REVIEW
Regulation of Cytokine Signaling by the SOCS
and Spred Family Proteins
Akihiko Yoshimura

Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan;
Japan Science and Technology Agency (JST), CREST, Tokyo, Japan(Received for publication on September 3, 2008)(Revised for publication on October 4, 2008)(Accepted for publication on October 9, 2008)

Abstract
Various cytokines are involved in the regulation of the immune system and of hematopoiesis. Most cytokines utilize the so-called JAK-STAT pathway, but others activate the RasERK pathway, which is more important than the STAT pathway for the proliferation of hematopoietic cells. Dysregulation of cytokine signaling can cause a variety of diseases, including allergy, inflammation, and cancer. We have identified two important regulator families involved in cytokine signaling: the SOCS proteins and the Spred proteins. Suppressors
of cytokine signaling (SOCS) proteins bind to JAK and to certain receptors, thereby suppressing further signaling events. Spred family proteins interact with Ras and Raf, thereby
suppressing ERK activation. Studies have shown that SOCS and Spred proteins are key
physiological regulators of immunity, hematopoiesis, and angiogenesis. Evidence is also
emerging for the involvement of these proteins in human diseases. (Keio J Med 58 (2) : 73－
83, June 2009)

Keywords: cytokine, signal transduction, negative regulation
Introduction

JAK and/or STAT proteins; for example, pro-inflammatory cytokine IL-6 binds to the IL-6 receptor α chain andto gp130, both of which mainly activate JAK1 and
STAT3. IFNγ utilizes JAK1 and JAK2, although it mainly activates STAT1. Interestingly, anti-inflammatory cytokine IL-10 also activates STAT3. STAT4 and STAT6
are essential for Th1 and Th2 development since these
are activated by IL-12 and IL-4, respectively.
In addition, the Ras-ERK pathway is activated throughadaptor proteins such as SHP2 and Gab-1 (Fig. 1). This
pathway is essential for the proliferation of hematopoietic cells through hematopoietins including IL-3, IL-5,
erythropoietin (EPO), and granulocyte colony-stimulating factor (G-CSF).3, 4 For example, STAT3 activated by
G-CSF suppresses granulopoiesis induced by G-CSF,
while the Ras-ERK pathway is shown to play an essential role in promoting proliferation and anti-apoptosis of
granulocyte progenitors.5
Although our understanding of the intracellular signaling molecules that mediate the functional outcome of cytokine-receptor activation has increased profoundly, the

Cytokines play several essential roles in the development, differentiation, and function of myeloid and lymphoid cells. Some of them, including interleukins, interferons (IFNs), and hematopoietic growth factors, activate
the Janus kinase/Signal Transducers and Activators ofTranscription (JAK/STAT) pathway. In this pathway, cytokine binding induces receptor oligomerization, which
initiates signaling from cytokine receptors. This signaling brings associated JAK kinases (JAK1, JAK2, JAK3,
and Tyk2) into close apposition and allows their crossphosphorylation and activation (Fig. 1).1, 2 The activated
JAKs phosphorylate the receptor cytoplasmic domains,which creates docking sites for SH2-containing signaling
proteins. Among the substrates of tyrosine phosphorylation are members of the Signal Transducers and Activators of Transcription family of proteins (STATs).1, 2 Although this pathway was initially found to be activatedby IFNs, it is now known that a large number of cytokines, growth factors, and hormonal factors also activate

Reprint requests to: Akihiko Yoshimura, M.D., Department of Microbiology and Immunology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan, Tel: +81-3-5363-3483, Fax: +81-3-5360-1508, E-mail: yoshimura@a6.keio.jp73

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Yoshimura A: Cytokine Negative Regulators, SOCS and Spred

Fig. 1 Signal transduction of the cytokine receptors
Most cytokines activate receptor-associated JAK tyrosine kinases,
thereby activating STAT transcription factors and the Ras-ERK
pathway.

most recent research has placed increasing emphasis on
the mechanisms by which cytokine signals are terminated. A number of mechanisms have been proposed, including tyrosine phosphatases and transcription suppressors. In addition to these, we have previously identified
two more potential mechanisms: the large CIS/SOCS
and Spred/Sprouty families of proteins.6, 7 At the time of
their discovery, the SOCS proteins were recognized as
an important mechanism for the negative regulation of
the cytokine-JAK-STAT pathway, but recent studies using gene-disrupted (KO) mice have revealed that theyplay additional, unexpected, and profound roles in many
immunological processes. The Spred/Sprouty family
proteins, meanwhile, are more specific to the Ras-ERK
pathway, although it has been shown that they are very
important regulators for development, angiogenesis andneural networks. Furthermore, their relationship to human diseases has been recently discovered.
CIS/SOCS Family
Suppressor of cytokine signaling (SOCS) proteins andcytokine inducible SH2-containing (CIS; also known as
CISH) protein molecules comprise a family of intracellular proteins, several of which have been shown to regulate the responses of immune cells to cytokines. 8- 1 0
There are eight CIS/SOCS family proteins; CIS, SOCS1,
SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7,
each of which has a central SH2 domain, an amino-ter-minal domain of variable length and sequence, and acarboxy-terminal 40-amino-acid module known as the
SOCS box (Fig. 2A). The SOCS box is also found inother miscellaneous proteins. The SOCS box interacts
with elongin B and elongin C, cullin 5, and the RINGfinger-domain-only protein RBX2 (which recruits E2
ubiquitin–transferase).11 CIS/SOCS family proteins, aswell as other SOCS-box-containing molecules, probably
function as E3 ubiquitin ligases and mediate the degradation of proteins that are associated with these family
members through their N-terminal regions (Fig. 2A).
The best characterized SOCS-family members are CIS,
SOCS1, SOCS2 and SOCS3.
In addition to their ability to suppress signaling by
ubiquitin-mediated degradation of the signaling complex, both SOCS1 and SOCS3 can inhibit JAK tyrosine
kinase activity directly through their kinase inhibitory
region (KIR), which has been proposed to function as a
pseudosubstrate and which is important for the suppression of cytokine signals (Fig. 2).1 2 The KIR peptides
have been shown to inhibit JAK2-mediated phosphorylation of STAT1.
The central SH2 domain determines the target of each
SOCS and CIS protein. The SH2 domains of CIS,
SOCS2, and SOCS3 bind to phosphorylated tyrosine
residues on activated cytokine receptors.1 3, 1 4 SOCS3
binds to gp130-related cytokine receptors, including thephosphorylated tyrosine 757 (Tyr757) residue of gp130
and the Tyr800 residue of IL-12 receptor β2 (Fig. 2B).15
The SH2 domain of SOCS1 directly binds to the activation loop of JAKs.12 The SH2 domain of SOCS3, in contrast, does not have a high affinity to the activation loopof JAKs; yet the KIR of SOCS3 has a higher affinity to
the kinase domain of JAK2 than that of SOCS1 has.1 6
Therefore, SOCS3 might also use the same strategy offirst binding with high affinity to the receptor before inhibiting JAKs through KIR (Fig. 2B).
Physiological Functions of SOCS
SOCS1 is an essential negative regulator of IFNγ
Although SOCS1 knockout (KO) mice are normal atbirth, they exhibit stunted growth and die within 3 weeks
of birth, with a syndrome characterized by severe lymphopenia, activation of peripheral T cells, fatty degeneration and necrosis of the liver, and macrophage infiltration of major organs.17 The neonatal defects exhibited by
SOCS1–/– mice appear to occur primarily as a result ofunbridled IFNγ signaling, since SOCS1–/– mice that alsolack the IFNγ gene or the IFNγ receptor gene do not die
neonatally.10, 17 Constitutive activation of STAT1 as wellas constitutive expression of IFNγ-inducible genes wasobserved in SOCS1 KO mice. These data strongly suggest that the excess IFNγ is derived from the abnormallyKeio J Med 2009; 58 (2):73－ 83

75

Fig. 2 The structure and function of SOCS proteins(A) Schematic structure of the CIS/SOCS family proteins. The SOCS box is conserved in all CIS/SOCS family proteins. The
function of the SOCS box is the recruitment of the ubiquitin-transferase system; thus the CIS/SOCS family proteins, as well asother SOCS-box-containing molecules, function as E3 ubiquitin ligases and mediate the degradation of proteins with which they areassociated through their amino (N)-terminal regions. SOCS1 (also called JAB) and SOCS3 (also called CIS3) inhibit the tyrosine
kinase activity of JAK directly, as they contain a kinase inhibitory region (KIR) immediately upstream of the central SH2 domain,which is proposed to function as a pseudosubstrate. (B) Mechanism of suppression by CIS (also called CIS1), SOCS1 (also called
JAB), and SOCS3 (also called CIS3). All of these are induced by cytokine stimulation. CIS1 binds to the STAT5 activating receptors,
thereby suppressing further activation of STAT5. SOCS1 binds to JAKs, and SOCS3 binds to the receptor through the SH2 domain,
but both inhibit JAK activity through KIR. These complexes may be degraded by ubiquitination and proteasomal degradation recruited
through the SOCS-box.

activated T cells in SOCS1–/– mice.
Using liver-specific SOCS1-conditional knockout
mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on NKT cells
and IFNγ.18 The pro-apoptotic signals, including STAT1
and JNK activation, were enhanced in SOCS1-deficient
mice compared to those in wild-type (WT) mice. In contrast, SOCS1 overexpression in the liver by adenoviralgene transfer prevented ConA-induced liver injury bysuppressing STAT1 activation. These findings indicatethat SOCS1 plays an important negative role in fulminant hepatitis and that forced expression of SOCS1 istherapeutic in preventing hepatitis.18
We have recently demonstrated that SOCS1 is essential for helper T cell differentiation. Most SOCS1–/–CD4naïve T cells differentiated into Th1, even under skewing
conditions, while Th17 differentiation was strongly suppressed. This was also dependent on IFNγ, since Th17
was normally developed in SOCS1–/–IFNγ–/– T cells. As aresult, T cell specific SOCS1-deficient mice were very
sensitive to dextran sulfate sodium (DSS)-induced colitis(Th1 type disease)1 9 but resistant to experimental autoimmune encephalomyelitis (EAE), a typical Th17 typedisease.20 Taken together, these data indicate that SOCS1
negatively regulates IFNγ signaling in various types ofcells, thus fulfilling an important function for the suppression of Th1-type inflammatory diseases.
Physiological functions of SOCS3 defined by gene targeting at various tissues
SOCS3 knockout mice die during the embryonic stageof development due to placental function defects. In other words, deletion of SOCS3 causes embryonic lethality;
these embryos can be saved, however, by a tetraploid
rescue approach. These observations demonstrate
SOCS3’s essential role in placental development andnon-essential role in embryo development. Rescued
SOCS3-deficient mouse embryos exhibit prenatal lethality with cardiac hypertrophy, which suggests that SOCS3
is essential for regulating either LIF receptors or gp130
signaling.21
Conditional-KO mice studies have demonstrated that

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Yoshimura A: Cytokine Negative Regulators, SOCS and Spred

SOCS3 is an important negative regulator of IL-615 andG-CSF.22 Mice in which the SOCS3 gene was deleted inall hematopoietic cells developed neutrophilia and aspectrum of inflammatory pathologies. When stimulatedwith G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3
activation and enhanced cellular responses to G-CSF;2 2
SOCS3-deficient mice injected with G-CSF in vivo displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed
inflammatory neutrophil infiltration into multiple tissues
and consequent hind-leg paresis. Interestingly, conditional STAT3-deletion in neutrophils also resulted in hyper-response to G-CSF,23 suggesting that a major role of
STAT3 in neutrophils is the induction of SOCS3. It
seems likely that the ERK pathway induced by G-CSF
play a major role in the proliferation and survival of neutrophils.
The essential roles of SOCS3 in endocrine systems
have also been clarified in recent years. Administrationof leptin to neural cell-specific SOCS3 conditional KO
mice greatly reduces their food intake and causes enhanced body weight loss compared to WT mice, indicating that SOCS3 in the brain negatively regulates leptin
signaling.2 4 Moreover, the SOCS3-deficient mice wereresistant to high fat diet-induced weight gain and hyperleptinemia, and their insulin-sensitivity was retained.24, 25
These data indicate that SOCS3 is a key regulator of diet-induced leptin as well as of insulin resistance. In addition, SOCS3 deficient adipocytes generated from SOCS3
KO fibroblasts are significantly protected from TNF-α-induced insulin resistance, mainly due to reduced proteasomal degradation of IRS proteins by TNF-α, suggesting that SOCS3 is an important mediator of insulin resistance in vivo.26 Consistent with these ideas is the observation that the loss of SOCS3 in the liver apparently improved insulin sensitivity.27 Unexpectedly, however, liver-specific SOCS3 cKO mice exhibited obesity and systemic insulin resistance with age, due to constitutive activation of STAT3 which mimics chronic inflammation.
Collectively, these results indicate that SOCS3 can be apotential therapeutic target for human metabolic disorders such as obesity and diabetes, although long-term
treatment may cause inconvenient side effects.
SOCS and Immunity
The role of SOCS1 in innate immunity and inflammatory
diseasesToll-like receptor (TLR) signals that initiate innate immune responses to pathogens must be tightly regulated
to prevent excessive inflammatory damage in the host.
TLR ligands, such as LPS and CpG DNA, are potent inducers of SOCS1 and SOCS3; therefore the role of

SOCS1 and SOCS3 in TLR responses has been extensively investigated.
SOCS1-deficient mice are hypersensitive to LPS,
which leads to increases in tumour necrosis factor (TNF)
and IL-12 production.2 8 The hyperactivation of macrophages in SOCS1–/– mice might be due in part to a stronger responsiveness of SOCS1–/– cells to IFNγ comparedwith wild-type cells. However, since IFNγ–/–SOCS1–/–
mice are still sensitive to LPS-induced shock, IFNγ-independent mechanisms probably exist. A direct effect of
SOCS1 on the TLR–NF-κB (nuclear factor-κB) pathway
has been proposed.2 9 Moreover, Kimura et al. indicatethat LPS can activate JAK2 and STAT5, which are involved in IL-6 induction, and that SOCS1 selectively inhibits this process.30
SOCS1 also negatively regulates LPS- and IL-4-induced dendritic cell (DC) maturation. SOCS1-deficientDCs secrete larger amounts of IFNγ, IL-6, IL-12, andTNF in response to LPS and CpG compared with wildtype cells.31, 32 SOCS1-deficient DCs express higher levels of MHC class II and co-stimulatory molecules. 3 2Therefore, SOCS1 must be deeply involved in the development, maturation, and activation of DCs.
These SOCS1-deficient DCs seem to be responsible
for the development of systemic autoimmunity in aged
SOCS1-deficient mice. Immunization with normal DCs
can activate autoreactive T cells but rarely causes autoimmune pathology, indicating that self-tolerance is still
maintained in the vaccinated hosts. However, an adoptive transfer of SOCS1–/– DCs to wild-type recipients resulted in the induction of autoantibodies through enhanced expression of B-cell-activating factor (BAFF) by
the donor DCs.3 1 This breaking of self-tolerance might
be due to hyper-production of IL-12 by SOCS1-deficientDCs.3 2 These data indicate that SOCS1 is an essential
negative regulator for T-cell activation by DCs and forthe maintenance of immunological tolerance.
Depletion of the SOCS1 protein in DCs, therefore,
may enhance anti-tumour immunity. Indeed, we haveshown that adoptive transfer of antigen-loaded SOCS1–/–
BMDCs can prevent B16 melanoma growth in mice.3 2Similarly, Chen and colleagues reported that silencing
SOCS1 using small interfering RNA (siRNA) technology
in antigen-presenting DCs strongly enhanced antigenspecific anti-tumour immunity.33 A transfer of DCs treated with ovalbumin (OVA)-pulsed SOCS1-siRNA enhanced the proliferation and function of OVA-specificcytotoxic T cells (CTLs) compared with control DCs.
TLR signaling pathways are essential for macrophagedependent inflammatory bowel diseases, and SOCS1 has
an important role in the development of colitis. SOCS1–/–
Tcra–/– mice develop very severe colitis and die within
two months of birth.35 This very severe colitis is dependent on both IFNγ and IL-4. In addition, NF-κB and
MAP kinases were strongly activated in the colons of

Keio J Med 2009; 58 (2):73－ 83

these mice; interestingly, however, this severe colitis wascompletely abolished when the mice were derived in
germ-free conditions. This is consistent with previous
observations that eventually all colitis models in miceoccur in non-germ-free conditions. These findings suggest that TLR signaling is prerequisite for pro-inflammatory cytokine production and that these two signals mustbe mutually activated to promote colitis. Furthermore,
SOCS1 has an important role in the negative regulationof both the JAK/STAT signaling cascade and the TLR
pathway during the development of inflammatory bowel
diseases.
SOCS3 and regulation of TLR signaling
SOCS3 has now been shown to be a key regulator forthe divergent activity of IL-6 and IL-10 following TLR
stimulation.1 5 IL-6 is a pro-inflammatory cytokine thatassumes a progressive role in many inflammatory diseases, while IL-10 is an immunoregulatory cytokine thathas potent anti-inflammatory activity, including the suppression of gene activation through TLR signaling pathways. While it was known that STAT3 is essential for the
biological actions of both IL-6 and IL-10, it was unclear
how these two cytokines could have such precisely opposing functions. SOCS3 protein is strongly induced byboth IL-6 and IL-10 in the presence of LPS, but IL-6 signaling is selectively inhibited due to the binding of
SOCS3 to the IL-6R subunit gp130 (Tyr759), but not to
the IL-10 receptor (IL-10R). Therefore, STAT3 activation is transient in response to IL-6, but is sustained for along period in response to IL-10. Furthermore, a gp130
mutant lacking the SOCS3 binding site (Tyr759Phe), as
well as any heterologous cytokine receptors that are mutated to induce sustained STAT3 activation by deleting
the SOCS3 binding sites, can elicit an anti-inflammatory
effect indistinguishable from that induced by IL-10.
Therefore, the anti-inflammatory response is a genericcytokine signaling pathway dependent on STAT3 but notunique to the IL-10. This idea is consistent with recent
studies showing that constitutively activated STAT3(STAT3c) is sufficient for the suppression of LPS-induced TNF and IL-6 production in macrophages. We
proposed that this sustained activation of STAT3 is essential for the anti-inflammatory effect, while transient
activation of STAT3 promotes inflammation.
SOCS3 and DC-mediated T-cell differentiation
Recently, we examined the antigen-presenting cell(APC) function of SOCS3-deficient DCs. Like SOCS3–/–
macrophages, SOCS3–/– DCs showed constitutive activation of STAT3 and expressed low levels of MHC class II
molecules, co-stimulatory molecules, and IL-12.36 Surprisingly, adoptive transfer of SOCS3–/– DCs suppressed

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EAE. SOCS3 –/– DCs are poor activators of effector
CD4＋ T cells, but they selectively expand forkhead box
P3 (FOXP3)＋ regulatory T cells (Tregs), which can suppress EAE. We have shown that IL-10 and TGFβ1 aretarget genes of STAT3.3 7, 3 8 FOXP3＋ T-cell expansion
can be blocked by TGFβ-specific antibody, and indeed
SOCS3 –/– DCs produced higher levels of TGFβ thanwild-type DCs did. Thus, high STAT3 activation in DCs
without SOCS3 results in increased production of IL-10
and TGFβ, thereby inducing FOXP3＋ Tregs. By contrast, reduced STAT3 expression due to the overexpression of SOCS3 in SOCS3-transduced DCs results in reduced production of IL-12, IFNγ, and IL-23, thereby inducing Th2 cells.39 These results suggest that the expression of SOCS3 by DCs might have a crucial role in thebalance between effector Th2 cells and Tregs.
SOCS3 and inflammatory diseases
A growing body of evidence suggests that, in pathological situations, SOCS3 could suppress inflammatory
reactions in which IL-6-related cytokines play important
progressive roles. This is because SOCS3 is a relatively
specific inhibitor of gp130, as described above. STAT3
activation and high SOCS3 expression levels have beenfound in epithelial and lamina propria cells in the colonof intestinal bowel disease (IBD) model mice, as well as
in human ulcerative colitis and CD patients40 and in synovial fibroblasts of RA patients.4 1 STAT3 activation
preceded SOCS3 expression, which is consistent with
the idea that SOCS3 is part of the STAT3 negative-feedback loop. 4 0 We have shown that overexpression of
SOCS3 by adenovirus gene transfer could prevent thedevelopment of experimental arthritis.4 1 Therefore, theIL-6/STAT3 pathway promotes the progression of thechronic status of diseases by contributing to cytokine andgrowth factor production, tissue hyperplasia, synovial fibroblast proliferation, fibrosis, and osteoclast activation.
Modulation of the gp130/JAK/STAT pathway therefore
represents a reasonable strategy for new anti-inflammatory drug development. In addition, STAT3 is now
known to play an essential role in the development ofTh17 cells, which are extremely inflammatory andpathogenic. SOCS3 has been shown to negatively regulate Th17 development by suppressing STAT3 activatedby IL-6 or IL-23.2 0, 4 2 Thus, again, enforced expressionof SOCS3 in T cells may also ameliorate Th17-mediatedinflammatory diseases such as RA.
In contrast, enhanced action of SOCS3 may promoteallergic responses, since a recent analysis has indicatedthat transgenic SOCS3 expression in T cells inhibits Th1development and promotes Th2 development.4 3 Indeed,
the same report also proposes that the degree to which
SOCS3 expression in T cells is increased correlates withthe severity of human allergic diseases such as asthma

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Yoshimura A: Cytokine Negative Regulators, SOCS and Spred

Fig. 3 Role of SOCS1 and SOCS3 in inflammation-associated tumourigenesis
Two-step carcinogenesis model associated with inflammation. STAT1/SOCS1 are involved in the early stage of inflammation whichleads to tissue damages, resulting in increased cellular turnover. NO and ROS from inflammatory cells may induce DNA damage,which increases the possibility of the emergence of cells possessing a high risk of malignant transformation. STAT3/SOCS3 are
involved in the late stage, in which tumour promotion occurs by cellular and extracellular signals activated by cytokines frominflammatory cells or stromal cells. This step raises immortalized cells that are resistant to growth-inhibitory signals, apoptosis, andanti-tumour immunity.

and atopic dermatitis. Modulation of SOCS3 levels in T
cells could help to regulate Th1/Th2 balance for thetreatment of autoimmune inflammatory diseases.
SOCS and Inflammation-associatedTumourigenesis
It has been estimated that more than 20% of all malignancies are initiated or exacerbated by inflammation; forexample, most human hepatocellular carcinomas (HCCs)
are a consequence of hepatitis C virus (HCV) infection.
The expression of SOCS1 is often silenced in these tumours by hypermethylation of CpG islands of the SOCS1
promoter.44 SOCS1 is one of the most frequently methylated genes (65%) in HCCs, and the deletion of SOCS1 intumour cells might enhance IL-6-mediated cell proliferation. Therefore, SOCS1 is considered to be an anti-oncogene because it is a suppressor of signals induced by agrowth factor (IL-6). SOCS1＋/– mice are consistently
shown to be hypersensitive to dimethylnitrosamine-induced hepatocarcinogenesis.44
The full story, however, may not be so simple. We
found that silencing of SOCS1 was frequently observed
even in pre-malignant HCV-infected patients.44 Liver injury is associated with hyperactivation of STAT1 and reduced activation of STAT3.1 8, 4 5 Therefore, reduced expression of SOCS1 might enhance tissue injury and inflammation by hyperactivation of STAT1, promoting theturnover of epithelial cells and enhancing their susceptibility to oncogenesis. The importance of SOCS1 for in-hibition of inflammation-associated tumour development
is supported by the recent finding that SOCS1–/– transgenic mice, in which SOCS1 expression is deleted in all
types of cells except T and B cells, developed chronic
colitis and colon tumours.46 This study strongly suggeststhat chronic activation of the IFNγ–STAT1 pathway that
occurs in the absence of SOCS1 causes colitis-inducedcolon tumours. Therefore, SOCS1 is a unique anti-oncogene that prevents carcinogenesis by suppressing chronic
inflammation (Fig. 3).
SOCS3 might also be involved in the development andprogression of malignancies. Unlike SOCS1, SOCS3 expression levels were high in infected non-tumour areasof patients infected with HCV.45 Reduced expression of
SOCS3 has been observed in various human cancers andis associated with constitutive STAT3 activation.4 7 Indeed, the levels of SOCS3 were inversely correlated with
STAT3 activation in regions of human livers with andwithout HCC.4 5 Numerous studies have shown that hyperactivation of STAT3 can contribute to tumourigenesis
by inducing multiple tumour-promoting genes.47
Thus, we propose a two-hit model of inflammationassociated tumourigenesis (Fig. 3). In this model, initiation occurs in the form of an activating or disabling mutation in one of the molecules that regulate the cellular
circuits and capacitors controlling cell division, survival,
and senescence. Persistent inflammation leads to tissue
damage, resulting in increased cellular turnover. Nitricoxide (NO) and reactive oxygen species (ROS) from inflammatory cells may induce DNA damage, which in-Keio J Med 2009; 58 (2):73－ 83

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Fig. 4 Structure of the Spred/Sprouty family proteins and relationship to NCFC syndrome(A) Conserved N-terminal tyrosine motifs of Sproutys and other domains are illustrated. S/T rich: serine/threonin rich region, SPR:
Sprouty-related region, KBD: c-kit-binding domain, EVH1: Ena/VASP homology 1, WASP: Wiskott-Aldrich syndrome protein.
(B) The growth factor-induced Ras-ERK pathway is illustrated and the component proteins involved in each syndrome are shown.

creases the possibility of the emergence of cells possessing a high risk of malignant transformation. STAT1 plays
a positive role in non-tumour inflammatory regions inthis early stage, and SOCS1 silencing in pre-tumour cells
results in strong and persistent STAT1 activation, which
induces apoptosis and tissue damage, leading to DNA
damage and cell regeneration which may promote theemergence of malignant cells. Then, promotion occurs
by cellular and extracellular signals activated by cytokines from inflammatory cells or stromal cells, leading
to immortalized cells that are resistant to growth-inhibitory signals, apoptosis, and anti-tumour immunity. Constitutive STAT3 activation in tumour cells contributes toan expansion of tumour cells by promoting cell proliferation, survival, angiogenesis, and tissue remodeling.
SOCS3 silencing is one of the mechanisms for constitutive STAT3 activation. However, the mechanism of the
reduction of SOCS3 expression in tumours has not been
established.
Spred and Sprouty
Finding of the Spred/Sprouty family
Sprouty was originally identified in Drosophila as anegative regulator of FGF (fibroblast growth factor) signaling during tracheal development. Now it is regarded

as a general inhibitor of the growth factor-induced RTK(receptor tyrosine kinase)-dependent Ras/Raf/ERK signaling pathways involved in development and organogenesis. In mammals, four Sprouty orthologues havebeen identified (Sprouty1, -2, -3, and -4).4 8 In addition,
we have found three Sprouty-related genes, which wecall Spreds (Sprouty-r―elated E
na/VASP homology 1 ―do―――
main-containing proteins) (Fig. 4A).7, 4 9 Spreds bind to
Ras and Raf, thereby suppressing activation of Raf. 7
Gene targeting and overexpression studies have demonstrated that mammalian Sproutys also inhibit growth factor-induced cellular responses by inhibiting the RTK-dependent ERK signaling pathway (Fig. 4B).50-52
Spred and hematopoiesis
Spred-1 has been implicated in hematopoiesis eversince it was observed that bone marrow-derived mast
cells and eosinophils from Spred-1–/– mice were moresensitive to IL-3 and IL-5, respectively, than those from
WT mice.53, 54 In Spred-2–/– mice, embryonic hematopoiesis in the aorta-gonad-mesonephros (AGM) region wasenhanced compared with that observed in WT mice.55
Spred-1–/– adult animals appeared to be healthy andshowed no apparent abnormalities in most organs. Studies in a murine allergic asthma model of Spred-1-deficient mice demonstrated that Spred-1 negatively regu-

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lates allergen-induced airway eosinophilia, hyperresponsiveness, and mucus production, without affecting helper
T cell differentiation. Biochemical assays demonstrated
that Spred-1 suppresses IL-5-dependent cell proliferationand ERK activation. This indicates that Spred-1 negatively controls eosinophil numbers and functions bymodulating IL-5 signaling in allergic asthma.54
Spred-1 deficient mice developed myeloproliferative
diseases with age. RT-PCR analysis showed high levelsof Spred-1 expression in hematopoietic stem cells andbone-marrow-derived mast cells (BMMCs), erythroid
cells, and B-cells, but low levels in megakaryocytes andmacrophages. Spred-1 is expressed in a particular subset
of mature hematopoietic cells and is inducible by IL-3.
In IL-3 dependent Ba/F3 cells expressing c-kit, forced
expression of Spred-1 resulted in a reduced proliferation
rate and ERK activation in response to not only SCF but
also IL-3.5 6 In Spred-1-deficient bone-marrow-derived
mast cells, proliferation and ERK/MAP kinase activation
was increased in response to IL-3 or SCF.5 6 Therefore,
Spred-1 inhibits not only growth-factor-induced ERK
activation but also cytokine-induced ERK activation.
Spred and lymphangiogenesis
Although the individual physiological roles of Spred-1
and Spred-2 have been investigated using gene-disrupted
mice, the overlapping functions of Spred-1 and Spred-2
have not been clarified. We demonstrate that the deletionof both Spred-1 and Spred-2 resulted in embryonic lethality at E12.5-15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled witherythrocytes.5 7 The phenotype of these mice resembled
that of Syk–/– and SLP-76–/– mice, with defects in the separation of lymphatic vessels from blood vessels. The
numbers of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells were markedly increased in
Spred-1/2 -deficient embryos compared with wild-type
embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or
differentiation. In cultured cells, the overexpression of
Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor(VEGFR)-3-mediated ERK activation, while Spred-1/
2-deficient cells were extremely sensitive to VEGFR-3
signaling. Thus, Spreds play an important role in lymphatic vessel development by negatively regulating
VEGF-C/VEGFR-3 signaling. Interestingly, microRNA
miR-126 has been shown to enhance the proangiogenic
actions of VEGF and FGF, and to promote blood vessel
formation by repressing the expression of Spred-1.5 8Thus, Spreds may also be involved in the regulation ofangiogenesis.

Yoshimura A: Cytokine Negative Regulators, SOCS and Spred

Spred-1 and human diseases
Neurofibromatosis type 1 (NF1), or von Recklinghausen disease, is an autosomal dominant condition characterized by multiple café-au-lait spots, axillary freckling,
Lisch nodules in the iris, and tumours of the nervous system. Other frequently observed features are short stature,
macrocephaly, and learning and behavioral problems.
Most NF1 is caused by inactivating mutations in the NF1tumour suppressor gene encoding neurofibromin, a positive regulator of RAS inactivation. NF1 was the first human disorder shown to originate from germline mutations in a gene encoding a component of the RAS-ERK
pathway. Subsequently, mutations in genes encoding
other components of this pathway were implicated indisorders showing some phenotypic overlap with NF1,
for example, PTPN11, KRAS, and SOS1 were implicatedin Noonan syndrome, PTPN11 in LEOPARD syndrome,
HRAS in Costello syndrome, and KRAS, BRAF, MEK1,
and MEK2 in cardio-facio-cutaneous (CFC) syndrome(Fig. 4B). These disorders, now known as the ‘neurocardio-facial-cutaneous’ (NCFC) syndromes, present
with a variable degree of cognitive impairment, facial
dysmorphism, congenital heart defects, and skin abnormalities.59
We reported germline loss-of-function mutations in
SPRED1 resulting in a newly identified autosomal dominant human disorder (Fig. 5A).60 The clinical features of
the reported disorder resemble those of neurofibromatosis type 1; they consist of multiple café-au-lait spots, axillary freckling, and macrocephaly. All mutations arefound to be the loss-of -function type (Fig. 5B). To ourknowledge, this is the first report of mutations in thisfamily of genes causing human disease, and the existence and nature of this disorder strongly support our notion that Spred is a negative regulator of the RAS-ERK
pathway.
Furthermore, Spreds are now considered to be potential tumour suppressors, as is NF-1. It has been reported
that Spred-1 and Spred-2 expression was reduced in human HCC.61 In addition, overexpression of Spred-1 canefficiently suppress tumourigenesis in nude mice.62 Further study is underway to identify mutations or loss ofexpression of Spreds/Sproutys in cancer and to establish
Spreds as a therapeutic target.
Concluding Remarks
In the past decade, following the discovery of the
SOCS and Spred protein families, we have extended ourunderstanding of the structure and function of these proteins. SOCS proteins not only act as simple negativefeedback regulators, but also play a part in the fine tuning of the immune response and in the cross-talk of thecomplicated cytokine signal networks. Spreds/Sproutys

Keio J Med 2009; 58 (2):73－ 83

81

Fig. 5 Mutations in SPRED1 gene found in NF1 patients (A) and suppression of the FGF-induced Raf1 kinase activity bymutant Spred1 proteins (B). 293 cells transfected with various mutant SPRED1 genes were stimulated with FGF, then Raf1 was
immunoprecipitated. In vitro Raf1 kinase assay was performed using MEK1 as substrate (B).

are also very important for development, hematopoiesis,
angiogenesis, and neurogenesis. Since various cytokines
and stimuli are constantly in the microenvironment of
immune cells, hematopoietic cells, neural cells, and endothelial cells, signal regulation by those proteins andother regulators must be important to maintain the proper
homeostasis. Therefore, further investigation into thefunction of SOCS and Spred proteins might provide us
with an unexpected role for these proteins in the regulation of signaling pathways in life.
Acknowledgements
We thank Ms. Y. Nishi for preparing the manuscript.
This work was supported by special grants-in-aid from
the Ministry of Education, Science, Technology, Sports,
and Culture of Japan and the Program for Promotion of
Fundamental Studies in Health Sciences of the National
Institute of Biomedical Innovation (NIBIO).
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