Center for Biorestoration of Oral Health and Department of Periodontics, Prevention and Geriatrics, University of Michigan, Ann Arbor, Michigan 48109-1078
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ABSTRACT |
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The delivery of platelet-derived
growth factor (PDGF) for tissue engineering of skin and periodontal
wounds has become an active area of interest. However, little is known
regarding the extended effects of PDGF on cell signaling via gene
therapy and how such an approach facilitates the exiting of cells from
growth arrest and entry to competence required for cell cycling. We
show in vitro expression and secretion of PDGF-AA by recombinant
adenovirus encoding the PDGF-A gene (Ad-PDGF-A). The bioactive
PDGF-AA protein released induces sustained downregulation of PDGFR
that is encoded by a growth arrest-specific (gas)
gene. Ad-PDGF-A induces sustained phosphorylation of PDGF
R as well
as prolonged phosphorylation of downstream extracellular
signal-regulated kinase 1/2 and Akt signaling pathways. Furthermore,
the phosphorylation of PDGF
R is abolished by cotransducing cells
with adenovirus encoding a dominant negative mutant of the PDGF-A gene
that disrupts PDGF bioactivity. These findings demonstrate the
prolonged effects of adenoviral delivery of PDGF and aid in the better
understanding of sustained PDGF signaling.
platelet-derived growth factor; growth arrest; extracellular signal-regulated kinase; Akt/protein kinase B
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INTRODUCTION |
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PLATELET-DERIVED
GROWTH FACTOR (PDGF) is a potent mitogen for cells of
mesenchymal origin and possesses pluripotential effects on wound
healing (15, 17). Recently, multiple investigators have studied the promotion of tissue repair using plasmid DNA and
adenoviral approaches for PDGF gene delivery (5, 9, 10,
12). In addition to the well-described PDGF-A and -B chains, two
new members of this family, PDGF-C and PDGF-D, were recently identified
(2, 23, 24). Two PDGF receptors (PDGFRs) also have been
comprehensively described, PDGFR and PDGF
R (6, 15).
In this work we have focused on PDGF-AA, which selectively binds to and
activates PDGF
R receptor homodimerization and the triggering of
several signaling cascades (15, 17).
PDGFR protein binds to PDGF-AA, -AB, -BB, and -CC isoforms and has a
central role in mediating the state of competence induced by PDGF
(6, 7). In addition to its traditional role as a receptor
tyrosine kinase, PDGF
R was later identified as being encoded by a
growth arrest-specific (gas) gene (25).
Gas genes are preferentially expressed when cell division in
culture is prevented by serum deprivation. In principle, gas
genes encode products that cause growth arrest or, conversely, can be
expressed persistently during the growth-arrested state
(G0) because factors required for cell cycling are lacking
(3, 28). It has been shown that expression of PDGF
R,
which is important in cellular mitogenic responses and early stage
embryogenesis following growth arrest (27), may facilitate
the cell cycling that occurs following the addition of PDGF
(25).
In regard to cell signaling, it is traditionally believed that growth
factor-stimulated signaling events including PDGF signal transduction
occur transiently. However, it is difficult to reconcile how PDGF as a
mitogen contributes to cell cycle entry, which occurs many hours later.
Harrington et al. (14) first demonstrated the possibility
of "late" PDGF receptor phosphorylation following PDGF exposure
under specific experimental conditions. More recent cumulative evidence
suggests that growth factor-dependent signaling is not restricted
to the 1- to 2-h time frame subsequent to stimulation as has been
generally thought for many years. (1, 19, 20). PDGF
triggers a second wave of "late" phosphorylation (i.e.,
phosphatidylinositol 3-kinase and protein kinase C activities) that
occur ~2-7 h after PDGF exposure. The second phase of
phosphorylation events appears critical for cellular proliferation
(32). In our study, we observed time periods beyond the
late phase of phosphorylation for up to 96 h after PDGF treatment.
We utilized gene transfer of adenovirus encoding PDGF-A (Ad-PDGF-A) to
continuously produce PDGF-AA protein to study the signaling events
and PDGFR expression as a measure of traversing cell cycle
growth arrest. We have shown that Ad-PDGF-A prolongs
phosphorylation of extracellular signal-regulated kinase (ERK), Akt,
and the correlative sustained downregulation of PDGF
R, a
gas gene product.
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MATERIALS AND METHODS |
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Adenovirus construction. We have previously described the construction of adenoviruses encoding the PDGF-A and PDGF-1308 (dominant negative mutant of PDGF-A) genes (37). In brief, the full-length murine PDGF-A or PDGF-1308 cDNA (gifts of Dr. C. D. Stiles, Boston, MA) was subcloned into a shuttle plasmid (obtained from Genzyme, Cambridge, MA) under the control of the cytomegalovirus promoter. The precut viral backbone DNA Ad2/EGFP (encoding green fluorescent protein) and the shuttle plasmid containing either PDGF-A or PDGF-1308 cDNA were linearized by restriction enzyme digestion. The linearized shuttle plasmid and viral backbone DNA were cotransfected into 293 packaging cells. Recombination between the shuttle plasmid and the GFP viral backbone resulted in replacement of the GFP cDNA with PDGF-A or PDGF-1308 cDNA. Subsequent recombinant viral plaques were identified, picked, and purified. Titers of the virus stocks were determined on 293 cells by plaque assay and expressed as the number of plaque-forming units per ml.
Cell culture and gene transfer. Primary cultures of rat dermal fibroblasts (DFs; kindly donated by Dr. R. B. Rutherford, University of Michigan) were utilized as previously described for ex vivo gene transfer (22). Low-passage cells were plated in six-well culture dishes containing DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, and 2 mM glutamine. Subconfluent cultures were then brought to a stage of quiescence by washing with PBS and reducing the serum concentration to 0.1% for 48 h prior to adenovirus transduction or recombinant human PDGF-AA (rhPDGF-AA; Upstate Biotechnology, Lake Placid, NY) treatment. In all experiments, cells were treated either with recombinant adenoviruses (Ad-GFP, Ad-PDGF-A or Ad-PDGF-1308) for 4-5 h at a multiplicity of infection (MOI) of 100 or with 20 ng/ml rhPDGF-AA. Cells were incubated for various time periods (see below) depending on the experiment and were harvested at 8, 24, 48, and 96 h.
Measurement of PDGF-AA after transduction with Ad-PDGF-A.
To determine the production of PDGF-AA protein following gene transfer,
we infected cells with Ad-PDGF-A as described in Cell culture and
gene transfer. The conditioned cell culture medium was
harvested 24 and 96 h after Ad-PDGF-A transduction and was centrifuged to remove cell debris. Phenylmethylsulfonyl fluoride (PMSF;
1 mM; Sigma Chemical, St. Louis, MO) was added to the conditioned medium. The medium was then transferred to molecular porous membrane tubing (Spectrum Laboratories, Rancho Dominguez, CA) and dialyzed at 4°C overnight while deionized H2O containing 0.1 mM
PMSF was gently stirred in. The samples were lyophilized and then
suspended in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) containing -mercaptoethanol for subsequent analysis by Western blotting techniques as previously described (11). In
brief, samples were resolved on SDS-polyacrylamide gels and
electrophoretically transferred to polyvinylidene difluoride membranes.
Membranes were blocked for 1 h in PBS containing 5% nonfat dry
milk, followed by incubation for 1 h with polyclonal anti-PDGF-A
as the primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Donkey anti-rabbit Ig-horseradish peroxidase conjugate (Amersham Life
Science, Amersham, UK) was added as the secondary antibody. In
experiments in which a monoclonal antibody was used as primary
antibody, sheep anti-mouse Ig-horseradish peroxidase conjugate
(Amersham Life Science) was added as the secondary antibody. Specific
immunoreactive protein bands were detected by using an enhanced
chemiluminescence reagent (ECL; Amersham Pharmacia Biotech,
Piscataway, NJ).
Measurement of PDGFR expression.
Cells were washed twice with ice-cold PBS and then lysed in
radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris · HCl, pH 7.3, 150 mM NaCl, 0.25 mM EDTA, pH 8.0, 1% Triton X-100, 1% C24H39O4Na, 0.2% NaF, 0.1%
Na3VO4, 1 mM PMSF, 5 µg/ml aprotinin, and 20 µM leupeptin). Protein concentrations of cell lysates were normalized
using a protein assay kit (Bio-Rad). Equal amounts of proteins were
processed for Western blotting analysis with PDGF
R (Santa Cruz
Biotechnology) as primary antibody. In addition,
-tubulin (Sigma)
was measured to show the corresponding equal loading of cell lysates.
Densitometric scanning was performed by normalizing PDGF
R expression
to
-tubulin to demonstrate relative changes in PDGF
R expression
following PDGF-AA exposure, using NIH Image analysis software (National
Institutes of Health, Bethesda, MD).
Measurement of phosphorylation of PDGFR, ERK1/2, and Akt.
For phosphotyrosine analysis of immunoprecipitated PDGF
R, 200 µg
of total protein from cell lysates were incubated with 1 µg of
PDGF
R antibody (Upstate) for 2 h at 4°C. Subsequently, 30 µl of protein A-agarose suspension beads were added to capture the
immunocomplexes for 1 h at 4°C. Agarose immunocomplexes were then centrifuged and resuspended for phosphotyrosine Western blot analysis. Anti-phosphotyrosine antibodies PY20 (Santa Cruz
Biotechnology) and 4G10 (Upstate) were combined and used to detect the
presence of tyrosine phosphorylated PDGF
R. The activated form of
ERK1/2 was tested by using a specific anti-phospho-ERK1/2 antibody
(Cell Signaling Technology, Beverly, MA) that recognized only the
activated forms phosphorylated on Thr202 and
Tyr204, and the activated form of protein kinase B
(PKB)
/Akt was tested by using a specific anti-phospho-Akt antibody
(Cell Signaling) that recognized only the activated forms
phosphorylated on Ser473. ERK1, PKB
/Akt (BD Transduction
Laboratories, Lexington, KY), and
-tubulin proteins were measured to
show the relative protein loading for each of the lanes.
Cell cycle regulation by Ad-PDGF-A: flow cytometry and assessment of cyclin D1 and cyclin E. To determine cell cycle distribution, DF cell monolayers were harvested by trypsinization, washed once with cold PBS, stained and hypotonically treated with 500 µl of propidium iodide hypotonic lysis buffer [0.1% sodium citrate, 0.1% Triton X, 100 µg/ml RNase A (Sigma), and 50 µg/ml propidium iodide (Sigma)], and analyzed by flow cytometry as previously described by using 488-nm excitation (19). In addition, cell lysates in RIPA buffer were tested for the expression of cyclin D1 and cyclin E with anti-cyclin D1 or anti-cyclin E antibodies (Santa Cruz Biotechnology) by using Western blotting analysis.
Coinfection of cells with Ad-PDGF-A and Ad-PDGF-1308.
To determine the reversibility and specificity of Ad-PDGF-A on signal
transduction, we coinfected DFs with Ad-GFP or Ad-PDGF-1308 with
Ad-PDGF-A. The cells were initially transduced with Ad-PDGF-1308 or
Ad-GFP (MOI = 300) 24 h before infection with Ad-PDGF-A or Ad-GFP (MOI = 100) for an additional 24 h. Cell lysates were
harvested, and PDGFR expression and PDGF
R tyrosine
phosphorylation were assessed by immunoprecipitation using PDGF
R antibody.
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RESULTS |
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Expression of PDGF-AA by Ad-PDGF-A gene transfer. To examine whether Ad-PDGF-A-transduced cells produce and secrete bioactive PDGF-A protein, we collected conditioned media from transduced cells and then immunoprobed for PDGF-A by Western blot analysis. To choose the appropriate media collection time for maximal protein release, we performed a fluorescence-activated cell sorting (FACS) time course using the control virus Ad-GFP to measure protein expression kinetics. FACS analysis revealed initial GFP fluorescence as early as 6-8 h after Ad-GFP exposure, with >95% of the cells exhibiting GFP protein by 20-24 h after transduction with a MOI of 100 (data not shown). Therefore, conditioned media were analyzed at 24 and 96 h for PDGF-AA protein secretion.
Figure 1 demonstrates a Western blot of protein that was concentrated and lyophilized from medium that was probed with a monospecific PDGF-A antibody (Santa Cruz Biotechnology). Ad-PDGF-A-transduced cells produced immunoreactive protein that exhibited the same electrophoretic mobility as purified PDGF-A peptide on
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Prolonged downregulation of PDGFR by Ad-PDGF-A gene transfer.
To determine whether continuous treatment with PDGF via adenovirus
results in prolonged effects, we examined the downregulation of
PDGF
R that results from sustained ligand binding and activation. Cells treated with the control virus Ad-GFP (Fig.
2, lanes 1-6), serum-free
medium (data not shown) or Ad-PDGF-1308 (data not shown) did not
demonstrate any measurable changes in PDGF
R expression over the 96-h
observation period. PDGF-AA treatment revealed a potent reduction in
PDGF
R protein levels within 2-3 h after treatment (data not
shown), and this diminution was sustained for at least 24 h before
it returned to baseline levels at 48 h (Fig. 2, lanes 7-12). On the other hand, cells treated with Ad-PDGF-A
resulted in prolonged downregulation of PDGF
R for at least 96 h
(Fig. 2, lanes 13-18). This reduction of PDGF
R was
first noted at 24 h and was sustained for 96 h. The lag in
PDGF
R downregulation by Ad-PDGF-A was presumably due to the time
required for PDGF-AA protein expression (Fig. 2, lanes
13-18). These results suggest that the brief exposure of
cells to PDGF-AA results in a transient (~24 h) reduction in PDGF
R
levels, whereas adenoviral delivery results in constant downregulation
of the PDGF
R for at least 96 h.
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Coordination of sustained phosphorylation of ERK and Akt pathways
with the sustained tyrosine phosphorylation of PDGFR.
The goal of these experiments was to identify the effects on signaling
elicited by continuous PDGF-AA treatment. Binding of ligand to
tyrosine receptor kinase results in an elevation of activity of a
receptor kinase and a triggering of downstream signal transduction
pathways. Because the prolonged adenoviral protein expression was shown
previously, we sought to determine whether the adenoviral PDGF-A
protein is as biologically active as rhPDGF-AA and whether
adenoviral delivery could extend intracellular signaling events; we
therefore evaluated the phosphorylation of PDGF
R and several key
pathways involved in PDGF signaling. Our results show that Ad-PDGF-A
induces the phosphorylation of PDGF
R by 8 h after gene delivery
and that the phosphorylation increases gradually and is maintained over
96 h (Fig. 3A,
lanes 7-12). The phosphorylation induced by PDGF-AA
peptide was sustained and weakened gradually by 72 h (Fig.
3A, lanes 1-6).
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Competition between PDGF-1308 and PDGF-A on receptor tyrosine
phosphorylation.
To investigate the reversibility of PDGF-A signaling events, we
performed a competition study on receptor tyrosine phosphorylation between PDGF-1308 and PDGF-A. In this experiment, DFs were infected with Ad-PDGF-1308 24 h before Ad-PDGF-A or Ad-GFP transduction to
"prime" the cells with PDGF-1308 protein. Figure
4 demonstrates that PDGF-1308 (MOI = 300) completely abolished the phosphorylation of PDGFR induced by
Ad-PDGF-A (MOI = 100), whereas Ad-PDGF-1308 (MOI = 100-200) only partially inhibited PDGF
R tyrosine
phosphorylation (data not shown). Interestingly, coinfection of
Ad-PDGF-1308 (MOI = 100-300) and Ad-PDGF-A (MOI = 100)
could not affect PDGF
R protein levels elicited by Ad-PDGF-A (data
not shown). This datum suggests that the mechanisms of PDGF-1308
antagonism with native PDGF-A chains may not prevent dimer association
with the PDGF
R to alter receptor expression but does disrupt the
ability of PDGF-A isoforms to elicit functional PDGF
R tyrosine
phosphorylation.
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DISCUSSION |
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Despite the active interest in utilizing long-term PDGF delivery
to stimulate tissue repair, there is minimal information available
regarding the signal transduction mechanisms involved following PDGF
gene transfer. Our observations have provided evidence suggesting that
prolonged PDGF-A expression by adenovirus allows for continued
downregulation of PDGFR, which results from sustained activation by
ligand binding. Our findings also illustrate the sustained stimulation
of several signaling pathways; notably, activation of ERK1/2 and Akt,
which coincides with prolonged activation of PDGF
R, may contribute
to the mechanisms of growth-arrest exiting. In addition, this is the
first study to demonstrate the sustained effects of PDGF-AA on signal
transduction by Ad-PDGF-A and its antagonist, Ad-PDGF-1308.
In serum-free conditions, cells are induced to leave the cell cycle and
enter a state of growth arrest, where they remain unless triggered to
reenter the cycle by mitogenic signals. Certain genes are known to be
preferentially or selectively expressed during growth arrest and are
called gas genes (3, 28). PDGFR was
shown to be a gas gene by use of the lacZ gene to
identify chromosomal loci that are transcriptionally active during the growth arrest of NIH/3T3 cells (25) or cell cycle
repression genes by microarray (8). Lih et al.
(25) demonstrated that PDGF
R is not induced in NIH/3T3
cells cultured in serum-deprived medium when transformed with
erb2, src, or raf, all of which block growth arrest under conditions of serum deprivation. Their
results indicated that regulation of PDGF
R expression is governed by cell cycling, rather than the presence or absence of serum growth factors per se. The addition of PDGF-AA protein to growth-arrested cells enables progression in the cell cycle. While such PDGF-stimulated cells become "competent" for cycling, progression through
G1 and entry into S phase require additional factors
usually present in serum or in platelet-poor plasma in
"traditional" cell types such as BALB/c 3T3 cells
(15). However, recently it was found that in certain cell
types such as AKR-2B (32) and NIH/3T3 (1, 19,
20), PDGF-BB alone could function not only as a competence factor but also as a progression factor when it was continuously present in the medium. Our studies have shown that the DF is more like
a traditional cell type, where PDGF-AA functions solely as competence factor. Our data show that adenovirus produces PDGF-AA dimers continuously and that this prolonged expression of PDGF activates and downregulates PDGF
R for extended time periods. Nevertheless, this sustained PDGF expression by adenovirus cannot drive
cells to progress beyond the G1/S boundary without
progression factors. Using propidium iodide staining, we failed to
demonstrate cell cycle progression into S phase following treatment
with PDGF-AA for up to 16 h unless either serum or IGF-I was added
to the medium (data not shown). Although PDGF alone did not elicit DNA
synthesis, we believe these data support the potential efficacy of PDGF
gene delivery for tissue repair. In these studies serum deprivation conditions were used to specifically examine effects of long-term PDGF
delivery in vitro (which avoids the effects of other growth factors or
cytokines in serum). However, in vivo gene therapy approaches would
result in much different results under physiological conditions, where
there are various other growth factors, cytokines, etc., that
collaborate with PDGF in cell cycle progression. Furthermore, this
study focused on mitogenic signal transduction events elicited by PDGF.
PDGF is known to be not only a strong mitogen but also a
chemoattractant, a potent stimulator of matrix biosynthesis and
granulation tissue formation (17). Therefore, the
pleotropic effects of PDGF highlight its efficacy as a strong promoter
of wound repair in vivo using gene therapy (5, 9, 10).
It is well documented that sustained receptor activation by ligand
induces receptor downregulation. This phenomenon results mainly from
receptor internalization such as receptor endocytosis and intracellular
degradation process as effects of a ligand autocrine system. The
downregulation may provide a mechanism for desensitizing the cells to
subsequent stimulation by that ligand (4, 16, 18, 21, 33).
The sustained downregulation of PDGFR by Ad-PDGF-A in our study is
therefore an expected result of sustained activation of PDGF
R by
continuous PDGF-AA production and binding. However, several unexpected
findings show that sustained stimulation of cells by mitogenic growth
factors or certain transforming oncogenes induce receptor
downregulation not only at the protein level but also at the mRNA level
via reduction of mRNA transcription (25, 26, 34, 36). It
has been suggested that the state of growth arrest (G0) is
associated with enhanced expression of PDGF
R and/or PDGF
R with
corresponding repression of these receptors at both the mRNA and
protein levels in normal cells after growth factor stimulation or in
oncogene-transformed cells (25, 34). For instance, Vaziri
and Faller (34) tested the regulation of PDGF
R in
BALB/c 3T3 cells by using basic fibroblast growth factor (FGF)-2 instead of PDGF and found that both mRNA and protein of PDGF
R were
downregulated. We also noted similar results on the downregulation of
PDGF
R by FGF-2 in our primary DFs by semiquantitative PCR (data not
shown). Together, these findings are important to elucidate the role of
PDGF
R in cell cycling and better understand the phenomenon of
PDGF
R downregulation.
We and others have shown that PDGF stimulates sustained ERK activity
(35). Induction of activated MEK (mitogen-activated protein kinase/ERK kinase) also has been revealed to elevate cyclin D1
levels (35). Whether activated MEK alone is sufficient to trigger DNA synthesis needs further investigation. Our results showed
that there was the expected early induction of cyclin D1 and cyclin E
protein levels as early as 8 h after treatment with either
Ad-PDGF-A or PDGF-AA; however, these levels remained relatively constant throughout the 96-h observation period (data not shown). Cyclin D1 and cyclin E are characterized as G1 proteins
because they increase when reentering the cell cycle from
G0. Cyclins D1 and E are further degraded after their
active combination with their specific cyclin-dependent kinases
(13, 31). Given the lack of significant alterations in
cyclin D1 and E levels over time, we speculate that minimal DNA
synthesis occurs when DFs are exposed to PDGF-AA or Ad-PDGF-A in the
absence of other serum or plasma factors despite the fact that
Ad-PDGF-A prolongs activation of ERK1/2 and Akt. Here we have shown
that the extended ERK and Akt activities correlate with the sustained
tyrosine phosphorylation of PDGFR and the continued downregulation
of PDGF
R in G1, suggesting a necessary link to
mitogen-induced progression through G1. However, such
signaling events are insufficient to commit cells beyond the
G1/S boundary. Further study of other more downstream
signaling molecules following PDGF exposure may aid in the better
understanding of S phase entry in the cell cycle. Moreover, future gene
delivery approaches using other vectors and evaluating additional
growth factors would be important to a better understanding of
sustained signaling using viral vectors or plasmid DNA. To date, little has been evaluated to determine what other effects these vector strategies may play in modulating signal transduction in vitro and in vivo.
Furthermore, we have shown that a dominant negative form of PDGF-A,
PDGF-1308, can compete with the wild-type growth factor in PDGFR
activation. PDGF-1308, generated by site-directed mutagenesis (cysteine-129 to serine), reduces both homo- and heterodimer secretion of PDGF by forming inactive or unstable heterodimers with wild-type A
and B subunits (29, 30). We used adenoviral infection to show the antagonistic effect of PDGF-1308 in terms of receptor activation. We found that coinfection of Ad-PDGF-A and PDGF-1308 failed
to reverse the downregulation of the PDGF
R elicited by continuous
PDGF-A exposure (data not shown). However, when we evaluated the
perturbation in receptor tyrosine phosphorylation, we
demonstrated that Ad-PDGF-A blocked the Ad-PDGF-A mediation of
PDGF
R downregulation. Hence, PDGF-1308 acts by preventing the
transduction of a signal while having no effect on altering PDGF
R
protein levels.
In conclusion, the results from this study suggest that delivery of the
PDGF-A gene by adenovirus elicits the production of PDGF-AA protein
with extended effects on PDGFR downregulation and tyrosine
phosphorylation. Furthermore, Ad-PDGF-A also results in significant
prolongation of phosphorylation of two key signaling molecules, ERK1/2
and Akt, over time.
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ACKNOWLEDGEMENTS |
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This study was funded by National Institute of Dental and Craniofacial Research Grants DE-11960 and DE-13397 (to W. V. Giannobile). FACS experiments were performed at the University of Michigan Flow Cytometry Facility.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. V. Giannobile, Center for Biorestoration of Oral Health and Dept. of Periodontics, Prevention and Geriatrics, Univ. of Michigan, 1011 N. Univ. Ave., Ann Arbor, MI 48109-1078 (E-mail: wgiannob{at}umich.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.
10.1152/ajpcell.00419.2001
Received 29 August 2001; accepted in final form 3 November 2001.
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