically, cAMP is thought to mediate the vast majority
of its intracellular effects by binding and activating cAMP-depen-
dent protein kinase (PKA), which controls the phosphorylation
status and activity of multiple intracellular substrates (53).
However, another family of intracellular cAMP sensors
termed e xchange proteins directly activated by cAMP”
(Epacs) have been recently identified (10). Epac1 and -2
function as cAMP-activated guanine nucleotide exchange
factors specific for the Rap family of small G proteins and,
thus, promote the accumulation of active GTP-bound Rap1
and -2 (5, 10). Interestingly, a role for Epac in EC function
was recently revealed by the finding that the Epac-mediated
activation of Rap1 and the subsequent formation of adher-
ens junctions contribute toward the ability of cAMP to en-
hance endothelial barrier function, an important aspect of
its anti-inflammatory effects in vascular ECs (9, 15).
To begin identifying potential mechanisms by which cAMP
could impact on cytokine signaling, we have been examining
the effects of cAMP elevation on IL-6 receptor signaling in
vascular ECs and assessing its functional significance. Our ob-
servations suggest that cAMP-mediated inhibition of IL-6 re-
ceptor signaling occurs independently of SHP-2 but requires
induction of SOCS-3. Moreover, the induction of SOCS-3 and
the subsequent inhibition of STAT phosphorylation occur in-
dependently of PKA, instead requiring Epac-mediated activa-
tion of Rap1.
MATERIALS AND METHODS
Materials. Recombinant human leptin, IL-6, and sIL-6R were obtained from
R&D Systems Europe, Ltd. (Abingdon, United Kingdom). Forskolin, rolipram,
H89, monoperoxo(picolinato)oxovanadate (mpV), U0126, polymyxin B, and
JAK inhibitor I were obtained from Merck Biosciences (Nottingham, United
Kingdom). Prostaglandin E
2
(PGE
2
) and -melanocyte-stimulating hormone
(MSH) were from Sigma-RBI (Dorset, United Kingdom). The Epac-selective
activator 8-(4-chlorophenylthio)-2-O-methyladenosine-3,5-cyclic monophos-
phate (8-pCPT) was from the Biolog Life Science Institute (Bremen, Germany).
Human umbilical vein endothelial cells (HUVECs) and human aortic endothe-
lial cells (HAECs) were purchased from Cambrex Biosciences Nottingham, Ltd.
(Nottingham, United Kingdom). The MCP-1 sandwich enzyme-linked immu-
nosorbent assay (ELISA) kit was from Promocell (Heidelberg, Germany). Endo-
Porter delivery reagent and morpholino antisense oligonucleotides against
SOCS-3 and C/EBP were purchased from Gene Tools, LLC (Philomath, OR),
while control nontargeting (catalog no. D-001210-0120) and Epac1-targeted (cat-
alog no. M-007676-00) small interfering RNAs (siRNAs) were purchased from
Dharmacon (Lafayette, CO). Wild-type (SOCS-3
/
) and SOCS-3
/
murine
embryonic fibroblasts (MEFs) were generously provided by Akihiko Yoshimura
(Kyushu University, Japan), while functionally null SHP-2
46-110
3T3 fibroblasts
and SHP-2
46-110
cells in which function had been rescued by stable expression
of full-length wild-type SHP-2 (59) (SHP-2
/
cells) were generously provided
by Benjamin Neel (Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, MA). Human SOCS-1 and SOCS-3 expression constructs were
generously provided by Doug Hilton (Walter and Eliza Hall Institute for Medical
Research and Co-operative Research Centre for Cellular Growth Factors,
Parkville, Victoria, Australia) and Jim Johnston (Centre for Cancer Research
and Cell Biology, Queen’s University, Belfast, United Kingdom). Expression
constructs encoding hemagglutinin-tagged Val12Rap1a and myc-tagged
Leu61Cdc42 were generously donated by Johannes Bos (University Medical
Center, Utrecht, Netherlands) and Martin Schwartz (Mellon Prostate Cancer
Institute, University of Virginia, Charlottesville, VA), respectively. Antibodies
were from Cell Signaling Technology, Inc. (Beverly, MA) except those against
Epac1 (characterized in reference 26) (generously supplied by Johannes Bos),
SOCS-1 and SOCS-3 (sc-7005 and sc-7009, respectively; Santa Cruz Biotechnol-
ogy, Santa Cruz, CA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(ab9485; Abcam) and -tubulin (T9026; Sigma-Aldrich). Sources of other ma-
terials have been described elsewhere (39).
Cell culture. HUVECs and HAECs were propagated at 37°C in a humidified
atmosphere containing 5% (vol/vol) CO
2
in ECM-2 medium (cambrex) supple
-
mented with 2% (wt/vol) fetal bovine serum, hydrocortisone, ascorbate, and
recombinant growth factors as recommended by the supplier. MEFs, 3T3 cells,
and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium sup-
plemented with 10% (vol/vol) fetal bovine serum, 1 mM
L-glutamine, 100 U/ml
penicillin, and 100 g/ml streptomycin.
Transient transfection, siRNA, and morpholino delivery in HUVECs. Endotoxin-
free cDNA expression constructs were prepared using a Wizard Purefection plas-
mid DNA purification system (Promega United Kingdom, Ltd., Southampton,
United Kingdom). These were introduced into HUVECs by nucleofection per
the manufacturer’s instructions (Amaxa, Cologne, Germany). Briefly, 1 10
6
HUVECs per sample were resuspended in 100 l of nucleofection buffer con-
taining 1 g of pMaxGFP (to assess transfection efficiency) and 4 g of either
pcDNA3 (vector control) or the desired construct indicated in the figures. Plas-
mid DNA was introduced into the nucleus of the cells using an Amaxa Nucleo-
fector set at program U-O1. For antisense morpholino delivery, HUVECs plated
in a six-well dish were incubated with 10 M SOCS-3-specific antisense mor-
pholino (5-GAAACTTGCTGTGGGTGACCATG-3) or a control morpholino
against C/EBP (5-AGTTAGAGTTCTCCCGGCATGGCGA-3), which is not
expressed at detectable levels in these cells (data not shown), in combination
with 6 M Endo-Porter delivery reagent for 48 h prior to removal and subse-
quent experimental treatment. For introduction of siRNAs, 1 10
6
HUVECs/
well plated in a six-well dish were treated twice over a period of 48 h using 20 M
siRNA oligonucleotides and 3 l Oligofectamine as described by Kooistra et
al. (26).
6334 SANDS ET AL. MOL.CELL.BIOL.
Immunoblotting. Confluent cells in six-well plates were treated as described in
the figure legends prior to washing in ice-cold phosphate-buffered saline (PBS)
and solubilization by scraping into 50 l/well detergent lysis buffer (50 mM
sodium HEPES [pH 7.5], 150 mM sodium chloride, 5 mM EDTA, 10 mM
sodium fluoride, 10 mM sodium phosphate, 1% [vol/vol] Triton X-100, 0.5%
[wt/vol] sodium deoxycholate, 0.1% [wt/vol] sodium dodecyl sulfate [SDS], 0.1
mM phenylmethylsulfonyl fluoride, 10 g/ml soybean trypsin inhibitor, 10 g/ml
benzamidine, and EDTA-free complete protease inhibitor mix). Following brief
vortexing, insoluble material was removed by microcentrifugation, and the su-
pernatant was assayed for protein content using a bicinchonic acid assay. Sam-
ples equalized for protein content (typically 10 to 20 g/sample) were fraction-
ated by SDS-polyacrylamide gel electrophoresis (PAGE) on 10 or 12% (wt/vol)
resolving gels. Following transfer to nitrocellulose, membranes were blocked for
1 h at room temperature in blocking solution (5% [wt/vol] skim milk in PBS
containing 0.1% [vol/vol] Tween 20). Membranes were then incubated either
overnight at 4°C or for1hatroom temperature with primary antibody diluted in
fresh blocking buffer. Primary antibodies were each used at used at a final
concentration of 1 g/ml. Following three washes in blocking solution, mem-
branes were incubated for1hatroom temperature with appropriate horseradish
peroxidase-conjugated secondary antibody at a 1-in-1,000 dilution. After further
washes with blocking buffer and PBS, immunoreactive proteins were visualized
by enhanced chemiluminescence. For phosphospecific antibodies, a similar pro-
tocol was used except that the primary antibodies were diluted in Tris-buffered
FIG. 1. Effect of cAMP elevating agents on STAT3 phosphoryla-
tion by sIL-6R/IL-6 and leptin. (A) HUVECs were pretreated for 5 h
with or without 10 M Fsk and/or 10 M Roli prior to the addition of
vehicle or 25 ng/ml sIL-6R/5 ng/ml IL-6 for a further 30 min as
indicated. Soluble cell extracts equalized for protein content were then
fractionated by SDS-PAGE for immunoblotting with anti-Tyr705
phospho-STAT3 and total STAT3 antibodies. Quantitative analysis
from three experiments is presented (
*
, P 0.05 versus the response
observed with sIL-6R/IL-6 alone). (B) HUVECs were pretreated for
5 h with 10 M Roli and either 3 M PGE
2
,10M CGS21680, or 1
M MSH prior to the addition of vehicle or 2.5 ng/ml sIL-6R/0.5
ng/ml IL-6 for a further 30 min as indicated. Soluble cell extracts
equalized for protein content were then fractionated by SDS-PAGE
for immunoblotting with anti-Tyr705 phospho-STAT3 and total
STAT3 antibodies. Quantitative analysis from three experiments is
presented (
*
, P 0.05 versus the response observed with sIL-6R/IL-6
alone). (C) HUVECs were pretreated for 5 h with or without
FskRoli prior to the addition of vehicle or 100 ng/ml leptin for a
further 30 min as indicated. Soluble cell extracts equalized for protein
content were then fractionated by SDS-PAGE for immunoblotting
with anti-Tyr705 phospho-STAT3 and total STAT3 antibodies, as de-
scribed for panel A. Quantitative analysis from three experiments is
presented (
*
, P 0.05 versus the response observed with leptin alone).
Veh, vehicle.
VOL. 26, 2006 cAMP/Epac INDUCTION OF SOCS-3 6335
saline, pH 7.5, containing 5% (wt/vol) immunoglobulin G-free bovine serum
albumin and 0.1% (vol/vol) Tween 20, and all washes were with Tris-buffered
saline/0.1% (vol/vol) Tween 20. Quantification was by densitometric scanning of
nonsaturating exposed films using TotalLab imaging software (Phoretix).
Quantitative reverse transcription-PCR analysis of SOCS-3 mRNA. Total
RNA was isolated from stimulated HUVECs using TRIzol reagent. Two micro-
grams/sample was then utilized for cDNA synthesis using oligo(dT) primers in
combination with avian myoblastosis virus reverse transcriptase (Promega,
Southampton, United Kingdom) in accordance with the manufacturer’s instruc-
tions. The cDNA for SOCS-3 was amplified and quantified using DyNAmo
SYBR Green qPCR reagents (Finnzymes, Braintree, United Kingdom) using the
forward primer 5-CTTCAGCTCCAAGAGCGAGT-3 and reverse primer 5-
CAGGTTCTTGGTCCCAGACT-3, which generated a predicted 208-bp prod-
uct, as verified by agarose gel electrophoresis. PCRs (25 l, final volume) were
performed in quadruplicate using a DNA Engine Opticon II real-time two-color
PCR detection system (Bio-Rad Laboratories, Hemel Hempstead, United King-
dom) and employed 36 cycles of denaturation at 94°C (30 s), annealing at 50°C
(20 s), and extension at 72°C (10 s). Melting curves were determined using the
following parameters: 95°C cooling to 70°C, and ramping to 90°C at 0.2°C/s.
Known amounts of pcDNA3/human SOCS-3 cDNA (0.1 fg to 1 ng) were am-
plified in parallel to allow quantification of the copy number from which induc-
tions (n-fold) over basal levels were determined.
Sandwich ELISA for MCP-1. HUVECs in 96-well culture plates (10
4
cells/
well) were treated in triplicate as indicated in Results. Aliquots of medium from
each well were then assayed for MCP-1 accumulation using a sandwich ELISA
kit in accordance with the manufacturer’s instructions.
Statistical analysis. Data are presented in the text as means standard errors
for the number of experiments indicated, while representative experiments are
shown in the figures. Statistical significance was assessed by analysis of variance
with an probability of 0.05.
RESULTS
Effect of cAMP elevation on cytokine receptor activation of
the JAK-STAT pathway in HUVECs. The effects of IL-6 in ECs
are mediated by interaction of sIL-6R/IL-6 complexes with
gp130 dimers at the cell surface (30). This activates the JAK-
STAT signaling pathway, which mediates many of the effects of
IL-6 exposure on target gene expression and EC function (19,
37). Preliminary experiments revealed that a 30-min exposure
to maximally effective concentrations of sIL-6R/IL-6 stimu-
lated the tyrosine phosphorylation of STAT3 24- 6-fold (P
0.05 versus the vehicle-treated control; n 3). Thus, to assess
the functional consequences of cAMP elevation on receptor
function, the effects of forskolin (Fsk) and rolipram (Roli)
pretreatment on sIL-6R/IL-6-stimulated phosphorylation of
STAT3 were assessed. Fsk directly binds and activates adenylyl
cyclase isoforms to increase cAMP synthesis, while Roli blocks
cAMP degradation by inhibiting the PDE4 family of phos-
phodiesterases (6, 21). While Roli alone had no effect on
optimal STAT3 phosphorylation, treatments with Fsk alone
and with Fsk and Roli together (FskRoli) produced a sub-
stantial inhibition of STAT3 phosphorylation in response to
either sIL-6R/IL-6 (67% 9% inhibition by FskRoli versus
vehicle-pretreated controls; P 0.05; n 3) without altering
total levels of STAT3 protein (Fig. 1A). Importantly, similar
inhibitory effects on STAT3 phosphorylation in response to
sIL-6R/IL-6 were observed upon activation of endogenous G
protein-coupled receptors for PGE
2
(via EP
2
and EP
4
recep
-
FIG. 2. Effect of SHP-2 inhibition on cAMP-mediated inhibition of
STAT3 phosphorylation by sIL-6R/IL-6. (A) HUVECs were incu-
bated with or without the protein Tyr phosphatase inhibitor mpV (100
M) for 5 h prior to treatment with or without 5 ng/ml vascular
endothelial growth factor (VEGF) for 1 h as indicated. Soluble cell
extracts equalized for protein content were then fractionated by SDS-
PAGE for immunoblotting with anti-phospho-Tyr antibody. The three
most prominent immunoreactive bands are indicated. preinc, preincu-
bation. (B) HUVECs were incubated with or without the protein Tyr
phosphatase inhibitor mpV (100 M) for 30 min prior to treatment
with or without FskRoli for 5 h and vehicle or 25 ng/ml sIL-6R/5
ng/ml IL-6 for a further 30 min as indicated. Soluble cell extracts
equalized for protein content were then fractionated by SDS-PAGE
for immunoblotting with anti-Tyr705 phospho-STAT3, total STAT3,
and SOCS-3 antibodies. (C) Upper panels, SHP-2
/
and SHP-2
46-110
3T3 fibroblasts were pretreated with or without FskRoli for 5 h prior
to the addition of vehicle or sIL-6R/IL-6 for a further 30 min as
shown in panel B. Soluble cell extracts equalized for protein content
were then fractionated by SDS-PAGE for immunoblotting with anti-
Tyr705 phospho-STAT3 and total STAT3 antibodies. Lower panel,
lysates from SHP-2
/
and SHP-2
46-110
3T3 fibroblasts were also
immunoblotted with anti-SHP-2 antibody to identify wild-type (WT)
and mutated SHP-2
46-110
, which migrates with enhanced mobility on
SDS-PAGE due to the introduced deletion.
6336 SANDS ET AL. M
OL.CELL.BIOL.
tors), CGS21680 (A
2A
adenosine receptor [A
2A
AR]) and
MSH (melanocortin receptor 1), each of which are known to
activate adenylyl cyclase and elevate cAMP (11, 14, 33, 44)
(Fig. 1B). In addition, the inhibitory effect of each agonist was
potentiated by the inclusion of rolipram (Fig. 1B). To deter-
mine whether the effect of cAMP elevation was receptor specific,
the effects of Fsk with or without Roli were also tested on STAT
phosphorylation by leptin, which binds and activates the leptin
receptor Ob-Rb in ECs (58). Leptin stimulated the tyrosine phos-
phorylation of STAT3 by 11- 1-fold (P 0.05 versus the
vehicle-treated control; n 3) and, similar to the effect on sIL-
6R/IL-6 responses, FskRoli pretreatment inhibited this pro-
cess (69% 15% inhibition versus vehicle-pretreated controls; P
0.05; n 3) without altering total levels of STAT3 protein (Fig.
1C). Thus, cAMP could significantly blunt the ability of both
sIL-6R/IL-6 and leptin to activate the JAK-STAT pathway in
vascular ECs.
Effects of blocking protein tyrosine phosphatase activity on
cAMP-mediated inhibition of STAT3 phosphorylation. Several
distinct molecular events serve to down-regulate class I cyto-
kine receptor signaling (19). Since cAMP elevation specifically
inhibited STAT3 phosphorylation in response to both sIL-
6R/IL-6 and leptin without altering STAT3 expression, a
common postreceptor inhibitory mechanism involving either
protein tyrosine phosphatases, such as T-cell protein tyrosine
phosphatase and SHP-2 and/or SOCS proteins, was likely to be
operative (19, 58). With respect to the former possibility, it has
been demonstrated by others that SHP-2 can be phosphory-
lated by PKA in vitro, resulting in an increase in its tyrosine
phosphatase activity (35). Thus, to examine a role for tyrosine
phosphatases in mediating the effects of cAMP, two distinct
approaches were taken. First, HUVECs were pretreated with
FIG. 3. Effect of blocking SOCS-3 induction on cAMP-mediated
inhibition of STAT3 phosphorylation by sIL-6R/IL-6. (A) HUVECs
were incubated with or without the protein synthesis inhibitor emetine
for 30 min prior to treatment with or without FskRoli for5hand
vehicle or 25 ng/ml sIL-6R and 5 ng/ml IL-6 for a further 30 min as
indicated. Soluble cell extracts equalized for protein content were then
fractionated by SDS-PAGE for immunoblotting with anti-Tyr705
phospho-STAT3, total STAT3, and SOCS-3 antibodies. (B) HUVECs
were treated with SOCS-3 morpholino antisense oligonucleotide or a
control morpholino for 48 h prior to treatment with or without 10 M
Fskfor5hasindicated and preparation of soluble cell extracts.
Following equalization for protein content, samples were fractionated
by SDS-PAGE for immunoblotting with anti-SOCS-3 and anti-GAPDH
antibodies, the latter serving as a loading control. (C) HUVECs were
treated with SOCS-3 morpholino antisense oligonucleotide or a control
morpholino for 48 h prior to pretreatment with or without 10 M Fsk for
5 h prior to the addition of vehicle or 25 ng/ml sIL-6R/5 ng/ml IL-6 for
a further 30 min as indicated. Soluble cell extracts equalized for protein
content were then fractionated by SDS-PAGE for immunoblotting with
anti-Tyr705 phospho-STAT3 and total STAT3 antibodies. (D) SOCS-
3
/
and SOCS-3
/
MEFs were treated with FskRoli for the indicated
times prior to the preparation of soluble cell extracts. Following equal-
ization for protein content, samples were fractionated by SDS-PAGE for
immunoblotting with anti-SOCS-3 and anti-tubulin antibodies, the latter
serving as a loading control. (E) SOCS-3
/
and SOCS-3
/
MEFs were
pretreated with or without FskRoli for 5 h prior to the addition of
vehicle or 25 ng/ml sIL-6R/5 ng/ml IL-6 for a further 30 min as indicated.
Soluble cell extracts equalized for protein content were then fractionated
by SDS-PAGE for immunoblotting with anti-Tyr705 phospho-STAT3
and total STAT3 antibodies.
V
OL. 26, 2006 cAMP/Epac INDUCTION OF SOCS-3 6337
the protein tyrosine phosphatase inhibitor mpV prior to
FskRoli exposure and assessment of sIL-6R/IL-6-stimu-
lated phosphorylation of STAT3. At a concentration (100 M)
at which mpV was effective in potentiating basal levels and
vascular endothelial growth factor-stimulated increases in the
Tyr phosphorylation of multiple intracellular proteins (Fig. 2A),
no significant alteration in the inhibitory effect of FskRoli was
detectable (Fig. 2B) (mpV potentiated the inhibitory effect of
FskRoli by 34% 44%; n 3; P 0.05 [not significant
{NS}]). In a second approach, experiments were also per-
formed using SHP-2
/
and SHP-2
46-110
3T3 fibroblasts (59)
to address a specific contribution of this tyrosine phosphatase
in mediating the effects of cAMP. Interestingly in SHP-2
/
cells, no significant inhibition of sIL-6R/IL-6-stimulated
STAT3 phosphorylation by FskRoli was detectable. In con-
trast, the absence of functional SHP-2 in SHP-2
46-110
cells was
associated with a robust FskRoli-mediated inhibition of
STAT3 phosphorylation (Fig. 2B) (0% 10% inhibition in
SHP-2
/
cells versus 77% 12% inhibition in SHP-2
46-110
cells; n 3; P 0.05).
Effects of blocking SOCS-3 induction on cAMP-mediated
inhibition of STAT3 phosphorylation. The expression of SOCS
proteins is induced de novo upon cellular exposure to multiple
stimuli (2, 27, 50, 51). Consequently, the inhibition of protein
synthesis should exclude any contribution of SOCS induction
toward mediating the effects of cAMP. Consistent with this
hypothesis, the inhibition of protein synthesis by emetine pre-
treatment substantially reduced the ability of FskRoli to in-
hibit sIL-6R/IL-6-stimulated phosphorylation of STAT3
(Fig. 3A) (100 M emetine reduced the inhibitory effect of
FskRoli treatment by 61% 17%; n 4; P 0.05). We also
noted that STAT3 phosphorylation by sIL-6R/IL-6 was re-
duced by emetine treatment in the absence of any change in
total STAT3 levels, suggesting that the expression levels of
JAKs and/or gp130 are reduced in the absence of any protein
synthesis. This is consistent with observations that both gp130
and JAKs can be targeted for degradation in a cytokine-stim-
ulated manner (3, 46). However, most importantly, parallel
immunoblotting experiments demonstrated that FskRoli
treatment was capable of promoting the accumulation of
SOCS-3 protein, an effect that was abolished by emetine pre-
treatment (Fig. 3A).
A specific role for SOCS-3 in mediating the inhibitory effects
of cAMP was confirmed using two separate experimental ap-
proaches. The first involved using morpholino antisense oligo-
FIG. 4. Effect of cAMP elevation on SOCS-3 induction and STAT3
phosphorylation by sIL-6R/IL-6 in HAECs. HAECs were pretreated
for 5 h with or without 10 M forskolin prior to the addition of vehicle
or 25 ng/ml sIL-6R and 5 ng/ml IL-6 for a further 30 min as indicated.
Soluble cell extracts equalized for protein content were then fraction-
ated by SDS-PAGE for immunoblotting with either anti-Tyr701 phos-
pho-STAT1 and total STAT1 antibodies (A) or anti-Tyr705 phospho-
STAT3 and total STAT3 antibodies (B). Quantitative analysis from
three experiments is presented (
*
, P 0.05 versus the response ob-
served with sIL-6R/IL-6 alone). (C) HAECs were pretreated for 5 h
with 10 M Fsk prior to the preparation of soluble cell extracts.
Following equalization for protein content, samples were fractionated
by SDS-PAGE for immunoblotting with anti-SOCS-3 and anti-
GAPDH antibodies, with the latter serving as a loading control.
6338 SANDS ET AL. M
OL.CELL.BIOL.
nucleotides to test the effect of selectively blocking SOCS-3
induction on inhibition of STAT phosphorylation by cAMP in
HUVECs. Under conditions in which Fsk-mediated induction
of SOCS-3 was abolished (Fig. 3B), the ability of Fsk to inhibit
sIL-6R/IL-6-stimulated phosphorylation of STAT3 was al-
most completely attenuated (Fig. 3C) (70% 15% inhibition
in control morpholino-treated versus 15% 17% inhibition in
SOCS-3 morpholino-treated HUVECs; n 3; P 0.05). The
second approach involved comparing the inhibitory effect of
cAMP elevation in SOCS-3
/
and SOCS-3
/
MEFs. Inter
-
estingly, both basal and sIL-6R/IL-6-stimulated levels of
phosphorylated STAT3 were elevated in SOCS-3
/
MEFs,
suggesting that low-level basal expression of SOCS-3 may play
a role in dampening STAT3 activation under unstimulated
conditions. Importantly, however, cAMP elevation was capable
of strongly increasing SOCS-3 expression and inhibiting sIL-
6R/IL-6-stimulated phosphorylation of STAT3 in SOCS-3
/
cells (Fig. 3D and E). Both effects were abolished in SOCS-
3
/
cells (Fig. 3D and E) (51% 20% inhibition in SOCS-
3
/
MEFs versus 1% 8% inhibition in SOCS-3
/
MEFs;
n 3; P 0.05).
Taken together, these experiments argue against a role for
SHP-2 and other protein tyrosine phosphatases in mediating
the inhibitory effects of cAMP elevation on STAT3 phosphor-
ylation by sIL-6R/IL-6 and instead suggest that cAMP-medi-
ated up-regulation of the inhibitory regulator SOCS-3 is the
mechanism responsible for this effect.
Inhibition of STAT phosphorylation in aortic ECs. It has
been well documented that ECs from different vascular origins
can display considerable variation in their biochemical and
immunological properties, although they exhibit many overlap-
ping functions (1). Thus, it was important to determine whether
cAMP-mediated induction of SOCS-3 and subsequent inhibition
of STAT phosphorylation was a phenomenon unique to HUVECs.
To achieve this, the presence of the cAMP/SOCS-3 inhibitory
pathway in HAECs was also assessed (Fig. 4). These experiments
demonstrated that a 5-h pretreatment with Fsk inhibited sIL-
6R/IL-6-mediated STAT phosphorylation to an extent similar to
that observed with HUVECs (Fig. 4A and B) (65% 13%
inhibition of STAT1 phosphorylation and 46% 15% inhibition
of STAT3 phosphorylation; n 3; P 0.05 versus vehicle-
pretreated controls). Fsk treatment also promoted the accu-
mulation of SOCS-3 under these conditions (Fig. 4C). Thus,
the ability of cAMP to inhibit sIL-6R/IL-6-mediated STAT
phosphorylation and induce SOCS-3 expression is a feature
common to ECs from both venous and arterial origins.
Characterization of cAMP-mediated induction of SOCS-3
expression in HUVECs. Exposure to Fsk produced a time-
dependent accumulation of SOCS-3 protein that peaked after
4 h, was sustained for at least 24 h, and was temporally pre-
ceded by a transient induction of SOCS-3 mRNA (Fig. 5A and
B). Importantly, SOCS-3 accumulation was observed not only
with Fsk but also upon activation of endogenous cAMP-mobi-
lizing G protein-coupled A
2A
ARs by the selective agonist
CGS21680 (Fig. 5C). Moreover, the effect appeared to be
restricted to SOCS-3, as no induction of the otherwise closely
related protein SOCS-1 was detectable in response to either
CGS21680, Fsk, or FskRoli under conditions in which re-
combinant SOCS-1 was readily detectable (Fig. 5D).
FIG. 5. Characterization of SOCS-3 induction following cAMP ele-
vation in HUVECs. (A) HUVECs were treated with Fsk for the indicated
times prior to the preparation of RNA and analysis of SOCS-3 mRNA by
quantitative real-time reverse transcription-PCR. Results from one of
four experiments is presented. (B) HUVECs were treated with Fsk for the
indicated times prior to the preparation of soluble cell extracts. Following
equalization for protein content, samples were fractionated by SDS-
PAGE for immunoblotting with anti-SOCS-3 antibody. (C) HUVECs
were treated with the A
2A
AR-selective agonist CGS21680 (5 M) for the
indicated times prior to the preparation of soluble cell extracts. Following
equalization for protein content, samples were fractionated by SDS-
PAGE for immunoblotting with anti-SOCS-3 antibody. (D) HUVECs
were treated with or without Fsk, FskRoli, and CGS21680 for 5 h prior
to the preparation of soluble cell extracts. Following equalization for
protein content, samples were fractionated by SDS-PAGE for immuno-
blotting with anti-SOCS-1 antibody. Soluble extract (10 g) prepared
from HEK293 cells transiently expressing Flag epitope-tagged SOCS-1
(HEK293/Flag-SOCS-1) was run in parallel as a positive control for anti-
body immunoreactivity (ve).
V
OL. 26, 2006 cAMP/Epac INDUCTION OF SOCS-3 6339
Effect of blocking PKA activity on SOCS-3 induction and
inhibition of STAT phosphorylation. PKA is the most exten-
sively characterized mediator of cAMP’s intracellular effects
(53) and thus represented an important potential effector of
cAMP’s inhibitory effects on STAT phosphorylation. However,
under conditions in which pretreatment with FskRoli sub-
stantially inhibited subsequent phosphorylation of STAT1 and
STAT3 (77% 5% and 81% 4% inhibition, respectively,
versus vehicle-pretreated controls; P 0.05 for each; n 3)
(Fig. 6A and B), the effect could not be significantly reversed
by coincubation with the PKA inhibitor H89 (7% 9% and
8% 5% reversal of STAT1 and STAT3 phosphorylation,
respectively; P 0.05 [NS]; n 3) (Fig. 6A and B). Similarly,
the ability of Fsk to promote the accumulation of SOCS-3 was
not significantly inhibited by H89 pretreatment (2% 25%
inhibition; P 0.05 [NS]; n 3) (Fig. 6C). These observations
could not be explained by ineffective inhibition of PKA activity
under these conditions, as the concentration of H89 employed
(5 M) abolished Fsk-stimulated PKA-mediated CREB phos-
phorylation on Ser133 (Fig. 6D). Thus, the ability of cAMP
elevation to induce SOCS-3 and inhibit sIL-6R/IL-6-stimu-
lated STAT phosphorylation occurs via a PKA-independent
mechanism.
Contribution of Epac toward mediating SOCS-3 induction.
The lack of any involvement of PKA in mediating cAMP’s
effect on SOCS-3 induction and inhibition of STAT phosphor-
ylation suggested that alternative intracellular cAMP sensors
were responsible. To test any contribution of Epac toward
mediating these effects, the ability of the Epac-selective cAMP
analogue 8-pCPT to induce SOCS-3 expression in HUVECs
was initially tested. These experiments demonstrated that
8-pCPT could promote a concentration- and time-dependent
accumulation of SOCS-3 (Fig. 7A and B). Epac functions as a
guanine nucleotide exchange factor for the Rap family of small
G proteins (5, 10). Thus, if Epac was involved in SOCS-3
induction, the activation of Rap should also be sufficient to
trigger the effect. To test this hypothesis, the ability of consti-
tutively active GTPase-deficient Val12Rap1a to promote the
synthesis of SOCS-3 in HUVECs was assessed following tran-
sient expression over 48 h and pretreatment of cells with pro-
teasome inhibitor MG132 to block SOCS-3 degradation by the
proteasome at this time point. These experiments demon-
FIG. 6. Effect of blocking PKA activity on cAMP-mediated
SOCS-3 induction and inhibition of STAT3 phosphorylation by sIL-
6R/IL-6. HUVECs were incubated with or without the PKA inhibitor
H89 (5 M) for 30 min prior to treatment with FskRoli for5hand
vehicle or 25 ng/ml sIL-6R/5 ng/ml IL-6 for a further 30 min as
indicated. Soluble cell extracts equalized for protein content were then
fractionated by SDS-PAGE for immunoblotting with anti-Tyr705
phospho-STAT3 and total STAT3 (A) or anti-Tyr701 phospho-STAT1
and total STAT1 antibodies (B). (C) HUVECs were incubated with or
without the PKA inhibitor H89 (5 M) for 30 min prior to treatment
with 10 MFskfor5hasindicated. Soluble cell extracts equalized for
protein content were then fractionated by SDS-PAGE for immuno-
blotting with anti-SOCS-3 antibody. (D) HUVECs were incubated
with or without the PKA inhibitor H89 (5 M) for 30 min prior to
treatment with 10 M Fsk for 30 min as indicated. Soluble cell extracts
equalized for protein content were then fractionated by SDS-PAGE
for immunoblotting with anti-Ser133 phospho-CREB antibody.
6340 SANDS ET AL. M
OL.CELL.BIOL.
FIG. 7. Contribution of Epac toward SOCS-3 induction and inhibition of STAT3 phosphorylation by sIL-6R/IL-6. (A) HUVECs were treated
with the indicated concentrations of the Epac-selective activator 8-pCPT for 5 h prior to the preparation of soluble cell extracts. Following
equalization for protein content, samples were fractionated by SDS-PAGE for immunoblotting with anti-SOCS-3 and antitubulin antibodies, with
the latter serving as a loading control. Veh, vehicle. (B) HUVECs were treated with 50 M 8-pCPT for the indicated times prior to the preparation
of soluble cell extracts. Following equalization for protein content, samples were fractionated by SDS-PAGE for immunoblotting with anti-SOCS-3
antibody. (C) HUVECs were transiently transfected with empty vector or expression constructs encoding Leu61Cdc42 and Val12Rap1a as
indicated. Forty-eight hours posttransfection, cells were treated with or without 6 M MG132 for 6 h prior to the preparation of soluble cell
extracts. Following equalization for protein content, samples were then fractionated by SDS-PAGE for immunoblotting with anti-SOCS-3 and
anti-ERK1/2 antibodies, with the latter serving as a loading control. Quantitative analysis from three experiments is presented (
*
, P 0.05 versus
the response observed with vector plus MG132). (D) HUVECs were pretreated for 5 h with or without 50 M 8-pCPT prior to the addition of
vehicle, 2.5 ng/ml sIL-6R, and 0.5 ng/ml IL-6 for a further 30 min as indicated. Soluble cell extracts equalized for protein content were then
fractionated by SDS-PAGE for immunoblotting with anti-Tyr705 phospho-STAT3 and total STAT3 antibodies. (E) HUVECs were transfected
twice over 48 h with nontargeting control and Epac1-specific siRNAs prior to treatment with Fsk for5hasindicated. Soluble cell extracts equalized
for protein content were then fractionated by SDS-PAGE for immunoblotting with anti-Epac1, SOCS-3, and GAPDH antibodies. (F) HUVECs
were transfected with control and Epac1-specific siRNAs as described for panel E prior to pretreatment with or without FskRoli for 5 h followed
by exposure to 25 ng/ml sIL-6R and 5 ng/ml IL-6 for 30 min as indicated. Soluble cell extracts equalized for protein content were then fractionated
by SDS-PAGE for immunoblotting with anti-Tyr705 phospho-STAT3 and total STAT3 antibodies.
V
OL. 26, 2006 cAMP/Epac INDUCTION OF SOCS-3 6341
strated that expression of Val12Rap1a was sufficient to induce
a 3.3- 1.5-fold increase in SOCS-3 levels over vector-trans-
fected controls in the presence of proteasome inhibitor (Fig.
7C). In contrast, the expression of constitutively active Leu61-
mutated Cdc42, a Rho family G protein, failed to increase
SOCS-3 expression over vector controls (37% 30% inhibi-
tion of expression levels versus the vector-transfected control).
To determine whether the increase in SOCS-3 expression in-
duced by Epac was functionally relevant, we also tested the
ability of 8-pCPT to inhibit STAT phosphorylation by sIL-
6R/IL-6. Under these conditions, a 5-h pretreatment with 50
M 8-pCPT caused an 81% 13% inhibition of STAT3 phos-
phorylation (Fig. 7D) (P 0.05 versus vehicle-pretreated con-
trols; n 3). Together, these data demonstrate that selective
activation of the Epac/Rap pathway is sufficient to recapitulate
the ability of cAMP to induce the accumulation of SOCS-3 and
inhibit sIL-6R/IL-6 signaling. To determine whether Epac
activation was absolutely necessary to observe these effects, an
siRNA strategy was used to specifically deplete cellular Epac1
expression (Epac2 is not expressed to a significant extent in
HUVECs) (26). Under conditions where Epac1 expression was
reduced by 87% 3%, the ability of FskRoli to induce
SOCS-3 was reduced by 61% 12% (P 0.05 versus control
cells; n 3) (Fig. 7E), and inhibition of STAT3 phosphoryla-
tion was completely blocked (84% 13% inhibition in control
cells versus 3% 21% inhibition in Epac1-depleted cells; P
0.05 versus controls; n 3) (Fig. 7F). Therefore, the pres-
ence of Epac1 is necessary and sufficient for cAMP elevation to
induce SOCS-3 and thus inhibit sIL-6R/IL-6-mediated
STAT3 phosphorylation.
Effect of Epac activation on sIL-6R/IL-6-stimulated accu-
mulation of MCP-1. To assess the physiological significance of
the cAMP/Epac/SOCS-3 pathway in inhibiting sIL-6R/IL-6-
stimulated phosphorylation of STATs, it was important to de-
termine whether cAMP elevation and Epac activation could
suppress the induction of an important STAT-regulated gene
product. Due to its key role as a chemoattractant responsible
for leukocyte recruitment during the early stages of atherogen-
esis, the chemokine MCP-1 was chosen for analysis (37, 54).
While exposure of HUVECs to a maximally effective concen-
tration of sIL-6R/IL-6 produced a 5.2- 2.7-fold increase in
MCP-1 accumulation after 24 h, pretreatment with FskRoli
for 5 h prior to sIL-6R/IL-6 exposure substantially inhibited
MCP-1 release (Fig. 8). Importantly, this inhibition could be
largely reproduced by the selective activation of Epac with 50
M 8-pCPT (Fig. 8), a concentration at which SOCS-3 induc-
tion was readily detectable (Fig. 7A). Thus, Epac-mediated
up-regulation of SOCS-3 represents an inhibitory mechanism
capable of suppressing sIL-6R/IL-6-mediated up-regulation
of an important STAT-responsive gene product involved in the
progression of disease.
Identification of a cAMP-activated signaling pathway lead-
ing to SOCS-3 accumulation. One potentially trivial reason
that SOCS-3 induction was observed in response to FskRoli
and 8-pCPT could have been contamination of these reagents
with LPS, which has been shown to up-regulate multiple SOCS
proteins in macrophages and HL60 cells (51). However, pre-
incubation of each ligand with polymyxin B, which binds and
inactivates LPS, failed to inhibit SOCS-3 induction (data not
shown), thereby excluding the possibility that LPS contamina-
tion was responsible.
SOCS-3 induction in response to sIL-6R/IL-6 binding to
gp130 is due to the activation of the JAK-STAT pathway and
the subsequent binding of phosphorylated STAT dimers to a
GAS element within the SOCS-3 promoter (18). Moreover,
there is some evidence to suggest that G protein-coupled re-
ceptors, such as the thrombin receptor PAR-1 (28), are capa-
ble of directly activating the JAK-STAT pathway. Thus, to
assess whether cAMP elevation triggered this pathway to up-
regulate SOCS-3, the effects of JAK inhibition on FskRoli-
and sIL-6R/IL-6-mediated induction of SOCS-3 were com-
pared. This demonstrated that while SOCS-3 induction in
response to sIL-6R/IL-6 was essentially abolished by prein-
cubation with a selective JAK inhibitor (96% 5% inhibition
versus the vehicle-pretreated control; P 0.05; n 3), the
response to FskRoli remained largely intact (15% 12%
inhibition versus the vehicle-pretreated control; P 0.05 [NS];
n 3) (Fig. 9A). Consistent with this observation, we have
been unable to detect any FskRoli stimulation of STAT1 or
STAT3 tyrosine phosphorylation over a wide range of incuba-
tion times, spanning 15 min to 5 h (Fig. 1 to 3 and data not
shown). Thus, cAMP elevation up-regulates SOCS-3 expres-
sion via a mechanism distinct from the JAK-dependent path-
way utilized by sIL-6R/IL-6. However, Fsk-mediated induc-
tion of SOCS-3 was significantly reduced by preincubation with
the MEK inhibitor U0126 (79% 16% inhibition versus the
vehicle-pretreated control; P 0.05; n 3) (Fig. 9B). Indeed,
the treatment of HUVECs with Fsk for 15 min produced a
marked increase in ERK phosphorylation, equivalent to that
produced by the protein kinase C activator phorbol 12-myris-
tate 13-acetate (PMA) (45) (Fig. 9C). To determine whether
ERK activation alone was sufficient to induce SOCS-3, we
tested the ability of PMA treatment to induce SOCS-3. This
demonstrated that PMA was able to induce SOCS-3 (6.6-
0.9-fold over the vehicle-treated control; P 0.05; n 3) and
that induction was blocked by U0126, in contrast to the STAT-
mediated induction of SOCS-3 produced by sIL-6R/IL-6,
FIG. 8. Effect of Epac activation on the accumulation of MCP-1.
HUVECs were pretreated with either vehicle (Veh), FskRoli, or 50
M 8-pCPT for 5 h prior to treatment with 25 ng/ml sIL-6R and 5
ng/ml IL-6 for 24 h as indicated. Conditioned medium was then re-
moved for measurement of MCP-1 levels. Data from three experi-
ments are presented (
*
, P 0.05 versus sIL-6R/IL-6-stimulated
MCP-1 levels). Basal MCP-1 levels were 0.69 0.42 ng/ml (n 3).
6342 SANDS ET AL. M
OL.CELL.BIOL.
which was resistant to inhibition by U0126 (Fig. 9D). Thus,
cAMP elevation in HUVECs can activate the ERK pathway,
an event that appears to be sufficient to observe up-regulation
of SOCS-3.
DISCUSSION
The prototypical second messenger cAMP occupies a cen-
tral role in endothelial cell function by virtue of its ability to
limit vascular permeability and attenuate pro-inflammatory
events in response to multiple cytokines (4, 12, 15, 31). In this
study, we have demonstrated that cAMP elevation can inhibit
activation of the JAK-STAT pro-inflammatory signaling path-
way in response to a sIL-6R/IL-6 trans-signaling complex by
specifically promoting the accumulation of SOCS-3 via a novel
PKA-independent mechanism that can be mimicked by activa-
tion of the recently identified cAMP sensor Epac. Although
Epac was identified approximately 8 years ago, the biological
effects it controls have only recently begun to emerge. These
include positive regulation of integrin-mediated cell adhesion
(11, 17), stimulation of insulin secretion via activation of ryano-
dine-sensitive calcium channels, and control of vesicle exocy-
tosis (20, 24). Several studies have also recently identified a
central role for Epac in controlling the ability of cAMP to
reduce vascular permeability in part by potentiating the for-
mation of VE-cadherin-mediated cell-cell interactions (9, 15,
26). Thus, our observation that Epac activation can stimulate
the accumulation of SOCS-3, thereby resulting in an inhibition
of gp130-mediated downstream signaling, is of interest from at
least two perspectives. First, SOCS-3 induction represents a
new function for Epac that is consistent with the predomi-
nantly anti-inflammatory effects of cAMP elevation observed
for ECs (4). Second, our observations identify a second role for
Epac in EC biology in addition to its effects on barrier function
and would thus suggest that Epac is an especially important
mediator of cAMP’s effects in these cells. Therefore, selective
manipulation of the cAMP-Epac-Rap pathway might prove to
be a useful strategy for limiting multiple aspects of endothelial
FIG. 9. Identification of a signaling pathway controlling cAMP-
mediated induction of SOCS-3. (A) HUVECs were pretreated for 30
min with or without 0.1 M JAK inhibitor prior to the addition of
either 25 ng/ml sIL-6R and 5 ng/ml IL-6 or FskRoli for a further 3
or5hasindicated. Soluble cell extracts equalized for protein content
were then fractionated by SDS-PAGE for immunoblotting with anti-
SOCS-3 and anti-GAPDH antibodies, the latter serving as a loading
control. Quantitative analysis from three experiments is presented (
*
,
P 0.05 versus sIL-6R/IL-6-stimulated SOCS-3 expression in the
absence of JAK inhibitor). (B) HUVECs were pretreated for 30 min
with or without MEK inhibitor U0126 (0.1 M) prior to the addition
of 5 M Fsk for a further5hasindicated. Soluble cell extracts
equalized for protein content were then fractionated by SDS-PAGE
for immunoblotting with anti-SOCS-3 antibody. (C) HUVECs were
pretreated with or without 0.1 M U0126 prior to the addition of 5 M
Fsk or 1 M PMA as indicated. Soluble cell extracts equalized for
protein content were then fractionated by SDS-PAGE for immunoblot-
ting with anti-Thr202/Tyr204 phospho-ERK1/2 antibody. (D) HUVECs
were pretreated with or without 0.1 M U0126 prior to the addition of
1 M PMA or 25 ng/ml sIL-6R and 5 ng/ml IL-6 for5hasindicated.
Soluble cell extracts equalized for protein content were then fraction-
ated by SDS-PAGE for immunoblotting with anti-SOCS-3 and
GAPDH antibodies. Veh, vehicle; pre-inc, preincubation.
V
OL. 26, 2006 cAMP/Epac INDUCTION OF SOCS-3 6343
dysfunction associated with disease. Interestingly, while others
have observed that SHP-2 is positively regulated by cAMP via
phosphorylation by PKA (35), we found that the ability of
cAMP elevation to inhibit sIL-6R/IL-6 phosphorylation of
STAT3 was markedly potentiated in cells expressing an SH2
domain-mutated SHP-2
46-110
. One explanation for this find
-
ing is that since the mutated SHP-2 in these cells is incapable
of binding phospho-Tyr759 on activated gp130, more of this
docking site is available for binding the SOCS-3 induced by
cAMP elevation. It also suggests that cellular levels of SHP-2
may be an important determinant of the magnitude with which
cAMP-mediated SOCS-3 induction can inhibit activation of
the JAK-STAT pathway.
The ability of cAMP elevation to induce SOCS protein ex-
pression has been noted for other nonendothelial cell types, as
evidenced by the strong induction we have observed with
MEFs, and that others have noted with FRTL5 thyroid cells
and neutrophils (16, 34). However, our study is the first to
show that the induction of SOCS-3 is required for the inhibi-
tion of downstream signaling to occur and the first to define
the novel PKA-independent molecular pathway responsible.
Also, while cAMP has been reported to stimulate the accumu-
lation of both SOCS-1 and SOCS-3 in FRTL-5 cells (34), we
were unable to detect any accumulation of SOCS-1 in response
to a panel of cAMP-elevating agents under conditions in which
SOCS-3 induction was readily detectable, implying that cell
type-specific differences may determine the pattern of SOCS
isoform induction. Moreover, cAMP elevation increased Tyr705
phosphorylation of STAT3 in FRTL-5 cells (34), whereas the
ability of cAMP to induce SOCS-3 in HUVECs is JAK indepen-
dent and is not associated with any detectable increase in the Tyr
phosphorylation of either STAT1 or STAT3. Thus, while the
ability of cAMP elevation to promote the accumulation of SOCS
proteins appears to be conserved between multiple cell types, the
exact mechanisms involved may vary.
Interestingly, SOCS-3 accumulation in response to cAMP
elevation in HUVECs was sustained for up to 24 h despite a
transient increase in SOCS-3 mRNA being detectable only
between 1 and 2 h (Fig. 5A and B). A similar transient appear-
ance of SOCS-3 mRNA followed by sustained accumulation of
SOCS-3 protein over 24 h has also been observed for HUVECs
stimulated with oncostatin M which, like IL-6, signals via gp130
to activate the JAK-STAT pathway (29). In fact, we found
destabilization of SOCS-3 only to be a major issue in
Val12Rap1a transfection experiments (Fig. 7C), which were
performed over a 48-h period and which necessitated the in-
clusion of MG132 to observe any reproducible accumulation of
SOCS-3 protein. The sustained accumulation of SOCS-3 ob-
served with endothelial cells contrasts with other cell types in
which the accumulation of SOCS-3 is much more transient due
to its rapid proteasomal degradation (40, 48). Degradation of
SOCS-3 is triggered, at least in part, by phosphorylation of
Tyr204 and/or -221 within the C-terminal SOCS box by either
Src family kinases (SFKs) or JAKs (48). The related protein
SOCS-1 is also sensitive to phosphorylation within its N-ter-
minal region by the Ser/Thr kinases Pim1 and Pim2, which has
the effect of stabilizing the protein (7). Finally, induction of an
N-terminally truncated SOCS-3 due to alternative transla-
tion initiation at Met12, and thus rendered resistant to poly-
ubquitylation and proteasomal degradation, is another poten-
tial mechanism by which sustained SOCS-3 protein levels
could remain elevated (40). However, we found no evidence
that cAMP elevation could promote the accumulation of de-
tectable levels of truncated SOCS-3. Instead, it is more likely
that SOCS-3 is subject to posttranslational modifications or
protein-protein interactions within endothelial cells that limit
either its phosphorylation by JAKs/SFKs or its degradation
following phosphorylation. In this respect, other investigators
have noted that the stability of SOCS-3 varies markedly in a
cell type-specific manner (40), although the factors responsible
for this variability remain uncharacterized.
Also of particular interest in our study is the observation that
the activation of ERK appeared to be sufficient to observe
SOCS-3 induction by cAMP. While the mechanism by which
Ras triggers the ERK pathway via interaction with Raf kinases
has been well characterized, the steps leading from Rap to
ERK activation are controversial; some investigators have pro-
vided evidence for a positive interaction between GTP-bound
Rap1 and B-Raf (57), while others have shown that the path-
way can potentially be more indirect, involving sequential ac-
tivation of a Rap2b/phospholipase C (PLC)-ε/RasGRP path-
way leading to the accumulation of GTP-bound Ras (25).
Rap1a is incapable of stimulating PLC-ε under conditions
where Rap2b is active (13) and so, presumably, this pathway is
not involved in SOCS-3 accumulation. It should also be noted
that a sensitivity of SOCS-3 induction to inhibition of the ERK
pathway does not necessarily exclude a contribution of other
Epac-activated events in the control of SOCS-3 accumulation.
Cumulatively, our observations support the presence of a
novel PKA-independent anti-inflammatory effect of cAMP el-
evation in vascular ECs that involves Epac-mediated accumu-
lation of GTP-bound Rap1a, leading to the ERK-dependent
up-regulation of SOCS-3, which then down-regulates receptor
activation of the JAK-STAT pathway by sIL-6R/IL-6 and
leptin. Further investigation will be required to identify the
steps linking ERK activation to the accumulation of SOCS-3
transcript and to determine any significance of the Epac/
SOCS-3 pathway in regulating the activity of other SOCS-3
binding partners, such as IRS1 (38, 49), that play critical roles
in EC function in disease states (47).
ACKNOWLEDGMENTS
This work was supported by project grants from the British Heart
Foundation, Heart Research UK, and the Biotechnology and Biolog-
ical Sciences Research Council (to T.M.P.) and Ph.D. studentships
from the British Heart Foundation (H.D.W.) and the UK Biotechnol-
ogy and Biological Sciences Research Council (G.R.M.).
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    • "Elevation of intracellular SOCS3 levels blocks insulin signaling through ubiquitin-mediated degradation of IR substrate proteins, which share signaling cascades of JAK2/Stat3 with leptin receptors [32, 33]. In human aortic endothelial cells, increased cAMP induces SOCS3 protein expression, leading to leptin inhibition and IL-6-stumulated phosphorylation of Stat3 [34]. In mouse model of insulin resistance induced by high-fat diet, liver-specific knockdown of FGF-21 resulted in increased glycogenolysis and gluconeogenesis by activating glucose-6-phosphatase and phosphoenolpyruvate carboxykinase via the Stat3/SOCS3 pathway, which ultimately led to exacerbation of hepatic insulin resistance [35] . "
    [Show abstract] [Hide abstract] ABSTRACT: Diabetes is the most common and complex metabolic disorder, and one of the most important health threats now. MicroRNAs (miRNAs) are a group of small non-coding RNAs that have been suggested to play a vital role in a variety of physiological processes, including glucose homeostasis. In this study, we investigated the role of miR-185 in diabetes. MiR-185 was significantly downregulated in diabetic patients and mice, and the low level was correlated to blood glucose concentration. Overexpression of miR-185 enhanced insulin secretion of pancreatic β-cells, promoted cell proliferation and protected cells from apoptosis. Further experiments using in silico prediction, luciferase reporter assay and western blot assay demonstrated that miR-185 directly targeted SOCS3 by binding to its 3'-UTR. On the contrary to miR-185's protective effects, SOCS3 significantly suppressed functions of β-cell and inactivated Stat3 pathway. When treating cells with miR-185 mimics in combination with SOCS3 overexpression plasmid, the inhibitory effects of SOCS3 were reversed. While combined treatment of miR-185 mimics and SOCS3 siRNA induced synergistically promotive effects compared to either miR-185 mimics or SOCS3 siRNA treatment alone. Moreover, we observed that miR-185 level was inversely correlated with SOCS3 expression in diabetes patients. In conclusion, this study revealed a functional and mechanistic link between miR-185 and SOCS3 in the pathogenesis of diabetes. MiR-185 plays an important role in the regulation of insulin secretion and β-cell growth in diabetes. Restoration of miR-185 expression may serve a potentially promising and efficient therapeutic approach for diabetes.
    Full-text · Article · Feb 2015
    • "SOCS proteins function as classical negative feedback inhibitors of cytokine signalling, since most SOCS proteins are themselves cytokine-inducible (Figure 1). Cytokines shown to induce SOCS3 include the gp130 signalling cytokines (e.g., IL-6, oncostatin M), IL-2, IL-3, IL-4, IL-10, type I and type II interferons (IFNs) and leptin as well as Toll-like receptor (TLR) agonists (e.g., lipopolysaccharide (LPS), CpG-DNA), growth hormone (GH), prolactin and cyclic AMP-mobilising hormones10111213. Upon induction, SOCS3 regulates the magnitude, kinetics, and quality of JAK/STAT signalling initiated from multiple receptors. "
    [Show abstract] [Hide abstract] ABSTRACT: SOCS3 is an inducible inhibitor of multiple pathways responsible for the neo-intimal hyperplasia (NIH) that results in failure of stenting and coronary artery bypass graft (CABG) reconstructive procedures. However the potential for SOCS3 to limit NIH is compromised by its rapid turnover by the proteasome following ubiquitylation. Our original hypothesis is that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemical analysis of human saphenous vein (HSV) sections derived from CABG patients revealed that while SOCS3 was expressed throughout the media, its expression within the neo-intima was reduced. We have shown that the L189A SOCS box mutant was ubiquitylated despite an inability to interact with the elongin B and C components of the E3 ligase machinery, suggesting that SOCS3 is not auto-ubiquitylated. Moreover, studies using a panel of SOCS3 truncation mutants revealed that SOCS3 is predominantly ubiquitylated within a C-terminal, 44 amino acid region in which Lys173 was the only potential target residue for ubiquitylation. Consistent with a role for this residue, ubiquitylation of a K173R-mutated SOCS3 mutant was markedly reduced versus WT SOCS3. K173 may therefore represent the critical site of ubiquitin attachment important for proteasomal turnover of SOCS3. Finally, we have pursued several approaches to identify the E3 ubiquitin ligase(s) and deubiquitinases that control SOCS3 sensitivity to proteasomal degradation. One of these has involved identification by mass spectrometry of proteins co-immunoprecipitating with SOCS3 and we will present the initial findings of this analysis.
    Full-text · Article · Dec 2014
    • "SOCS proteins function as classical negative feedback inhibitors of cytokine signalling, since most SOCS proteins are themselves cytokine-inducible (Figure 1). Cytokines shown to induce SOCS3 include the gp130 signalling cytokines (e.g., IL-6, oncostatin M), IL-2, IL-3, IL-4, IL-10, type I and type II interferons (IFNs) and leptin as well as Toll-like receptor (TLR) agonists (e.g., lipopolysaccharide (LPS), CpG-DNA), growth hormone (GH), prolactin and cyclic AMP-mobilising hormones10111213. Upon induction, SOCS3 regulates the magnitude, kinetics, and quality of JAK/STAT signalling initiated from multiple receptors. "
    [Show abstract] [Hide abstract] ABSTRACT: The realisation that unregulated activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is a key driver of a wide range of diseases has identified its components as targets for therapeutic intervention by small molecule inhibitors and biologicals. In this review, we discuss JAK-STAT signalling pathway inhibition by the inducible inhibitor "suppressor of cytokine signaling 3 (SOCS3), its role in diseases such as myeloproliferative disorders, and its function as part of a multi-subunit E3 ubiquitin ligase complex. In addition, we highlight potential applications of these insights into SOCS3-based therapeutic strategies for management of conditions such as vascular re-stenosis associated with acute vascular injury, where there is strong evidence that multiple processes involved in disease progression could be attenuated by localized potentiation of SOCS3 expression levels.
    Full-text · Article · Jun 2014
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