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The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 9 1049
PERSPECTIVE SERIES
E. Helene Sage, Series Editor
Matricellular proteins
Expressed during many stages of development in a vari-
ety of organisms, the matricellular protein SPARC
(secreted protein acidic and rich in cysteine, also known
as osteonectin or BM-40) is restricted in adult vertebrates
primarily to tissues that undergo consistent turnover or
to sites of injury and disease (1). The capacity of SPARC
to bind to several resident proteins of the ECM, to mod-
ulate growth factor efficacy, to affect the expression of
matrix metalloproteinases, and to alter cell shape as a
counteradhesive factor, supports the idea that SPARC
acts to regulate cell interaction with the extracellular
milieu during development and in response to injury
(Figure 1; see also ref. 1). SPARC is a member of a gene
family whose members share structural similarities in one
or more protein domains (1). In addition to the numer-
ous studies in cultured cells, the function of SPARC in
vivo has been examined primarily in three evolutionarily
diverse organisms — Caenorhabditis elegans, Xenopus laevis,
and mice. These systems have been used to study the
effects of increased or inappropriate SPARC expression,
as well as diminished activity resulting from the inactiva-
tion of SPARC mRNA, the blocking of protein activity, or
mutation of the SPARC gene (Table 1). This Perspective
will integrate results from studies in vitro with findings
in vivo in an attempt to clarify the current information
and to propose functions for SPARC in living tissues.
SPARC in ECM organization
Vertebrate SPARC binds to a number of different ECM
components including thrombospondin 1, vitronectin,
entactin/nidogen, fibrillar collagens (types I, II, III, and
V), and collagen type IV, the prevalent collagen in base-
ment membranes (1). Therefore, SPARC has the poten-
tial to contribute to the organization of matrix in con-
nective tissue as well as basement membranes.
Interestingly, SPARC is expressed abundantly in base-
ment membranes and in capsules that surround a vari-
ety of organs and tissues. In this regard, SPARC-null
mice display early cataractogenesis, a phenotype with
100% penetrance (2). Transmission electron microscopy
of lens epithelial cells in SPARC-null mice shows an
intrusion of cellular processes into the basement mem-
brane of the lens capsule, whereas wild-type lens epithe-
lial cells exhibit a precise border at the cell-matrix inter-
face (3). We have proposed that this phenotype reflects
aberrant cell behavior or differentiation resulting from
altered composition or structure of the basement mem-
brane formed in the absence of SPARC.
SPARC, a matricellular protein that functions
in cellular differentiation and tissue response to injury
Amy D. Bradshaw and E. Helene Sage
Department of Vascular Biology, The Hope Heart Institute, Seattle, Washington, USA
Address correspondence to: E. Helene Sage, The Hope Heart Institute, 1124 Columbia Street, Suite 720, Seattle, Washington 98104, USA.
Phone: (206) 903-2026; Fax: (206) 903-2044; E-mail: hsage@hopeheart.org.
Figure 1
Structure of SPARC protein. A ribbon diagram
derived from crystallographic data shows the
three modular domains of SPARC. Representa-
tive activities attributed to each domain are
shown beneath the designated amino acids. The
follistatin-like domain contains the peptide 2.1
(see ref. 1) shown in green and the (K)GHK
angiogenic peptide (amino acids 114–130)
shown in black. The E-C domain contains the
amino acids 255–274 (peptide 4.2) shown in yel-
low. Modified from Hohenester et al. (29) and
the Brookhaven Protein Database, accession no.
1BM0 from Ref. 1, with permission.
With respect to connective tissue, preliminary trans-
mission electron microscopy of dermal collagen fibers
also revealed differences between wild-type and SPARC-
null mice. Whereas collagen fibrils from wild-type skin
exhibit a variety of large and small diameters, as
observed previously in normal adult animals, SPARC-
null fibrils are smaller and more uniform in diameter
(A.D. Bradshaw et al., unpublished results). Differences
in collagen fibril size are consistent with our primary
observation that the skin of SPARC-null mice is more
easily stretched and weaker in tensile strength than
that of wild-type mice. Apparently the absence of
SPARC affects collagen fibrillogenesis, most likely dur-
ing development, although confirmation of this idea
awaits completion of experiments in which a develop-
mental time course of collagen fibril assembly will be
analyzed. Whether SPARC acts to affect fibrillogenesis
directly through its collagen-binding capacity or by
another mechanism is unknown. However, in connec-
tive tissues of mov-13 mice, which are deficient in col-
lagen I, SPARC is not distributed in specific matrices
that are known sites of SPARC deposition in wild-type
embryos (1). In developing Xenopus, SPARC appears to
be concentrated within the intersomitic furrows, a loca-
tion known to be rich in collagen fibers (4).
Further evidence for the importance of SPARC in con-
nective tissue is found in the curly tails of SPARC-null
mice, a characteristic reminiscent of thrombospondin
2–null mice (5). Thrombospondin 2 is another matri-
cellular protein with potential collagen-binding activi-
ty, as thrombospondin 2–null mice also display aberrant
collagen fibrils in the skin and an abnormally flexible
tail (5). In fact, a variety of ECM-associated components
have been implicated in collagen fibrillogenesis by virtue
of the phenotypic abnormalities observed in transgenic
mice with targeted deletions of the genes for decorin,
fibromodulin, lumican, and osteopontin, among others
(6–8). Since some of these proteins and proteoglycans
are known to affect collagen fibrillogenesis in vitro, phe-
notypic abnormalities in col-
lagen fibrils were not unex-
pected in these animals.
Others, such as SPARC and
thrombospondin 2, proved
to be more surprising. Clear-
ly the assembly and regula-
tion of collagen fibril size is
a complex process about
which a great deal remains
to be learned.
Similar to vertebrate
SPARC, C. elegans SPARC is
encoded by a single gene,
ost-1. Although there are
both structural and func-
tional differences between
vertebrate and nematode
SPARC, such as a reduced affinity for Ca2+, binding to
both collagen types I and IV is conserved (1). SPARC in
C. elegans is expressed primarily by body wall and sex
muscle cells, although SPARC protein is also associat-
ed with the basement membrane of the pharynx, a
tissue in which SPARC mRNA is not detected (9). A
similar disparity between sites of synthesis and depo-
sition has been noted in C. elegans for collagen IV, and
in mouse, for SPARC (1). Thus, nematode SPARC
appears to be transported extracellularly to basement
membranes at some distance from its sites of synthe-
sis, an observation suggesting a function for this pro-
tein in matrix organization or activity.
The capacity of SPARC to bind to a number of differ-
ent ECM proteins provides a basis for the association of
SPARC with both basement membranes and fibrous
connective tissue. During development, when many
ECMs are being laid down, higher levels of SPARC are
observed (1). Subsequently, the expression of SPARC is
restricted to sites of ECM turnover and is virtually unde-
tectable in normal cells within their established ECM.
In fact, Damjanovski et al. observed in Xenopus embryos
that expression of SPARC decreased precipitously upon
morphological differentiation of specific tissues (10).
Interestingly, SPARC is a substrate of transglutami-
nase, an enzyme that establishes covalent cross-links
between proteins (11). Although the function of tissue
transglutaminase in the stabilization of the ECM is not
completely understood, strong evidence for its impor-
tance in matrix assembly and cell interaction with ECM
is emerging (12). Possibly, expression of SPARC in
response to injury or tissue remodeling is necessary to
facilitate production of an ECM permissive for cell
migration, proliferation, and differentiation. SPARC
might substitute for other transglutaminase substrates
that provide structural support in the ECM: for exam-
ple, cross-linking of SPARC instead of fibronectin,
another substrate of transglutaminase, could result in
a more malleable matrix.
1050 The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 9
PERSPECTIVE SERIES
E. Helene Sage, Series Editor
Matricellular proteins
Table 1
Phenotypic abnormalities resulting from inactivation of SPARC in vivo
Organism Technique Phenotype Reference
C. elegans cRNA interference Embryonic lethality 9
Lack of gut granules
Nonfunctional gonads
X. laevis Antibody block Bent and shortened embryonic axes 28
Abnormal eye development
Mus musculus Transgenic-null Cataractogenesis 2 (and references therein)
Accelerated dermal wound healing A.D. Bradshaw et al., unpublished
Aberrant dermal collagen fibrils A.D. Bradshaw et al., unpublished
Curly tails
Osteopenia 17
Increased fat deposition A.D. Bradshaw and E.H. Sage,
unpublished
SPARC and growth factors
SPARC binds to and decreases the mitogenic potency
of both PDGF and VEGF, in part by its abrogation of
growth factor–receptor interaction (1, 2). In addition,
SPARC can counteract the proliferative capacity of
bFGF on smooth muscle cells, although physical inter-
action of SPARC with bFGF remains to be determined
(1, 2). Furthermore, addition of SPARC to myoblasts
promotes differentiation through inhibition of bFGF-
induced proliferation (K. Motamed et al., unpublished
results). That SPARC binds ECM components and
modulates the growth factor–induced proliferation
(and, in at least one case, differentiation) of at least
three separate mitogens adds another dimension to the
function of SPARC in the regulation of cell behavior.
In addition to PDGF, VEGF, and bFGF, SPARC can
also modulate the activity of TGF-β, as seen in recent
studies with SPARC-null mesangial cells. In culture,
these cells express decreased levels of TGF-βmRNA
and protein in comparison with wild-type mesangial
cells (1). Addition of recombinant SPARC to either
wild-type or SPARC-null cells increases TGF-βmRNA
and protein and is associated with an increase in colla-
gen I production. Interestingly, TGF-βcan interact
with the ECM through the binding of latent TGF-β
binding protein (LTBP-1), which is cross-linked to the
ECM in a transglutaminase-dependent manner and
facilitates activation of latent TGF-βin endothelial
cells (13). The lack of SPARC might lead to aberrant
assembly of ECM that in turn affects the localization
and subsequent activation of TGF-β.
SPARC as a counteradhesive protein
The capacity for SPARC to modulate growth factor
activity has far-reaching implications for many aspects
of cell behavior that include proliferation, migration,
and differentiation. Perhaps equally important, how-
ever, is the counteradhesive activity associated with
SPARC. For over a decade, SPARC has been appreciat-
ed as a modifier of cell shape (1). Addition of purified
SPARC to many cell types in culture induces cell round-
ing, a process thought to be distinct from the inhibi-
tion of cell cycle by SPARC. Cell rounding activity is
sensitive to tyrosine kinase inhibitors, whereas inhibi-
tion of proliferation depends in part upon a pertussis
toxin–sensitive pathway, at least in vascular smooth
muscle cells (1). Focal adhesions dissociate upon addi-
tion of purified SPARC to endothelial cell cultures,
although the mechanism by which SPARC influences
cell shape is not completely understood (14). Whether
SPARC acts upon cell surfaces through a specific recep-
tor or acts by blocking adhesive interactions is unclear.
Indeed these possibilities might each apply in different
situations and would thereby allow cells of various
types to respond to SPARC in a characteristic manner
(see Murphy-Ullrich, this Perspective series, ref. 15).
Cell shape, like growth factor activity, can influence
cell proliferation, migration, and differentiation. The
evidence that SPARC acts to modulate such process-
es in vivo is becoming substantially more credible,
based on experiments performed in C. elegans, Xenopus,
and transgenic mice that do not express SPARC. The
counteradhesive function of SPARC appears to be
conserved in the diverse organisms that express the
protein. Thus, overexpression of SPARC in C. elegans
leads to an uncoordinated phenotype that probably
reflects the compromised ability of the body wall
muscle cells to attach to the adjacent basement mem-
brane and to generate force (1). Huynh et al. (16) have
tested the effects on vertebrate development of a pep-
tide representing the COOH-terminal Ca2+-binding
domain of SPARC (Figure 1, E-C domain). This pep-
tide mimics the full-length protein in its ability to
provoke changes in cell shape (1). When injected into
developing Xenopus embryos, it caused the rounding
of migrating mesodermal cells and severe develop-
mental abnormalities — incomplete gastrulation and
a reduction in anterior structures (16). The severe
cataracts seen at early ages in SPARC-null mice might
also arise because of abnormalities in cell shape. As
shown in Figure 2a, differentiating lens epithelial cells
(shown in cross sections from the cortex of the lens
from 3-month-old animals) normally exhibit an
ordered structure of uniformly shaped cells. Age-
matched SPARC-null lens cells (Figure 2b) display
irregular shapes and alignment. Whether the changes
in cell shape that occur during the normal develop-
ment of the lens are due to the direct action of SPARC
or to a secondary effect occurring later in the differ-
entiation process is currently unknown.
The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 9 1051
PERSPECTIVE SERIES
E. Helene Sage, Series Editor
Matricellular proteins
Figure 2
SPARC-null lens epithelial cells display changes in
cell shape and alignment in comparison with
wild-type cells. Sections from similar areas of
wild-type and SPARC-null lens were stained with
hematoxylin and eosin. Cortical fibers from a 3-
month-old wild-type lens (a) are regular and pre-
cisely aligned, whereas those from a SPARC-null
lens (b) are irregular in shape and alignment.
Magnification, ×220.
A recent study of proteins that influence morphine-
induced locomotor sensitivity in the amygdala identi-
fied SPARC as important in this process. It is believed
that prolonged exposure to morphine leads to physio-
logical changes in the brain that might be dependent
upon synaptic rearrangement. Ikemoto et al. found
expression of SPARC in the amygdala, the region of the
brain known to control locomotor sensitivity, in rats
exposed to morphine (17). In fact, administration of
recombinant SPARC directly to the amygdala resulted
in locomotor sensitivity in the absence of morphine.
The authors also showed that SPARC induces shape
changes in neurons, as previously reported for several
other cell types (1). Perhaps the counteradhesive activi-
ty of SPARC facilitates neuronal rearrangement in vivo,
including synaptic plasticity, with its attendant physio-
logical and behavioral implications.
SPARC and differentiation
The body wall muscle dysfunction seen in nematodes
with aberrant expression of SPARC has been pursued by
cRNA interference to block the production of SPARC in
these organisms. The resulting phenotypes ranged from
embryonic lethality to surviving adults with significant
morphological abnormalities, including smaller adults,
a lack of gut granules, and no functional gonads (Table
1; ref. 9). Because the major site of SPARC mRNA tran-
scription in C. elegans occurs in the body wall muscle
cells, embryonic death has been attributed to defects in
muscle function, as no obvious differences in morphol-
ogy are evident at this stage. Smaller adults might result
from a nutritional defect caused by the lack of gut gran-
ules. Whether the absence of SPARC affects the ECM
upon which precursor cells migrate and receive appro-
priate signals for differentiation into gut granules, or
whether another developmental mechanism is dis-
turbed, remains to be determined. Clearly, functional
interruption of SPARC activity in both C. elegans and
Xenopus (Table 1) has severe consequences that most cer-
tainly involve differentiation, although the molecular
mechanisms are currently undefined.
Lens epithelial cells differentiate at specific points
along the lens periphery, during which cells lose contact
with the capsular basement membrane, elongate anteri-
orly and posteriorly, lose their organelles, and eventual-
ly form the highly ordered, transparent structure of the
lens. As SPARC is a component of the lens capsule, the
lack of SPARC might result in a significantly altered
ECM with respect to structure and/or growth factor dep-
osition (2). A hitherto unidentified function of SPARC
might also be critical for these highly specialized cells,
such as the regulation of intercellular adhesive proteins,
e.g., certain of the cadherins, and gap junction proteins,
which are critical for the differentiation of lens fibers.
In vertebrates, SPARC is a major noncollagenous com-
ponent of bone. Delaney et al. recently reported a severe
bone phenotype associated with SPARC-null mice that
was manifested as adult osteopenia (18). The absence of
SPARC led to substantial decreases in bone mass, the
severity of which increased with age. Interestingly, we
have observed significant increases in subcutaneous fat
in SPARC-null relative to wild-type mice, a difference
that also appears to increase with age. Other fat deposits
in SPARC-null mice are increased in size as well, in com-
parison with those of wild-type animals, although over-
all body weight is not substantially different. Both
osteoblasts and adipocytes originate from common pro-
genitor cells present in the bone marrow (19). The
decrease in bone mass and the increase in fat deposition
might reflect differentiation events that favor the for-
mation of adipocytes over that of osteoblasts. Indeed,
SPARC might act directly to influence cell fate in this
example; conversely, the absence of SPARC might affect
ECM assembly and/or localization of growth factors
that in turn could influence differentiation. Rodriguez
et al. recently reported an analysis of the differentiative
capacity of mesenchymal stem cells isolated from post-
menopausal women with and without osteoporosis (20).
Cells from osteoporotic patients produced less TGF-β
and collagen I and exhibited an increased incidence of
adipogenic differentiation in culture. Seemingly, the
deficiencies in TGF-βand hence the capacity to synthe-
size a collagen I–rich ECM favored adipose formation
over bone formation. Since a decrease in TGF-βand col-
lagen I is observed in SPARC-null mesangial cells versus
wild-type cells, a similar mechanism might be in place to
influence mesenchymal stem cell differentiation as well.
That SPARC-null mice develop to adulthood and
reproduce with a grossly normal skeleton and fat
deposits argues against a direct influence of SPARC in
decisions regarding cell fate. A more plausible hypoth-
esis is that subtle differences in ECM configuration
and/or growth factor activity in animals lacking
SPARC lead eventually to tissue failure, especially when
the animal is challenged with injury or disease.
SPARC and wound healing
Dermal wound healing involves many processes that
could be affected by the absence of SPARC: ECM
turnover and reassembly, cell migration, cell prolifera-
tion, and recruitment/differentiation of connective tis-
sue cells. Perhaps surprisingly, we have found that exci-
sional dermal wounds produced in SPARC-null mice
close more rapidly, relative to those in wild-type mice
(A.D. Bradshaw et al., unpublished results). The mech-
anisms by which SPARC could influence dermal clo-
sure and/or healing are many. One possibility is the
capacity of SPARC to influence the activity of TGF-β.
Recently, Ashcroft et al. reported that mice deficient in
Smad-3 (a downstream signaling molecule in the TGF-
βpathway) also display accelerated dermal wound heal-
ing, in comparison with wild-type mice (21). Although
TGF-βhas been shown to augment healing when
administered topically, the signaling pathways in vivo
1052 The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 9
PERSPECTIVE SERIES
E. Helene Sage, Series Editor
Matricellular proteins
are complex. Local and probably cell-specific inhibition
of TGF-βsignaling apparently enhances dermal heal-
ing. Thus, the absence of SPARC might decrease the
levels of active TGF-β, and consequently its availability
to certain cells, during dermal healing and would there-
by accelerate the process. In addition, the capacity of
SPARC to inhibit PDGF, bFGF, and VEGF, three fac-
tors that have been shown to improve healing, might
also contribute to the enhancement of wound closure
in the absence of SPARC.
The structure of the dermis in SPARC-null mice, in
particular the collagen fibrils, is clearly different from
that in wild-type mice. The decrease in size of the col-
lagen fibrils, perhaps with other alterations in the
ECM, might result in a more permissive milieu for cel-
lular infiltration and healing. It is likely that the
SPARC-null ECM would be more easily degraded by
matrix metalloproteinases. In addition, overall wound
contraction might be more easily achieved with a
SPARC-null matrix, given that it appears to be less
structured and hence possibly easier to contract than
that of wild-type mice.
Another contribution to enhanced wound closure
might be the increase in subdermal fat that we have
observed in SPARC-null mice. Many of the cells that
invade the wound bed to repair the injured tissue have
been shown to originate in the subdermal fat and mus-
cle layers (22). Perhaps an increase in the amount of fat
enhances the availability of cells that contribute to
wound closure and thus accelerates the healing
process. Moreover, Frank et al. reported recently that
leptin, a factor produced by adipose tissue, enhances
dermal wound healing in mice (23). An increase in the
subcutaneous fat layer in SPARC-null mice most like-
ly results in increased levels of leptin in the skin. Thus
accelerated closure might reflect an increase in leptin
levels at the wound site.
Cancer and angiogenesis
Similar to wound healing, substantial ECM turnover
and rearrangement also occur during tissue invasion by
tumor cells. High levels of SPARC are often associated
with metastatic tumors (1). In fact, SPARC has been
proposed as a diagnostic marker of invasive menin-
giomas (24). Inhibition of SPARC expression by anti-
sense RNA diminished both adhesive and invasive
capacities of human melanoma cells in vitro and in
vivo. Moreover, melanoma cells with suppressed
expression of SPARC were no longer able to generate
tumors when injected into athymic mice, whereas con-
trol melanoma cells showed 100% tumorigenicity (25).
Angiogenesis, the growth of new vessels from extant
vasculature, is a major factor in tumor growth and
metastasis (26). Since neovascularization includes
endothelial cell invasion and ECM remodeling, it was
not surprising to find that SPARC is expressed by
endothelial cells in culture and in tissues (1). Notably,
SPARC has been shown to be associated with growing
vessels in the chick chorioallantoic membrane, a high-
ly vascular structure that provides nutrients and gas
exchange for the growing embryo (1). Interestingly, the
authors reported the generation of proteolytic frag-
ments from SPARC that were localized to the tips of
growing vessels. The Cu2+-binding peptide GHK, previ-
ously characterized as an angiogenic compound in
plasma, as well as the more potent stimulator KGHK,
could be derived from SPARC (Figure 1; see also ref. 1).
To date, SPARC is the only extracellular protein found
to contain the sequence KGHK, which is conserved
throughout vertebrate SPARC (1). The existence of
regions in SPARC that can be released by proteolysis
and that display activities different from those of the
native, intact protein somewhat complicates our
understanding of the functions of SPARC, but at the
same time offers new possibilities for modulation of
cell-matrix interactions (27).
We have recently performed a sponge invasion assay
in SPARC-null mice to determine whether angiogene-
sis in the dermis is abnormal. Animals lacking SPARC
displayed a substantial increase in the amount of
fibrovascular invasion of subcutaneous polyvinyl alco-
hol sponges in comparison with that of wild-type mice
(A.D. Bradshaw et al., unpublished results). Whether an
increase in invasion and angiogenesis reflects a more
permissive milieu for cell migration and proliferation,
due to an altered ECM, or an increase in the availabili-
ty of angiogenic growth factors such as PDGF and
VEGF, remains to be determined. Also of interest is the
use of SPARC-null mice in selected tumor models to
determine whether metastatic potential or tumor
angiogenesis is affected by the absence of SPARC.
Concluding remarks and future directions
That SPARC is expressed by organisms as diverse as C.
elegans and humans is testimony to the fundamental
importance of this protein to multicellular life. Since
invertebrate SPARC does not bind hydroxyapatite, the
SPARC gene has apparently become specialized for an
additional function unique to vertebrates, i.e., bone
formation. The existence of other SPARC family
members, such as SC1, provides another layer of com-
plexity with regard to compensatory activities (1, 2).
SPARC might influence cell behavior through its
interactions with cell surfaces, growth factors, and
ECM. Elucidating the mechanisms by which SPARC
influences cell migration, proliferation, and differen-
tiation will contribute to our understanding of a vari-
ety of different biological processes including devel-
opment, wound healing, angiogenesis, and cancer.
Although significant progress has been made in the
characterization of SPARC, a substantial amount of
information remains to be unearthed, with results
which undoubtedly will prove to be as provocative as
they are gratifying.
The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 9 1053
PERSPECTIVE SERIES
E. Helene Sage, Series Editor
Matricellular proteins
Acknowledgments
We would like to thank the members of the Sage labo-
ratory for helpful discussions and ideas. Qi Yan con-
tributed the photograph in Figure 2. This work was sup-
ported by NIH grants GM-40711 and HL-59475 to E.H.
Sage, and DK-07467 to A.D. Bradshaw.
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1054 The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 9
PERSPECTIVE SERIES
E. Helene Sage, Series Editor
Matricellular proteins