(Hypertension. 1999;33:298-302.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Novel Cis Element for Tissue-Specific Transcription of Rat Platelet-Derived Growth Factor ß-Receptor Gene
From The Second Department of Internal Medicine, Ehime University School of Medicine, Ehime, Japan.
Correspondence to Yutaka Kitami, MD, The Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-0295, Japan. E-mail kitamiyk{at}m.ehime-u.ac.jp
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Abstract |
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Abstract—Platelet-derived growth factor (PDGF) and its receptors are widely expressed in several tissues in the stage of cellular growth and development. In adulthood, PDGF ß-receptor (PDGFßR) is mainly detected in pathological conditions such as atherosclerotic lesions and injured vascular wall. The purpose of the present study was to elucidate the underlying mechanism of PDGFßR gene expression under pathological conditions in vascular smooth muscle cells (VSMC) and to identify the important cis elements responsible for tissue-specific gene transcription. Gel mobility shift assay and supershift assay indicated that the CCAAT motif located at -67 (C67) was mainly interacted with NF-YC, and this element drove the basal promoter activity of the gene as a putative promoter. On the other hand, another important sequence essential for the basal transcription was found at a 30-bp region (R30) spanning -150 to -121. To test whether R30 actually regulates the tissue-specific transcription of PDGFßR gene, electromobility shift pattern was compared between VSMC and hepatoma cell line (HTC). We obtained the result that DNA-protein complex seen only in nuclear extracts from HTC suppressed the promoter activity in HTC in a tissue-specific manner. Furthermore, cis element decoy transfection experiments for C67 and R30 also revealed that both elements were functionally important in mRNA expression of PDGFßR in VSMC. From these results, we concluded that the basal activity of PDGFßR gene expression was transactivated by the interaction or coordination of both C67 and R30, and the latter one mainly controlled the tissue-specific gene expression in VSMC.
Key Words: muscle, smooth, vascular • platelet-derived growth factors • promoter • CCAAT box • NF-Y family • cis element • gene transcription
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Introduction |
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The growth of vascular smooth muscle occurs as an adaptive process in response to long-term changes in hemodynamic conditions and humoral factors. In the context of hypertension, the vascular structural changes in smooth muscle may play an important role in the pathogenesis, amplification, and perpetuation of high blood pressure.1 In response to hemodynamic forces, physical injury or circulating factors, cells in the vessel wall are activated to release growth factors including platelet-derived growth factor (PDGF), thereby participating in the process of vascular remodeling.2 PDGF and its receptors are widely expressed in several tissues in the stage of cellular growth and development, and their expression is also developmentally regulated. Whereas PDGF
-receptor (PDGFR) plays a pivotal role early in development, PDGF
ß-receptor (PDGFßR) becomes the predominant receptor type later
on.3 In adulthood, PDGFßR is mainly detected in
pathological conditions such as wound healing,4 diabetic
proliferative retinopathy,5
glomerulonephritis,6 and fibrosis.7 In the
vessel wall, both PDGF-BB and PDGFßR are known to be upregulated in
the atherosclerotic lesion8 or injured vascular
endothelial cells after balloon
injury.9 Thus, control of PDGFßR expression appears to be critically important under pathological conditions in vivo, and it is important to elucidate the underlying mechanism of PDGFßR gene expression. Previously, we have isolated the 5'-flanking region of the rat PDGFßR gene and identified 2 regulatory elements essential for its basal promoter activity in vascular smooth muscle cells (VSMC) by using both reporter assay and electromobility shift assay (EMSA).10 One is a CCAAT box located at -67 upstream of the transcription start site (C67) and the other is an upstream control element (UCE) spanning -310 to -121. In the present study, we further characterized the nature of DNA-binding proteins that interacted with these 2 elements and newly identified the regulatory domain on UCE, which was mainly responsible for the tissue-specific transcriptional control of the PDGFßR gene in VSMC.
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Methods |
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Cell Culture and Northern Blot Analysis
All procedures were in accordance with institutional guidelines for animal research. VSMC and fibroblasts were isolated from the thoracic aortas and the lung of male Sprague-Dawley rats according to the enzyme-disperse method and explant procedure, respectively. Rat hepatoma cell line (HTC) was purchased from Dainippon Laboratories, Osaka, Japan. All cells were maintained in Dulbeco's modified Eagle's medium with 10% heat-inactivated fetal calf serum, and total cellular RNA was isolated from cells with the use of ISOGEN (Wako Pure Chemical Industries). Northern blot analysis for PDGFßR mRNA was performed by the method described previously.11 For the quantitative measurement of hybridized signals, the membrane was exposed to an imaging plate and then analyzed by Bio-Imaging Analyzer BAS 1000 (Fuji Photo Film Co).
Plasmid Constructs and Transient Transfection
Plasmid constructs used for luciferase assay are summarized in
Figure 2
. Luc-1,681, Luc-310, and Luc-120 correspond to Luc-1,
Luc-8, and Luc-9, which have been designated previously.10
Other 5'-deletions, which had the nucleotide sequences
starting at -270, -230, -190, and -150 upstream of the
transcription start site, were selectively generated by polymerase
chain reaction, and the resultant plasmids were designated Luc-270,
Luc-230, Luc-190, and Luc-150, respectively. Transient transfection was
performed by the method of DEAE-dextran, and the luciferase activity
was determined by the Dual-Luciferase Reporter Assay System (Promega).
Promoter activity of each plasmid construct was calculated as a
firefly/renilla-luciferase activity ratio and was finally
presented as relative activity with reference to the activity
of Luc-1,681, which was set to 100%. Although the substrate
specificity of renilla-luciferase (originated from Renilla
reniformis) is different from that of firefly-luciferase,
enzymatic activity and stability of both luciferases are almost
equivalent.
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P<0.05,
significant difference between VSMC and HTC. Data are expressed as
mean+SEM of 4 separate assays.
Preparation of Nuclear Extracts and EMSA
Nuclear extracts were prepared from VSMC and HTC according to
the method described by Dignam et al.12 For EMSA, 2
double-stranded oligodeoxynucleotide (ODN) probes were
prepared. One was a probe for the sequence spanning -76 to -52, which
contained a consensus sequence for the CCAAT box located at -67 (C67),
and the other for the 30-bp upstream region (R30) spanning -150 to
-121. EMSA was performed according to the method described
previously.10 11 In brief, C67 and R30 probes were
end-labeled with [
-32P]ATP with T4
polynucleotide kinase, and 1.0x104
cpm of each labeled probe was added to 2 µg of nuclear extracts. A
100-fold molar excess of unlabeled competitor or 1 µL of antibodies
against members of mouse NF-Y family (generous gifts of Dr Shigekazu
Nagata, Department of Genetics, Biomedical Research Center, Osaka
University Medical School) was incubated with nuclear extracts for 15
minutes before adding the labeled probe. The reaction mixture was
analyzed by 4% polyacrylamide gel electrophoresis
under nondenaturing condition.
Cis Element "Decoy" Double-Stranded
ODN
Phosphorothioate double-stranded ODN was prepared for C67 or R30
as a cis element decoy. In addition, scramble decoy ODN was
also prepared for C67 and was used as a control. Each decoy ODN was
mixed with LIPOFECTAMINE Plus (GIBCO BRL) and was directly added to the
culture medium at a final concentration of 1 µmol/L. After
incubation for 3 hours, cells were washed with PBS and cultured for 24
hours before isolation of RNA.
Statistical Analysis
Statistical evaluation was performed by ANOVA (2x8 factorial
design for Figure 2
and Kruskal-Wallis H test for
Figure 5), and multiple comparisons between treatments or
between groups were evaluated by Duncan's new multiple-range test for
Figure 2 and 2-sided analysis of Mann-Whitney
U test for Figure 5. All data are expressed as
mean+SE, and statistical significance is defined as
P<0.05.
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Results |
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Differential Expression of PDGFßR Gene
The level of endogenous PDGFßR mRNA expression was determined by Northern blot analysis with 3 different types of cells: VSMC, HTC, and pulmonary fibroblasts (Figure 1
). PDGFßR mRNA was expressed highly in
both VSMC (lane 1) and pulmonary fibroblasts (lane 2) but not
in HTC (lane 3). To further clarify the differential expression of
PDGFßR mRNA, promoter-luciferase constructs were functionally tested
by transient transfection experiments on both VSMC and HTC (Figure 2). In VSMC (closed columns), Luc-1,686
through Luc-150 showed significantly greater promoter activities than
did the promoterless luciferase vector, pGLB. In contrast, the promoter
activity was drastically decreased on going from Luc-150 to Luc-120 to
a level almost equal to that of pGLB. In HTC (open columns), all
plasmid constructs did not show significantly greater promoter
activities than did pGLB.
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EMSA With Nuclear Extracts From VSMC
To address the nature of DNA-binding proteins on both C67 and R30,
EMSA was performed with the use of nuclear extracts from VSMC (Figure 3). Labeled C67 probe was shifted by
nuclear extracts from VSMC, generating 2 specifically shifted bands, B1
and B2 (lane 1). Both bands were competed out almost completely by
adding a 100-molar excess of unlabeled C67 probe (lane 2), and only B2
was selectively competed out by adding a 100-molar excess of unlabeled
R30 probe (lane 3). On the other hand, labeled R30 probe was shifted by
nuclear extracts from VSMC, generating a single broad band, B3 (lane
4). The B3 was competed out almost completely by adding a 100-molar
excess of either unlabeled R 30 (lane 5) or C67 (lane 6) probe. The B1
through B3 were not competed out by adding a 100-molar excess of other
CCAAT consensus sequences such as CCAAT-binding transcription
factor/NF-1 (CTF/NF-1) and CCAAT/enhancer-binding protein (C/EBP)
probes (data not shown). To further characterize the nature of
DNA-binding proteins for C67, supershift assay was performed by using
antibodies against members of mouse NF-Y family (Figure 4). The B1 was supershifted mainly by
antibodies against NF-YC (lane C) and not significantly by antibodies
against NF-YA (lane A) and NF-YB (lane B), whereas B2 was not
supershifted by any antibodies.
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Effects of C67 and R30 "Decoy" ODN on PDGFßR mRNA
Expression
To further determine the functional significance of both C67 and
R60 in the basal promoter activity of PDGFßR gene, we investigated
effects of C67 and R30 decoy ODN on endogenous expression
of PDGFßR mRNA in VSMC (Figure 5).
Either C67 (lane 2) or R30 (lane 3) decoy ODN significantly reduced a
basal expression level of PDGFßR mRNA by 
60% or 45%,
respectively.
Comparison of EMSA Pattern Between VSMC and HTC
EMSA pattern for C67 or R30 was compared between nuclear extracts
from VSMC and HTC (Figure 6). EMSA
pattern using C67 probe was almost identical between nuclear extracts
from VSMC (lane 2) and HTC (lane 1), generating 2 bands (B1 and B2). In
contrast, EMSA pattern with the use of R30 probe was clearly different
between both nuclear extracts. Labeled R30 probe was shifted by nuclear
extracts from VSMC (lane 4), generating a single band, B3, whereas it
was shifted by those from HTC (lane 3), generating a closely shifted
band (marked asterisk) in addition to B3.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have previously identified 2 important regulatory elements essential for the basal transcriptional activity of the rat PDGFßR gene in cultured VSMC.10 One is the CCAAT box located at -67 (C67) and the other is the UCE spanning -310 to -120. In addition, we have also suggested that the former one is mainly responsible for the basal transcriptional activity of the gene as a putative promoter and the latter one enhances or positively regulates the activity in a tissue-specific manner. In this study, we characterized the nature of the DNA-binding proteins interacted with C67 and UCE and found that the basal promoter activity of the PDGFßR was driven by the interaction or cooperation of these 2 elements. Furthermore, we newly identified a 30-bp regulatory domain (R30) spanning -150 to -121 in UCE, which is mainly responsible for tissue-specific transcriptional control of the PDGFßR gene.
Although several transcriptional factors such as NF-Y, CTF/NF-1, and
C/EBP are known to bind to the CCAAT motif, NF-Y is the most frequent
CCAAT-binding factor that can bind to a 60- to 80-bp upstream region of
the transcription start site.13 NF-Y (also called CBF in
the rat) binds to the CCAAT motif as a heterotrimeric complex of NY-YA
(CBF-B), NF-YB (CBF-A1), and NF-YC (CBF-C) subunits.14
Ballagi et al15 have previously reported the 5'-flanking
region and its promoter function of the mouse PDGFßR gene and shown
that a CCAAT motif, which is identical to C67 presented herein,
is also found at -60 in the mouse promoter region. In addition,
Ishisaki, et al16 have reported that this CCAAT box seen
in the mouse gene is essential for the basal promoter activity of the
gene and demonstrated that NF-Y mainly binds to this motif and controls
PDGFßR gene expression in the mouse NIH3T3 fibroblast cells. They
used antibodies against just 2 members, NF-YA and NF-YB, for the
supershift assay and concluded that both NF-YA and NF-YB were mainly
responsible for the DNA-protein complex formation on the CCAAT box. In
contrast, we used antibodies against all members of the NF-Y family for
the supershift assay and clearly showed that mainly NF-YC (CBF-C) and
partially NF-YA (CBF-B) or NF-YB (CBF-A1) were responsible for the
DNA-protein complex formation in VSMC (Figure 4). Although the
discrepancy between the 2 studies may be due to differences in
antibodies, tissues, or animals used for each study, we believe our
results are correct because NF-Y is known to become active on formation
of the heterotrimeric complex.
On the other hand, another pivotal element for the basal
transcriptional activity of the PDGFßR gene has been identified as a
UCE spanning -120 to -310. Using VSMC, we have demonstrated that UCE
specifically enhances the basal transcriptional activity driven by the
smooth muscle–specific gene promoter such as -actin gene promoter
but not SV40 virus promoter.10 This strongly postulates
the hypothesis that UCE contains a novel cis element that is
mainly responsible for the tissue-specific transcription of the gene.
Therefore we newly prepared progressive 5'-deletions between Luc-310
and Luc-120 and determined their promoter activities in both VSMC and
HTC (Figure 2). We then identified a functionally active domain
(R30) in UCE. Although R30 did not contain well-defined consensus
sequences, basal levels of PDGFßR mRNA were significantly decreased
by treatment with R30 decoy ODN (Figure 5, lane 3). This
strongly indicates that R30 as well as C67 is essential for the basal
transcription activity of the PDGFßR gene in VSMC. Furthermore,
competition experiments for EMSA revealed that the B2 generated by
labeled C67 probe was competed out selectively by unlabeled R30 probe,
and the B3 generated by labeled R30 probe were also competed out
completely by either unlabeled C67 or R30 (Figure 3). These
results indicate that the PDGFßR gene expression is
transactivated by the interaction or coordination of the 2
regulatory elements, C67 and R30. To elucidate the role of R30 on the
tissue-specific gene transcription, we compared EMSA pattern by using
R30 probe between nuclear extracts from VSMC and HTC and obtained the
result that nuclear extracts from HTC specifically generated a closely
shifted band that was not observed in those from VSMC (Figure 6, lane 4). This strongly suggests that HTC differentially expresses
nuclear factors responsible for the additional band formation, and
these nuclear factors may suppress the basal promoter activity of the
PDGFßR gene in HTC in a tissue-specific manner.
In conclusion, we identified 2 pivotal elements, C67 and R30, essential for the basal transcriptional activity of the rat PDGFßR gene. NF-YC (also called CBF-C) mainly binds to C67 and promotes the basal promoter activity in VSMC. On the other hand, since the DNA binding factors specific for the R30 element appear to be restricted to HTC, this element may be a major site for tissue-specific regulation of PDGFßR gene expression.
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Acknowledgments |
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This work was supported in part by a Grant-in-Aid of Scientific Research from the Ministry of Education, Science, Culture, and Sports, Japan (No. 09670723), a Japan Heart Foundation Grant for Research on Hypertension and Vascular Metabolism, and Takeda Medical Research Foundation. We thank Dr Shigekazu Nagata (Department of Genetics, Biomedical Research Center, Osaka University Medical School) for the generous gift of antibodies against mouse NF-YA, B, and C. Dr Takata and Dr Fukuoka are equivalent first authors. Dr Kitami is their supervisor, and Dr Okura performed preparation and maintenance of cells used in this study. Prof Hiwada is a director of our department.
Received September 16, 1998; first decision October 12, 1998; accepted October 23, 1998.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Folkow B, Hallbäck M, Lundgren Y, Sivertsson R, Weiss L. Importance of adaptive changes in vascular design for establishment of primary hypertension, studies in man and in spontaneously hypertensive rats. Circ Res. 1973;32(suppl I):I-2–I-16.
2.
Dzau VJ, Gibbons GH, Morishita R, Pratt RE. New
perspectives in hypertension research: potentials of vascular biology.
Hypertension. 1994;23:1132–1140.
3. Bowen-Pope DF, van Koppen A, Schwatteman G. Is PDGF really important? Testing the hypotheses. Trends Genet. 1991;7:413–418.[Medline] [Order article via Infotrieve]
4. Robson MC, Phillips LG, Thomason A, Robson LE, Pierce GF. Platelet-derived growth factor BB for the treatment of chronic pressure ulcers. Lancet. 1992;339:23–25.[Medline] [Order article via Infotrieve]
5.
Robbins SG, Mixon RN, Wilson DJ, Hart CE, Robertson
JE, Westra I, Planck SR, Rosenbaum JT. Platelet-derived growth
factor ligands and receptors immunolocalized in proliferative retinal
diseases. Invest Ophthalmol Vis Sci. 1990;35:3649–3663.
6. Gesualdo L, Di Paolo S, Milani S, Pinzani M, Grappone C, Ranieri E, Pannarale G, Schena FP. Expression of platelet-derived growth factor receptors in normal and diseased human kidney: an immunohistochemistry and in situ hybridization study. J Clin Invest. 1994;1:50–58.
7. Smits A, Funa K, Vassbotn FS, Beausang-Linder M, af Ekenstam F, Heldin CH Westermark B, Nistér M. Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin. Am J Pathol. 1992;140:639–648.[Abstract]
8. Heldin C-H. Structural and functional studies on platelet-derived growth factor. EMBO J. 1992;11:4251–4259.[Medline] [Order article via Infotrieve]
9. Lindner V, Reidy MA. Platelet-derived growth factor ligand and receptor expression by large vessel endothelium in vivo. Am J Pathol. 1995;146:1488–1497.[Abstract]
10. Kitami Y, Fukuoka T, Okura T, Takata Y, Maguchi M, Igase M, Kohara K, Hiwada K. Molecular structure and function of rat platelet-derived growth factor ß-receptor gene promoter. J Hypertens. 1998;16:437–445.[Medline] [Order article via Infotrieve]
11.
Kitami Y, Inui H, Uno S, Inagami T. Molecular structure
and transcriptional regulation of the gene for the platelet-derived
growth factor receptor in cultured vascular smooth muscle
cells. J Clin Invest. 1995;96:558–567.
12.
Dignam JD, Lebovitz RM, Roeder RG. Accurate
transcription initiation by RNA polymerase II in a soluble extract
isolated from mammalian nuclei. Nucl Acids Res. 1983;11:1475–1489.
13. Bucher P. Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J Mol Biol. 1990;212:563–578.[Medline] [Order article via Infotrieve]
14.
Maity SN, Sinha S, Ruteshouser EC, de Crombrugghe B.
Three different polypeptides are necessary for DNA binding of the
mammalian heteromeric CCAAT binding factor. J Biol
Chem. 1992;267:16574–16580.
15. Ballagi AE, Ishizaki A, Nehlin J-O, Funa K. Isolation and characterization of the mouse PDGF ß-receptor promoter. Biochem Biophys Res Commun. 1995;210:165–173.[Medline] [Order article via Infotrieve]
16. Ishisaki A, Murayama T, Ballagi AE, Funa K. Nuclear factor Y controls the basal transcriptional activity of the mouse platelet-derived-growth-factor ß-receptor gene. Eur J Biochem. 1997;246:142–146.[Medline] [Order article via Infotrieve]
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