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RESEARCH ARTICLE
The role of osteopontin in inflammatory processes
Susan Amanda Lund &Cecilia M. Giachelli &
Marta Scatena
Received: 15 May 2009 / Accepted: 9 September 2009 /Published online: 2 October 2009
#The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract Osteopontin (OPN) is a matricellular protein that
mediates diverse biological functions. OPN is involved in
normal physiological processes and is implicated in the
pathogenesis of a variety of disease states, including
atherosclerosis, glomerulonephritis, cancer, and several
chronic inflammatory diseases. Through interactions with
several integrins, OPN mediates cell migration, adhesion,
and survival in many cell types. OPN also functions as a
Th1 cytokine, promotes cell-mediated immune responses,
and plays a role in chronic inflammatory and autoimmune
diseases. Besides its function in inflammation, OPN is also
a regulator of biomineralization and a potent inhibitor of
vascular calcification.
Keywords Inflammation .Matricellular protein .
Osteopontin
Abbreviations
DC Dendritic cell
IFN Interferon
MMP Matrix metalloproteinase
OPN Osteopontin
PBMCs Peripheral blood mononuclear cells
Introduction
OPN is a secreted phosphorylated glycoprotein that
mediates diverse biological functions. Originally isolated
from bone, OPN was later shown to have a wider
distribution (Brown et al. 1992). In adults, OPN expression
is normally limited to the bone, kidney, and epithelial
linings, and is secreted in bodily fluids including milk,
blood and urine (Chen et al. 1993). In contrast to its
restricted distribution in normal tissue, OPN is strikingly
upregulated at sites of inflammation and tissue remodeling
(Liaw et al. 1998;O’Brien et al. 1994). OPN exists both as
a component of the extracellular matrix and as a soluble
cytokine. Physiologically OPN is thought to regulate
biomineralization in bone tissue, and to reduce growth
and aggregation of calcium crystals in epithelial tissues
(Wesson et al. 2003). OPN has also been implicated in a
variety of disease states, where it mediates diverse cellular
functions such as adhesion, migration, and survival of
several different cell types, including regulating and
propagating inflammatory responses of macrophages, T-
cells, and dendritic cells. The pleiotropic nature of OPN
may reflect the various isoforms, post-translational mod-
ifications, and diversity of cell types which OPN can
interact with. Clinically, OPN plasma levels are correlated
with chronic inflammatory diseases such as Crohn’s disease
(Agnholt et al. 2007), cancer (El-Tanani et al. 2006),
atherosclerosis, aortic abdominal aneurysms (Golledge et
al. 2007), and autoimmune diseases including lupus
(Kariuki et al. 2009), multiple sclerosis (Comabella et al.
2005), and rheumatoid arthritis (Sennels et al. 2008). In this
review we will focus on the role of OPN in inflammation
biology.
OPN structure
OPN was originally isolated from bone and was later
independently identified as secreted phosphoprotein I
S. A. Lund :C. M. Giachelli :M. Scatena (*)
Department of Bioengineering, University of Washington,
Box 358056, Seattle, WA 98195, USA
e-mail: mscatena@u.washington.edu
J. Cell Commun. Signal. (2009) 3:311–322
DOI 10.1007/s12079-009-0068-0
(SppI) and early T-lymphocyte activation 1 (Eta-1) (Senger
et al. 1989; Patarca et al. 1989). OPN is a member of the
SIBLING (Small Integrin-Binding Ligand, N-linked Gly-
coprotein) family of proteins, which map to human
chromosome 4 (Fisher et al. 2001). OPN is synthesized as
an approximately 32 kDa protein, but due to extensive post-
translational modifications its apparent molecular mass
ranges from 45 to 75 kDa (Kazanecki et al. 2007). OPN
possesses a negative charge due to a preponderance of
acidic amino acids and serine phosphorylation. OPN also
contains calcium binding sites and two putative heparin
binding domains (Kon et al. 2008). OPN can interact
directly with extracellular matrix proteins including fibro-
nectin (Mukherjee et al. 1995) and collagen type I (Chen et
al. 1992; Martin et al. 2004).
Adhesion motifs
OPN has multiple functional adhesive motifs, which allows
interactions with many cell types including smooth muscle
cells, endothelial cells, and inflammatory cells, thus
mediating a broad range of biological functions. The OPN
protein is poorly conserved among species (63% human to
mouse, 30% human to chicken) however its functional
domains are conserved. The highly conserved motifs
include: abundance of acidic residues, the RGD integrin
binding domain, similar phosphorylation and glycosylation
motifs, and at least one site of controlled proteolysis
(Bellahcene et al. 2008). The adhesive RGD domain of
OPN mediates interactions via α
v
β
1,
α
v
β
3
,α
v
β
5,
α
v
β
6,
α
8
β
1
, and α
5
β
1
integrins (Liaw et al. 1995; Yokosaki et al.
2005; Denda et al. 1998; Hu et al. 1995). Immediately C-
terminal to the RDG motif is a cryptic SVVYGLR
(SLAYGLR in mice) sequence that becomes exposed upon
cleavage with thrombin and mediates interactions with
α
9
β
1,
α
4
β
1
, and α
4
β
7
integrins (Yokosaki et al. 1999; Ito et
al. 2009; Green et al. 2001). Finally, the ELVTDFTDLPAT
(human OPN) domain of OPN has also been described to
bind to α
4
β
1
(Bayless and Davis 2001).
In addition to interacting with integrins, OPN has also
been reported to interact with CD44, the hyaluronic acid
receptor (Weber et al. 1996). A number of different CD44
isoforms exist due to alternative splicing of the 10 variant
exons and while OPN can bind some CD44 splice variants,
notably v6 and v7, OPN does not bind to the standard
isoform, CD44H (Smith et al. 1999; Katagiri et al. 1999).
Further, interactions between OPN and CD44 appears to be
mediated via β
1
integrins in an RGD independent manner
(Katagiri et al. 1999). Other studies indicate interactions
between the C-terminal fragment of thrombin cleaved OPN
and a CD44 variant (Weber et al. 2002). However, the
precise domain of OPN that interacts with CD44 has not
been identified. In addition, growing evidence suggests that
OPN is a major regulator of CD44 surface expression,
especially in osteoclasts (Chellaiah et al. 2003; Marroquin
et al. 2004).
Post-translational modifications
OPN is subject to extensive post-translational modifica-
tions including serine and threonine phosphorylation.
Phosphorylation is cell specific with phosphorylation
levels varying depending on the tissue type. OPN in
milk is highly phosphorylated with human milk OPN
containing 36 phosphate sites (Christensen et al. 2005).
Phosphorylation tends to occur in clusters separated by
stretches of unmodified residues. Normal rat kidney cells
can secrete both phosphorylated and non-phosphorylated
forms, indicating regulated control of phosphorylation
(Singh et al. 1990). In some cases, OPN function is tightly
controlled by phosphorylation state. Calcification of
smooth muscle cells in vitro is inhibited by native OPN,
but desphosphorylated or recombinant bacterially pro-
duced OPN has no effect on calcification (Jono et al.
2000). Similarly, in vivo in OPN-null mice, phosphorylat-
ed OPN, but not non-phosphorylated, prevents ectopic
calcification in a subcutaneous model of bioprosthetic
valve mineralization (Ohri et al. 2005). In contrast, the
adhesive activity of OPN is not dependent on post-
translational modifications, since bacterially derived re-
combinant OPN has been shown to support cell adhesion
in a wide variety of cell types (Gao et al. 2004;Xuanetal.
1994). OPN is also subject to sulfation (Nagata et al.
1989), glycosylation (Sorensen et al. 1995)andtrans-
glutamination (Beninati et al. 1994). Interestingly, poly-
merization of OPN by transglutaminase 2 has been
reported to increase the adhesive activity of OPN via the
α
9
β
1
integrin, independent of the SVVYGLR adhesion
domain (Nishimichi et al. 2009). Precise regulation of
OPN post-translational modifications may represent a
mechanism to control OPN function.
Proteolytic processing
The bioactivity of OPN can be further regulated by
proteolytic processing. OPN is a substrate for thrombin
and the matrix metalloproteinases, MMP-3 (stromelysin-1),
MMP-7 (matrilysin), MMP-2 and MMP-9 (Agnihotri et al.
2001; Dean and Overall 2007; Takafuji et al. 2007). Human
OPN contains three cleavage sites for MMPs; Gly
166
-
Leu
167
, Ala
201
-Tyr
202
, and Asp
210
-Leu
211
. The thrombin
cleavage site generating a RGD and SVVYGLR
(SLAYGLR in mice) is conserved in human and mice.
Thrombin cleavage of OPN, Arg
168
-Ser
169
in humans and
Arg
153
-Ser
154
in mice, reveals a cryptic SVVYGLR binding
domain capable of fostering interactions with α
9
β
1
(Smith
312 S.A. Lund et al.
and Giachelli 1998; Smith et al. 1996) and α
4
β
1
(Bayless
and Davis 2001) integrins. Thus, the known adhesive
functional domains of OPN are located in the thrombin
cleaved N-terminal fragment. Little is known about the role
of C-terminal fragments; however, as described above, it
has been suggested to contain a CD44 binding domain
(Weber et al. 2002; Takafuji et al. 2007).
Proteolytic processing may represent a way to locally
regulate the function of OPN as the functional properties of
cleaved OPN differ from those of the intact molecule. Of
particular interest, rather than mediating degradation and
inactivating OPN-mediated functions, proteolytic process-
ing of OPN can increase the biological activity of the
molecule (O’Regan et al. 1999). OPN and MMPs are co-
localized during wound healing and tumorigenesis, indicat-
ing there may be an in vivo role for proteolyzed forms of
OPN (Senger et al. 1994). Few studies suggest that OPN
fragments may play a functional role in vivo. Enhanced
production of the thrombin cleaved form of OPN is found
in the synovial fluid of patients with rheumatoid arthritis
(Ohshima et al. 2002) and the cryptic SLAYGLR motif was
found to play an essential role in a murine and a primate
model of rheumatoid arthritis (Yamamoto et al. 2003,
2007). An antibody against the SLAYGLR sequence
inhibited inflammatory cell influx into arthritic joints and
attenuated the severity of disease. The human OPN derived
peptide SVVYGLR has also been found to induce
angiogenesis in vitro and in vivo (Hamada et al. 2003,
2007). Several in vitro studies have demonstrated that the
N-terminal fragments generated both by thrombin cleavage
and MMP cleavage induced enhanced adhesion when
compared to the full length molecule. This appears to be
due mostly to increased activity of the RGD site, perhaps
an indication of conformational change resulting in higher
affinity binding (Senger and Perruzzi 1996; Smith and
Giachelli 1998; Agnihotri et al. 2001). The SVVYGLR
cryptic domain exposed following thrombin cleavage is
also able to induce adhesion and migration through the α
4
and α
9
integrins (Smith and Giachelli 1998). However, the
α
9
-dependent adhesion and migratory functions are com-
pletely lost in the N-terminal MMP generated fragment, as
Arg
168
seems to be required for α
9
-dependent binding
(Yokosaki et al. 2005; Ito et al. 2009). In contrast, α
4
-
dependent adhesion and migratory functions are only
partially lost in the N-terminal MMP generated fragment
(Ito et al. 2009). The C-terminal fragment of OPN
generated by thrombin and MMP cleavage does not contain
any integrin adhesive domains, it does not mediate
adhesion when presented in immobilized form to cells
and, in contrast, it appears to suppress OPN mediated
adhesion and migration in monocyte-derived cells (Gao et
al. 2004; Smith et al. 1996; Maeda et al. 2001; Takahashi et
al. 1998)
Intracellular OPN
An intracellular form of OPN (iOPN) has been reported to
be expressed in dendritic cells and macrophages (Shinohara
et al. 2006,2008a,b; Zohar et al. 2000). Studies by
Shinohara et al. suggest that the intracellular form of OPN
is generated due to translation initiation downstream of the
usual start site in bone marrow-derived DCs and transfected
293T cells. Utilization of this downstream start site
generates the truncated iOPN form that lacks the N-
terminal signal sequence and consequently localizes to the
cytoplasm, where it may associate with TLR9 and the
MyD88 adaptor molecule (Shinohara et al. 2008a).
The interaction of iOPN with MyD88 appears to activate
the transcription factor IRF7 and to induce expression of
IFN-αultimately leading to Th1 cell-mediated immunity
and pro-inflammatory responses (Shinohara et al. 2006).
The same group has also suggested that iOPN expression in
conventional DCs is permissive for Th17 T cell responses
(Shinohara et al. 2008b). Th17 T cells are a subset of T
helper cells producing IL-17. They are considered devel-
opmentally distinct from Th1 and Th2 cells and are thought
to play a key role in autoimmune diseases including the
tissue injury associated with these conditions (Steinman
2007). Thus iOPN expression may allow for autoimmune
type disease progression. Indeed, OPN accelerates the
progression of experimental autoimmune encephalomyelitis
(EAE), a model of multiple sclerosis (Chabas et al. 2001;
Hur et al. 2007; Jansson et al. 2002; Shinohara et al. 2006,
2008b). Others have found that iOPN may associate with
the intracellular domain of CD44 and with the ezrin/
radixin/moesin (ERM) protein ezrin. iOPN in this context
may modulate cytoskeletal rearrangements important for
macrophage migration and osteoclast fusion (Zhu et al.
2004; Zohar et al. 2000).
A summary of the structural features of OPN is shown in
Fig. 1.
The role of OPN in inflammation
OPN regulates the immune system at many different levels. It
serves as a chemotactic molecule to promote the migration of
inflammatory cells to the wound site and acts as an adhesive
protein to retain cells at the site. OPN also functions as a pro-
inflammatory cytokine and can modulate the immune
response by enhancing expression of Th1 cytokines and
matrix degrading enzymes (Weber et al. 2002; Bruemmer et
al. 2003). OPN plays a pivotal role in T cell and macrophage
responses during cell mediated immune responses against
bacterial and viral pathogens (Ashkar et al. 2000). More
recently, OPN has also been shown to modulate dendritic
cell responses and neutrophil chemotaxis.
The role of osteopontin in inflammatory processes 313
Macrophages
OPN is not expressed in circulating monocytes, but is
dramatically upregulated during macrophage differentiation
and constitutes one of the major macrophage products
(Krause et al. 1996). OPN is known to be induced in
macrophages by several inflammatory cytokines, including
TNF-α,IL-1β,IFN-γ, and IL-6, and other factors
including angiotensin-II, oxidizedLDL, and phorbol-ester
are known inducers of OPN in macrophages (Nakamachi et
al. 2007; Ogawa et al. 2005; Bruemmer et al. 2003). More
recently, Liver X Receptor and Peroxisome proliferator-
activated receptor αantagonists have been shown to
suppress OPN expression (Nakamachi et al. 2007; Ogawa
et al. 2005). Functionally OPN plays a key role in
macrophage biology by regulating migration, survival,
phagocytosis, and pro-inflammatory cytokine production
(Bruemmer et al. 2003; Nyström et al. 2007).
We and others have shown that OPN serves as a potent
chemoattractant for macrophages (Bruemmer et al. 2003;
Persy et al. 2003; Giachelli et al. 1998; Panzer et al. 2001).
Functional inhibition of OPN and genetic ablation of OPN
in mice greatly impair macrophage recruitment in several
models of acute inflammation. In OPN-null mice acute
macrophage infiltration was greatly diminished com-
pared to wild-type mice in an obstructed kidneys model
(Ophascharoensuk et al. 1999), and in a thioglycollate
induced peritonitis model (Bruemmer et al. 2003). Further,
purified OPN induced macrophage accumulation when
injected in rat dermis and following intradermal injection
of N-formyl-met-leu-phe (FMLP), a potent macrophage
chemotactic peptide. The OPN effect was neutralized by
an anti-OPN blocking antibody (Giachelli et al. 1998). In a
rat model of heart injury OPN was highly expressed in the
granulation tissue associated macrophages, and it was
downregulated with healing progression and formation of
the fibrotic scar (Murry et al. 1994). Wound healing studies
in mice also indicate that OPN is expressed during the
acute inflammatory phase at very high levels in infiltrating
leukocytes and other cell types where it appears to regulate
leukocyte infiltration and activation as well as proper matrix
organization (Liaw et al. 1998). Interestingly, downregulation
of OPN at the wound site with antisense mRNA diminished
macrophage infiltration and accelerated wound healing
(Mori et al. 2008).
OPN also modifies chronic inflammatory responses.
Chronic inflammation is characterized by the persistence
of macrophages at sites of injury and disease. Deficits in
macrophage accumulation have been noted in OPN-null
mice when challenged with chronic inflammatory condi-
tions, including atherosclerosis, delayed-type hypersensi-
tivity (Yu et al. 1998; Ashkar et al. 2000), granulomatous
disease (Weber et al. 2002; Nau et al. 1999), and
biomaterial implantation (Steitz et al. 2002; Tsai et al.
2005). These data suggest that OPN may be particularly
important in promoting migration and retention of macro-
phages at sites of acute and chronic inflammation. We have
also shown that OPN regulates foreign body giant cell
(FBGC) formation in vitro and in vivo. In a recent paper we
described that despite the defect in macrophage recruitment,
OPN-null mice formed more FBGCs on the surface of the
implant. In vitro, OPN inhibited macrophage fusion to form
FBGCs in a dose dependent manner (Tsai et al. 2005).
In vitro, OPN-null macrophages exhibit reduced basal
migration and impaired migration towards MCP-1, despite
the fact that wild type and OPN-null macrophages express
comparable levels of CCR-2, the MCP-1 receptor. This may
be a consequence of a lack of a permissive pro-migratory
substrate, and the reduced expression of CD44 observed in
OPN-null macrophages. CD44 is well known to be
essential for macrophage migration (Marcondes et al.
2008), and its expression is upregulated by OPN in
macrophages (Chellaiah et al. 2003). Macrophages from
OPN-null mice are also more susceptible to programmed
cell death (Bruemmer et al. 2003). Together with impaired
migration, macrophage apoptosis may further contribute the
N
H2COOH
1169 300
Ca2+ binding
domain
RGD
(αvβ3, αvβ5,
αvβ1, α8β1)
Thrombin cleavage site
SVVYGLR
(α9β1, α4β1, α4β7)
MMP cleavage site
(CD44)
P P P P P P P P P PP
Ca2+ binding
domain
Fig. 1 OPN structural features.
The cell adhesive domains are
indicated in color. The known
specific integrin receptors for
each adhesion domain are also
shown in color. The calcium
(Ca
2+
) binding domains are
shown in yellow and other ma-
trix binding domains are in
black. Also shown are known
phosphorylation sites (P).
Arrows indicate known cleavage
sites for thrombin and MMPs.
Thrombin cleavage site in blue
and MMPs cleavage site in
green
314 S.A. Lund et al.
impaired macrophage accumulation observed in OPN-null
mice in response to acute and chronic inflammatory stimuli.
In addition to regulating macrophage migration, OPN
can also modulate the cytokine production by macro-
phages. OPN stimulates production of IL-12 while
inhibiting the production of IL-10, thereby promoting
Th1 cell mediated responses (Weber et al. 2002;Ashkar
et al. 2000). Interestingly, these results were mediated by
different receptors. IL-12 production was mediated via an
N-terminal fragment interaction with α
v
β
3
integrin, while
IL-10 was inhibited via a C-terminal fragment, possibly
via the CD44 receptor. OPN regulation of IL-12 and IL-10
were also demonstrated in vivo in an angiotensin II
(AngII)-accelerated model of atherosclerosis. In this
model, Bruemmer at al. showed reduced expression of
IL-12 and an increased expression of IL-10 in ApoE-/-
OPN-/- AngII treated mice compared to ApoE-/-OPN+/+
AngII treated control mice by RT-PCR of whole mouse
aortas. These results correlated with less macrophage rich
lesions and lower expression of the macrophage marker
CD68 (Bruemmer et al. 2003).
While OPN is generally classified as a pro-inflammatory
cytokine, it also appears to have anti-inflammatory effects.
OPN is a potent trans-repressor of inducible nitric oxide
synthase (iNOS) expression in macrophages (Rollo et al.
1996). OPN represses inducible nitric oxide synthase
(iNOS) by increasing Stat1 ubiquitination and proteasome
mediated degradation of Stat1, consequently inhibiting
Stat1 mediated iNOS transcription and protein expression
(Gao et al. 2007). NO feedback inhibits its own synthesis
by increasing transcription of OPN (Guo et al. 2001). OPN
inhibition of NO may be particularly relevant for tumor cell
evasion of inflammation (Wai et al. 2006; Crawford et al.
1998).
Finally, a recent study suggests that OPN may play a role in
macrophage differentiation. Using siRNA to stably silence the
expression of OPN in RAW 264.7 cells, Nystrom et al.
showed that OPN silenced cells displayed an altered pheno-
type with monocyte-like characteristics (Nystrom et al. 2007).
Further, OPN silenced cells had decreased expression of
macrophage scavenger receptor A type 1 (Msr-1), a
macrophage differentiation marker. While these studies are
intriguing, the phenotype could not be rescued by the
addition of exogenous OPN suggesting non-receptor medi-
ated effects or iOPN may be involved.
Together, these data suggest that OPN may be
particularly important in promoting migration and
retention of macrophages at sites of acute and chronic
inflammation by regulating multiple macrophage func-
tions. These studies also emphasize the importance of
macrophage-derived OPN in the regulation of OPN’s
functions, suggesting that macrophages are both a
source and target of OPN.
Neutrophils
While much is known about the role of OPM
in macrophage biology, relatively few studies have ex-
plored the function of OPN in neutrophils. Neutrophils
express low levels of OPN (Koh et al. 2007). However,
OPN is important for the recruitment and migration of
neutrophils, as neutrophils from OPN-null mice display
reduced chemotaxis toward fMLP and in vivo the recruit-
ment of neutrophils to the peritoneal cavity in response to
sodium periodate is impaired in OPN-null mice (Koh et al.
2007). OPN-null mice also have impaired neutrophil
infiltration into liver when challenged with concanavalin
A induced hepatitis (Diao et al. 2004). Despite these defects
in migration and chemotaxis, OPN-null neutrophils do not
display reduced destructive capacity in terms of phagocy-
tosis, the generation of reactive oxygen species, or cytokine
production (Koh et al. 2007). Recent reports indicate that
polymeric OPN interacts with α
9
β
1
on neutrophils and
serves as a potent neutrophil chemoattractant (Nishimichi et
al. 2009).
T-cells
OPN is also known as Eta-1 (early T lymphocyte activation
gene 1) for its high expression in activated T cells and it
plays an important role in the induction of cell mediated
immune responses through the regulation of T cells.
Following activation, naïve CD4 T cells can differentiate
towards Th1, Th2, or Th17 cells which differ in effector
function. The development of Th1 cells leads to cell-
mediated immunity while development of Th2 cells
provides humoral immunity. Th17 cells are associated with
autoimmunity. OPN is not expressed in naïve T-cells but it
is strongly upregulated in response to T cell receptor
ligation (Shinohara et al. 2005). OPN functions in T cells
by mediating migration, adhesion, and co-stimulating T cell
proliferation (O’Regan et al. 1999; Patarca et al. 1993).
Shinohara et al. have shown that OPN gene expression in T
cells is controlled by T-bet, a transcription factor that
promotes CD4+ T helper cell lineage commitment to Th1
(Shinohara et al. 2005). Further, T-bet-dependent expres-
sion of OPN in T cells is essential for efficient skewing of
CD4 T and CD8 T cells toward the Th1 and type 1 CD8 T
cell (Tc1) pathway, respectively (Shinohara et al. 2005). In
vivo, OPN-null mice display impaired Th1 responses to the
intracellular bacterium Listeria monocytogenes and the viral
pathogen HSV1 (herpes simplex virus type 1) both of
which depend on the induction of IL-12 for protection
(Ashkar et al. 2000). Indeed, mice deficient in OPN have
decreased IL-12 and IFN-γproduction, while IL-10 levels
are enhanced (Bruemmer et al. 2003). Further studies have
shown that OPN regulates CD3-mediated T cell expression
The role of osteopontin in inflammatory processes 315
of IFN-γand CD40L (O’Regan et al. 2000), which in turn
stimulates IL-12 production from leukocytes. Together
these findings suggest that OPN may play a role in
polarizing early Th1 responses. More recently, OPN has
also been shown to regulate IFN-γand IL-17 production by
T cells in an α
v
β
3
-dependent manner and to dampen IL-10
in a CD44-dependent manner (Murugaiyan et al. 2008).
This appears to be important for the progression of
experimental autoimmune encephalomyelitis (EAE), a
murine model of multiple sclerosis. The same authors
found that during EAE, OPN expression was elevated in
DCs both in the periphery and in the CNS (Murugaiyan et
al. 2008). This correlated with the increased expression of
OPN in DCs and increased expression of OPN receptors
CD44 and α
v
β
3
on T cells isolated from patients affected
by multiple sclerosis (Murugaiyan et al. 2008). These
results may indicate that OPN produced by DCs in EAE is
linked to the production of IL-17 (Murugaiyan et al. 2008;
Shinohara et al. 2008b). Recent studies indicate that OPN
enhances the survival of autoreactive T cells in a NF-κB-
dependent manner. Enhanced OPN-induced T cell survival
also appears to promote progression of EAE (Hur et al.
2007). In addition, an anti-OPN antibody has been shown
to promote apoptosis of activated T cells, particularly CD4+
T cells, by inhibiting activation of NF-κB and by altering
the balance between the proapoptotic proteins, Bim and
Bax, and the antiapoptotic protein, Bcl-2, in a model of
rheumatoid arthritis (Fan et al. 2008).
Finally, a subset of lymphocytes bridging innate and
adaptive immunity called invariant natural killer T (iNKT)
cells has also been shown to be regulated by OPN. Diao et
al. found that the number of peripheral iNKT cells was
significantly reduced in OPN-deficient mice compared with
wild-type mice. This appeared to be consequence of
impairment of intrathymic iNKT cell maturation and
significant alteration of iNKT cell function as well (Diao
et al. 2004).
Dendritic cells
OPN plays a key role in DC maturation, migration, and
polarization. Interestingly, immature IL-4 and GM-CSF
differentiated DCs express high levels of OPN, and OPN
production decreases when DCs are stimulated with LPS
and CD40L (Kawamura et al. 2005; Schulz et al. 2008).
These findings suggest differential regulation of OPN in
DCs and macrophages. During macrophage differentiation,
OPN expression increases as monocytes adhere and
differentiate into macrophages (Krause et al. 1996).
In vitro OPN stimulates DC migration in a dose
dependent manner in both the presence and absence of
divalent cations, and studies with blocking antibodies have
indicated a role for the OPN receptors α
v
β
3
and CD44 in
OPN induced DC migration (Weiss et al. 2001). In vivo,
DCs in OPN-null mice display a defect in DC trafficking to
the lymph nodes resulting in reduced contact hypersensi-
tivity responses (Weiss et al. 2001). Despite the influence of
OPN on DC migratory capacity, treatment with recombi-
nant OPN does not affect the expression of CCR5 and
CCR7, chemokine receptors which are involved in DC
migration (Schulz et al. 2008). Recently, it has also been
suggested that OPN modulates different subset of DCs in
the airway hypersensitivity reaction (a model of asthma). It
appears that OPN increased the reaction during primary
sensitization but decreased the reaction in the challenge
phase, perhaps by mediating differential recruitment of
different DCs (plamacytoid and conventional) subsets to the
lymph nodes (Xanthou et al. 2007).
OPN also influences DC cytokine production. In co-
culture systems, OPN induces DC secretion of TNF-αand
IL-12p70 which stimulates secretion of IFN-γby T-cells
(Renkl et al. 2005). By augmenting DC production of IL-
12, OPN can enhance Th1 polarization.
As mentioned earlier, recent studies illustrate the critical
role of intracellular OPN in IFN-αproduction by plasma-
cytoid DCs, a specialized subset of DCs which produce
high levels of type I interferons upon stimulation
(Shinohara et al. 2006). IFN-αproduced by pDCs activates
NK cells, and consequently OPN deficient mice display
impaired IFN-αdependent natural killer cell responses.
Further, iOPN appears to decrease IL-27 in conventional
DCs, leading to increases in Th17 responses (Shinohara et
al. 2008b).
Other biological functions of OPN
Wound healing
OPN is a key cytokine regulating tissue repair. OPN is
present at sites of wound healing where it serves as a
chemotactic molecule to recruit inflammatory cells to the
site of injury. Wound healing studies in OPN-null mice
have elucidated the role of OPN in tissue repair. Compared
to wild type mice, incisional wounds made in OPN-null
mice displayed alterations in the matrix architecture
especially collagen fiber diameter, and had more residual
debris (Liaw et al. 1998). Furthermore, macrophages at the
wound site in OPN-null mice expressed higher levels of
mannose receptor, suggesting OPN may contribute to
macrophage polarization and thus regulate healing
responses. Mannose receptor expression in macrophages is
associated with reduced pro-inflammatory (IL-1, IL-6,
IL-12, and TNF-α) cytokine secretion, upregulation of
pro-healing molecules (IL-10 and TGF-β), and certain
phagocytic receptors (Sica et al. 2008).
316 S.A. Lund et al.
A more recent study by Mori et al. explored the use of
local OPN knockdown at the site of wound healing by
delivering antisense oligodeoxynucleotides from a drug
delivery polymer gel. Consistent with the finding in OPN-
null mice, in OPN knockdown wounds the diameter of
collagen fibrils was also smaller than in control wounds.
Further, OPN knockdown hindered the migration of
inflammatory cells to the wound site and resulted in
accelerated healing and a reduction in granulation tissue
formation and scarring (Mori et al. 2008). Whether the
infiltrated macrophages in OPN knockdown wounds also
had a less pro-inflammatory and more pro-healing pheno-
type was not established, however, this may be behind the
observed accelerated healing. The reduced size of the
collagen fibers that was observed in the OPN-null and
OPN knockdown wounds may be related to the finding that
OPN has been shown to bind directly to collagen type I
(Chen et al. 1992), and to interact with collagen types II,
III, IV, and V (Bulter et al. 1995). Further, it has recently
been shown that OPN is necessary for TGF-β1-induced
myofibroblast differentiation and OPN-null fibroblasts
exhibited less spreading, less resistance to detachment,
and a reduction in collagen gel contraction (Lenga et al.
2008). These studies indicate a role for OPN in promoting
proper collagen organization and regulating ECM and
myofibroblast interactions.
Vascular disease
To date, several studies have investigated the role of OPN in
the progression of atherosclerosis. In hyperlipidemic apoE-
deficient mice, Matsui et al, showed that osteopontin
deficiency significantly reduces atherosclerotic lesion size in
female ApoE-/-OPN-/- mice compared to ApoE-/-OPN+/+
mice after 36 weeks on a normal chow diet (Matsui et al.
2003). Similarly, studies in ApoE/LDLreceptor/OPN triple
knockout mice showed that OPN deficiency resulted in
decreased atherosclerotic lesion size and an increase in the
number of apoptotic cells in lesions (Strom et al. 2004).
Bone marrow transplantation studies in an angiotensin II-
accelerated model of atherosclerosis indicated that leukocyte
derived OPN contributes to OPN mediated development of
atherosclerosis (Bruemmer et al. 2003). These studies
suggest that OPN promotes macrophage accumulation and
retention in the atherosclerotic lesions, thus contributing to
the chronicity of the disease.
OPN also modulates other vascular cells associated with
vascular disease. In human atherosclerotic lesions, OPN is
expressed in smooth muscle cells (SMC), angiogenic endo-
thelial cells, and macrophages and it is re-expressed in SMCs
associated with human restenotic lesions (Giachelli et al.
1993; Panda et al. 1997). Consistently, animal models have
confirmed the role of OPN in promoting SMC migration and
proliferation (Liaw et al. 1994; Isoda et al. 2002). All these
data indicate that during injury, OPN enhances the prolifer-
ation, migration, and accumulation of smooth muscle and
endothelial cells involved in repair and remodeling processes
of the vasculature.
Cancer
OPN is highly expressed in transformed cells and is found
in a variety of cancers (Wai and Kuo 2008). OPN
overexpression can confer metastatic phenotype to non-
metastatic, benign transformed cells, and increased OPN
expression correlates with tumor progression, poor progno-
sis and increased invasiveness. In metastatic models, OPN
has been shown to induce matrix proteases MMP2 and uPA
in an integrin-dependent manner (Mi et al. 2006). Further,
OPN has been shown to bind and activate MMP3. Thus, the
ability of OPN to stimulate migration and matrix break-
down could contribute to invasiveness and to the metastatic
potential of tumors cells. OPN may also promote tumori-
genesis and metastasis by inhibiting apoptosis of tumor
cells (Zhao et al. 2008), and by stimulating neovasculariza-
tion (Wai and Kuo 2004). Finally, OPN is widely expressed
by macrophages, which infiltrate tumor tissue (Brown et al.
1994; Chambers et al. 1996). Macrophage-derived OPN
functions as a chemoattractant and was associated with
reduced tumor burden while tumor-derived OPN appeared
to inhibit macrophage function and enhance tumor growth
(Crawford et al. 1998). It is possible that tumor cell-derived
OPN may enhance cancer cell survival by downregulating
iNOS expression and NO production in macrophages. OPN
is currently being studied as a potential biomarker for
cancer and there is interest in targeting OPN as a
therapeutic treatment for cancer.
Biomineralization
OPN is one of the most abundant non-collagenous proteins
in bone. Because of its abundance in bone, OPN has been
studied as a regulator of biomineralization. OPN is a potent
inhibitor of mineralization, prevents ectopic calcification,
and is an inducible inhibitor of vascular calcification (Steitz
et al. 2002). OPN binds hydroxyapatite and calcium ions
thereby physically inhibiting crystal formation and growth
in vivo.Studies in OPN-null mice have shown that OPN-/-
bones are hypermineralized, with increased mineral content
and crystal size (Boskey et al. 2002). OPN also plays a role
in osteoclast differentiation and osteoblast recruitment and
function (Rittling et al. 1998). OPN functions in osteoclast
migration to sites of resorption and is crucial for normal
resorption and bone turnover (Chellaiah et al. 2003).
OPN appears also to be an important regulator of vascular
calcification and is associated with mineralized deposits in
The role of osteopontin in inflammatory processes 317
humans (Giachelli et al. 1993). In mice, OPN levels are
greatly elevated in the spontaneously mineralizing arteries of
MGP
-/-
mice and we have recently shown that OPN is major
inducible inhibitor of arterial medial calcification in this
system (Steitz et al. 2002). Vascular calcification is now
recognized as a marker of atherosclerotic plaque burden as
well as a major contributor to loss of arterial compliance and
increased pulse pressure seen with age, diabetes, and renal
insufficiency. These findings suggest that OPN may be an
important inhibitor of arterial mineral deposition under
conditions of injury and disease, and that strategies to
replenish OPN might be useful to prevent or treat ectopic
calcification, including vascular calcification.
Conclusions
OPN is emerging as a key regulator of immune cell biology.
Most of the evidence indicates that OPN is transiently
expressed in leukocytes during acute inflammation. How-
ever, persistence of OPN expression by immune cells
exacerbates chronic inflammatory diseases. Clinically, this
is manifested by increased OPN plasma levels in Crohn’s
disease (Agnholt et al. 2007), cancer (El-Tanani et al.
2006), atherosclerosis, and autoimmune diseases including
lupus (Kariuki et al. 2009), multiple sclerosis (Comabella et
al. 2005), and rheumatoid arthritis (Sennels et al. 2008).
Mechanistically, OPN appears to regulate innate immune
cells (macrophages and DCs) and adaptive immune cells
(T cells) at multiple levels (see Fig. 2). Recent data point to
a role for OPN in the regulation of cross-talk between DCs
and T cells and their subsequent polarization in Th1 and
more recently in Th17 cells.
In vitro and in vivo studies show that both the thrombin
and MMP proteolytically cleaved OPN fragments possess
higher activity than the full-length form. In addition, at least
the thrombin cleaved fragment also gains a new cell
interacting domain (SVVYGLR). Antibodies specifically
reacting toward the SVVYGLR (human and primate) or the
SLAYGLR (murine) sequences have been shown effective
in ameliorating rheumatoid arthritis symptoms in non-
human primates and mice (Yamamoto et al. 2003,2007).
INFγ Th1 polarization
OPN
DCs
T-Cells
macrophages
migration
activation
survival
Accumulation
IL-12
IL-10
Th1 polarization
migration
activation
IL-12 TNFα
Th1 polarization
survival
IL-17
Th17 polarization
EAE autoimmunity
iOPN
INFα
Th1 polarization
OPN fragments
N-terminal OPN
SVVYGLR
Rheumatoid
Arthritis
Angiogenesis
C-terminal OPN
?
migration
proliferation
Fig. 2 OPN regulation of immune and inflammatory cells. OPN is
secreted and modulates the function of macrophages, DCs and T
Cells. OPN may induce macrophage accumulation by promoting
migration and survival. Further, OPN induces IL-12 and inhibits IL-10
in macrophages, thus, propagating a Th1 response. In DCs OPN
appears to modulate their function as an extracellular soluble cytokine
and also as an intracellular molecule (iOPN). Both OPN forms appear
to induce Th1 polarization. Extracellular OPN appears to induce
expression of IL-12 and TNF-α, and iOPN appears to regulate the
production of INF-α. In T cells OPN induces migration, proliferation,
survival, and IL-17 secretion. These two latter functions have been
correlated with Th17 responses and autoimmunity. Further, OPN
appears to induce IFN-γsecretion by T cells thus propagating a Th1
response. Finally, the N-terminal OPN fragment (containing the
activated adhesive domain SVVYGLR) may be important in the
propagation of rheumatoid arthritis
318 S.A. Lund et al.
Therefore, during inflammation, it is likely that the
secreted, less potent, full-length OPN is rapidly cleaved
and thus activated. Understanding differences in the
mechanisms and structure/function relationships governing
the proinflammatory properties of OPN could help create
specific therapeutics aimed at targeting chronic inflamma-
tory diseases selectively.
Acknowledgements Dr. Giachelli’s laboratory is funded by NIH
grants HL62329, HL081785, and HL18645, and grant #2361524 from
the Washington State Life Science Discovery Fund.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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