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Regular Articles |
From the Centre for Thrombosis and Vascular Research,*
The University of New South Wales, Sydney, Australia; and Johnson and
Johnson Research Pty. Limited,
Sydney, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early growth response factor-1 is the product of an immediate-early
gene and is a prototype member of the zinc finger superfamily of
transcription factors.5-7
The gene appears on human
chromosome 5 (5q23–31) and encodes a protein with three tandem
Cys2-His2 zinc finger motifs.5
Egr-1 interacts with the major groove of DNA8
and
associates with the promoter regions of a large number of genes
implicated in the pathogenesis of vascular occlusive lesions. These
include genes encoding cytokines, adhesion molecules, components of the
coagulation cascade, and growth factors such as basic fibroblast growth
factor, transforming growth factor-ß and platelet-derived growth
factor9-11
(reviewed in Refs. 10 and 12
). Recent studies
have implicated a regulatory role for Egr-1 in the control of cell
proliferation. For example, transforming
growth factor-
enhancement of cyclin D1 transcription may involve
the interaction of Egr-1 with a cis-regulatory element in
the D1 promoter.13
Similarly, a dominant negative form to
Egr-1 blocks progression of the cell cycle from late G1 to
the S-phase.14
In the normal rat vessel wall, Egr-1 is
expressed at low or undetectable levels. However, it is dramatically
induced following balloon dilatation of the carotid
artery15
or on denudation of arterial
endothelium.10,16
Since Egr-1 positively regulates the
expression of multiple genes implicated in the proliferative response
to injury, targeting this transcription factor in SMCs may ultimately
be useful in efforts to reduce the incidence of restenosis after
angioplasty.
Gene expression can be suppressed by exploiting specificity conferred by Watson-Crick base pairing. Synthetic antisense oligonucleotides (ODNs) can bind to target mRNA and block gene expression by presenting the mRNA as a heteroduplex such that it is a substrate for endogenous RNase H. Alternatively, the duplex can effect a steric block as the translational machinery moves along the RNA. Synthesis of the target protein is suppressed thereby compromising the ability of the factor to participate in biological processes. In this paper, we used the antisense approach targeting Egr-1 in cultured vascular SMCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphorothioate (PS)-ODNs were synthesized commercially and
purified by HPLC to greater than 90% purity (Oligos, Etc.).
PS-ODNs were 5'-end labeled with [
32P]ATP
(Bresatec) using T4 polynucleotide kinase (New
England Biolabs). Unincorporated label was separated by
centrifugation using Chromaspin-10 columns (Clontech).
Cell Culture
Rat aortic SMCs (used between passages 3–7) (Cell Applications, Inc.) were cultured in Waymouth's MB752/1 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 30 µg/ml L-glutamine, 10 units/ml penicillin and 10 µg of streptomycin at 37°C and 5% CO2. Cells were passaged by rinsing the monolayers with phosphate-buffered saline (PBS) and trypsinization.
32P-PS-ODN Uptake
Subconfluent SMCs were arrested overnight in serum-free medium (SFM). The 32P-labeled PS-ODN was incubated with the cells for various times before washing with cold PBS and solubilization in 0.1 mol/L NaCl, 0.01 mol/L Tris-HCl, pH 7.6, 1 mmol/L EDTA, pH 8.0, 1 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonyl fluoride (PMSF). Cell-associated radioactivity in the lysate was quantitated in a ß-scintillation counter (Packard).
Fluorescence Microscopy
SMCs were seeded onto chamber slides (Nunc InterMed) and grown to 80% confluency. Fluoresceinated (FITC) PS-ODN was incubated with the cells 6 hours after the change of medium to serum-free and allowed to incubate for a further 24 hours. The cells were washed with PBS before fixation with 2% paraformaldehyde for 30 minutes at 22°C. The fixed cells were then treated with permeabilizing solution (0.5% Triton X-100, 50 mmol/L NaCl, 3 mmol/L MgCl2, 10 mmol/L PIPES, pH 6.8) for 1 minute at 4°C. The slides were mounted in anti-fade mounting medium (0.1% p-phenylenediamine, 10% PBS, 90% glycerol) before fluorescence imaging using a laser scanning microscope (Olympus).
Assay of DNA Synthesis
DNA synthesis in SMC was assessed using a modification of a protocol described previously.17 SMCs were seeded into the wells of a 96-well titer plate (3000 cells/well) and incubated for 48 hours. The cells were incubated with SFM for 6 hours before addition of PS-ODN and incubation for a further 18 hours. The cells were incubated for 24 hours with fresh PS-ODN in medium containing a concentration of FBS sufficient to stimulate [3H]thymidine incorporation into DNA submaximally. The cells were pulsed with [3H]thymidine for 6 hours. The cells were washed three times with cold PBS to remove unincorporated label. The cells were then fixed with cold 5% trichloroacetic acid and washed with cold absolute ethanol. Plates were dried at 37°C for 10 minutes and the cells were lysed with 0.1 mol/L NaOH. ACSII scintillant was added to tubes containing the lysate and radioactivity was quantitated using a ß-scintillation counter (Packard).
Total Cell Counts
SMCs were seeded into the wells of a 96-well titer plate (3000 cells/well) in complete medium and incubated for 72 hours before incubation in SFM. After 6 hours, PS-ODNs were added to the wells and incubated for a further 18 hours. FBS sufficient to stimulate cell replication submaximally was added together with fresh PS-ODN and the incubation was continued for a further 72 hours. The cells were rinsed with PBS, trypsinized, and the cell suspension quantitated by hemocytometry or Coulter counter.
Western Immunoblot Analysis
Subconfluent SMCs in Petri dishes were incubated with SFM for 6 hours before addition of PS-ODN and a further incubation for 18 hours. Medium containing FBS and fresh PS-ODN was added and incubated for the times indicated. The cells were washed in cold PBS and extracted in 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mmol/L EDTA, 1% trasylol, 10 µg/ml leupeptin, 1% aprotinin, 2 mmol/L PMSF). Ten-µg protein samples were loaded onto 10% denaturing SDS-polyacrylamide gels and electroblotted onto PVDF nylon membranes (NEN-DuPont). Membranes were air dried before blocking with nonfat skim milk powder in PBS containing 0.1% Tween 20. The membrane was incubated with the antibodies to Egr-1 (Santa Cruz Biotechnology, Inc.) or PDGF-A (Genzyme) then with horseradish peroxidase-linked mouse anti-rabbit Ig secondary antiserum. Bands were visualized by chemiluminescent detection (DuPont, NEN). Where indicated, the membrane was stripped reprobed with antibodies to Sp1 (Santa Cruz).
In Vitro Injury Model
SMCs were grown to confluence in slide chambers and the medium was changed to serum-free. Six hours subsequently, the cells were exposed to 1 µmol/L of PS-ODN and incubated for an additional 18 hours. Two hours before scraping the cells with a sterile toothpick, mitomycin C (20 µmol/L, final concentration) and fresh PS-ODN (1 µmol/L) were added. The cells were incubated for 72 hours before fixation with 4% (v/v) formaldehyde and staining with hematoxylin/eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Certain regions of any given mRNA are more susceptible to PS-ODN
binding and inhibition than others. The basis for this variation in
accessibility may reside in regional differences in the conformation of
the mRNA. Areas of low theoretical secondary structure were determined
for Egr-1 mRNA by application of the MFOLD program to consecutive 50
base-overlapping windows of 250 bases within the mRNA.18
Regions containing low levels of local secondary structure in all
folded structures were selected as potential target sites, and a series
of antisense phosphorothioate-modified ODNs of 15 base length were
synthesized (Table 1)
.
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The capacity of an PS-ODN to inhibit gene expression by antisense
mechanism(s) is contingent on its ability to enter the cell. We first
determined whether a candidate PS-ODN, E11, whose sequence is
complementary to a region within Egr-1 mRNA encoding the third zinc
finger (Figure 1)
, could localize within
SMCs. To facilitate these studies, E11 was fluorescein-labeled at its
5' end and incubated with cultured SMCs. After 24 hours exposure, the
PS-ODN was readily detectable apparently within the cytoplasm and, to a
lesser extent, the nucleus (Figure 2
,
bottom). FITC-E1, a size-matched, nonspecific PS-ODN, E1, was taken up
with similar efficiency (Figure 2
, center). Autofluorescence was not
observed in cultures in which PS-ODN was omitted (Figure 2
, center).
These results indicate that SMCs incorporate PS-ODN from the culture
media and accumulate the nucleic acid intracellularly. PS-ODN
accumulation in the extranuclear environment is consistent with PS-ODN
localization previously observed in the absence of
carrier.19-22
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To determine the temporal pattern with which E11 associates with
SMCs, the PS-ODN was 5' end-labeled with 32P by kinase
reaction and incubated with the cells for various times.
32P-E11 was taken up most efficiently within the first 4
hours of exposure and optimally after 6 hours (Figure 3)
. Interestingly, 32P-E11
was still associated with the cells even after 24 hours (Figure 3)
.
Electrophoretic analysis on denaturing gels revealed that
cell-associated 32P-E11 was not fragmented even after 24
hours, since laddering was not observed (data not shown).
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E11 and the entire antisense PS-ODN panel (Table 1)
were assessed
for the ability to inhibit SMC proliferation inducible by serum. SMCs
were exposed to the PS-ODNs 6 hours after the removal of serum from the
medium to induce growth arrest. PS-ODNs were added a second time 18
hours subsequently with a change of medium and the addition of serum.
In preliminary titration studies, we determined that the maximal
tolerated dose of the nonspecific PS-ODN, E1, was 1 µmol/L (data not
shown). Further experiments revealed that PS-ODNs E11, and AS2, whose
sequence is complementary to the translation initiation region (Figure 1)
, inhibited [3H]thymidine incorporation into DNA at
this concentration in cells exposed to serum by over 50% (Figure 4)
. In contrast, E1 and antisense PS-ODNs
E6, E7, and E9 (Figure 4)
failed to inhibit serum-inducible DNA
synthesis in SMCs. Antisense PS-ODNs E3/4, E8, and E10 were also unable
to influence this mitogenic response (data not shown). Neither AS2,
E11, nor any other PS-ODN used in this study possessed any more than
three contiguous guanosines in their respective sequences (Table 1)
.
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To confirm findings from thymidine incorporation experiments, we
measured the total numbers of cells in each treatment. AS2 and E11 each
suppressed serum-inducible SMC proliferation by over 50% (Figure 5A)
. In contrast, E1 had no effect
(Figure 5A)
. PS-ODNs AS2C and E11C (Table 1)
, with base composition
similar to AS2 and E11, respectively, also failed to inhibit
proliferation (Figure 5A)
. In addition, neither E11S, the sense
counterpart of E11, nor E11M, bearing a 3 nucleotide mismatch (Table 1)
could attenuate SMC replication (Figure 5B)
. These findings indicate
that antisense Egr-1 PS-ODNs are able to inhibit SMC replication in a
sequence-specific manner and are consistent with the capacity of
antisense PS-ODNs to inhibit DNA synthesis and cell proliferation
despite localization in the cytoplasm.23
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Antisense Egr-1 PS-ODNs Inhibit Egr-1 Induction in SMCs Exposed to Serum
We performed Western blot analysis to determine whether antisense
PS-ODN inhibition of SMC proliferation was a consequence of the ability
of these molecules to suppress the induction of Egr-1 itself. Initial
experiments established that Egr-1 protein was strongly induced in
growth-arrested SMCs exposed to serum within 1.5 hours (Figure 7
, top). Subsequent studies revealed that
incubation of E11 or AS2 before the addition of serum inhibited the
induction of Egr-1 protein (Figure 7
, center) by 60% and 75%,
respectively (Figure 7
, bottom). In contrast, identical exposure to E1
had no effect (Figure 7
, center). When the blot was stripped and
reprobed with antibodies directed toward Sp1, we observed that E11 or
AS2 did not affect levels of this zinc finger protein (Figure 7
, center
and bottom). PS-ODNs E11 and AS2 can thus inhibit the activation and
nuclear accumulation of Egr-1 protein. Additional experiments revealed
that E11C, E11S, or E11M could inhibit serum-inducible Egr-1 synthesis
(data not shown).
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.
Preincubation with PS-ODN E1 did not affect the induction of PDGF-A,
whereas AS2 inhibited the increase in PDGF-A (Figure 8)
. Inhibition
by E11 was less profound than AS2, consistent with the relative effects
of these PS-ODNs on the induction of Egr-1 protein (Figure 7)
. Thus,
PS-ODN inhibition of Egr-1 can influence gene expression dependent on
this transcription factor.
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To determine whether these molecules also had the capacity to
modulate regrowth of SMCs after injury, we exposed the PS-ODN panel to
SMCs that had been injured by scraping. E1, E11, E11C, or no PS-ODN
were added 18 hours before and immediately after the cells were
scraped. SMCs not exposed to any PS-ODN migrated from the wound edge
into the denuded zone and completely covered this area within 72 hours
(Figure 9A)
. Inclusion of E1 in the
culture medium had no effect (Figure 9A)
. In contrast, SMC regrowth was
attenuated in wells containing E11 (Figure 9, A and B)
. E11C, like E1,
failed to affect the reparative response (Figure 9, A and B)
. SMC
repair was also inhibited by AS2, whereas AS2C, E11S, and E11M had no
effect (data not shown). When fresh medium without PS-ODN was added to
the cultures, SMCs previously inhibited by E11 recovered the denuded
zone within a further 72 hours (Figure 9A)
. Thus, as well as
suppressing the serum-induction of Egr-1 and cell proliferation,
PS-ODNs targeting Egr-1 can inhibit the reparative response to injury
in vitro in a sequence-specific and reversible manner.
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, is suppressed by
antisense PS-ODNs targeting Egr-1.
Finally, we determined whether PS-ODN potency could be augmented by
facilitation of nuclear, rather than extranuclear localization under
passive conditions (Figure 2)
. Using a dendrimeric carrier, E11
suppressed serum-inducible SMC proliferation at 0.3 µmol/L (Figure 10A)
. At this concentration, E11 was
unable to inhibit in the absence of carrier (Figure 10A)
. Fluorescence
microscopy revealed that PS-ODN accumulation in SMC nuclei when carrier
was used (Figure 10B)
, which contrasts with the spatial pattern
observed under passive conditions (Figure 2)
. These results support the
notion that PS-ODN potency is influenced by its localization within the
cell.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While many groups have applied PS-ODNs to the study and treatment of
restenosis in experimental models (c-myb,26,30
c-myc,31,32
nuclear factor-
B p65
subunit,33
cdc2, and PCNA34
), there has been
considerable speculation as to the precise mechanisms and specificity
of the observed effects. A contributory factor to the controversy is
the phosphorothioate group in the ODN. Substitution of the
phosphodiester backbone of native DNA with phosphorothioate moieties
markedly augments resistance to nuclease digestion.35,36
Phosphorothioates, however, have also been found to bind with high
affinity to a large number of proteins37,38
presumably the
result of alterations in the presentation of negative charges. Without
appropriate controls, sequence-independent effects caused by
phosphorothioate chemistry can be mistakenly attributed to a true
antisense effect. Scrambled PS-ODNs serve as useful controls since
these have identical net charge as their parent counterparts. Sense and
mismatch PS-ODNs help provide further evidence of sequence specificity.
The field is confounded by observations that have shown that ODNs
bearing four consecutive G nucleotides can inhibit SMC proliferation in
a G quartet-specific or sequence-independent30,39
manner.
While such inhibitory ODNs still have clinical potential, these
observations raise concerns over the mechanism(s) of action. It was,
therefore, essential in this study to determine the levels at which
PS-ODNs exerted sequence-independent suppression of SMC growth and,
based on such information, to use antisense molecules and appropriate
controls at permissible concentrations. We observed that the maximum
tolerated dose of an PS-ODN before sequence-independent inhibition of
SMC proliferation was 1 µmol/L. The random sequence PS-ODN, E1, as
well as size-matched PS-ODNs with scrambled, sense, and mismatch
sequences served as important controls. Inhibition was not due to
differences in PS-ODN uptake, since an inhibitory and noninhibitory
PS-ODN localized intracellularly with similar spatial pattern. Use of
various controls in the present study suggests that antisense Egr-1
PS-ODN inhibition of SMC replication is sequence-specific.
The focus for target regions in the antisense literature is typically
in the vicinity of the translation initiation codon (such as the AS2
target site; Figure 1
) or the 3' untranslated region. In this paper,
regions of accessibility within Egr-1 mRNA were selected based on
prediction of areas of low free energy.18
Clearly, site
selection was not perfect as only a subset of the PS-ODN panel could
inhibit SMC proliferation at 1 µmol/L. Nevertheless, this method
yielded E11, targeting a region within Egr-1 mRNA encoding the third
zinc finger (Figure 1)
. A sequence homology search (FastA; EMBL and
GenBank) revealed no significant homology between the E11 and AS2
target sites in Egr-1 mRNA and the mRNA sequences of Egr-2, Egr-3, or
Egr-4. Improved methods for secondary structure prediction are
emerging40,41
and may be useful in identifying sites that
are structurally more accessible. While secondary structure is likely
to remain a key determinant in the efficiency with which antisense ODNs
bind to their target and inhibit gene expression, the importance of
other variables still to be characterized, such as the role of
sequence-dependent serum binding proteins and mechanisms of cellular
uptake may also emerge as influential factors.
While Egr-1 is expressed at low or undetectable levels in the normal rat artery wall, it is dramatically induced following mechanical injury.10,15 Interestingly, recent studies indicate that Egr-1 is expressed by SMCs as they migrate from the media toward the intima after injury.16 Egr-1 binds to the proximal promoters of many genes implicated in the proliferative and chemotactic response to injury.10,12 Its activation by injury and multiple other stimuli6,12 combined with its ability to drive the expression of an increasing number of pathophysiologically relevant genes clearly implicates the transcription factor as a potential therapeutic target. That inducible Egr-1 expression in SMCs can be selectively inhibited by the antisense PS-ODNs in this study provides a valuable research tool to delineate further the biological functions of Egr-1. Moreover, the capacity of these PS-ODNs to inhibit SMC proliferation and regrowth after injury in a sequence-specific manner indicates an important regulatory role for Egr-1 in SMC regeneration and suggests that these molecules may be useful in future strategies directed at compromising neointima formation after injury.
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Footnotes |
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Supported in part by a Strategic Partnership with Industry (Research and Training) grant from the Australian Research Council (L.M.K.) and a Health Research & Development Infrastructure grant from the N.S.W. Department of Health. L.M.K. is supported by an R. Douglas Wright Fellowship from the National Health and Medical Research Council of Australia.
Accepted for publication May 10, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhances cyclin D1 transcription through the binding of early growth response protein to a cis-regulatory element in the cyclin D1 promoter. J Biol Chem 1997, 272:33181-33190This article has been cited by other articles:
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