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, 283 (22), 15003-14

Transforming Growth Factor Beta Up-Regulates Cysteine-Rich Protein 2 in Vascular Smooth Muscle Cells via Activating Transcription Factor 2

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Transforming Growth Factor Beta Up-Regulates Cysteine-Rich Protein 2 in Vascular Smooth Muscle Cells via Activating Transcription Factor 2

Da-Wei Lin et al. J Biol Chem.

Abstract

CRP2 (cysteine-rich protein) is a vascular smooth muscle cell (VSMC)-expressed LIM-only protein. CRP2 associates with the actin cytoskeleton and interacts with transcription factors in the nucleus to mediate smooth muscle cell gene expression. Using Csrp2 (gene symbol of the mouse CRP2 gene)-deficient mice, we previously demonstrated that an absence of CRP2 enhances VSMC migration and increases neointima formation following arterial injury. Despite its importance in vascular injury, the molecular mechanisms controlling CRP2 expression in VSMC are largely unknown. Transforming growth factor beta (TGFbeta), a key factor present in the vessel wall in the early phases of arterial response to injury, plays an important role in modulating lesion formation. Because both CRP2 and TGFbeta are mediators of VSMC responses, we examined the possibility that TGFbeta might regulate CRP2 expression. TGFbeta significantly induced CRP2 mRNA and protein expression in VSMCs. Promoter analysis identified a conserved cAMP-responsive element (CRE)-like site (TAACGTCA) in the Csrp2 promoter that was critical for basal promoter activity and response to TGFbeta. Gel mobility shift assays revealed that mainly ATF2 bound to this CRE-like element, and mutation of the CRE sequences abolished binding. TGFbeta enhanced the activation of ATF2, leading to increased phospho-ATF2 levels within the DNA-protein complexes. Furthermore, ATF2-transactivated Csrp2 promoter activity and TGFbeta enhanced this activation. In addition, a phosphorylation-negative ATF2 mutant construct decreased basal and TGFbeta-mediated Csrp2 promoter activity. Our results show for the first time in VSMC that TGFbeta activates ATF2 phosphorylation and Csrp2 gene expression via a CRE promoter element.

Figures

FIGURE 1.
FIGURE 1.
TGFβ increases CRP2 expression in VSMCs. A, TGFβ induces CRP2 protein expression. VSMCs were exposed to TGFβ (10 ng/ml), and protein extracts were harvested at the indicated time points. Total protein was also extracted from unstimulated control cells. Western blot analyses were performed using 20 μg of total protein/lane. After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated with a polyclonal primary antiserum specific for CRP2-(91–98) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The blot was visualized with enhanced chemiluminescence and exposed to film. Blots were subsequently probed for α-tubulin for normalization. CRP2 protein induction is expressed relative to control without TGFβ stimulation at 0 h. Values are mean ± S.E. of three experiments. B, up-regulation of CRP2 mRNA by TGFβ. VSMCs were treated with TGFβ (10 ng/ml) for the indicated times. Northern blot analysis was performed with 10 μg of total RNA/lane. After electrophoresis, RNA was transferred to nitrocellulose filters and hybridized with a random primed 32P-labeled mouse CRP2 cDNA probe that hybridized to a 1.0-kb CRP2 message. The blots were subsequently hybridized with a 32P-labeled 18 S oligonucleotide to verify loading. The signal intensity of each RNA sample hybridized to the CRP2 probe was divided by that hybridized to the 18 S probe. The normalized intensities were expressed relative to control without TGFβ treatment at 0 h. Values are mean ± S.E. of 3–5 experiments. C, TGFβ treatment reduces wild type but not Csrp2–/– VSMC migration in response to PDGF-BB. Wild type (+/+) and Csrp2–/– (–/–) VSMCs were serum-starved for 24 h, treated without or with TGFβ (10 ng/ml) for 12 h and then plated in triplicate in 6-well transwell plates for migration assays using PDGF-BB (10 ng/ml) as a chemoattractant. Cells migrating through the filters after 2 h were quantified and normalized to the cell number of wild type without TGFβ treatment. Values are mean ± S.D. of two experiments.
FIGURE 2.
FIGURE 2.
TGFβ induction of CRP2 mRNA does not alter CRP2 mRNA half-life or require new protein synthesis. A, TGFβ does not alter CRP2 mRNA half-life. VSMCs were pretreated with vehicle (95% ethanol) or transcriptional inhibitor actinomycin D (AcD) (10 μg/ml) for 30 min and then stimulated without or with TGFβ (10 ng/ml) for 4 h and analyzed as in Fig. 1B. TGFβ induced CRP2 mRNA expression in the absence of AcD. In comparison, AcD blocked the CRP2 mRNA induction by TGFβ. B, TGFβ does not alter CRP2 mRNA half-life. VSMCs were stimulated with or without TGFβ (10 ng/ml) for 4 h, and then AcD (10 μg/ml) was administered to the cells. Total RNA was extracted at the indicated times after administration of AcD, and Northern blot analyses were performed as in Fig. 1B. The normalized intensity was then plotted as a percentage of the 0 h value (in log scale) against time. C, CRP2 induction by TGFβ does not require new protein synthesis. VSMCs were pretreated with vehicle (DMSO) or protein synthesis inhibitor cyclohexamide (CHX) (10 μg/ml) for 30 min and then stimulated without or with TGFβ (10 ng/ml) for 4 h. RNA was harvested, reverse transcribed, and analyzed by real time quantitative PCR assays. Cyclohexamide did not prevent the induction of CRP2 mRNA by TGFβ. Values are mean ± S.E. of three experiments.
FIGURE 3.
FIGURE 3.
The Csrp2 promoter region bp –480 to –438 is important for TGFβ induction. VSMCs were transiently transfected with Csrp2 promoter-luciferase reporter plasmids (500 ng/well) in triplicate using FuGENE 6. All wells received 500 ng of pCMVβ to normalize for transfection efficiency. Two hours after transfection, cells were treated with or without TGFβ (10 ng/ml) for 24 h and harvested for luciferase and β-galactosidase activity assays. A, 5′ deletion constructs of Csrp2 promoter-luciferase reporter plasmids. TGFβ induction is expressed relative to control without TGFβ of each construct. Values are mean ± S.E. of three experiments. B, the –795 Csrp2 wild type –795 and mutant –795Δ(480/438) (an internal deletion of bp –480 to –438) promoter constructs are schematically depicted in the top panel. Luciferase activity is expressed relative to –795 without TGFβ treatment. Values are mean ± S.E. of three experiments.
FIGURE 4.
FIGURE 4.
The CRE-like site is functionally important for basal and TGFβ induction of Csrp2 promoter activity. A, conservation of the putative CRE site among species. Sequence alignment of corresponding regions of human and rat promoter sequences to the mouse promoter. A putative CRE site (TAACGTCA) is in boldface type and underlined in the mouse sequence. B, the putative CRE site is important for Csrp2 promoter activity. The –795 Csrp2 wild type (wt) and CRE mutant (mut) promoter constructs are schematically depicted in the left panel. VSMCs were transiently transfected with Csrp2 luciferase reporter constructs (500 ng/well) containing –795 or –795CREmut with putative CRE site mutated and pCMVβ (500 ng/well) to normalize for transfection efficiency in triplicate using FuGENE 6 transfection reagent. Two hours after transfection, cells were treated with or without TGFβ (10 ng/ml) for 24 h. Cells were then harvested for luciferase and β-galactosidase activity assays. Luciferase activity is expressed relative to –795 without TGFβ treatment.
FIGURE 5.
FIGURE 5.
Nuclear proteins binding to the CRE-like element of Csrp2 promoter. A, oligonucleotide sequences used in the electrophoretic mobility shift assays (EMSAs) are shown. The core sequence of CRE-like and consensus CRE sites are in boldface type, and mutated sequence is underlined. EMSAs were performed with double-stranded oligonucleotides corresponding to bp –467 to –448 of the Csrp2 promoter. The addition of nuclear extracts (10 μg) from VSMCs to the 32P-labeled Csrp2 probe resulted in two major retarded DNA-protein complexes, designated I and II (arrows on left)(lane 2). A nonspecific band (ns) is indicated. Complexes I and II were abolished by the addition of unlabeled identical (Csrp2, lane 3) or consensus CRE (lane 5) oligonucleotides as competitors but not by the addition of three bases mutated (mut) oligonucleotides (lane 4). Conversely, EMSAs using 32P-labeled mutated oligonucleotides did not result in specific complex formation (lane 7). As a comparison, EMSAs using 32P-labeled CRE oligonucleotides were performed. The addition of nuclear extracts to the CRE probe resulted in the formation of complexes I and II (lane 9), which were competed away by identical unlabeled oligonucleotides (CRE; lane 10). The addition of unlabeled Csrp2 mainly abolished complex I and to a lesser degree complex II (lane 11). B, oligonucleotides used in the EMSAs are indicated. Csrp2 oligonucleotides contain the core sequence of CRE-like site (in boldface type), whereas mut1 has a one-base mutation (underlined) in the core and mut2 has two bases mutated (underlined). As in A, complex I and II were abolished by the addition of unlabeled identical (Csrp2, lane 3) oligonucleotides as competitors. mut1 partially competed away the complexes (lane 4), whereas mut2 did not compete away the complexes (lane 5). EMSAs using 32P-labeled mut1 oligonucleotides resulted in low intensity complex I and II formation (lane 6), whereas 32P-labeled mut2 oligonucleotides did not result in specific complex formation (lane 7). C, nuclear extracts from control (lanes 2–5) or TGFβ treated for 15 min (lanes 6–9) VSMCs were incubated with 32P-labeled Csrp2 probe without the addition of antibodies (lanes 2 and 6) or antibodies specific to ATF2 (lanes 3 and 7), CREB (lanes 4 and 8), or c-Jun (lanes 5 and 9). ATF2 antibody completely supershifted complex I to an upper band (*, lanes 3 and 7), whereas CREB antibody supershifted complex II to an upper complex (•, lanes 4 and 8). Incubation with c-Jun antibody did not produce supershifted bands (lanes 5 and 9).
FIGURE 6.
FIGURE 6.
TGFβ increases the phosphorylation of ATF2 but not CREB. VSMCs were stimulated with TGFβ (10 ng/ml) and activation of ATF2 and CREB was determined using cell lysates harvested at the indicated time points by Western blot analysis. Phosphorylation of ATF2 and CREB was detected by using phospho-ATF2 (p-ATF2) and p-CREB antibodies, respectively. To verify equal loading, the blots were probed with total ATF2 and CREB antibodies. A representative of three independent experiments is shown.
FIGURE 7.
FIGURE 7.
TGFβ enhances phosphorylation of ATF2 within the DNA-protein complex. A, EMSAs were performed with double-stranded oligonucleotides corresponding to bp –467 to –448 of the Csrp2 promoter. The addition of nuclear extracts (NE; 10 μg) from control or TGFβ-treated for 15 min VSMCs to the 32P-labeled Csrp2 probe resulted in retarded DNA-protein complex I and II (lanes 2 and 5). To detect phosphorylation of ATF2 and CREB within the complexes, nuclear extracts were incubated with antibodies (Ab) specific to phospho-ATF2 or phospho-CREB before the reactions. Phospho-ATF2 (p-ATF2) antibody supershifted complex I to a larger complex (p-ATF2→, lanes 3 and 6), whereas phospho-CREB antibody disrupted complex II (lanes 4 and 7). B, quantitative ChIP assays. VSMCs were cross-linked with formaldehyde and DNA fragmented by sonication. Chromatin was immunoprecipitated with normal rabbit IgG, CREB, or p-CREB antibodies. Aliquots of samples equivalent to 1% of initial cell lysate for each reaction were processed, and DNA was purified to use as input DNA control. Quantitative PCR was performed to amplify a 165-bp fragment flanking the mouse Csrp2-CRE (Csrp2) and a 92-bp fragment flanking the mouse cylin D1-CRE (cyclin D1). As an additional negative control, a 164-bp fragment within intron 1 of the Csrp2 gene (intron 1) was also amplified by quantitative PCR. Binding activity is expressed as percentage relative to input DNA. Values are mean ± S.D. of two or three experiments.
FIGURE 8.
FIGURE 8.
ATF2 up-regulates Csrp2 promoter activity via the CRE site in VSMCs. A, VSMCs were transiently co-transfected with luciferase reporter plasmid –795Csrp2-luc and expression plasmid pCMV-CREB or pCMV· SPORT6-ATF2 in triplicate using FuGENE 6 transfection reagent. Empty pCMV vector was added to keep constant the total content of DNA. Two hours following transfection, cells were treated with or without TGFβ (10 ng/ml) for 24 h. Cells were then harvested for luciferase activity and total protein assays. Csrp2 promoter activity was plotted as -fold induction compared with activity of –795 with empty vector. Values are mean ± S.E. of three experiments. B, the reporter plasmid –795Csrp2-luc and phosphorylation-negative ATF2 mutant ATF2-AAA or the empty vector pFLAG-CMV5 were cotransfected into VSMCs and similarly treated as in A. Csrp2 promoter activity was plotted as percentage activity of –795 cotransfected with vector but without TGFβ. Values are mean ± S.D. C, VSMCs were transiently co-transfected with Csrp2 luciferase reporter plasmid –795 or –795CREmut and expression plasmid pCMV·SPORT6-ATF2 or empty vector in triplicate using FuGENE 6 transfection reagent. Cells were harvested 24 h later for luciferase activity and total protein assays. Csrp2 promoter activity was plotted as -fold induction compared with activity of –795 without ATF2. Values are mean ± S.E.
FIGURE 9.
FIGURE 9.
Dominant negative Smad3 decreases Csrp2 promoter activity in the absence and presence of TGFβ. A, VSMCs were transiently cotransfected with luciferase reporter plasmid –795Csrp2-luc and empty vector pRK5 or expression plasmid pRK5-Smad3ΔC. Two hours following transfection, cells were treated with or without TGFβ (10 ng/ml) for 24 h. Cells were then harvested for luciferase activity and total protein assays. Csrp2 promoter activity was plotted as percentage of activity of –795 with empty vector without TGFβ. Values are mean ± S.E. of three experiments. B, VSMCs were transiently cotransfected with 3TP-Lux reporter plasmid and empty vector pRK5 or expression plasmid pRK5-Smad3ΔCasin A. 3TP-Lux luciferase activity was plotted as -fold induction compared with activity of vector without TGFβ. Values are mean ± S.E.

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