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2Department of Molecular Genetics (B.V.L., F.-E.M., L.F.L.), University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607-7170; Munin Corporation (J.A.G.), Chicago, Illinois 60612; Mouse Cancer Genetics Program (N.G.C., D.J.G., N.A.J.), National Cancer Institute-Frederick, Frederick, Maryland 21702; and Department of Microbiology (S.R.R.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6142
Address all correspondence and requests for reprints to: Lester F. Lau, Ph.D., Department of Molecular Genetics, University of Illinois at Chicago College of Medicine, 900 South Ashland Avenue, Chicago, Illinois 60607-7170. E-mail: lflau{at}uic.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Encoded by a growth factor-induced immediate-early gene, Cyr61 is a 40-kDa secreted cysteine-rich protein (4, 5). Due to its strong heparin-binding activity, Cyr61 is tightly associated with the ECM and the cell surface immediately after secretion and is not found in the cell culture medium (5). Purified Cyr61 protein promotes the adhesion, migration, and proliferation of both endothelial cells and fibroblasts (6, 7). The expression of Cyr61 during mouse embryogenesis is tightly correlated with mesenchymal condensations as they differentiate into chondrocytes and with the developing vasculature (8). Consistent with these findings, purified Cyr61 promotes chondrogenic differentiation in mouse limb bud mesenchymal micromass cultures (9) and induces neovascularization in vivo (10). Moreover, expression of Cyr61 enhances the tumorigenicity of human tumor cells in immunodeficient mice by increasing tumor size and vascular density. These observations indicate that Cyr61 may play important roles in angiogenesis and chondrogenesis during embryonic development.
Cyr61 is a ligand of, and binds directly to, the integrin
Vß3 on endothelial
cells and the integrin
IIbß3 on blood
platelets (11, 12). In fibroblasts, Cyr61 mediates cell
adhesion through the integrin
6ß1 with the
requirement of cell surface heparan sulfate proteoglycans as
coreceptors (13). Fibroblast adhesion to immobilized Cyr61
induces an array of adhesive signaling events, including actin
cytoskeleton reorganization, formation of filopodia and lamellipodia
concomitant with formation of integrin
6ß1-containing focal
complexes, activation of intracellular signaling molecules including
focal adhesion kinase, Rac, p42/p44 mitogen-activated protein kinases,
and up-regulation of matrix metalloproteinases 1 and 3
(14). The finding that Cyr61 interacts with multiple
integrins put its functional diversity into a mechanistic context.
Integrins are heterodimeric, cell surface adhesion receptors that are
capable of transducing extracellular signals into intracellular
responses that regulate cell adhesion, migration, proliferation,
differentiation, and survival (15, 16). Thus, many of the
activities of Cyr61 may be explained through integrin-mediated
signaling pathways.
An immediate-early gene, Cyr61 is transcriptionally activated rapidly in fibroblasts by serum, basic fibroblast growth factor, platelet-derived growth factor, and transforming growth factor-ß1 without requiring de novo protein synthesis (4, 17, 18, 19). The mouse Cyr61 promoter has been studied in cultured fibroblasts in transient transfection assays (20). It was found that a serum response element (SRE) located approximately 2 kb upstream of the transcription start site is necessary and sufficient to confer inducibility by serum, basic fibroblast growth factor and platelet-derived growth factor (20). The Cyr61 SRE includes a canonical CArG box, which has been implicated in expression of genes specific to the cardiovascular system (21, 22).
In this study, we have defined the functional Cyr61 promoter in vivo, mapped the mouse Cyr61 gene, and characterized the expression of Cyr61 during embryogenesis and wound healing. Our results show Cyr61 expression in the developing placenta and cardiovascular, skeletal, and nervous systems, and revealed the nonessential nature of the Cyr61 SRE for expression in the cardiovascular system. Further, a role for Cyr61 in cutaneous wound repair is supported by its induced expression in granulation tissue and ability to promote healing of a wounded monolayer in culture. Together with the demonstrated activities of Cyr61 (6, 9, 10, 14), these results provide insights into the biological functions of Cyr61 in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histological analysis
Imunohistochemistry was carried out as described
(7) and staining for ß-galactosidase activity was
performed as described (25).
Wound healing assays
To identify animals carrying the transgene, tails of mice were
excised for extraction of DNA for Southern blotting (25).
The tail wounds thus created were also used to monitor expression of
the transgene during wound healing by histological analysis. Similar
results were also obtained in excisional back wounds.
Monolayers of fibroblasts were wounded as described (26, 27) to simulate aspects of the healing process in cell culture. Cyr61 protein was purified as described (6). Tissue culture dishes (60-mm diameter) were coated overnight at 4 C with either 10 µg/ml Cyr61, 10 µg/ml fibronectin (Life Technologies, Inc., Rockland, MD), or Cyr61 storage buffer as described previously, and blocked with 1% BSA for 1 h as described (6). NIH 3T3 cells were plated at 3 x 106 cells per dish in DMEM containing 10% calf serum, and incubated at 37 C, 10% CO2 to allow them to form confluent monolayers. After reaching confluence, the cell medium was changed to serum-free DMEM. Confluent monolayers were wounded with a razor blade to create a linear wound and denuding cells on one side of the wound edge, as described previously (26, 27). After wounding, the cells were refed DMEM containing 0.2% FBS and incubated 20 h at 37 C, 10% CO2. Cells were then fixed with absolute methanol and stained with Giemsa-Wright stain (Harleco, Kansas City, MO). Cell migration into the simulated wound area was quantified by counting (under x100 magnification in three different fields) and summing the number of cells at various distances from the wound edge as described (28). The data reported were obtained from four independent experiments.
Interspecific mouse backcross mapping
Interspecific backcross progeny were generated by mating
(C57BL/6J x Mus spretus) F1
females and C57BL/6J males as described (29). A total of
205 N2 mice were used to map the Cyr61
locus (see Results). DNA isolation, restriction
enzyme digestion, agarose gel electrophoresis, Southern blot transfer,
and hybridization were performed essentially as described
(30). All blots were prepared with
Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The probe, an approximately 6.6
EcoRI fragment of mouse genomic DNA (20) was
labeled with [-32P]dCTP using a nick
translation labeling kit (Roche Molecular Biochemicals, Indianapolis, IN); washing was performed to
a final stringency of 0.8 x single-strand conformational
polymorphism, 0.1% SDS, 65 C. Fragments of 4.0, 2.3, 1.9, 1.7, 0.8,
and 0.6 kb were detected in HincII-digested C57BL/6J DNA and
fragments of 6.0, 4.0, 1.9, and 1.5 kb were detected in
HincII-digested M. spretus DNA. The presence or
absence of the 6.0- and 1.5-kb HincII M.
spretus-specific fragments, which cosegregated, was followed in
backcross mice. A description of the probes and RFLPs for the loci
linked to Cyr61 including Lmo4 and
Rabggtb has been reported previously (31, 32).
Recombination distances were calculated using Map Manager, version
2.6.5 from the Roswell Park Cancer Institute, Buffalo, NY,
http://mapmgr.roswellpark. org/classic.html. Gene order was determined
by minimizing the number of recombination events required to explain
the allele distribution patterns.
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Results |
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All 2lacZ transgenic mice, both established and transient
lines, show expression in developing cardiovascular and nervous systems
(Fig. 1
, A and B). In addition,
expression of the transgene was detected in the developing skeleton in
two of the three established lines. All tissues that express the
transgene have been confirmed to express endogenous Cyr61 in
previous studies (7, 8) and in this study, and all loci of
lacZ expression in this study have been confirmed by
immunohistochemical staining of the endogenous Cyr61 protein (Figs. 2C, 3, and 4, and data not shown). In no case was
inappropriate expression observed.
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). Expression of the transgene continued
to follow development of the cardiovascular system and somites (Fig. 1B). In the E11.5 embryo (Fig. 1B), expression was evident in the major
blood vessels and the heart; expression in these tissues persisted
throughout embryonic development (data not shown). Highly dynamic and
restricted expression of the transgene was also observed in the nervous
system. At E9.5, transgene expression was detected in the midbrain and
at the base of telencephalon, consistent with data obtained by
in situ hybridization (8) (Fig. 1A). By E11.5,
expression in the midbrain was down-regulated while the forebrain
expressed a high level of Cyr61 promoter activity, which was
sustained throughout embryonic development (Fig. 1B and data not
shown). By E14.5, the ventral spinal cord, dorsal ganglia, and nerves
innervating limbs all expressed ß-galactosidase (Fig. 2B). Endogenous
Cyr61 protein was also detected in the ventral spinal cord as judged by
immunohistochemical staining (Fig. 2C).
Expression of the transgene in the skeletal system began in the
developing notochord (Fig. 1A). By E11.5, expression of the transgene
in precursors of the axial skeleton, the sclerotomes, is evident (Fig. 1B). Precursors of the appendicular skeleton also express the
transgene, for example, the condensing mesenchyme of the E12.5 embryo
forming the digits in the forelimb (Fig. 2, D and E). At E16.5, the
transgene was expressed in newly formed bones as well (data not
shown).
Expression of the transgene in embryos was particularly prominent in
the placenta (Fig. 3A), consistent with levels of Cyr61 messenger RNA
and protein (7, 8). In the placenta, transgene expression
was localized in the trophoblastic giant cells (Fig. 3B) and in
endothelial cells of developing blood vessels (Fig. 3C). Because the
placenta is an intensely angiogenic organ whereby maternal and
embryonic blood must exchange nutrients, oxygen and waste, the
angiogenic activities of Cyr61 may play a role in the development of
the placental vasculature.
Taken together, these data show that the -2065/+65 fragment of the Cyr61 promoter is sufficient to mediate expression of the reporter gene in a manner that mirrors the expression pattern of endogenous Cyr61 in vivo. This includes extraembryonic expression in the placenta, and embryonic expression in the cardiovascular, skeletal, and central and peripheral nervous systems.
The SRE is not required for cardiovascular expression of
Cyr61
The SRE located at nucleotide -1933/-1921 was identified as an
essential regulatory element in the Cyr61 promoter for
transcriptional activation by serum growth factors in cultured
fibroblasts (20). To test the function of this sequence
in vivo, a construct was prepared (termed
1.4lacZ) that contains 1.4 kb of the Cyr61
promoter, deleting about 0.6 kb of uptream DNA from the
2lacZ construct and thus removing the SRE. Two transgenic
lines carrying this construct were analyzed. Both lines expressed the
transgene in the cardiovascular and central nervous systems in a
pattern similar to that of the 2lacZ lines (Fig. 1C).
Therefore, the 1.4-kb fragment of the Cyr61 promoter is
sufficient to direct tissue-specific transgene expression in the
cardiovascular and central nervous systems. Because the CArG box
sequence within the SRE has been implicated in mediating cardiovascular
gene expression in addition to its role in growth factor-mediated
activation (21, 22), it is somewhat unexpected that the
CArG box is dispensable for Cyr61 expression in the
cardiovascular system.
Neither 1.4lacZ transgenic line expressed the transgene in
the skeletal system (Fig. 1C). Because only two of the three
established 2lacZ lines exhibited skeletal expression of the
transgene, possibly due to integration effects, we cannot conclude
whether the distal 0.6-kb promoter sequence is required for skeletal
expression.
Cyr61 expression is induced in granulation tissue during wound
repair
The angiogenic, chemotactic, and proliferation-enhancing
activities of Cyr61 suggest its possible function in wound repair. To
investigate whether Cyr61 promoter activity is induced in
response to wounding, cutaneous wounds were inflicted on
2lacZ transgenic mice, and transgene expression was assessed
at various times after wounding. Transgene expression was not detected
in the unwounded dermis, but became detectable by 3 days after wounding
(data not shown). The number of dermal fibroblasts expressing the
transgene steadily rose and peaked 10–12 days post wounding (Fig. 4B).
As the wound healed by day 21, expression was down-regulated and only a
few fibroblasts were still expressing the transgene (data not shown).
Most of these transgene-expressing cells were in the area of newly
formed ECM as detected by histochemical staining for connective tissue,
suggesting that induction of Cyr61 is associated with the
ECM remodeling phase of wound healing. We confirmed that the Cyr61
protein is induced in healing wounds by immunohistochemical staining
(Fig. 4D). It is notable that at 1 day post wounding, Cyr61 was not
detected in the layer of keratinocytes migrating to reepithelialize the
wounded area, and in keratinocytes immediately adjacent to the wounds
(Fig. 4C).
Purified Cyr61 promotes fibroblast migration into a wounded
monolayer
As noted above, the biological activities of Cyr61 suggest its
possible roles in wound healing. We investigated this possibility
further using a cell culture system to assess migration of fibroblasts
into a simulated wound (27, 28). NIH 3T3 fibroblasts were
grown to confluence on plates precoated with Cyr61 or fibronectin. A
fibroblast monolayer was wounded using a razor blade to mark the dish
and to denude cells on one side of the wound edge. We then measured the
number of cells migrating over various distances across the wounded
edge 20 h after wounding (Fig. 5).
Like fibronectin, Cyr61 was able to promote the migration of
fibroblasts from the wound edge to the denuded area (Fig. 5E).
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), up to
165 mice were typed for some pairs of markers. Each locus was analyzed
in pairwise combinations for recombination frequencies using the
additional data. The ratios of the total number of mice exhibiting
recombinant chromosomes to the total number of mice analyzed for each
pair of loci and the most likely gene order are as follows:
centromere-Lmo4-2/165-Cyr61-4/160-Rabggtb.
The recombination frequencies (expressed as genetic distance in
centimorgans ± SE) are as follows:
Lmo4-1.2 ± 0.9-Cyr61-2.4 ±
1.2-Rabggtb. The distal region of mouse chromosome 3 shares
a region of homology with human chromosome 1p, consistent with the
reported localization of the human ortholog of CYR61 to
lp31–1p22 (33).
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Discussion |
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Previous studies identified a 2-kb Cyr61 promoter fragment
that is necessary and sufficient for serum inducibility in transient
transfection assays in fibroblasts (20). Within this
promoter fragment, an SRE containing a CArG box located at
-1933/-1921 was shown to mediate transcriptional response to serum,
fibroblast growth factor, and platelet- derived growth factor
(20). The CArG box is known to be involved in both growth
factor-induced transcriptional activation and in
cardiovascular-specific gene expression (21, 22).
Surprisingly, deletion of this CArG box sequence from the
Cyr61 promoter did not alter expression of the transgene in
the cardiovascular system (Fig. 1C). Clearly, sequences other than the
CArG box are sufficient to mediate Cyr61 expression in the
heart and blood vessels. Identification of these sequences in future
studies will help to understand the diversity of regulatory elements
that can specify gene expression in the cardiovascular system.
The major sites of Cyr61 expression as determined by
in situ messenger RNA hybridization (8) and
immunohistochemistry (7), including the placenta and
developing embryonic blood vessels and cartilage, were also detected as
sites of transgene expression in the present study. In addition,
because of the high sensitivity of the ß-galactosidase assay, this
study revealed details of spatial and temporal regulation of
Cyr61 that were not observed in previous studies. Briefly
summarized, Cyr61 promoter-driven expression in
2lacZ transgenic lines could be detected in a wide range of
tissues, including the following: 1) the placenta, including
trophoblastic giant cells and placental vessels (Fig. 3); 2) the entire
cardiovascular system including the heart, major arteries, and veins
(Figs. 1 and 3), as well as the lung (data not shown); 3) the embryonic
skeletal system, including the notochord, sclerotomes, limbs (Figs. 1 and 2), and developing bone (data not shown); 4) the developing nervous
system, including the ventral spinal cord, dorsal root ganglia, parts
of the mesencephalon and telencephalon (Figs. 1 and 2), and the
olfactory bulb (data not shown); and 5) the embryonic and adult
epidermis, in the inner root sheath of hair follicles (Fig. 4 and data
not shown), and in granulation tissue during cutaneous wound healing
(Fig. 4).
What are the functional implications of this complex Cyr61
expression pattern? It has been shown that Cyr61 promotes endothelial
cell adhesion, migration, and proliferation in culture, and induces
neovascularization in vivo (6, 7, 10). Thus,
Cyr61 is an angiogenic factor that may play important roles in the
development of the embryonic and placental vasculature. The expression
of Cyr61 in endothelial cells is sustained after the blood
vessels are formed, suggesting a role for Cyr61 in vessel maintenance.
Moreover, the angiogenic activity of Cyr61 may be important in tissue
remodeling in the adult, as indicated by the expression of
Cyr61 in granulation tissue during cutaneous wound healing
(Fig. 4). The 2-kb Cyr61 promoter is sufficient to drive
this inducible expression in transgenic mice. In addition, purified
Cyr61 protein promotes the migration of fibroblasts into the wounded
area of a monolayer (Fig. 5), supporting a role in wound healing. In
this context, Cyr61 has been shown to induce angiogenesis, to
up-regulate expression of metalloproteinases (14), to
induce chemotaxis, and to promote mitogenesis in endothelial cells and
fibroblasts (6, 7, 10). Thus, it is possible to propose
that Cyr61 may play important roles in wound healing by inducing
angiogenesis, stimulating matrix degradation and remodeling, and
promoting fibroblast chemotaxis and proliferation in the granulation
tissue.
Fibroplasia, a key component of the wound healing process in
vivo, includes the migration of fibroblasts into the wound area.
In this regard, it is noteworthy that Cyr61 expression increases
steadily during the early stages of wound healing, peaks around day 12
post wounding during a stage characterized by extensive fibroplasia,
and gradually declines to the basal (uninduced) level by 21 days post
wounding, by which time fibroplasia has been essentially completed
(Fig. 4 and data not shown). Serial sections revealed that during the
peak of Cyr61 expression most of the dermal fibroblasts in the
granulation tissue expressed the protein, and that the subsequent
decline in Cyr61 expression was mirrored by a decline in the number of
dermal fibroblasts in the granulation tissue that expressed the
protein. This observation suggests that Cyr61 is involved in a function
common to most of the dermal fibroblasts in the granulation tissue
during fibroplasia. It is interesting to note that another member of
the CCN family, CTGF, has also been shown to be induced in the
granulation tissues of healing wounds (34) and to promote
fibrosis (35). Although we detected Cyr61 expression in
normal epidermis, this expression is down-regulated during the early
phase of reepithelialization (Fig. 4C). Whether Cyr61 secreted by some
other cell type promotes keratinocyte migration or whether Cyr61 is not
involved in this process, or even negatively regulates the process,
remains to be elucidated.
We have demonstrated previously that Cyr61 acts as a chondrogenic
differentiation factor in micromass cultures of limb bud mesenchymal
cells (9). As noted above, we detected Cyr61
expression during skeletal development (Figs. 1 and 2, and data not
shown). Thus, Cyr61 is expressed in mesoderm that gives rise to
cartilage and bone, indicating that Cyr61 likely plays a critical role
in chondrogenesis in vivo. The proposed role of Cyr61 in the
development of cartilage and bone likely reflects several of its known
activities. For example, its ability to promote cell-matrix adhesion
and/or cell-cell aggregation of cultured limb bud mesenchymal cells
(9) could play a role in the initiation of the
chondrogenic differentiation pathway. Cyr61 protein is also found in
hypertrophic cartilage and ossified bone (7), suggesting
that the angiogenic activity of Cyr61 may be necessary for
endochondro-ossification at later stages of skeletal development
(10). In this context, it is noteworthy that Cyr61
expression is induced following bone fracture repair (36),
again indicating a role for Cyr61 in the formation of the skeleton.
This study has also revealed prominent expression of Cyr61 in the
developing embryonic central and peripheral nervous systems (Figs. 1 and 2). However, the specific activity of Cyr61 upon neuronal cells has
not yet been elucidated. Neuronal cells of the developing central and
peripheral nervous systems undergo extensive directed migration, a
process critically dependent on the ECM that interacts with these cells
(37, 38). Given the known adhesive and chemotactic
activities of Cyr61, it is tempting to speculate that Cyr61 may mediate
neuronal cell-ECM interactions and play a role in directing axonal
outgrowth and neuronal migration (14). In this regard, it
is interesting to note that the carboxy-terminal domain of the Cyr61
protein exhibits 25% amino acid sequence homology with the
Drosophila protein Slit, which is a key regulator of axon
guidance, axonal branching, and cell migration (39, 40, 41).
Cyr61 is also induced during differentiation of the
immortalized hippocampal neuronal cell line H19-7 in culture
(42), suggesting a role for Cyr61 in neuronal
differentiation. Furthermore, Cyr61 expression has been
detected in the cerebral cortex of the adult rat (43) and
mouse (Latinki, B. V., and L. F. Lau, unpublished
data) brain, and is induced by muscarinic acetylcholine receptor
signaling in culture (43). Together, these findings
suggest that Cyr61 may play a role in both embryonic neuronal
development and cholinergic regulation of synaptic plasticity in the
adult. However, the specific roles of Cyr61 in these processes remain
to be determined.
We have mapped Cyr61 to the distal end of chromosome 3 in
mice (Fig. 6). We have compared our interspecific map of chromosome 3
with a composite mouse linkage map that reports the map location of
many uncloned mouse mutations (provided by Mouse Genome Database, a
computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Cyr61 mapped in a region
of the composite map that lacks mouse mutations with a phenotype that
might be expected for an alteration in this locus (data now shown).
In summary, we have identified the Cyr61 promoter sequence necessary and sufficient for accurate expression in transgenic mice. The specific sites of Cyr61 expression can be interpreted in terms of the known activities of the Cyr61 protein as determined in various assays, suggesting that these activities contribute to specific functional roles for Cyr61 in the development of the cardiovascular, skeletal, and neuronal systems during embryogenesis, as well as in tissue reconstruction such as wound healing. Further analysis using mutant mice with targeted disruption of the Cyr61 gene will likely provide new insights into the biological functions of Cyr61 in these processes.
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Acknowledgments |
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Footnotes |
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2 Present address: Division of Developmental Biology, National
Institute for Medical Research, Mill Hill, London NW7 1AA, United
Kingdom
Received January 10, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BV, Kolesnikova TV, Chen
C-C, Yang GP, Abler AS, Lau LF 1997 Cyr61 and Fisp12 are both
signaling cell adhesion molecules: comparison of activities,
metabolism, and localization during development. Exp Cell Res 233:63–77[CrossRef][Medline]
Vß3. J Biol Chem 273:3090–3096IIbß3. J
Biol Chem 274:24321–243276ß1 and cell surface
heparan sulfate proteoglycans. J Biol Chem 275:24953–24961 BV, O’Brien TP, Lau LF 1991 Promoter function and structure of the growth factor-inducible
immediate early gene cyr61. Nucleic Acids Res 19:3261–3267This article has been cited by other articles:
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C.-C. Chen, F.-E Mo, and L. F. Lau The Angiogenic Factor Cyr61 Activates a Genetic Program for Wound Healing in Human Skin Fibroblasts J. Biol. Chem., December 7, 2001; 276(50): 47329 - 47337. [Abstract] [Full Text] [PDF]
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