From the Cardiovascular Biology Laboratory, The CD44 gene codes for a family of alternatively
spliced, multifunctional adhesion molecules that participate in
extracellular matrix binding, lymphocyte activation, cell migration,
and tumor metastasis. In a mouse model of transplant-associated
arteriosclerosis, CD44 protein was induced in the neointima of
allografted vessels and colocalized with a subset of proliferating
vascular smooth muscle cells (SMC). To elucidate the molecular
mechanisms regulating CD44 expression in this model, we investigated
the regulation of CD44 gene expression by interleukin (IL)-1 Vascular smooth muscle and the associated connective tissue matrix
are central to blood vessel integrity and function, and activation of
vascular smooth muscle cells is characteristic of arteriosclerosis and
hypertension. After vessel wall injury, smooth muscle cells are
transformed from a contractile, quiescent phenotype to a proliferative,
migratory phenotype that secretes abundant extracellular matrix (1).
Vascular smooth muscle cells are subject to complex regulation by
soluble extracellular signals provided by growth factors, cytokines,
and vasoactive agents as well as cell-cell and cell-matrix interactions
(1). Therefore, factors controlling smooth muscle cell behavior,
including migration, proliferation, and lipid metabolism, are critical
to the pathogenesis of cardiovascular disease. Cell surface signal
transduction and adhesion molecules such as vascular cell adhesion
molecule-1 (2) and the The CD44 transmembrane glycoprotein exists in a variety of isoforms
generated by alternative splicing of one (or more) of 10 variable exons
in the extracellular domain (6, 7). CD44 is the principal cell receptor
for hyaluronic acid (8) and interacts with other extracellular matrix
molecules including osteopontin, collagen, and fibronectin (9, 10).
Present on many cell types, CD44 has been correlated with cell
proliferation (11, 12) and oncogenic transformation (13). Ligation of CD44 stimulates cytokine release by monocytes/macrophages (14, 15), and
it may modulate T lymphocyte activation signals (16, 17). In a variety
of cell systems CD44 imparts a novel cellular adhesive and/or migratory
phenotype to transfected cells (9, 18, 19), and an isoform containing
the sixth variable exon (v6) confers metastatic potential to rat
pancreatic carcinoma cells (20). The importance of CD44 in
vivo has also been demonstrated in a mouse model in which an
antisense CD44 transgene is expressed selectively under the control of
a keratinocyte-specific promoter (21). Suppression of CD44 expression
inhibits keratinocyte proliferation and results in abnormal hyaluronate
metabolism in the skin. Moreover, CD44 is induced on smooth muscle
cells after vascular injury, and it may mediate the proliferative
effects of hyaluronate (5).
Our laboratory has developed a mouse model of transplant-associated
arteriosclerosis in which a carotid artery loop is transplanted between
inbred strains in syngeneic and allogeneic combinations (22). Lesion
development depends on an acquired immune response and begins with
infiltration of inflammatory cells, after which follows accumulation of
smooth muscle cells in the neointima (23). In the present study
we evaluated CD44 cell surface protein expression in vivo
during the pathogenesis of transplant arteriosclerosis in order to
understand the role of CD44 in modulating vascular smooth muscle cell
phenotype. To elucidate the molecular mechanisms regulating CD44
expression in vascular smooth muscle cells after injury, we studied the
effect of interleukin
(IL)11 Immunocytochemistry--
Carotid artery transplantation was
performed as described (22). In brief, a carotid artery loop was
transplanted between two strains of inbred mice incompatible in the H-2
region. B.10A(2R) (H-2h2) mice were used as donors in the
allograft group, and C57BL/6J (H-2b) mice were used as
donors in the isograft group. C57BL/6J (H-2b) mice were
used as recipients in both groups. Transplant samples were prepared and
immunostained as described by Shi et al. (22). Grafts were
harvested and processed in methyl Carnoy's fixative, embedded in
paraffin, and cut in a microtome in 4-µm cross-sections. After
removal of the paraffin, tissue sections were incubated with 10%
normal serum for 20 min at room temperature.
Department of Medicine,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
.
Treatment of rat aortic SMC with IL-1
resulted in a 5.3-fold
increase in cell surface CD44 expression. Northern analysis showed
that IL-1
promoted a dose- and time-dependent
induction of CD44 mRNA which reached 6.6-fold after 48 h, and
nuclear run-on analysis showed that IL-1
increased the rate of CD44
gene transcription within 8 h of stimulation. In transient
reporter gene transfection experiments in rat aortic SMC, a
1.4-kilobase fragment of the mouse CD44 5'-flanking sequence mediated
this response to IL-1
. Regulation of CD44 gene expression by the
proinflammatory cytokine IL-1
may contribute to SMC phenotypic modulation in the pathogenesis of arteriosclerosis.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 and
3 integrins (3, 4) have been
implicated in the modulation of smooth muscle cell function. Previous
studies from our laboratory indicate that the proteoglycan CD44 may
also mediate vascular smooth muscle cell activation during vascular remodeling (5).
on CD44 gene
expression in cultured rat aortic smooth muscle cells (RASMC).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-actin
staining. Sections stained for CD44 and PCNA were labeled with
avidin-biotin enzyme complex peroxidase (Vector Laboratories) and
developed in 3,3'-diaminobenzidine. Sections stained for
-actin were
labeled with avidin-biotin enzyme complex alkaline phosphatase and
developed with VectorRed (Vector Laboratories). Sections treated for
CD44 and PCNA were counterstained with 1% methyl green, and sections
treated for
-actin were counterstained with Verhoeff's stain for
elastic tissue.
Cell Culture--
RASMC were harvested from the thoracic aorta
of adult male Sprague-Dawley rats (200-250 g) by enzymatic
dissociation according to the method of Gunther et al. (24).
The cells were cultured in Dulbecco's modified Eagle's medium (JRH
Biosciences, Lenexa, KS) and supplemented with 10% fetal calf serum,
penicillin (100 units/ml), streptomycin (100 µg/ml), and 25 mM Hepes (pH 7.4). RASMC were passaged every 4-7 days, and
experiments were performed on cells four to seven passages from primary
culture. Recombinant IL-1 and platelet-derived growth factor-BB were
obtained from Collaborative Biomedical (Bedford, MA). Tumor necrosis
factor-
and interferon-
were obtained from Life Technologies,
Inc.
Flow Cytometry Analysis--
Cultured RASMC were treated with
vehicle or IL-1 (10 ng/ml), harvested with 0.05% trypsin/0.2
mM EDTA in phosphate-buffered saline (PBS), washed, and
resuspended in 0.2% bovine serum albumin in PBS. Cells
(106) were incubated with antibody OX49 (anti-rat CD44,
IgG2a; PharMingen) or mouse IgG2a negative control antibody
(Biosource International, Camarillo, CA) for 30 min at 4 °C. Labeled
cells were washed twice with 0.2% bovine serum albumin in PBS and
incubated with fluorescein isothiocyanate-conjugated secondary antibody
(goat F(ab)' anti-mouse IgG; Biosource International). Flow cytometry
analysis was performed on an Ortho 2150 cytofluorograph
(Cyonics/Uniphase, Sunnyvale, CA) equipped with a Cyclops data
acquisition and analysis system (Cyclops Software, Cytomation, Fort
Collins, CO).
RNA Blot Hybridization--
Total RNA was obtained from cultured
cells by guanidinium isothiocyanate extraction and centrifuged through
cesium chloride (25). The RNA was fractionated on a 1.3%
formaldehyde-agarose gel and transferred to nitrocellulose filters. The
filters were hybridized with a random primed, 32P-labeled
rat CD44 cDNA probe as described (5). The hybridized filters were
autoradiographed with Kodak XAR film at 80 °C or stored on
Phosphor screens. To correct for differences in RNA loading, the RNA
filters were rehybridized with an 18-s oligonucleotide probe. The
filters were scanned, and radioactivity was measured on a
PhosphorImager running the ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
Nuclear Run-on Analysis--
Confluent RASMC were either treated
with PBS (control) or stimulated with IL-1 for 8 h. The cells
were then lysed, and nuclei were isolated as described (26). The
nuclear suspension (200 µl) was incubated with a 0.5 mM
concentration each of CTP, ATP, and GTP and with 125 µCi of
[
-32P]UTP (3,000 Ci/mmol; NEN Life Science Products).
The samples were then extracted with phenol/chloroform, precipitated,
and resuspended at equal counts/min/ml in hybridization buffer
(1.4 × 106 cpm/ml). Denatured probes (1 µg)
dot-blotted on nitrocellulose filters were hybridized at 40 °C for 4 days in the presence of formamide. cDNAs for the CD44 and
-actin
genes were used as probes. The filters were scanned, and radioactivity
was measured on a PhosphorImager running the ImageQuant software. The
amount of sample hybridizing the CD44 probe was divided by that
hybridizing the
-actin probe, and the corrected density was recorded
as a percentage increase from the control density.
Isolation of the CD44 5'-Flanking Sequence--
A fragment of
the 5'-flanking region of the mouse gene encoding CD44 was amplified by
using the PromoterFinder Kit (CLONTECH, Palo Alto,
CA). Primers were designed according to the published mouse CD44
sequence (27, 28). A CD44-specific primer
(5'-GCAAGAGGCAAAGTCCCCAAGCTGT-3') and a nested primer
(5'-CCAAGCTGTGTGCCACCAAAACTTG-3') were used in the primary and
secondary polymerase chain reactions, respectively, to amplify a
1,400-base pair fragment from mouse genomic DNA. The polymerase chain
reaction fragment was subcloned and sequenced by the dideoxy chain
termination method (25) with Sequenase 2.0 DNA polymerase (U. S.
Biochemical Corp.). A fragment of the mouse CD44 5'-flanking sequence
was radiolabeled with [-32P]dCTP and used to screen a
phage library of mouse genomic DNA in the
FixII vector
(Stratagene, La Jolla, CA) as described (29). Hybridizing clones were
isolated and purified, and phage DNA was prepared according to standard
procedures (25). Restriction fragments derived from the mouse CD44
genomic phage clones were subcloned into pSP72 (Promega, Madison, WI)
and sequenced with Thermo Sequenase DNA polymerase (Amersham Pharmacia
Biotech). Sequence analysis was performed with the GCG software package (Genetics Computer Group, Madison, WI).
Primer Extension--
Primer extension analysis to map the
transcription start site of the mouse CD44 gene was performed as
described (29). A synthetic oligonucleotide primer
(5'-AAGGGCAACGAGGGTGAATGG-3') complementary to the
5'-flanking sequence of the mouse CD44 cDNA was end labeled with
[-32P]ATP and hybridized to 50 µg of total RNA,
which was then subjected to reverse transcription using avian
myeloblastosis virus-reverse transcriptase (Promega). Extension
products were analyzed by electrophoresis on an 8% denaturing
polyacrylamide gel. C2C12 cells were obtained from the American Type
Culture Collection (Rockville, MD). S49 cells were the gift of Dr.
Jayne Lesley (The Salk Institute, La Jolla, CA).
Plasmids--
Plasmid pGL2-Basic contained the firefly
luciferase gene with no promoter (Promega), and phagemid pOPRSVI-CAT
(Stratagene) contained the prokaryotic chloramphenicol
acetyltransferase (CAT) gene driven by the RSV-LTR (Rous sarcoma
virus-long terminal repeat) promoter. Reporter constructs containing
fragments of the mouse CD44 5'-flanking sequence were named according
to the location of the fragment from the transcription start site in
the 5' and 3' directions. A gene fragment amplified from mouse genomic
DNA containing 1,262 base pairs of the CD44 5'-flanking sequence
upstream and 109 base pairs downstream of the transcription initiation site was cloned into pGL2-Basic and named CD44 (1.3/+0.1 kb). A
larger fragment containing approximately 3,900 base pairs upstream and
109 base pairs downstream of the CD44 transcription initiation site was
cloned into pGL2-Basic and named CD44 (
3.9/+0.1 kb). All of the above
constructs were generated by polymerase chain reaction with
Pfu polymerase (Stratagene) and sequenced by the dideoxy
chain termination method to confirm the identity and orientation of the
insert.
Transfection and Luciferase Assay--
RASMC were transfected by
a DEAE-dextran method (30). In brief, cells were plated onto 100-mm
tissue culture dishes and allowed to grow for 48-72 h (until 80-90%
confluent). Luciferase plasmid DNA and pOPRSVI-CAT (to correct for
differences in transfection efficiency) were added (5 µg each) to
RASMC in a solution containing 500 µg/ml of DEAE-dextran. RASMC were
then shocked with a 5% dimethyl sulfoxide solution for 1 min and
allowed to recover in medium containing 10% fetal calf serum. 12 h after transfection, RASMC were placed in 2% fetal calf serum and
stimulated with vehicle (PBS) or IL-1 (10 ng/ml) for 48 h. Cell
extracts were prepared by a detergent lysis method (Promega), and
luciferase activity was measured in duplicate for all samples by using
the Promega luciferase assay system and an EG&G AutoLumat LB953
luminometer (Gaithersburg, MD). The CAT assay was performed by a
modified two-phase fluor diffusion method as described (30). The ratio of luciferase activity to CAT activity in each sample served as a
measure of normalized luciferase activity. Each construct was transfected at least five times, and data for each construct are presented as the mean ± S.E. Relative luciferase activity in
groups treated with vehicle was compared with that in groups treated with IL-1
by analysis of variance. Statistical significance was accepted at p < 0.05.
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RESULTS |
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Expression of CD44 in Transplant Arteriosclerosis--
To
understand how CD44 may modulate vascular smooth muscle cell phenotype
during vascular remodeling, we studied CD44 expression in a mouse model
of transplant-associated arteriosclerosis. Immunocytochemical analysis
was performed on histological sections from allografted (Fig.
1, left) and isografted (Fig.
1, right) carotid arteries harvested at day 15 after
transplantation, a time at which neointimal thickening is substantial
(22). Allograft sections displayed robust CD44 protein expression in
the neointima (Fig. 1A, brown), whereas
isografted sections showed minimal CD44 expression and little neointima
formation (Fig. 1B). The level of proliferation among
neointimal cells, as evidenced by staining for PCNA (Fig. 1C, brown), was significant in allograft
sections. PCNA staining was absent in the isograft control (Fig.
1D). Note that CD44 and PCNA exhibited a similar pattern of
induction in the neointima in day 15 allografted arteries. Vascular
smooth muscle cells predominated in the neointima, as evidenced by
staining for smooth muscle -actin (Fig. 1E,
pink). Taken together, these data suggest that proliferating vascular smooth muscle cells account for the majority of CD44 expression in the neointima during the development of
transplant-associated arteriosclerosis.
|
Induction of CD44 Protein and mRNA by IL-1 in RASMC--
To investigate signals regulating CD44 expression in vascular smooth
muscle cells, we tested the effect of the proinflammatory cytokine
IL-1
on CD44 protein and mRNA levels. RASMC were cultured in
0.4% fetal calf serum and treated with IL-1
(10 ng/ml) for 48 h. Cell surface CD44 expression was then assessed by antibody staining and flow cytometry. CD44 surface antigen was present on RASMC
under basal conditions (Fig. 2, Control)
and increased by 5.3-fold after IL-1
stimulation.
|
|
Regulation of CD44 mRNA Expression by Other Inflammatory
Cytokines--
To assess the specificity of the effect of IL-1 on
CD44 gene regulation, we tested the ability of other cytokines and
growth factors implicated in the development of arteriosclerotic
lesions to modulate CD44 mRNA expression. Serum-starved RASMC were
treated with tumor necrosis factor-
, interferon-
, and
platelet-derived growth factor-BB for various lengths of time, and the
CD44 message was measured by Northern analysis (Fig.
4). Tumor necrosis factor-
, an
inflammatory cytokine whose effects are often similar to those of
IL-1
, promoted a gradual increase in CD44 mRNA which reached 3.7-fold at 48 h. Interferon-
, which inhibits vascular smooth muscle cell growth (31, 32) yet stimulates macrophage (33) and
lymphocyte (34, 35) activation, had only a minimal effect on CD44
message levels. Platelet-derived growth factor-BB, a potent mitogenic and chemotactic agent for smooth muscle cells, produced a
3.5-fold induction of CD44 mRNA at 4 h which returned to base line by 12 h. In light of this experiment, we concentrated our efforts on IL-1
, which produced a dramatic and sustained induction of CD44 in vascular smooth muscle cells.
|
IL-1 Increases CD44 Gene Transcription--
To elucidate the
mechanism by which IL-1
elevates CD44 message levels, we performed
nuclear run-on experiments to assess the effect of IL-1
on the rate
of CD44 gene transcription. CD44 mRNA transcription in RASMC
increased by 3.7-fold within 8 h of IL-1
stimulation
compared with control (Fig. 5). Exposure
of RASMC to IL-1
did not significantly prolong the half-life of CD44
mRNA in the presence of the transcription inhibitor actinomycin D,
which was approximately 48 h (data not shown).
|
Isolation and Characterization of the Mouse CD44 5'-Flanking Sequence-- To investigate further the transcriptional mechanisms regulating the expression of CD44 in vascular smooth muscle cells, we cloned the 5'-regulatory sequences of the mouse CD44 gene. We used sequence information derived from a fragment of the mouse CD44 gene isolated with the PromoterFinder Kit (CLONTECH) to amplify a 1.4-kb fragment of CD44 5'-flanking sequence from mouse genomic DNA. Fig. 6A shows the nucleotide sequence of the mouse CD44 promoter.
|
IL-1 Induces CD44 Promoter Activity in RASMC--
We
constructed luciferase reporter plasmids driven by 1.4-kb and 4.0-kb
fragments of the CD44 5'-flanking sequence to analyze CD44 promoter
function in RASMC. Promoter constructs in pGL2-Basic were cotransfected
into RASMC with pOPRSVI-CAT, and corrected luciferase activity was
expressed as a percentage of activity in unstimulated controls. The
promoter activity of plasmid CD44 (
1.3/+0.1 kb) increased by 3.5-fold
upon stimulation with IL-1
(Fig. 7).
Similarly, IL-1
treatment produced a 2.8-fold increase in the
activity of plasmid CD44 (
3.9/+0.1 kb), which contains additional
upstream regulatory sequences. These data suggest that the most
important IL-1
regulatory elements are contained in the
1.3 to
+0.1 kb fragment of the CD44 5'-flanking sequence. The
1.3 to +0.1 kb
fragment cloned into pGL2-Basic in the antisense orientation had only
minimal luciferase activity that was not altered by treatment with
IL-1
(data not shown). In conjunction with the nuclear run-on
experiments, these experiments demonstrate that the induction of CD44
mRNA by IL-1
is due to a transcriptional regulatory
mechanism.
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DISCUSSION |
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In a variety of cell systems, functions have been proposed for CD44 in cellular activation and movement, processes that are central to the smooth muscle cell response to arterial wall injury. In our mouse model of transplant-associated arteriosclerosis, CD44 is expressed minimally in the medial layer of the normal vascular wall in vivo and is induced markedly on smooth muscle cells in the neointima of allografted carotid arteries. In addition, extracellular matrix ligands for CD44, including hyaluronate and osteopontin, are elaborated in developing atherosclerotic lesions and may be important in modulating vascular smooth muscle cell function (5, 37, 38). The coincident expression of the CD44 adhesion receptor and its ligands may facilitate biological processes such as smooth muscle cell replication and migration during vascular remodeling.
Although CD44 expression has been documented in a variety of cell types, little is known about the molecular regulation of the CD44 gene. CD44 transcription is up-regulated in ras-transformed rat embryonic fibroblasts in an AP-1-dependent fashion, and expression correlates with cellular metastatic potential (13). Epidermal growth factor acts through a novel cis-acting element to induce CD44 expression in mouse fibroblasts, which is accompanied by enhanced cell attachment to hyaluronic acid (39). In the context of lymphocyte activation during humoral immune responses, the CD44 promoter is activated by the EGR1 transcription factor after B cell antigen receptor stimulation (40). Our present studies give new insight into the mechanisms by which cytokines regulate CD44 gene expression in vascular smooth muscle cells.
Cytokines and growth factors regulate cellular functions that are
central to atherogenesis, including proliferation, chemotaxis, lipid
metabolism, and synthesis of extracellular matrix components (1, 41).
The proinflammatory cytokine IL-1 is present in arteriosclerotic
lesions and mediates changes in cellular gene expression which
correlate with the development of pathological smooth muscle cell
behavior (42). IL-1
is mitogenic for vascular smooth muscle cells
in vitro (43, 44), and it contributes to the regulation of
cytokine production, extracellular matrix deposition, adhesion molecule
expression, and matrix metalloproteinase secretion by these cells
(45-48). We demonstrate here that stimulation of primary cultured
RASMC with IL-1
induced CD44 protein and mRNA, which was
attributable to an increase in the rate of CD44 gene transcription.
Transient transfection experiments in RASMC showed that induction by
IL-1
was mediated by regulatory sequences that reside within a
1.4-kb fragment of the mouse CD44 promoter. We propose that IL-1
induction of CD44 gene expression contributes to a coordinated response
of smooth muscle cells to arterial wall injury. Thus, further insight
into the transcriptional control of the CD44 promoter may elucidate
regulatory mechanisms that modulate smooth muscle cell phenotype in the
pathogenesis of vascular disease.
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ACKNOWLEDGEMENTS |
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We extend our gratitude to Mu-En Lee for critically reading the manuscript and for support throughout this project. We thank Thomas McVarish for editorial assistance, Dorothy Zhang for histological studies, and Bonna Ith for technical assistance. The S49 cell line was graciously provided by Jayne Lesley (The Salk Institute).
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FOOTNOTES |
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* This work was supported by a grant from Bristol-Myers Squibb.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: Cardiovascular
Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-2273; Fax: 617-432-2980; E-mail:
perrella{at}cvlab.harvard.edu.
The abbreviations used are: IL, interleukin; RASMC, rat aortic smooth muscle cells; PCNA, proliferating cell nuclear antigen; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; kb, kilobase(s).
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REFERENCES |
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