Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2003;92:104-110
Published online before print December 2, 2002, doi: 10.1161/01.RES.0000049103.38175.1B
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/1/104    most recent
01.RES.0000049103.38175.1Bv1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Villacorta, L.
Right arrow Articles by Azzi, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Villacorta, L.
Right arrow Articles by Azzi, A.
Related Collections
Right arrow Mechanism of atherosclerosis/growth factors
Right arrow Cell signalling/signal transduction
Right arrow Gene expression
Right arrow Gene regulation
Right arrow Growth factors/cytokines
(Circulation Research. 2003;92:104.)
© 2003 American Heart Association, Inc.

Molecular Medicine

{alpha}-Tocopherol Induces Expression of Connective Tissue Growth Factor and Antagonizes Tumor Necrosis Factor-{alpha}–Mediated Downregulation in Human Smooth Muscle Cells

Luis Villacorta*, Aurélio V. Graça-Souza*, Roberta Ricciarelli*, Jean-Marc Zingg, Angelo Azzi

From the Department of Medical Biochemistry (A.V.G.-S.), Institute of Biomedical Sciences, Federal University of Rio de Janeiro-RJ, Brazil; Dip. Medicina Sperimentale (R.R.), University of Genova, Italy; and the Institute of Biochemistry and Molecular Biology (L.V., J.-M.Z., A.A.), University of Bern, Bern, Switzerland.

Correspondence to J.M. Zingg, PhD, Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland. E-mail zingg{at}mci.unibe.ch


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The effect of {alpha}-tocopherol treatment on gene expression in human aortic vascular smooth muscle cells was analyzed by gene expression arrays. The expression of the connective tissue growth factor (CTGF) gene was induced by {alpha}-tocopherol 1.8-fold in gene array experiments, and similar results were also obtained by RT-PCR (1.7-fold) and at the protein level (1.4-fold). The antioxidants ß-tocopherol and N-acetylcysteine did not induce CTGF gene expression, suggesting a nonantioxidant mechanism for {alpha}-tocopherol action. Protein kinase C (PKC) inhibition by {alpha}-tocopherol has been previously described. However, PKC downregulation did not prevent CTGF induction by {alpha}-tocopherol, and inhibition of PKC activity with several inhibitors did not increase its expression, suggesting an alternative pathway for the {alpha}-tocopherol effect. On the other hand, tumor necrosis factor-{alpha} reduced the expression of CTGF, an effect that was reversed by antioxidants. The data suggest that tumor necrosis factor-{alpha} inhibition of CTGF gene expression is prevented in an antioxidant-sensitive process and that {alpha}-tocopherol increases CTGF expression by a PKC-independent, nonantioxidant mechanism. Because CTGF stimulates the synthesis of extracellular matrix, the normalization of CTGF gene expression by {alpha}-tocopherol may accelerate wound repair and tissue regeneration during atherosclerosis.


Key Words: connective tissue growth factor • okadaic acid • protein kinase C • {alpha}-tocopherol • tumor necrosis factor-{alpha}


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Connective tissue growth factor (CTGF),1 a member of CCN family (CTGF, CYR61, and NOV),1 which is also named insulin-like growth factor binding protein 8 (IGFBP-8), belongs to the "immediate-early response genes" that are expressed after induction by serum, growth factors, and cytokines.2 CTGF has a low insulin-like growth factor (IGF) activity, but has also IGF-independent actions by acting as a chemotactic and mitogenic factor for connective tissue cells.3,4 CTGF is overexpressed at high levels during atherosclerosis and fibrotic disorders such as scleroderma.5,6 However, CTGF at low levels supports wound repair and plays a role in embryogenesis and uterine function.7–9 These beneficial processes are most likely mediated by the mitogenic, chemotactic, and matrix-inducing effects of CTGF, such as the ability to stimulate the expression of fibronectin, type I collagen, and {alpha}5 integrin.10

CTGF expression is upregulated by dexamethasone11 and by transforming growth factor-ß1 (TGF-ß) by means of a regulatory element located within the promoter sequence.12 Moreover, levels of TGF-ß and CTGF seem to correlate during wound repair.7 On the other hand, CTGF expression is inhibited by tumor necrosis factor-{alpha} (TNF-{alpha}) in aortic endothelial cells, fibroblasts, and vascular smooth muscle cells (SMCs).13,11,14 TNF-{alpha} is found in human atheroma and it leads to the production of oxygen-derived free radicals.15 The secretion of TNF-{alpha} by macrophages is enhanced on exposure to oxidized LDL that may accelerate the formation of atherosclerotic lesions.16 Moreover, TNF-{alpha} regulates cytoskeletal organization of SMCs by inducing depolymerization of actin stress fibers and dispersion of vinculin from sites of focal adhesion.17 {alpha}-Tocopherol is a well-known antioxidant and, therefore, could change the activity of transcription factors and signal transduction pathways modulated by pro- and antioxidant conditions.18 Moreover, {alpha}-tocopherol acts specifically as an inhibitor of protein kinase C (PKC) enzymatic activity19,20 and can also influence transcription.21–24 In SMCs, {alpha}-tocopherol specifically inhibits PKC-{alpha}, which is strongly involved in the proliferative signal transduction pathway.25

In this study, we investigated, using gene expression arrays containing 7075 genes, whether {alpha}-tocopherol modulates the expression of specific genes in human aortic SMCs. We have confirmed by RT-PCR and Western blots that the expression of one gene on the gene expression array, the connective tissue growth factor (CTGF) gene, was upregulated by {alpha}-tocopherol in cultured human vascular SMCs and in cultured human skin fibroblasts. {alpha}-Tocopherol promoted upregulation was independent of the known PKC inhibition by {alpha}-tocopherol because this effect was also observed in SMCs in which PKC was either downregulated or inhibited. We have provided evidence that the downregulation of CTGF expression promoted by TNF-{alpha} was reverted by {alpha}-tocopherol and other antioxidants, suggesting that this molecule can also modulate CTGF expression by acting as an antagonist of TNF-{alpha}.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
RRR-{alpha}-tocopherol and RRR-ß-tocopherol were obtained from Henkel. N-acetylcysteine, probucol, and phorbol 12-myristate 13-acetate (PMA) were from Sigma. Calphostin C was from Calbiochem; Gö 6976 and okadaic acid (OA) were from LC Laboratories. PKC412 was from Novartis Pharma. TNF-{alpha} was from Alexis Biochemicals. pAP-1-luc and pTAL were from Clontech.

Human aortic smooth muscle cells (T/G) (ATCC No. CRL-1999) and human female skin fibroblasts (sample 1, ATCC No. CRL-7648, 29 years old; sample 2, ATCC No. CRL-7739, 1 year old; sample 3, ATCC No. CRL-7601, 59 years old) were from the American Type Culture Collection (Rockville, Md). Human primary coronary artery smooth muscle cells (HCASMCs), medium 231, and smooth muscle growth supplement (SMGS) were from Cascade Biologics.

Cell Culture
Stock solutions of RRR-{alpha}-tocopherol and RRR-ß-tocopherol, N-acetylcysteine and probucol were dissolved in ethanol and the concentration of the stock solutions confirmed spectrophotometrically. T/G cells were cultured in DMEM/10% FCS and used between passages 4 and 10. Human female skin fibroblasts (sample 1, 29 years old; sample 2, 1 year old; sample 3, 59 years old) were cultured in DMEM/10% FCS and used between passages 2 and 8. HCASMCs were cultured in medium 231 supplemented with smooth muscle growth supplement (SMGS) and used between passages 5 and 10. Treatments were done either with subconfluent (T/G) or confluent cells (HCASMCs) as indicated in the text.

Gene Array Experiments
Human SMCs (T/G, passage 5) were grown to {approx}70% confluence in 18 Falcon 9-cm dishes in 10 mL DMEM/10% FCS. The medium was replaced with new medium 24 hours before the experiment. Nine dishes were treated with 50 µmol/L {alpha}-tocopherol (10 µL from 50 mmol/L stock of {alpha}-tocopherol in ethanol), and 9 control dishes were treated with 10 µL ethanol, both for exactly 8 hours. Therefore, final ethanol solvent was below 0.1%. Afterward, cells were washed twice with cold PBS and total mRNA was isolated by using twice the Oligotex Midi mRNA isolation kit (Qiagen). Analysis of differential mRNA expression was done on Unigem V gene expression arrays at GenomeSystems.

RT-PCR and Cloning
Total RNA was isolated using a RNA extraction kit from Qiagen. Semiquantitative RT-PCR was performed according to the RT-PCR kit from Perkin-Elmer. RT-PCR for CTGF expression was performed with primer CTGFfor (5'-CCAAGGACCAAACCGTGGT-3'), which anneals to exon 3 and primer CTGFrev (5'-TACTCCACAGAATTTAGCTCG-3'), which anneals to exon 4/5.12 PCR conditions were at 95°C, 30 seconds; 55°C, 30 seconds; and 72°C, 30 seconds for 22 cycles, and the expected fragment was 311 bp long. Control reactions were performed with primers GAP1 (5'-AGCCACATCGCTCAGACACC-3') and GAP2 (5'-TGAGGCTGTTGTCATACTTCTC-3'), which are specific for the human glyceraldehyde-3-dehydrogenase (GAPDH) exons 2 and 6, respectively. PCR conditions were at 95°C, 30 seconds; 68°C, 30 seconds; and 72°C, 30 seconds for 18 cycles, and the expected fragment was 465 bp long. To ensure being in the linear range of the PCR reaction, a low number of PCR cycles were used, and all PCR reactions were performed in the presence of 0.2 µCi {alpha}32P-ATP per tube. The amplification products were separated using a 6% polyacrylamide gel, dried, and exposed to Kodak X-Omat film. The autoradiograms were scanned with an image densitometer (BioRad). CTGF data are presented as mean±SD and were calculated after normalization to the GAPDH data. The identity of the PCR fragments was confirmed by increasing the cycle number for amplification and automated sequencing (ABI-PRISM).

For cloning the CTGF promoter sequence, PCR was performed with human genomic DNA (Roche) and primer CTPROCfor (5'-AGGAATTCCTGCTGTTTGCC-3') and primer CTPROBrev (5'-TGGAGCGCTGGCGGTGGT-3'), which both anneal to the CTGF promoter.26 PCR conditions were at 95°C, 30 seconds; 62°C, 30 seconds; and 72°C, 1 minute for 30 cycles. The PCR fragment was isolated with the JETsorb gel extraction kit (Genomed) and cloned into pT7Blue-3 (Novagen), and clones containing an insert were sequenced with automated sequencing (ABI-PRISM). The CTGF fragment was cloned into the pGL3-basic (sense, pCT-sb; antisense, pCT-asb) luciferase reporter plasmids (Promega). For cloning of TGF-ß response element (TGF-ßRE) into pTAL, the two oligonucleotides, CTGFREUP: 5'-CAGGAATGCTGAGTGTCAAGGGGTCAGGATCA- ATCCAGGAATGCTGAGTGTCAAGGGGTCAGGATCAATCCG-3' and CTGFRELO: 5'-CTAGCGGATTGATCCTGACCCCTTGACACT- CAGCATTCCTGGATTGATCCTGACCCCTTGACACTCAGCATTC- CTGAGCT-3', containing a direct repeat of the TGF-ß response element of the human CTGF promoter, were annealed and cloned into the SacI/NheI site of pTAL.

Western Blots
Western blot for CTGF was done according to standard methods. Briefly, SMCs were treated with {alpha}-tocopherol (50 µmol/L) or ethanol for 24 hours. HCASMCs were allowed to grow to confluence. Protein content was measured using the Pierce BCA Protein Assay Reagent kit. Proteins (40 µg per lane) were separated using a 10% SDS-PAGE and transferred onto PVDF membrane (NEN, DuPont).

Mouse anti-human CTGF antibody (Bachem, Switzerland), diluted at 1:500, was used as primary antibody to detect the presence of CTGF, and a horseradish peroxidase–labeled anti-mouse IgG antibody (Amersham-Pharmacia), diluted at 1:10 000, was used as secondary antibody. Monoclonal anti-{alpha}-actin antibody was used as primary antibody to control the amount of loaded protein.

For the Western blot analysis of PKC downregulation, T/G cells were treated either with 1 µmol/L PMA or vehicle (DMSO) for 24 hours. Proteins (20 µg per lane) were separated using an 8% SDS-PAGE gel and electroblotted onto PVDF membrane. An anti-classical PKC isoforms antibody, clone MC-5 (Santa Cruz), was used, followed by a horseradish peroxidase–labeled anti-mouse IgG antibody, diluted at 1:10 000. Rabbit anti-human {alpha}-actin antibody (Sigma) was used as primary antibody to control the amount of loaded protein, in addition to BCA protein assay (Pierce).

Proteins were detected by chemiluminescence (ECL, Amersham), exposure to film, and analyzed using a Lumi imager (Roche). Data are presented as mean±SD and were calculated after normalization to {alpha}-actin staining.

Transfection and Promoter Assays
CTGF promoter plasmids, pAP-1-luc, pTGF-ß-RE-luc, and Renilla luciferase control reporter vector (pRL-TK) were transfected into T/G cells and HCASMCs with SuperFect (Qiagen). Cells were then treated with compounds at concentrations indicated in the Figure legends. Promoter activity was measured using the Dual-Luciferase Assay Kit (Promega) with a TD-20/20 luminometer (Turner Designs). CTGF promoter–firefly-luciferase activity was normalized to the TK promoter–renilla-luciferase activity, and the activity of the ethanol-treated CTGF promoter was set to 100%.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Identification of {alpha}-Tocopherol Responsive Genes With Gene Expression Arrays
Human vascular smooth muscle cells (T/G, passage 5) were grown to {approx}70% confluence and treated with {alpha}-tocopherol (50 µmol/L) or ethanol as control solvent (0.1%) for 8 hours, and mRNA was isolated. The Unigem V gene expression analysis was done twice with T/G cells mRNA, from two independently performed experiments. Of the 7075 genes that were analyzed, few were responsive to {alpha}-tocopherol, probably due to the selection of one cell line and one time point. Among them, only a few showed a change of expression that was consistent in both experiments. The increase in the expression observed of one of these genes, the prostacyclin-stimulating factor, is in accordance with the known stimulatory effect of {alpha}-tocopherol on prostacyclin release in human endothelial cells.27 We decided to characterize the expression of the connective tissue growth factor (CTGF) gene, which was increased 1.8-fold on the gene array (Figure 1A), by a more detailed analysis.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. {alpha}-Tocopherol induces CTGF expression in human aortic smooth muscle cells and fibroblasts. A, Gene expression array of mRNA from human SMCs treated with {alpha}-tocopherol (50 µmol/L for 8 hours), or as control with solvent ethanol (0.1% for 8 hours). Panel shows 8 random neighboring genes and the CTGF gene in the center. B, Induction of CTGF gene expression by {alpha}-tocopherol (50 µmol/L, for the time indicated) in T/G human aortic SMCs as analyzed by RT-PCR. C, Induction of CTGF gene expression by {alpha}-tocopherol (+; 50 µmol/L, 8 hours) as compared with ethanol control (-; 0.1%, 8 hours) in cultured human skin fibroblasts from 3 different individuals as analyzed by RT-PCR. Top panels in B and C show representative experiments using primers specific for CTGF and GAPDH, respectively. Bottom graphs show expression data obtained from 4 independent experiments. Data were obtained from scanning densitometry of the autoradiographs and are presented as mean±SD after normalization of the CTGF to the GAPDH expression data.

{alpha}-Tocopherol Induces CTGF Gene Expression in T/G Cells and Skin Fibroblasts
To evaluate the effect of {alpha}-tocopherol on the CTGF expression and to further confirm the results obtained from the Unigem V gene expression arrays, we performed RT-PCR using specific primers for this gene. Figure 1B shows that incubation of human SMCs (T/G) with {alpha}-tocopherol (50 µmol/L) induced the expression of CTGF, confirming the data derived from the gene expression arrays. This induction was observed after 2 and 8 hours of incubation (73% ±4.7 and 60% ±8, respectively, compared with the control cells) and was also noted after 24 hours of incubation (data not shown). However, when human SMCs were incubated with ß-tocopherol, a molecule differing from {alpha}-tocopherol only on a methyl group but with similar radical-scavenging properties, no induction of CTGF gene expression was seen. We did not observe any alteration compared with the control cells in the ß-tocopherol–treated samples even at shorter (2 hours) or longer (24 hours) incubation times (data not shown), suggesting that the stimulation of CTGF expression promoted by {alpha}-tocopherol is not related to its antioxidant properties. Because it has been shown that fibroblasts are the major source of CTGF production, we tested for the ability of {alpha}-tocopherol to stimulate CTGF expression in that particular cell type. When different cell lines of human female skin fibroblasts were incubated with 50 µmol/L {alpha}-tocopherol for 8 hours, induction of CTGF expression was observed (209±13%, sample 1; 70±1.8%, sample 2; and 270±25%, sample 3) (Figure 1C). Hence, {alpha}-tocopherol stimulation of CTGF expression can occur also with cultured human fibroblasts, thus suggesting {alpha}-tocopherol as a putative stimulus for extracellular matrix deposition.

{alpha}-Tocopherol Stimulates CTGF Protein Expression in T/G cells and HCASMCs
The next step was to evaluate whether the stimulation of CTGF transcription by {alpha}-tocopherol was also reflected at the level of protein expression. In all cases studied, CTGF protein was very low, but sufficient to detect {alpha}-tocopherol–induced changes of its expression. Two different strains of SMCs, T/G and HCASMCs, which express SMC {alpha}-actin when confluent (data not shown), were used to further confirm the results observed at the transcriptional level. Cells were treated with 50 µmol/L {alpha}-tocopherol for 24 hours. When {alpha}-tocopherol was added to the culture medium, a significant increase in CTGF protein (139.4±8.6% for T/G and 184.3±10.1% for HCASMCs) was observed (Figure 2), indicating that the {alpha}-tocopherol stimulation of CTGF mRNA expression leads to induction of CTGF protein. In the conditioned media, no CTGF was detectable under the employed experimental conditions (24 hours treatment), as it was previously described for mesangial cells and fibroblasts.28,29



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. CTGF protein expression is stimulated by {alpha}-tocopherol. A, T/G and HCASMCs were treated with {alpha}-tocopherol (50 µmol/L) or ethanol (0.1%) as control for 24 hours. Total protein was extracted, separated by SDS-PAGE, and transferred to PVDF membrane. Blotted membrane was probed with anti-CTGF antibody and horseradish peroxidase coupled anti-mouse Ig antibody. B, Graphs show expression data obtained from 3 independent experiments. Data are presented as mean±SD quantified with scanning densitometry.

{alpha}-Tocopherol Induction of CTGF Expression Is a PKC-Independent Phenomenon
Because {alpha}-tocopherol inhibits PKC activity in human smooth muscle cells,30,31 and a recent study showed the effect of PKC on CTGF gene expression,32 we tested for the role of PKC on the {alpha}-tocopherol stimulation of CTGF expression. To this end, PKC was downregulated in T/G cells by prolonged exposure to PMA (1 µmol/L for 24 hours). Figure 3A shows that after this treatment the classical isoforms of PKC were undetectable by Western blot analysis. After this, the PKC-downregulated human SMCs were incubated in the presence of 50 µmol/L {alpha}-tocopherol for 8 hours, and CTGF expression was assessed by RT-PCR as described above. Under these conditions, we still observed 69±9.8% increased expression of CTGF, suggesting that the effect of {alpha}-tocopherol is not mediated by modulation of PKC (Figure 3B). In line with this, PKC downregulation did not lead to any change of CTGF protein expression in SMCs. We also incubated human SMC with several PKC inhibitors such as calphostin C (data not shown), the indolocarbazole Gö 6976 (20 nmol/L), and the staurosporine derivative PKC412 (1 µmol/L),33 and no effect on the basal level of CTGF protein expression was observed (Figure 3C). In summary, induction of CTGF expression appears not to be linked to the tocopherol-mediated inhibition of PKC.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. {alpha}-Tocopherol induction of CTGF expression is a PKC-independent phenomenon. A, Human SMCs were treated with either 1 µmol/L PMA (PMA-treated) or DMSO vehicle (untreated) for 24 hours. Total protein was analyzed by Western blotting with an anti-classical PKC isoforms ({alpha}, ßI, ßII, {gamma}) monoclonal antibody. The same membrane was analyzed with anti-{alpha}-actin antibody to confirm equal loading. B, PKC downregulated T/G cells were incubated in the presence of 50 µmol/L {alpha}-tocopherol or ethanol solvent (0.1%) for 8 hours. CTGF expression was analyzed by RT-PCR as described in Materials and Methods. Top shows a representative experiment using primers specific for CTGF and GAPDH, respectively. Bottom graph shows expression data obtained from 3 independent experiments. Data were obtained from scanning densitometry of the autoradiographs and are presented as mean±SD after normalization of the CTGF to the GAPDH expression data. C, Western blot of PKC-downregulated cells (PMA-treated) and SMCs treated with the PKC inhibitors, Gö 6976 and PKC412, showing CTGF protein expression.

TNF-{alpha} Downregulates CTGF Gene Expression and This Effect Is Reversed by {alpha}-Tocopherol and Other Antioxidants
TNF-{alpha} is a proinflammatory cytokine with pleiotropic cellular effects, and it is expressed by SMCs in atheromatous plaques. TNF-{alpha} has been previously described to downregulate CTGF gene expression,13 and suppresses the TGF-ß–induced expression of CTGF.14 In order to assess the role of {alpha}-tocopherol in this process, we studied the cytokine effect on CTGF mRNA levels together with the addition of different antioxidants. We compared the mRNA levels of CTGF with and without the addition of TNF-{alpha} (10 ng/mL) for 4 hours to the culture media. As shown in Figure 4, TNF-{alpha} downregulated CTGF mRNA expression up to 58.4±15%. When {alpha}-tocopherol (50 µmol/L) was added together with TNF-{alpha}, the effect of the latter was completely reversed. Similar results were found with ß-tocopherol, probucol, and N-acetylcysteine.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 4. TNF-{alpha} inhibition of CTGF expression is counteracted by {alpha}-tocopherol as well as by other antioxidants. RT-PCR analysis with total RNA isolated from human SMCs (T/G) treated with 10 ng/mL of TNF-{alpha} in the absence or presence of {alpha}-tocopherol ({alpha}-toc, 50 µmol/L), ß-tocopherol (ß-toc, 50 µmol/L), probucol (prob, 50 µmol/L), or N-acetylcysteine (NAC, 20 mmol/L). First lane corresponds to T/G cells treated with 0.1% ethanol treatment as a solvent control. Top shows a representative experiment using primers specific for CTGF and GAPDH, respectively. Bottom graph shows expression data obtained from 4 independent experiments. Data were obtained from scanning densitometry of the autoradiographs and are presented as mean±SD after normalization to the GAPDH expression data.

{alpha}-Tocopherol Stimulates CTGF Promoter Activity
To assess the mechanism of CTGF induction by {alpha}-tocopherol, the promoter of the CTGF gene was cloned upstream of the luciferase reporter gene (pCT-sb). T/G cells and HCASMCs were transfected with pCT-sb and treated for 24 hours with {alpha}-tocopherol. Luciferase activity was enhanced up to 33% in T/G cells and 265% in HCASMCs, showing that {alpha}-tocopherol may act via increasing the CTGF promoter activity (Figure 5A).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Effect of {alpha}-tocopherol on CTGF promoter activity in smooth muscle cells. A, pCT-sb was transiently transfected into T/G and HCASMC. Cells were then treated with 50 µmol/L {alpha}-tocopherol for 24 hours or ethanol (0.1%) as control solvent. B, T/G cells were transfected with pTGF-ß-RE-luc and pAP-1-luc, treated with compounds for 24 hours, and luciferase activity was measured. Columns and vertical bars denote mean±SD of 3 experiments.

Two previously described elements present in the CTGF promoter, TGF-ß-RE and AP-1,12 were cloned in front of a thymidine kinase promoter luciferase reporter construct. Transient transfection of these constructs in T/G cells shows that the two elements are mediating the effect of {alpha}-tocopherol on CTGF promoter activation (Figure 5B).

We previously showed that {alpha}-tocopherol increases the activity of the phosphatase PP2A25; therefore, we studied the effect of an inhibitor of PP2A, okadaic acid (OA) (25 nmol/L), on the CTGF promoter. Promoter activity was enhanced by OA 5-fold, showing that the stimulatory effect of {alpha}-tocopherol on CTGF is not due to PP2A activation. Moreover, when {alpha}-tocopherol was added together with OA, the effect of CTGF activation was partially reversed (Figure 6). On the other hand, TGF-ß-RE was still responsive to {alpha}-tocopherol despite that OA did not have an effect on this element (Figure 6).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 6. OA activation of CTGF expression is counteracted by {alpha}-tocopherol, and {alpha}-tocopherol stimulates TGF-ß-RE independent of PP2A. T/G cells were transfected with pCT-sb and pTGF-ß-RE-luc, treated with OA (25 nmol/L) and {alpha}-tocopherol (50 µmol/L) for 12 hours, and luciferase activity was measured. Columns and bars represent mean±SD values of at least 3 independent experiments performed in triplicates.

In general, we observed that the {alpha}-tocopherol–mediated CTGF induction was higher when PP2A was inhibited by OA (compare Figures 5 and 6). This may be explained in that {alpha}-tocopherol–stimulated PP2A activity normally counteracts the stimulatory effect of {alpha}-tocopherol on CTGF promoter, whereas in the presence of OA this effect does not occur. Taken together, these results show that the induction of the CTGF promoter by {alpha}-tocopherol may occur via the TGF-ß response element, whereas other elements are regulated by OA.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Several epidemiological studies have shown that {alpha}-tocopherol supplementation has preventive effects in atherosclerosis,34 whereas other studies showed no effect.35 These paradoxical results can be partially explained by the differences in the population groups (eg, gender, age, and other factors) and onset of supplementation in patients with late stages of the disease. Other studies suggest beneficial effects of {alpha}-tocopherol during wound repair.36 Whether these beneficial effects are mediated by the antioxidant function of {alpha}-tocopherol or by the nonantioxidant modulation of signal transduction pathways and/or gene expression is not known.

Using gene expression arrays, we have identified the connective tissue growth factor (CTGF) gene, among others, as being reproducibly modulated by {alpha}-tocopherol in human vascular SMCs. We focused in a further characterization on the CTGF gene because other studies showed a role of this factor in wound repair and atherosclerosis. In human cultured SMCs and skin fibroblasts, CTGF expression was stronger increased by {alpha}-tocopherol when compared with other antioxidants. Transient expression of CTGF is involved in normal repair processes such as wound healing and permanently overexpressed in pathological events.11,7 Moussad et al37 suggest that CTGF can play different roles depending on the site and timing of expression. The level of expression may also be important, because CTGF has been demonstrated to be highly overexpressed during fibrotic disease and atherosclerosis. The upregulation of CTGF by {alpha}-tocopherol seen in our study in cultured SMCs and fibroblasts is most likely not sufficient to generate pathological effects. Further in vivo experiments will be required to evaluate at what level of CTGF overexpression becomes causal for fibrotic diseases, and whether an upregulation of CTGF in atherosclerosis and wound repair could provide a benefit.

Searching for the mechanism of the {alpha}-tocopherol–mediated upregulation of CTGF expression, we found that TNF-{alpha}, a major inflammatory cytokine found in atheromas38 and wounds, suppresses CTGF expression at the mRNA level. These results are supported by recent reports about TNF-{alpha}, which show suppression of levels of CTGF in bovine aortic endothelial cells and fibroblasts.13,14 Inhibition of CTGF mRNA expression by TNF-{alpha} was partially reversed by {alpha}-tocopherol, suggesting that {alpha}-tocopherol can act like an antagonist of TNF-{alpha}–induced CTGF downregulation. However, the suppressive effect of TNF-{alpha} was also efficiently counteracted by ß-tocopherol and other antioxidants, suggesting that this event is primarily mediated by oxidative stress.

At the protein level, CTGF induction was of the same orders as that of its mRNA, thus excluding the possibility that {alpha}-tocopherol could result in lesion formation. In pathological situations, where stronger signals lead to higher CTGF expression, {alpha}-tocopherol may efficiently suppress them.39

Our results show that {alpha}-tocopherol does not modulate CTGF expression via inhibition of PKC, because CTGF mRNA levels were not affected when PKC was downregulated by the addition of PMA. Moreover, incubation with several PKC inhibitors did not change the basal expression of CTGF, suggesting other mechanisms of CTGF expression induction by {alpha}-tocopherol, possibly via the direct stimulation of an AP-1 response element or others present in the CTGF promoter. {alpha}-Tocopherol–mediated upregulation of CTGF may take place via modulation of the TGF-ß signaling pathway, because we observed that the TGF-ß response element located in the promoter is responsive to {alpha}-tocopherol treatment. TGF-ß stimulates CTGF expression in human aortic SMCs by interfering with the activity of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) and Smad3.40 On the other hand, TGF-ß decreases the expression of CD36 scavenger receptor by reducing the PPAR-{gamma} activity,41 and {alpha}-tocopherol inhibits CD36 expression possibly also by interfering with PPAR-{gamma}.22,42 Taken together, it can be speculated that {alpha}-tocopherol may interfere with the PPAR-{gamma} activity, thus leading to activation of CTGF expression, or reduction of CD36 expression.

When extrapolated to the in vivo situation, {alpha}-tocopherol may support connective tissue cells during wound repair and vessel damage by stimulating the synthesis of CTGF and thus extracellular matrix, either by interfering with TNF-{alpha} or by directly acting on the CTGF promoter. Upregulation of CTGF expression by {alpha}-tocopherol in SMCs may increase the resistance of vascular cells to vessel injuries, thus acting as an atherosclerosis-preventing agent. Moreover, the beneficial {alpha}-tocopherol effects in some epidemiological studies in patients with advanced atherosclerosis may be explained by transient CTGF overexpression. In fact, plaques with a soft lipid core, and thus prone to rupture and embolism, may become covered by a fibrous connective tissue cap, separating and stabilizing the plaque material from the luminal blood flow. CTGF is overexpressed in advanced atherosclerotic lesions, and CTGF expressing cells are localized in areas with increased connective tissue accumulation, such as the shoulder of the fibrous cap.6 Thus, {alpha}-tocopherol–mediated induction of CTGF expression may lead to enhanced deposition of extracellular matrix and thus stabilize the cap, possibly explaining its beneficial effect also in advanced atherosclerotic situations.


*    Acknowledgments
 
This study was supported by the Swiss National Science Foundation, the Foundation for Nutrition Research in Switzerland, and by Hoffmann-La Roche, AG. Aurelio V. Graça-Souza is recipient of a fellowship from Fundaçao de Coordenaçao de Aperfeiçoamento do Pessoal de Nivel Superior (CAPES-Brasil). We thank M. Feher for the excellent technical assistance.


*    Footnotes
 
*These authors contributed equally to this work. Back

Received July 26, 2002; revision received October 29, 2002; accepted November 13, 2002.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Bork P. The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett. 1993; 327: 125–130.[CrossRef][Medline] [Order article via Infotrieve]
  2. Kim HS, Nagalla SR, Oh Y, Wilson E, Roberts CT Jr, Rosenfeld RG. Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): characterization of connective tissue growth factor as a member of the IGFBP superfamily. Proc Natl Acad Sci U S A. 1997; 94: 12981–12986.[Abstract/Free Full Text]
  3. Bradham DM, Igarashi A, Potter RL, Grotendorst GR. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol. 1991; 114: 1285–1294.[Abstract/Free Full Text]
  4. Brigstock DR, Steffen CL, Kim GY, Vegunta RK, Diehl JR, Harding PA. Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids: identification as heparin- regulated Mr 10,000 forms of connective tissue growth factor. J Biol Chem. 1997; 272: 20275–20282.[Abstract/Free Full Text]
  5. Oemar BS, Luscher TF. Connective tissue growth factor: friend or foe? Arterioscler Thromb Vasc Biol. 1997; 17: 1483–1489.[Abstract/Free Full Text]
  6. Oemar BS, Werner A, Garnier JM, Do DD, Godoy N, Nauck M, Marz W, Rupp J, Pech M, Luscher TF. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation. 1997; 95: 831–839.[Abstract/Free Full Text]
  7. Igarashi A, Okochi H, Bradham DM, Grotendorst GR. Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell. 1993; 4: 637–645.[Abstract]
  8. Uzumcu M, Homsi MF, Ball DK, Coskun S, Jaroudi K, Hollanders JM, Brigstock DR. Localization of connective tissue growth factor in human uterine tissues. Mol Hum Reprod. 2000; 6: 1093–1098.[Abstract/Free Full Text]
  9. Ball DK, Surveyor GA, Diehl JR, Steffen CL, Uzumcu M, Mirando MA, Brigstock DR. Characterization of 16- to 20-kilodalton (kDa) connective tissue growth factors (CTGFs) and demonstration of proteolytic activity for 38-kDa CTGF in pig uterine luminal flushings. Biol Reprod. 1998; 59: 828–835.[Abstract/Free Full Text]
  10. Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR. Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol. 1996; 107: 404–411.[CrossRef][Medline] [Order article via Infotrieve]
  11. Dammeier J, Beer HD, Brauchle M, Werner S. Dexamethasone is a novel potent inducer of connective tissue growth factor expression. Implications for glucocorticoid therapy. J Biol Chem. 1998; 273: 18185–18190.[Abstract/Free Full Text]
  12. Grotendorst GR, Okochi H, Hayashi N. A novel transforming growth factor ß response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ. 1996; 7: 469–480.[Abstract]
  13. Lin J, Liliensiek B, Kanitz M, Schimanski U, Bohrer H, Waldherr R, Martin E, Kauffmann G, Ziegler R, Nawroth PP. Molecular cloning of genes differentially regulated by TNF-{alpha} in bovine aortic endothelial cells, fibroblasts and smooth muscle cells. Cardiovasc Res. 1998; 38: 802–813.[Abstract/Free Full Text]
  14. Abraham DJ, Shiwen X, Black CM, Sa S, Xu Y, Leask A. Tumor necrosis factor {alpha} suppresses the induction of connective tissue growth factor by transforming growth factor-ß in normal and scleroderma fibroblasts. J Biol Chem. 2000; 275: 15220–15225.[Abstract/Free Full Text]
  15. Goetze S, Xi XP, Kawano Y, Kawano H, Fleck E, Hsueh WA, Law RE. TNF-{alpha}-induced migration of vascular smooth muscle cells is MAPK dependent. Hypertension. 1999; 33: 183–189.[Abstract/Free Full Text]
  16. Jovinge S, Crisby M, Thyberg J, Nilsson J. DNA fragmentation and ultrastructural changes of degenerating cells in atherosclerotic lesions and smooth muscle cells exposed to oxidized LDL in vitro. Arterioscler Thromb Vasc Biol. 1997; 17: 2225–2231.[Abstract/Free Full Text]
  17. Jovinge S, Hultgardh-Nilsson A, Regnstrom J, Nilsson J. Tumor necrosis factor-{alpha} activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol. 1997; 17: 490–497.[Abstract/Free Full Text]
  18. Sen CK. Redox signaling and the emerging therapeutic potential of thiol antioxidants. Biochem Pharmacol. 1998; 55: 1747–1758.[CrossRef][Medline] [Order article via Infotrieve]
  19. Azzi A, Cantoni O, Özer NK, Boscoboinik D, Spycher S. The role of hydrogen peroxide and RRR-{alpha}-tocopherol in smooth muscle cell proliferation. Cell Death Differ. 1996; 3: 79–90.[Medline] [Order article via Infotrieve]
  20. Keaney JF Jr, Guo Y, Cunningham D, Shwaery GT, Xu A, Vita JA. Vascular incorporation of {alpha}-tocopherol prevents endothelial dysfunction due to oxidized LDL by inhibiting protein kinase C stimulation. J Clin Invest. 1996; 98: 386–394.[Medline] [Order article via Infotrieve]
  21. Ricciarelli R, Maroni P, Özer N, Zingg JM, Azzi A. Age-dependent increase of collagenase expression can be reduced by {alpha}-tocopherol via protein kinase C inhibition. Free Radic Biol Med. 1999; 27: 729–737.[CrossRef][Medline] [Order article via Infotrieve]
  22. Ricciarelli R, Zingg JM, Azzi A. Vitamin E reduces the uptake of oxidized LDL by inhibiting CD36 scavenger receptor expression in cultured aortic smooth muscle cells. Circulation. 2000; 102: 82–87.[Abstract/Free Full Text]
  23. Kim HS, Arai H, Arita M, Sato Y, Ogihara T, Inoue K, Mino M, Tamai H. Effect of {alpha}-tocopherol status on {alpha}-tocopherol transfer protein expression and its messenger RNA level in rat liver. Free Radic Res. 1998; 28: 87–92.[Medline] [Order article via Infotrieve]
  24. Aratri E, Spycher SE, Breyer I, Azzi A. Modulation of {alpha}-tropomyosin expression by {alpha}-tocopherol in rat vascular smooth muscle cells. FEBS Lett. 1999; 447: 91–94.[CrossRef][Medline] [Order article via Infotrieve]
  25. Ricciarelli R, Tasinato A, Clement S, Özer NK, Boscoboinik D, Azzi A. {alpha}-Tocopherol specifically inactivates cellular protein kinase C {alpha} by changing its phosphorylation state. Biochem J. 1998; 334: 243–249.[Medline] [Order article via Infotrieve]
  26. Xin LW, Martinerie C, Zumkeller W, Westphal M, Perbal B. Differential expression of novH and CTGF in human glioma cell lines. J Clin Pathol: Mol Pathol. 1996; 49: M91–M97.[Abstract/Free Full Text]
  27. Tran K, Chan AC. R,R,R-{alpha}-tocopherol potentiates prostacyclin release in human endothelial cells. Evidence for structural specificity of the tocopherol molecule. Biochim Biophys Acta. 1990; 1043: 189–197.[Medline] [Order article via Infotrieve]
  28. Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, Narins RG. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol. 2000; 11: 25–38.[Abstract/Free Full Text]
  29. Steffen CL, Ball-Mirth DK, Harding PA, Bhattacharyya N, Pillai S, Brigstock DR. Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro. Growth Factors. 1998; 15: 199–213.[Medline] [Order article via Infotrieve]
  30. Boscoboinik D, Szewczyk A, Hensey C, Azzi A. Inhibition of cell proliferation by {alpha}-tocopherol. Role of protein kinase C. J Biol Chem. 1991; 266: 6188–6194.[Abstract/Free Full Text]
  31. Tasinato A, Boscoboinik D, Bartoli GM, Maroni P, Azzi A. d-{alpha}-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties. Proc Natl Acad Sci U S A. 1995; 92: 12190–12194.[Abstract/Free Full Text]
  32. Fan WH, Karnovsky MJ. Activation of protein kinase C inhibits the expression of connective tissue growth factor. Biochem Biophys Res Commun. 2000; 275: 312–321.[CrossRef][Medline] [Order article via Infotrieve]
  33. Tenzer A, Zingg D, Rocha S, Hemmings B, Fabbro D, Glanzmann C, Schubiger PA, Bodis S, Pruschy M. The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C. Cancer Res. 2001; 61: 8203–8210.[Abstract/Free Full Text]
  34. Salonen JT, Nyyssonen K, Salonen R, Lakka HM, Kaikkonen J, Porkkala-Sarataho E, Voutilainen S, Lakka TA, Rissanen T, Leskinen L, Tuomainen TP, Valkonen VP, Ristonmaa U, Poulsen HE. Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study: a randomized trial of the effect of vitamins E and C on 3-year progression of carotid atherosclerosis. J Intern Med. 2000; 248: 377–386.[CrossRef][Medline] [Order article via Infotrieve]
  35. Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet. 2002; 360: 23–33.[CrossRef][Medline] [Order article via Infotrieve]
  36. Simon GA, Schmid P, Reifenrath WG, van Ravenswaay T, Stuck BE. Wound healing after laser injury to skin–the effect of occlusion and vitamin E. J Pharm Sci. 1994; 83: 1101–1106.[CrossRef][Medline] [Order article via Infotrieve]
  37. Moussad EE, Brigstock DR. Connective tissue growth factor: what’s in a name? Mol Genet Metab. 2000; 71: 276–292.[CrossRef][Medline] [Order article via Infotrieve]
  38. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol. 1990; 65: 297–302.[CrossRef][Medline] [Order article via Infotrieve]
  39. Kuemmerle NB, Brandt RB, Chan W, Krieg RJ Jr, Chan JC. Inhibition of transforming growth factor ß1 induction by dietary vitamin E in unilateral ureteral obstruction in rats. Biochem Mol Med. 1997; 61: 82–86.[CrossRef][Medline] [Order article via Infotrieve]
  40. Fu M, Zhang J, Zhu X, Myles DE, Willson TM, Liu X, Chen YE. Peroxisome proliferator-activated receptor {gamma} inhibits transforming growth factor ß-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3. J Biol Chem. 2001; 276: 45888–45894.[Abstract/Free Full Text]
  41. Han J, Hajjar DP, Tauras JM, Feng J, Gotto AM Jr, Nicholson AC. Transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 decrease expression of CD36, the type B scavenger receptor, through mitogen-activated protein kinase phosphorylation of peroxisome proliferator-activated receptor-{gamma}. J Biol Chem. 2000; 275: 1241–1246.[Abstract/Free Full Text]
  42. Devaraj S, Hugou I, Jialal I. {alpha}-Tocopherol decreases CD36 expression in human monocyte-derived macrophages. J Lipid Res. 2001; 42: 521–527.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Munteanu, M. Taddei, I. Tamburini, E. Bergamini, A. Azzi, and J.-M. Zingg
Antagonistic Effects of Oxidized Low Density Lipoprotein and {alpha}-Tocopherol on CD36 Scavenger Receptor Expression in Monocytes: INVOLVEMENT OF PROTEIN KINASE B AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-{gamma}
J. Biol. Chem., March 10, 2006; 281(10): 6489 - 6497.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
W. C. Duncan, S. G. Hillier, E. Gay, J. Bell, and H. M. Fraser
Connective Tissue Growth Factor Expression in the Human Corpus Luteum: Paracrine Regulation by Human Chorionic Gonadotropin
J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5366 - 5376.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/1/104    most recent
01.RES.0000049103.38175.1Bv1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Villacorta, L.
Right arrow Articles by Azzi, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Villacorta, L.
Right arrow Articles by Azzi, A.
Related Collections
Right arrow Mechanism of atherosclerosis/growth factors
Right arrow Cell signalling/signal transduction
Right arrow Gene expression
Right arrow Gene regulation
Right arrow Growth factors/cytokines

Circulation Research Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2003 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.