Vol. 281, Issue 6, H2738-H2746, December 2001
Differential effects of overexpression of two
forms of ephrin-A5 on neonatal rat cardiomyocytes
Yun You
Li1,
Zhibao
Mi2,
Yiqin
Feng1,
Charles F.
McTiernan1,
Renping
Zhou4,
Paul D.
Robbins2,
Simon C.
Watkins3, and
Arthur M.
Feldman1
1 Cardiovascular Institute and 2 Department of
Molecular Genetics and 3 Center for Biological Imaging,
University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
15213, and 4 Laboratory for Cancer Research, College
of Pharmacy, Rutgers University, Piscataway, New Jersey 08854
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ABSTRACT |
Eph receptors constitute the
largest family of receptor tyrosine kinases. Multiple transcripts of
ephrin-A5, the cognate ligand of the EphA3 receptor, were found in
neonatal rat cardiomyocytes. Two cDNA clones encoding the full-length
ephrin-A5 (ephrin-A5
) and a 27-amino acid deletion form
(ephrin-A5
) were isolated. To examine the role of ephrin-A5 in
cardiomyocytes, the cDNAs were inserted into adenoviral vectors, termed
Ad.ephrin-A5 and Ad.ephrin-A5, respectively, and overexpressed in
cardiomyocytes. The effect of ephrin-A5 on cardiomyocyte gene
expression was investigated using a cDNA expression array and Western
blot analysis. The results showed that both ephrin-A5 and
ephrin-A5 downregulated cyclin D2, cyclin-dependent kinase-4
proteins, and their cognate receptor EphA3, which were associated with
reduced bromodeoxyuridine incorporation in cardiomyocytes. Whereas
ephrin-A5 and ephrin-A5 also induced differential gene
expression, only ephrin-A5 significantly upregulated the
transcription of brain natriuretic peptide and downregulated ras-related protein RAB2, protein kinase C inhibitor protein-1, clusterin, and insulin-like growth factor-binding protein. The results suggest that the two forms of ephrin-A5 share similar function
while differ in regulating different sets of genes in cardiomyocytes.
Eph receptor tyrosine kinase; DNA synthesis; adenoviral gene
transfer; gene expression array
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INTRODUCTION |
THE EPH FAMILY OF
RECEPTORS constitute the largest subgroup of receptor tyrosine
kinases (31). Eph receptors have recently been classified
into two subfamilies, EphA and EphB, according to their preference for
either glycosylphophatidylinositol-anchored ephrin-A ligands or
transmembrane ephrin-B ligands (6, 36). While the function
of the Eph receptor tyrosine kinases is not fully defined, some may
play a role in signal transduction during differentiation, development,
neuronal axon guidance, bundle formation, and control of
cytoskeletal architecture (14, 15, 33). Ephrin-A1 (B61) was first identified as a proinflammatory cytokine-inducible endothelial cell product (16, 25, 27, 28) that plays a role in angiogenesis. Similarly, the activation of EphB receptors also
directs vascular network differentiation and assembly (1, 30,
32), which suggests additional roles for Eph receptors and their
ligands in both inflammation and cardiovascular development and diseases.
Recently, we (22) cloned and examined the regulation of
EphA3 in neonatal rat cardiomyocytes. EphA3 is downregulated by proinflammatory cytokines in neonatal rat cardiomyocytes. We
hypothesized that the downregulation of the EphA3 receptor may be
mediated by its cognate ligands. Of the ephrin ligands, EphA3 binds
ephrin-A5 with the highest affinity and vice versa (affinity for
EphA3 > EphA8 > EphA4 = EphA5) (7, 18,
19). Ephrin-A5, initially affinity purified using an EphA5-IgG
fusion protein column and named AL-1, is expressed as multiple
transcripts at high levels in the human brain, heart, and kidney
(17, 18, 33). It was reported that ephrin-A5 plays a role
in the fasciculation and guidance of axons during development of the
nervous system (9, 10). However, the downstream events
regulated by the activation of EphA3 receptor, the functional
significance of their activation, and the role of ephrin-A5 in the
cardiovascular system have not been defined. In addition, the nature of
the multiple transcripts is not clear. Direct activation of the
receptor using its cognate ligands may provide invaluable information
on the function of Eph receptors in cardiac myocytes.
To elucidate the role of Eph receptors and ephrin ligands in the
cardiovascular system, we studied the expression of ephrin-A5 and
isolated two forms of ephrin-A5 from cardiomyocytes. The effect of
ephrin-A5 overexpression on EphA3 expression and the role of ephrin-A5
in neonatal cardiomyocyte biology and development were further explored.
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MATERIALS AND METHODS |
Cell culture and cytokine treatment.
Cardiac myocytes were isolated from the ventricles of 1-day-old
Sprague-Dawley rats as previously described (24). After being plated on pronectin (Promega; Madison, WI)-coated tissue culture
plates, myocytes were grown at 37°C in 5% CO2 for
24 h. The medium was then changed, and the cells were allowed to
grow for an additional 48 h. Medium containing 100 U/ml tumor
necrosis factor- (TNF-; Biosource International; Camarillo, CA)
was then added to the cultures. The cardiac myocyte preparations
routinely contained >95% sarcomeric myosin-positive cells. All cell
preparation and culture procedures were carried out under sterile
conditions, and experiments conformed with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals (NIH
Publication No. 85-23, Revised 1996).
Northern blot analysis.
Total RNA was isolated from cultured cardiomyocytes via acid
phenol extraction (4). RNA samples were enriched for
polyadenylated species by oligo(dT)/magnetic bead capture with a
PolyATtract mRNA isolation system (Promega). Poly A+ mRNA
enriched from 150 µg of total RNA was resolved in
formaldehyde-agarose gels, transferred to nitrocellulose membrane
(Schleicher and Schuell; Keene, NH), and fixed by ultraviolet
cross-linking. There was no known sequence for rodent ephrin-A5 when
this cloning experiment was performed. Human AL-1, later named
ephrin-A5, was used to select a pair of degenerate primers for the
amplification of rat ephrin-A5. The amplified cDNA was sequenced and
used as a probe for Northern blot analysis. The rat ephrin-A5 probe was
prepared by PCR amplification of rat cardiomyocyte cDNA with rTth DNA
polymerase and a pair of degenerate primers (sense: 5'-TCT CTC CCC GGA
GTG GCG CGT C-3', antisense: 5'-GGA TCT CTG GTG TTC CAA GAC CC-3', corresponding to 
48 to 27 and 725 to 747 of the cloned full-length sequence, respectively). The PCR products were cloned into a TA vector
(Invitrogen; Carlsbad, CA). Clones homologous to human ephrin-A5 were
isolated and sequenced. The full-length ephrin-A5 cDNA clone was used
as a probe in Northern blot analysis. Prehybridization, hybridization,
and quantification of the signals were performed as previously reported
(22). After hybridization, the membranes were stripped
with 2 mM Tris · HCl (pH 8.0), 0.2 mM EDTA, and 0.1% SDS at
75°C for 1 h and rehybridized sequentially with radiolabeled glyceraldehyde-3-phosphate dehydrogenase probe.
For the analysis of brain natriuretic peptide (BNP) expression, 6 µg
of total RNA was resolved in formaldehyde-agarose gels and transferred
to nitrocellulose membrane, and Northern hybridization was performed as
described above using a BNP cDNA probe.
Cloning of rat heart ephrin-A5 cDNAs and construction of
recombinant adenovirus containing ephrin-A5 and ephrin-A5.
A pair of specific primers that flanked the ephrin-A5 coding sequence
with a XbaI or a BamHI site, respectively (sense:
5'-GGG TCT AGA ATG TTG CAC GTG GAG ATG TTG A-3', antisense: 5'-GGG GGA TCC CTA TAA TGT CAA AAG CAT CGC C-3', corresponding to 1-25 and 666-687 of the cloned full-length sequence, respectively), were used in the PCR amplification of ephrin-A5 in an attempt to isolate different forms of ephrin-A5. An ephrin-A5 with an 81-bp deletion (hereafter named as ephrin-A5) was isolated in addition to the full-length ephrin-A5 (named as ephrin-A5). The amplified cDNA products were sequenced and cut with XbaI and
BamHI, and the cDNAs were inserted into a pAdlox shuttle
vector (13). The recombinant adenoviral vector originates
from replication-deficient type 5 adenovirus (lacking E1 and E3 loci)
and was constructed through Cre-lox recombination (13,
34). The ephrin cDNAs and LacZ gene were inserted in
place of the E1 region, and expression is driven by the
cytomegalovirus promoter. High-titer suspensions of recombinant
adenoviruses were prepared by established methods (13).
Briefly, a confluent 10-cm dish with 1.6 × 107 Cre8
cells was split into five 6-cm dishes and incubated at 37°C for
4 h. Transfection of these cells with pAdlox recombinant plasmids was performed by the calcium phosphate precipitation method with 3 µg
of the construct digested with SfiI and 3 µg of 
5 helper virus DNA. The media was changed 16 h posttransfection. The
transfected Cre8 cells were carefully fed daily until large scales of
plaques appeared. The cells were then suspended in their media and
placed into a sterile tube. The viral lysate was obtained by
freezing/thawing three times. The recombinant virus was purified and
amplified by infecting two 10-cm dishes of Cre8 cells using 0.1 ml of
the lysate. One dish was used for a virus stock, and the other dish was
used to make DNA to confirm the identity of the virus. The viruses were
propagated in Cre8 cells, purified by cesium chloride density
centrifugation, and stored in aliquots at 80°C as previously described (34). The viral titer was equal to optical
density at 260 nm wavelength (OD260) times dilution and
divided by 9.09 × 10
13 particles/ml. A
hundred particles were assumed to be one plaque-forming unit. The viral
vectors containing ephrin-A5 and ephrin-A5 were named as
Ad.ephrin-A5 and Ad.ephrin-A5, respectively.
Adenoviral vector-mediated ephrin-A5 and ephrin-A5 gene
transfer.
After being plated, the cells were incubated for 24 h, the media
was then changed, and cardiac myocytes were transduced with Ad.LacZ,
Ad.ephrin-A5, or Ad.ephrin-A5 vectors (2 × 109
particles in 2 ml media for 1 × 106 cells),
respectively, and further incubated for 8-40 h before analysis.
In situ staining of ephrin-A5 and ephrin-A5 after gene
transfer.
After transduction with Ad.ephrin-A5 and Ad.ephrin-A5 for 24 h, cardiac myocytes were stained with the EphA5 receptor fused with
alkaline phosphatase (EphA5-AP) as described (5). Cells were incubated with media containing EphA5-AP for 2 h at room temperature and rinsed with Hank's balanced salt solution containing 0.5 mg/ml bovine serum albumin and 20 mM HEPES (pH 7.0). The cells were
fixed with 60% acetone and 3% formaldehyde in 20 mM HEPES (pH 7.5)
for 30 s, washed with wash buffer [150 mM NaCl and 20 mM HEPES
(pH 7.5)], and heated at 65°C for 15 min. Positive staining was
visualized with 0.17 mg/ml sodium 5-bromo-4-chloro-3-indolyl phosphate,
0.33 mg/ml nitro blue tetrazolium, and 2.24 mg/ml
L-homoarginine in color development buffer [100 mM
Tris · HCl, 100 mM NaCl, and 5 mM MgCl2 (pH 9.5)]
at 37°C.
cDNA array analysis of ephrin-A5- and ephrin-A5-responsive
genes in cardiomyocytes.
Total RNA was isolated from neonatal rat cardiomyocytes transduced with
Ad.LacZ, Ad.ephrin-A5, or Ad.ephrin-A5, respectively, for 40 h and treated with RQ1 RNase-free DNase I (Promega). Poly A+ mRNA was then isolated as described above. Total cDNA
probes were labeled directly by reverse transcription of the poly
A+ mRNA with specific oligo primers for the 588 genes in
the cDNA expression array (Clontech; Palo Alto, CA) in the presence of [-32P]dATP (3,000 Ci/mmol, NEN Life Science; Boston,
MA). The probes were purified with a Sephadex-G50 spin column.
Prehybridization was done in the ExpressHyb solution (Clontech) for
1 h. The solution was then replaced with fresh ExpressHyb solution
with the addition of radiolabeled cDNA probes. After overnight
hybridization at 68°C, the membranes were washed four times with 2×
saline-sodium citrate (SSC) and 1% SDS at 68°C and 0.1× SSC and
0.5% SDS and then exposed in a PhosphorImager cassette overnight, and
the results were scanned with a PhosphoImager (Molecular Dynamics;
Sunnyvale, CA). After each hybridization, the filters were
stripped and rehybridized with different probes. Hybridization results
of probes from Ad.LacZ-, Ad.ephrin-A5-, and
Ad.ephrin-A5-transduced cells were compared.
Western blot analysis.
The total protein of cultured neonatal cardiomyocytes was collected
through lysis of the cells into RIPA buffer (1× PBS, 1% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail, Sigma;
St. Louis, MO) after 8-40 h of transduction with the adenoviral
vectors. Protein concentration was determined using Bradford protein
assay with bovine IgG as a standard (Bio-Rad Laboratories; Hercules,
CA). Equal amounts of total protein were separated in a standard
SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes
(Micron Separations; Westborough, MA). The membrane was blocked with
3% fat-free dry milk in PBS and 0.05% Tween 20 for 1 h. EphA3,
ephrin-A5, cyclin D2, cyclin-dependent kinase (CDK)4, and extracellular
signal-regulated kinase (ERK)1 proteins were detected with specific
antibodies (all from Santa Cruz Biotechnologies; Santa Cruz, CA) at a
concentration of 0.3-0.5 µg/ml for 1 h at room temperature.
The membranes were then washed and incubated with horseradish
peroxidase-conjugated secondary antibodies. The reactions were
developed with enhanced chemiluminescence reagents (Pierce; Rockford,
IL), and the images were obtained by exposure to X-ray films.
Quantification of neonatal cardiomyocyte DNA synthesis.
For the quantification of bromodeoxyuridine (BrdU) incorporation,
the cardiomyocytes were incubated for 24 h, transduced with Ad.ephrin-A5 or Ad.ephrin-A5 for 12 h, and then pulsed with 20 µM BrdU for 24 h. The incorporated BrdU was stained using a FITC-conjugated monoclonal anti-BrdU antibody at a dilution of 1:20
(33284X, PharMingen; San Diego, CA). Myosin heavy chain staining with
MF20 at a dilution of 1:4 (developed by D. A. Fischman,
obtained from the Developmental Studies Hybridoma Bank maintained by
the University of Iowa, Department of Biological Sciences, Iowa City, IA) was used as a marker of cardiomyocytes. The cells were cultured in
the absence of a high concentration of BrdU (100 µM, used for inhibition of fibroblast growth in other routine studies). Because of
the absence of high concentrations of BrdU in the initial 24 h of
culture, fibroblasts presented at significantly higher proportions in
these cell preparations for the DNA synthesis studies. However, because
cardiomyocytes were counted separately under the microscope and were
clearly identified, the higher proportion of fibroblasts in the DNA
synthesis studies should not interfere with the result interpretation
for cardiomyocytes.
Flow cytometry.
After infection of the cultured cardiomyocytes with Ad.LacZ,
Ad.ephrin-A5, or Ad.ephrin-A5 for 24-40 h, the cells were
suspended using trypsin and stained with propidium iodide (50 µg/ml).
Flow cytometry was then performed by a specialist at the flow cytometry facility of the University of Pittsburgh using a FACScan flow cytometer
(Becton Dickinson; San Jose, CA). The percentages of cells at
sub-G0/G1, G0/G1, S,
and G2/M phases were recorded and compared.
Statistical analysis.
One-way analysis of variance was applied to compare EphA3,
cyclin D2, and CDK4 expression levels and cell counts in different experimental groups. Comparison among the means was performed with the
post hoc Student-Newman-Keuls test using SPSS statistical analysis
software (11). Results are presented as means ± SE. Statistical significance was considered at P < 0.05.
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RESULTS |
Expression of ephrin-A5 transcripts by cardiac myocytes.
At the time of these experiments, there was no known sequence of rodent
ephrin-A5. The full-length ephrin-A5 was amplified using a pair of
degenerate primers selected from human AL-1, sequenced, and used as a
probe for Northern blot analysis. Northern blot analysis revealed
multiple transcripts of ephrin-A5 at various levels of expression in
rat cardiomyocytes. Up to seven ephrin-A5 transcripts ranging in size
from 1.7 to 7.4 kb in the heart and cardiomyocytes were found.
Consistent with a previous report (17), the expression of
these transcripts was regulated by TNF- (Fig. 1). The significance of multiple
ephrin-A5 transcripts is not clear; therefore, we assessed whether
these transcripts might represent different gene products. A new pair
of primers were then selected from the full-length sequence and used to
amplify shorter forms of ephrin-A5. RT-PCR of cardiomyocyte and brain cDNA with rTth DNA polymerase identified two ephrin-A5 cDNA isoforms with different lengths. The nucleotide and deduced amino acid sequences
of both forms of ephrin-A5 are shown in Fig.
2. The full-length ephrin-A5 sequence was
deposited into the GenBank in 1997 (Accession No. RNU69279). The
full-length ephrin-A5 cDNA (designed as ephrin-A5) coding sequence
encompassed 687 bp in length, whereas the shorter cDNA (designed as
ephrin-A5) had a 81-bp deletion in the region coding for the spacer
domain of ephrin-A5. Eprhin-A5 was the only one found using this
strategy. Two cDNA isoforms with the same sequences were reported
recently (21). Therefore, of the multiple transcripts, at
least one splices in the coding region and generates a form of
ephrin-A5 with a deletion (ephrin-A5). Other transcripts of
different sizes may be due to differential polyadenylation of the mRNA,
which is very common among eukaryotic genes (12, 29).
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Fig. 1.
Expression of ephrin-A5 in cardiomyocytes. A:
Northern blot analysis showing multiple ephrin-A5 transcripts that were
upregulated by tumor necrosis factor (TNF)-. B: Western
blot result of ephrin-A5 proteins. Results are representative of 3 independent experiments. GADPH, glyceraldehyde-3-phosphate
dehydrogenase.
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Fig. 2.
Cloning of 2 forms of ephrin-A5 from the rat heart and
cardiomyocyte. The degenerative primers used for the isolation of the
full-length ephrin-A5 cDNA were located at 48 to 27 and 725 to 747, respectively. The specific primers used for cloning of the shorter cDNA
were located at 1-25 and 666-687, respectively. A:
RT-PCR amplification of ephrin-A5 cDNAs. Lane 1,
cardiomyocytes; lane 2, heart; lane 3, brain; M,
DNA marker. B: cDNAs were cloned and sequenced, and the
nucleotide and amino acid sequences of the full-length and deleted
forms of ephrin-A5 are shown. The 81-bp missing exon region of
ephrin-A5 is underlined.
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Overexpression of ephrin-A5 and ephrin-A5 induced
downregulation of EphA3.
To assess the physiological role of ephrin-A5 and the function
of ephrin-A5, cardiomyocytes were transduced with recombinant adenoviruses carrying each of the two forms of ephrin-A5 cDNAs. Overexpression of ephrin-A5 and ephrin-A5 was highly efficient. Both forms of ephrin-A5 expressed in cardiomyocytes specifically bound the EphA5 receptor, as demonstrated by staining with EphA5-AP. More than 95% of the cells were positively stained (Fig.
3), whereas none of those transduced with
Ad.LacZ was stained. Consistent with ligand-receptor interactions in
other ligand-receptor systems, extended (40 h) high-level expression of
ephrin-A5 by Ad.ephrin-A5 in cardiomyocytes induced downregulation of
the EphA3 receptor (P < 0.05 compared with Ad.LacZ;
Fig. 3). In addition, Ad.ephrin-A5 overexpression in cardiomyocytes
appeared to be more effective in downregulating EphA3 than
Ad.ephrin-A5 despite the fact that it harbored a deletion
(P < 0.05). Because the expression of ERK1 protein was
not changed by ephrin-A5 overexpression, it was used as a control for
protein loading in Western blot analysis of EphA3.
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Fig. 3.
Gene transfer and expression of ephrin-A5 and ephrin-A5 in
cardiomyocytes. EphA5-AP staining shows the expression of both
ephrin-A5 and ephrin-A5 in cardiomyocytes. A-C:
cardiomyocytes transduced with adenoviral LacZ (A),
ephrin-A5 (B), or ephrin-A5 (C).
D: downregulation of the EphA3 receptor by both ephrin-A5
and ephrin-A5. Extracellular signal-regulated kinase (ERK)1 was not
changed and was used as control of protein loading. E:
quantitative results of EphA3 protein levels; n = 4 each. *P < 0.05 compared with Ad.LacZ;
 P < 0.05 compared with
Ad.ephrin-A5.
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Both ephrin-A5 and ephrin-A5 downregulated the expression of
cyclin D2 and CDK4 and reduced neonatal cardiomyocyte DNA synthesis and
growth.
To further assess the molecular effects of ephrin ligand overexpression
on myocyte, we performed cDNA expression array analysis of
cardiomyocytes transduced with either ephrin-A5, ephrin-A5, or
the control vector Ad.LacZ. The expression of cyclin D2 was reduced in ephrin-A5- and ephrin-A5-transduced cells (Fig.
4). To further explore whether the
changes in the mRNA expression of cyclin D2 were associated with
changes at the protein levels and changes in the levels of functionally
associated CDK4, equal amounts of extracts from cardiomyocytes
transduced with Ad.ephrin-A5 or Ad.ephrin-A5 were examined using
Western blot analysis. The results showed that overexpression of either
ephrin-A5 or ephrin-A5 reduced the expression of cyclin D2 and
CDK4 proteins at 24 h postinfection. The representative results of
cyclin-D2 and CDK4 protein expression at 40 h after transduction
are shown in Fig. 4.
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Fig. 4.
Gene expression in cardiomyocytes overexpressing ephrin-A5s. cDNA
expression array analysis shows the regulation of cyclin D2 mRNA by
ephrin-A5 and ephrin-A5. Hybridization results of cDNA probes
radiolabeled by reverse transcription of mRNA were isolated from
Ad.LacZ-transduced cardiomyocytes (A),
Ad.ephrin-A5-transduced cardiomyocytes (B), and
Ad.ephrin-A5-transduced cardiomyocytes (C). Each paired
dot on the blot represents a specific gene. The intensity of the
hybridization signal corresponds to the abundance of transcript levels
of the individual gene. D and E: equal amounts of
protein extracts were analyzed using Western blots (top) for
the expression of cyclin D2 (D) and cyclin-dependent kinase
(CDK)4 proteins (E). Shown are the results after 40 h
of transduction. Bottom: summary of the quantitative results
of cyclin D2 and CDK4 protein expression. n = 4 each. *P < 0.05 compared with Ad.LacZ.
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Because cyclin D2 and CDK4 play an active role in DNA synthesis and
cell proliferation, we examined whether ephrin-A5- and ephrin-A5-induced cyclin D2 and CDK4 downregulation would have an
effect on cardiomyocyte DNA synthesis and growth. Indeed, the number of
neonatal cardiomyocytes with BrdU incorporation was significantly
reduced after overexpression of ephrin-A5 or ephrin-A5 (P < 0.05), suggesting decreased DNA synthesis and
growth (Fig. 5). In addition, FACScan
analysis of the cardiomyocytes transduced with either ephrin-A5 or
ephrin-A5 showed an increase in cell numbers in the
sub-G0/G1 phase and a significant decrease in
the number of cells in the G2/M phase (P < 0.05; Fig. 6).
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Fig. 5.
Overexpression of ephrin-A5 and ephrin-A5 reduced
bromodeoxyuridine (BrdU) incorporation into cardiomyocytes. Nuclei
were stained blue with 4'-6-diaminido-2-phenylindole
(DAPI), nuclei with DNA synthesis and the incorporation of BrdU
were stained green with anti-BrdU antibody, and cardiomyocytes were
stained red with MF-20 anti-myosin heavy chain
antibody. A-C: Ad.LacZ-transduced cells;
D-F: Ad.ephrin-A5-transduced cells; G-I:
Ad.ephrin-A5 transduced cells. C, F, and
I: overlay of DAPI, BrdU, and MF-20 staining.
Arrow, cardiomyocytes with active DNA synthesis. J:
there was a reduced number of BrdU-positive cardiomyocytes after
Ad.ephrin-A5 or Ad.ephrin-A5 transduction. *P < 0.05, compared with Ad.LacZ.
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Fig. 6.
Flow cytometry of propidium iodide-stained cells overexpressing
ephrin-A5. Cells stained with propidium iodide (50 µg/ml) and
apoptotic cells were quantified using a FACScan flow cytometer.
Compared with cells overexpressing LacZ (A),
ephrin-A5-overexpressing cells had an increased
sub-G0/G1 population (B and
C). G2/M phase cells were also significantly
reduced (D). The percentages of cells that are in the
sub-G0/G1 population and G2/M phase
are indicated. *P < 0.05 compared with Ad.LacZ.
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Differential regulation of cardiomyocyte gene expression by
ephrin-A5 and ephrin-A5.
To examine whether the two forms of ephrin-A5 have different effects on
cardiomyocytes, the expression of 588 genes was examined using a cDNA
expression array after transduction of the cells with Ad.LacZ,
Ad.ephrin-A5, or Ad.ephrin-A5. Genes with their expression
altered more than twofold by Ad.ephrin-A5 or Ad.ephrin-A5 relative to Ad.LacZ control are listed in Table
1. Whereas both Ad.ephrin-A5 and
Ad.ephrin-A5 downregulated the expression of cyclin D2 mRNA and
upregulated 27-kDa heat shock protein and macrophage migration
inhibitory factor, the expression of other genes in Ad.ephrin-A5-transduced cardiomyocytes differed from that in Ad.ephrin-A5-transduced cells. Among the genes that showed
significant alterations in their expression, platelet-derived growth
factor (PDGF)-associated protein, carboxypeptidase E, Ral A GTP-binding protein, ras-related protein RAB2, eukaryotic translation initiation factor 2 (subunit 1), and insulin-like growth factor-binding protein may be involved in the gene regulation and growth modulating effects of
ephrin-A5 (Table 1). Shown in Fig. 7 are
the representative results of the cDNA expression array analysis and
Northern blot analysis of BNP expression.
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Fig. 7.
Ephrin-A5 and ephrin-A5 differentially regulate genes in
cardiomyocytes. Hybridization results of cDNA probes radiolabeled by
reverse transcription of mRNA were isolated from Ad.LacZ-tranduced
cardiomyocytes (A), Ad.ephrin-A5-transduced
cardiomyocytes (B), and Ad.ephrin-A5-transduced
cardiomyocytes (C), respectively. The intensity of the
hybridization signal corresponds to the abundance of transcript levels
of the individual gene. Note the arrowheads indicating the changes in
brain natriuretic peptide (BNP) induced by ephrin-A5, and protein
kinase C (PKC) inhibitor protein-1 and clusterin suppressed by
ephrin-A5. D: Northern blot analysis demonstrates that
only Ad.ephrin-A5 upregulated BNP mRNA in cardiomyocytes. GAPDH
hybridization of the same blot was shown to verify equal loading.
*P < 0.05 compared with Ad.LacZ-transduced
cardiomyocytes.
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DISCUSSION |
The prototypic member of the family of ligands for Eph receptors,
ephrin-A1 (B61), was first identified as a protein whose expression was
upregulated in response to proinflammatory cytokines and may function
as a stimulator of angiogenesis (2, 16, 25, 27, 28). The
role of ephrin ligands in cardiovascular development is seen mainly in
vascular development (1, 23, 32). In the present study, we
demonstrated that two alternatively spliced forms of ephrin-A5
(full-length ephrin-A5 and the 27-amino acid-deleted form
ephrin-A5) were expressed in the rat heart. This finding is
consistent with earlier studies (8, 21) in mouse and rat
nervous tissues that demonstrated alternatively spliced transcripts of
ephrin-A5; however, it was not clear about their specific function and
whether they are expressed in the heart.
To elucidate the role of the different splice forms of ephrin-A5
in cardiomyocytes, we overexpressed the two forms of ephrin-A5 in
neonatal cardiomyocytes. The overexpression of both forms of ephrin-A5
resulted in downregulation of cyclin D2 and CDK4, which was associated
with reduced DNA synthesis in neonatal cardiomyocytes. Thus ephrin-A
ligands may have a unique function to withdraw those cells from the
cell cycle by regulating the expression of cyclin D2 and CDK4,
important regulators of cell cycling. These findings in cardiac cells
are consistent with an earlier study (3) demonstrating that activation of EphA5 receptor using an ephrin-A1 recombinant fusion
protein results in a marginal decrease in U-118 MG glioblastoma cell
proliferation. Similarly, spinal cord cells cocultured with ephrin-A5-expressing cells reduced both neurite length and neuron cell
density. Although the mechanisms responsible for neuronal cell loss are
not fully defined, the effect may be apoptosis, attributable to
ephrin-A5 expressed on the cell surface (35). Flow
cytometry study of ephrin-A5- or ephrin-A5-transduced
cardiomyocytes showed increased sub-G0/G1 cell
populations, which suggest increased apoptosis in those cells
(Fig. 6). Taken together, these results suggest a role of ephrin
ligands in cardiac cell growth and survival. However, mice with
ephrin-A5 gene knockout demonstrated defects in axon guidance in the
midbrain and dorsal midline structure, with anencephaly or open
crania with cleft nose and palate, but no obvious cardiac defects
(9, 26). The inability of ephrin-A5 knockout to induce
cardiac effects might suggest a large degree of redundancy in the
ephrin-mediated pathways, although cardiomyocyte changes have not been
assiduously evaluated in that model.
In the nervous system, ephrin-A5 is required for the proper guidance
and mapping of retinal axons in the mammalian midbrain by a two-step
mechanism of Eph receptor activation with distinct ligand binding and
ligand-independent receptor-receptor oligomerization events (9,
20). Those various functions of ephrin-A5 in different systems
may be achieved by different levels of its expression and differential
regulation of genes (17). Indeed, whereas ephrin-A5 and
ephrin-A5 share common effects such as downregulation of cyclin D2 and upregulation of 27-kDa heat shock protein and macrophage migration inhibitory factor, ephrin-A5 markedly regulated other growth-regulating genes such as eukaryotic translation initiation factor 2 (subunit 1) and insulin-like growth factor-binding protein. Ephrin-A5 may also be involved in the regulation of gene expression through differential regulation of genes such as PDGF-associated protein, carboxypeptidase E, Ral A GTP-binding protein, and ras-related protein RAB2.
In conclusion, multiple ephrin-A5 transcripts were found to be highly
responsive to TNF- in neonatal rat cardiomyocytes. Two isoforms of
ephrin-A5, ephrin-A5 and ephrin-A5, were identified in rat
cardiomyocytes. Overexpression of these two forms of ephrin-A5 reduces
BrdU incorporation in cardiomyocytes. In addition, ephrin-A5 and
ephrin-A5 regulated different sets of gene expression. These differential effects of ephrin-A5 and ephrin-A5 on cardiomyocyte gene expression suggest that these ligands may have additional different functions in cardiomyocytes.
| |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institutes of Health
Grants DK-44935 and AR6-2225 (to P. D. Robbins).
| |
FOOTNOTES |
Address for reprint requests and other correspondence: Y. Y. Li, Cardiovascular Institute, Univ. of Pittsburgh School of
Medicine, 1750 Biomedical Science Tower, 200 Lothrop St., Pittsburgh,
PA 15213 (E-mail: liyuny{at}pitt.edu).
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.
Received 2 May 2001; accepted in final form 10 August 2001.
| |
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INTRODUCTION
