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Abstract
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 β-receptor (PDGFβR) 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 PDGFβR 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 PDGFβR 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 PDGFβR in VSMC. From these results, we concluded that the basal activity of PDGFβR 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.
- muscle, smooth, vascular
- platelet-derived growth factors
- promoter
- CCAAT box
- NF-Y family
- cis element
- gene transcription
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 α-receptor (PDGFαR) plays a pivotal role early in development, PDGF β-receptor (PDGFβR) becomes the predominant receptor type later on.3 In adulthood, PDGFβR 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 PDGFβR are known to be upregulated in the atherosclerotic lesion8 or injured vascular endothelial cells after balloon injury.9
Thus, control of PDGFβR expression appears to be critically important under pathological conditions in vivo, and it is important to elucidate the underlying mechanism of PDGFβR gene expression. Previously, we have isolated the 5′-flanking region of the rat PDGFβR 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 PDGFβR gene in VSMC.
Methods
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 PDGFβR 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.
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 [γ-32P]ATP with T4 polynucleotide kinase, and 1.0×104 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 (2×8 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.
Results
Differential Expression of PDGFβR Gene
The level of endogenous PDGFβR mRNA expression was determined by Northern blot analysis with 3 different types of cells: VSMC, HTC, and pulmonary fibroblasts (Figure 1⇓). PDGFβR 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 PDGFβR 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.
Northern blot analysis of PDGFβR mRNA in VSMC, pulmonary fibroblasts, and HTC. Total cellular RNA (10 μg) obtained from quiescent VSMC (lane 1), pulmonary fibroblasts (lane 2), and HTC (lane 3) were analyzed by Northern blotting for PDGFβR mRNA (5.6 kb). Lower panel indicates ethidium bromide staining of 28S and 18S rRNA.
Plasmid constructs of promoter-luciferase fusion genes and their luciferase activities in VSMC or HTC. Luc-1,681 to Luc-120 indicate progressive 5′-deletions, which had nucleotide sequences starting at the positions, −1,681, −310, −271, −230, −190, −150, and −120, respectively. pGLB indicates a promoterless luciferase vector. Each construct was transiently cotransfected with pRL-CMV into VSMC (closed columns) or HTC (open columns) with the method of DEAE-dextran. Promoter activity was assessed in terms of its ability to drive firefly-luciferase cDNA expression after this had been normalized with respect to renilla-luciferase cDNA expression and was finally presented as relative luciferase activity with reference to the activity of Luc-1,681 which was set to 100%. *P<0.05, significant difference compared with Luc-1,681; †P<0.05, significant difference between VSMC and HTC. Data are expressed as mean+SEM of 4 separate assays.
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.
EMSA for CCAAT motif located at −67 and the R30 spanning −150 to −121 with nuclear extracts from VSMC. End-labeled probe for C67 (lane 1 to 3) or R30 (lane 4 to 6) was incubated with 2 μg of nuclear extracts from quiescent VSMC. For competition experiment, 100-molar excess of unlabeled C67 (lanes 2 and 6) or unlabeled R30 (lanes 3 and 5) was incubated with nuclear extracts for 15 minutes before adding labeled probe. Lanes 1 and 4 indicated no competitor. Positions of specific DNA-protein complex are indicated as B1-B3; free DNA probe as Free.
Supershift assay for CCAAT motif located at −67 with antibodies against NF-Y family. Labeled C67 probe was added to nuclear extracts (2 μg) from quiescent VSMC and was incubated with 1 μL of specific antibodies against NF-YA (A), NF-YB (B), and NF-YC (C) or preimmune serum (PI) for 15 minutes before electrophoresis. Positions of specific protein complexes were indicated as B1 and B2; supershifted bands as asterisks.
Effects of C67 and R30 “Decoy” ODN on PDGFβR mRNA Expression
To further determine the functional significance of both C67 and R60 in the basal promoter activity of PDGFβR gene, we investigated effects of C67 and R30 decoy ODN on endogenous expression of PDGFβR mRNA in VSMC (Figure 5⇓). Either C67 (lane 2) or R30 (lane 3) decoy ODN significantly reduced a basal expression level of PDGFβR mRNA by ≈60% or 45%, respectively.
Effect of cis element decoy ODN against C67 or R30 on endogenous PDGFβR mRNA expression. Phosphorothioate double-stranded ODN was prepared for C67 (lane 2) or R30 (lane 3) as cis element decoy. In addition, scramble decoy ODN (lane 1) was also prepared for C67 as control. Each decoy ODN was mixed with Lipofectamine Plus and was directly added to culture medium at 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. Northern blot analysis for PDGFβR mRNA was performed by same method described in Figure 1⇑. Amounts of endogenous PDGFβR mRNA were presented as relative expression levels with reference to level of control scramble decoy, which was set to 100%. Data are expressed as mean+SEM of 4 separate assays. †P<0.05, significant difference compared with level of control scramble decoy.
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.
Comparison of EMSA pattern between VSMC and HTC. EMSA pattern for C67 or R30 was compared between nuclear extracts from VSMC (lanes 2 and 4) and HTC (lanes 1 and 3). Nuclear extracts (2 μg) were incubated with labeled C67 probe (lanes 1 and 2) or labeled R30 probe (lanes 3 and 4) under same conditions as described in Figure 3⇑. Positions of specific DNA-protein complex observed in VSMC are indicated as B1 and B2; free DNA probe as Free. Closely shifted band (*) was specifically observed in HTC (lane 3) in addition to B3.
Discussion
We have previously identified 2 important regulatory elements essential for the basal transcriptional activity of the rat PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR 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 α-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 PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR gene expression.
Acknowledgments
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.
- Revision received October 12, 1998.
- Accepted October 23, 1998.
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