Connective Tissue Growth Factor Gene Regulation

REQUIREMENTS FOR ITS INDUCTION BY TRANSFORMING GROWTH FACTOR-beta 2 IN FIBROBLASTS*

Andrew LeaskDagger §, Alan Holmes, Carol M. Black, and David J. Abraham

From Dagger  Fibrogen, Inc., South San Francisco, California 94080 and the  Centre for Rheumatology, Royal Free and University College Medical School, Rowland Hill St., London NW3 2PF, United Kingdom

Received for publication, October 9, 2002, and in revised form, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In skin, the profibrotic protein connective tissue growth factor (CTGF) is not normally expressed. However, when skin cells are exposed to transforming growth factor-beta (TGF-beta ), CTGF is induced in fibroblasts but not in epithelial cells. We have begun to investigate the requirements for the fibroblast-selective induction of CTGF by TGF-beta . Previously we found that this response was Smad-dependent. Now we show that protein kinase C and Ras/MEK/ERK are necessary for the TGF-beta induction of the CTGF promoter but not of a generic Smad-responsive promoter (SBE-lux). Induction of the CTGF promoter is antagonized by c-Jun or by MEKK1, suggesting that a proper balance between the Ras/MEK/ERK and JNK MAPK cascades is necessary for TGF-beta induction of CTGF. We identify the minimal CTGF promoter element necessary and sufficient to confer TGF-beta responsiveness to a heterologous promoter and show that a tandem repeat of a consensus transcription enhancer factor binding element, 5'-GAGGAATGG-3', is necessary for this induction. This element has not been previously shown to play a role in TGF-beta induction of gene expression in fibroblasts. Gel shift analysis shows that this sequence binds nuclear factors that are greatly enriched in fibroblasts relative to epithelial cells. Thus Smads, Ras/MEK/ERK, protein kinase C, and fibroblast-enriched factors that bind GAGGAATGG act together to drive the TGF-beta -mediated induction of CTGF in fibroblasts.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As TGF-beta 1 promotes fibroblast proliferation and matrix synthesis, this cytokine has long been proposed to initiate and maintain fibrosis (e.g. Refs. 1-3). Indeed in acute drug- or surgery-induced animal models of fibrosis, neutralizing TGF-beta action reduces the deposition of collagen (for a review, see Ref. 1). Much interest has been devoted to developing anti-TGF-beta strategies, such as neutralizing anti-TGF-beta antibodies or soluble TGF-beta receptor fragments, to combat fibrosis; however, as TGF-beta possesses multiple roles in human physiology (4, 5), generally blocking its effects might be expected to have deleterious effects clinically. Thus, identifying downstream mediators of the profibrotic effects of TGF-beta might be useful in developing more clinically appropriate antifibrotic strategies.

Accordingly much interest has been recently devoted to the TGF-beta target gene CTGF. CTGF, a secreted protein (6) initially identified in the conditioned medium of cultured endothelial cells (7), is a member of the CCN (CTGF, Cyr61, and Nov) family of proteins that promote angiogenesis, cell migration, and cell adhesion (8). Recently CTGF knockout mice have been generated; mice homozygous for a deletion of the CTGF gene die soon after birth due to a defect in skeletal development characterized, in part, by reduced expression of bone-specific matrix genes (9). Pathologically CTGF seems to contribute to fibrotic disorders by mediating at least some of the profibrotic effects of TGF-beta (10). A CTGF response element exists in the type I collagen promoter (11). Furthermore, in mice, subcutaneous injection of TGF-beta results only in a transient fibrotic response; however, co-injection of CTGF and TGF-beta results in sustained, persistent fibrosis (12). Thus, CTGF and TGF-beta seem to synergize to promote chronic fibrosis. This phenomenon may result from the inherent stickiness of CTGF, which may enhance the activity or stability of TGF-beta at low TGF-beta concentrations (13). Recently we found that a small molecule inhibitor, Iloprost, reduces CTGF expression in vitro and in vivo and alleviates symptoms of fibrosis in vivo (14, 15). Thus understanding how to control CTGF expression would seem to be important in developing novel antifibrotic therapies (16).

In skin, CTGF is not normally expressed; however, in dermal fibroblasts exposed to TGF-beta , CTGF mRNA and protein expression are induced (17-21). The induction of CTGF by TGF-beta is cell-type specific, for example, occurring in connective tissue cells but not in epithelial cells (17-22). The regulation of CTGF expression by TGF-beta appears to be controlled primarily at the level of gene transcription (16-22). The induction of CTGF mRNA by TGF-beta is rapid, occurring within 30 min of ligand addition, and does not require de novo protein synthesis as this induction occurs even in the presence of an inhibitor of protein synthesis (17).

TGF-beta induction of gene expression has been extensively studied and generally involves the action of members of the Smad family of proteins (23). Activation of TGF-beta -mediated gene expression is generally mediated through Smad 2, 3, and 4. Smad 2 and 3 are normally present in the cytosol. Once activated by TGF-beta , Smad 2 and 3 become phosphorylated by type I TGF-beta receptor kinase and then, after forming a complex with Smad 4, translocate to the nucleus and activate expression of target genes. The inhibitory Smads, Smad 6 and 7, negatively regulate this process. Recent studies have identified a Smad-responsive promoter element, GTCTAGAC, that binds Smad 3 and 4 (23). Recently we identified a functional consensus Smad binding site in the CTGF promoter that is essential for the TGF-beta induction of CTGF in fibroblasts (20, 21). Cotransfection of Smad 3 and 4 enhanced CTGF expression significantly (20). TGF-beta induction of CTGF did not occur in embryonic fibroblasts cultured from Smad 3 knockout mice (20), suggesting that TGF-beta induction of CTGF occurs in a Smad 2-independent fashion. Intriguingly the Smad binding site of CTGF is insufficient to confer TGF-beta responsiveness to a heterologous promoter (21), suggesting that additional as yet unidentified factors must be required for the TGF-beta -mediated induction of CTGF gene expression in fibroblasts.

In this report, we elucidate requirements necessary for the TGF-beta induction of CTGF in fibroblasts but not in epithelial cells. We identify cis-acting promoter sequences and signaling pathways involved with this induction. To our knowledge this is the first investigation of the regulation of a fibroblast-selective TGF-beta -responsive promoter. Our results give new insights into fibroblast-selective TGF-beta signaling and suggest methods of developing new antifibrotic therapeutic strategies.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture, Transfections, and DNA Constructs-- Human dermal and mouse NIH 3T3 fibroblasts were cultured as described previously (19, 20). MvLu epithelial cells were purchased (ATCC) and cultured in 10% Dulbecco's modified Eagle's medium, 10% fetal bovine serum (Cellgro). Cells were transfected with LipofectAMINE plus (Invitrogen) or FuGENE (Roche Molecular Biochemicals) as described by their manufacturers. Compounds used (Go 6850, Go 6976, Go 6983, U0126, SB203580, and SP600125 (all from Calbiochem)) were added at the concentrations indicated 45 min before adding TGF-beta 2 (Celtrix) for 24 h. Expression vectors (1 or 0.5 µg as indicated) encoding dominant negative Ras (N17, Upstate Biotechnology), active MEKK1 (Stratagene), c-Jun (R. Tjian, University of California-Berkeley), dominant negative c-Jun (TAM67, M. J. Birrer, NCI, National Institutes of Health, Bethesda, MD), dominant negative MKK4 (R. Davis, University of Massachusetts Medical School, Worcester, MA), YAP65 (Research Genetics, GenBankTM accession number AW215560), or empty expression vector were transfected into each well of a six-well plate along with a full-length CTGF promoter/SEAP reporter expression vector (containing a promoter fragment between -805 and +17 of the CTGF promoter, 0.5 µg; Ref. 14) and CMV-beta -galactosidase (0.25 µg, Clontech) as an internal transfection control. An additional reporter construct containing multiple copies of the consensus Smad recognition sequence subcloned upstream of the luciferase reporter gene (SBE-lux) was from P. Ten Dijke (Ludwig Institute, Uppsala, Sweden). Promoter assays were performed and standardized to beta -galactosidase as described (Tropix). Values shown are the average (±S.D.) of at least three replicates and at least two independent trials. Statistical analysis was performed using the Student's t test (p < 0.05). A DNA construct containing nucleotides -805 to -23 of the CTGF promoter subcloned upstream of the herpes simplex virus minimal thymidine kinase (TK) promoter and SEAP reporter gene was as described previously (21). Additional promoter deletion constructs were generated using Pfu polymerase (New England Biolabs) and appropriate synthetic oligonucleotide primers (Sigma Genosys). The resultant DNA fragments were subcloned into TK-SEAP (Clontech). Linker scanning and TEF binding motif mutations were performed using oligonucleotides containing appropriate mutations (Sigma Genosys) and a mutagenesis kit (Stratagene). All constructs were fully sequenced before use. For Western blot analysis, a rabbit polyclonal anti-CTGF antibody was used as described previously (19), and a mouse anti-beta -tubulin antibody (Sigma) was utilized.

Gel Shift Analysis-- Nuclear extracts were prepared using a kit (Pierce), and protein concentration was determined (Bio-Rad). A double-stranded annealed oligomer spanning nucleotides -126 to -77 of the CTGF promoter (Sigma Genosys) was labeled with [32P]ATP (PerkinElmer Life Sciences) and polynucleotide kinase (New England Biolabs) and used as probe (60,000 cpm/reaction). As competitor oligomers, a 100-fold excess of wild-type probe or oligomers otherwise identical to the wild-type probe but bearing mutations in either TEF recognition element were used.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TGF-beta Induces CTGF Expression in Fibroblasts but Not in Epithelial Cells-- To verify previously published data showing that the TGF-beta induction of CTGF protein occurred in fibroblasts but not in epithelial cells (17), we cultured human foreskin fibroblasts or MvLu epithelial cells until confluence. Cells were then serum-starved for 18 h and treated with or without 25 ng/ml TGF-beta 2 for an additional 24 h. Cells were then harvested, and the resultant protein extracts were subjected to Western blot analysis with a rabbit anti-CTGF antibody (Fig. 1A, cell layer). Equal amounts of media were also subjected to Western analysis (Fig. 1A, media). As anticipated, in the absence of exogenously added TGF-beta , neither cell type expressed CTGF. However, TGF-beta 2 induced CTGF protein expression in fibroblasts but not in MvLu epithelial cells.


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Fig. 1.   TGF-beta 2 induces CTGF protein (A) and CTGF promoter (B) activity in fibroblasts but not in epithelial cells. A, human foreskin fibroblasts (HFF) and MvLu epithelial cells were serum-starved for 18 h prior to incubation with or without 25 ng/ml TGF-beta 2 for an additional 24 h. Cell layers were harvested, and the resultant protein extracts (25 µg/lane) were subjected to Western blot analysis with a rabbit anti-CTGF antibody (19) or an anti-beta -tubulin antibody to verify equal protein loading among samples. Equal amounts of conditioned media were also subjected to Western blot analysis with an anti-CTGF antibody. B, a construct containing a full-length CTGF promoter (spanning nucleotides -805 to +17) subcloned in front of the SEAP reporter gene or a construct containing 12 copies of a Smad binding element (CAGA) upstream of the luciferase reporter gene (1 µg/well of a six-well plate) was transfected into the cells described in A. After an 18-h serum starvation step, cells were treated with or without 25 ng/ml TGF-beta 2 for an additional 24 h. Cells were cotransfected with a control CMV promoter-driven beta -galactosidase expression plasmid (0.25 µg/well), which was used to adjust for differences in transfection efficiencies among samples. Average expression values (±S.D., n = 6) are shown. Results similar to those obtained with human foreskin fibroblasts were observed in NIH 3T3 fibroblasts (not shown).

To assess whether TGF-beta induction of CTGF was conferred by elements in the CTGF promoter, we transfected into cells a DNA construct containing a CTGF promoter fragment spanning nucleotides -805 and +17 that was subcloned in front of the SEAP reporter gene (19, 20). After an 18-h serum starvation step, cells were exposed for 24 h to 25 ng/ml TGF-beta 2. Media were then assayed for SEAP expression. We found that TGF-beta potently induced CTGF promoter activity in fibroblasts but not in MvLu cells (Fig. 1B). These results were consistent with previously published observations using a slightly larger CTGF promoter fragment (17). MvLu cells were capable of inducing a Smad-dependent transcriptional response to TGF-beta as TGF-beta was able to induce expression of a transiently transfected construct expressing a luciferase reporter gene under the control of multimers of a consensus Smad recognition sequence (SBE-luciferase; Fig. 1B). Thus, a CTGF promoter fragment between -805 and +17 responds to TGF-beta in a fibroblast-selective fashion.

TGF-beta Induction of CTGF Promoter Activity in Fibroblasts Involves Protein Kinase C and Ras/MEK/ERK and Is Suppressed by MEKK1/JNK/c-Jun-- We decided to examine known signaling pathways for their ability to modify the TGF-beta induction of CTGF in fibroblasts. For this analysis, we transfected our full-length CTGF promoter/SEAP reporter construct into fibroblasts. After an 18-h serum starvation step, cells were exposed for 45 min to commercially available signal transduction pathway inhibitors. Cells were then incubated for an additional 24 h with or without 25 ng/ml TGF-beta 2. Preincubation of fibroblasts with the protein kinase C (PKC) alpha , beta , gamma , delta , and epsilon  inhibitor Go 6850 (5 µM; bisindolylmaleimide, GF 109203X; Ref. 24) blocked the TGF-beta induction of CTGF promoter activity (Fig. 2, CTGF-SEAP). Smad-dependent TGF-beta induction of gene expression seemed not to generally require PKC as Go 6850 did not block the TGF-beta 2-mediated induction of SBE-luciferase (Fig. 2, SBE-LUX).


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Fig. 2.   Effect of signal transduction inhibitors on TGF-beta 2-induced CTGF promoter activity in fibroblasts. Inhibition of protein kinase C and Ras/MEK/ERK blocks the TGF-beta induction of the CTGF promoter but not the generic Smad-responsive promoter SBE-lux. PKC alpha , beta , gamma , delta , and epsilon  inhibitor Go 6850 (5 µM; bisindolylmaleimide, GF 109203X; Ref. 24) blocks the TGF-beta induction of CTGF promoter activity. PKC alpha  and beta  inhibitor Go 6976 (10 µM; Ref. 25) does not affect the TGF-beta 2-mediated induction of CTGF. PKC alpha , beta , gamma , delta , and zeta  inhibitor Go 6983 (10 µM; Ref. 26) blocks the TGF-beta induction of CTGF. A 10 µM concentration of the MEK inhibitor U0126 (28) blocks the TGF-beta 2 induction of CTGF (this figure and Ref. 15). A 20 µM concentration of the p38 inhibitor SB203580 (30) or a 10 µM concentration of the JNK inhibitor SP600125 (31) has no impact on the TGF-beta 2 induction of CTGF promoter activity in fibroblasts. The PKC and Ras/MEK/ERK cascades do not generally affect Smad-dependent TGF-beta signaling as the TGF-beta induction of the Smad-responsive reporter SBE-lux was not affected by Go 6850 or U0126. NIH 3T3 cells were transfected, processed, and analyzed as in Fig. 1. To assess the effect of inhibitors on the TGF-beta induction of CTGF, cells were preincubated for 45 min before an additional 24-h incubation with or without TGF-beta 2. Expression values represent averages (±S.D., n = 6). **, statistically significant difference in expression values (p < 0.05) relative to controls.

To further analyze the PKC dependence of the TGF-beta induction of CTGF in fibroblasts, we found that the PKC alpha  and beta  inhibitor Go 6976 (10 µM; Ref. 25) did not affect the TGF-beta 2-mediated induction of CTGF (Fig. 2). Conversely the PKC alpha , beta , gamma , delta , and zeta  inhibitor Go 6983 (10 µM; Ref. 26) blocked the TGF-beta induction of CTGF (Fig. 2). Thus, by comparing the selectivity of the different PKC inhibitors used, we deduced that PKC gamma  or delta  either alone or in combination is involved in the TGF-beta induction of CTGF in fibroblasts.

In many systems, MAP kinase pathways are involved in the TGF-beta induction of gene expression. For example, TGF-beta induces JNK and Ras/MEK/ERK kinase pathways in fibroblasts (Refs. 15 and 27 and not shown). We then investigated the role of MAP kinase pathways, namely the Ras/MEK/ERK, p38, and JNK cascades, in the TGF-beta induction of CTGF. We found that a 10 µM concentration of the MEK 1 and 2 inhibitor U0126 (28) blocked the TGF-beta 2 induction of CTGF (Fig. 2; Ref. 15). Similarly dominant negative Ras (N17; Ref. 29) blocked the TGF-beta 2 induction of CTGF promoter activity (Fig. 3, CTGF-SEAP). As for PKC, the Ras/MEK/ERK cascade did not seem to directly affect the ability of Smads to activate target gene expression as the TGF-beta induction of the Smad-dependent reporter SBE-lux was not affected by either U0126 or dominant negative Ras (Figs. 2 and 3, SBE-LUX).


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Fig. 3.   Effect of overexpressing wild-type or dominant negative signal transduction components on TGF-beta 2 induction of CTGF promoter activity in fibroblasts. NIH 3T3 fibroblasts were transfected and assayed for CTGF promoter activity as in Fig. 1B. The full-length CTGF promoter/SEAP reporter construct or SBE-lux (0.5 µg/well) was cotransfected with empty expression vector or expression vector encoding dominant negative Ras (dnRAS), dominant negative MKK4 (dnMKK4), dominant negative MEKK1 (dnMEKK1), dominant negative c-Jun (TAM67, dnc-jun), wild-type c-Jun (c-jun), or c-Jun with Smad3 and 4 (c-jun + Smad3/4; each at 0.5 µg/well) as indicated. All experiments were performed in six-well plates. Cells were cotransfected with a CMV promoter-driven vector encoding beta  galactosidase (0.25 µg) to control for differences in transfection efficiency. Values expressed are averages ± S.D. (n = 6). The effect of overexpressing c-Jun was rescued by overexpressing Smad 3 and 4 (0.5 µg/well). Also, overexpressing c-Jun blocked the Smad-dependent TGF-beta induction of SBE-lux suggesting that c-Jun generally antagonizes Smad-dependent signaling. **, statistically significant difference in expression values (p < 0.05) relative to controls.

We then continued our examination of MAP kinase signaling cascades in CTGF gene regulation. We found that a 20 µM concentration of the p38 inhibitor SB203580 (30) or a 10 µM concentration of the JNK inhibitor SP600125 (31) had no impact on the TGF-beta 2 induction of CTGF promoter activity in fibroblasts (Fig. 2). Similarly dominant negative MKK4 (an activator of p38 and JNK; Ref. 32) had no impact on the TGF-beta 2 induction of CTGF promoter activity (Fig. 3). However, overexpressing the JNK cascade mediator MEKK1 (33) blocked CTGF induction (Fig. 3). Furthermore, overexpressing the JNK target c-Jun reduced the TGF-beta induction of the CTGF promoter (Fig. 3). Similarly overexpressing dominant negative c-Jun (TAM67, which lacks the activation domain of c-Jun; Ref. 34) increased TGF-beta -induced CTGF promoter activity.

We then assessed whether the ability of c-Jun to suppress the TGF-beta induction of CTGF promoter activity could be via a Smad-dependent mechanism. First, we found that the ability of overexpressed c-Jun to suppress TGF-beta -induced CTGF promoter activity was rescued by overexpressing Smad 3 and 4 (Fig. 3, CTGF-SEAP). Second, we found that c-Jun seemed to generally suppress Smad-dependent activation of target gene expression as overexpressing c-Jun markedly attenuated the ability of TGF-beta to induce SBE-lux (Fig. 3, SBE-LUX). These results are consistent with the notion that activated c-Jun might suppress Smad-dependent gene activation by sequestering Smads and preventing them from activating gene expression of non-Ap-1-dependent promoters (see "Discussion"). In summary, our studies investigating the role of MAP kinase pathways in CTGF gene expression implied that the MAP kinase pathway Ras/MEK/ERK is necessary for the TGF-beta induction of CTGF promoter expression in fibroblasts and that the JNK MAP kinase cascade is refractory to this process. Therefore, a proper balance between activation of the Ras/MEK/ERK MAP kinase cascade relative to the JNK MAP kinase cascade is important in controlling CTGF induction in fibroblasts.

An Element in the CTGF Promoter Is Sufficient to Confer Fibroblast-specific Responsiveness to a Heterologous Promoter-- To further characterize the element in the CTGF promoter necessary for its fibroblast-specific induction by TGF-beta , we sought to identify elements in the CTGF promoter sufficient to confer fibroblast-specific TGF-beta responsiveness to a heterologous promoter. We subcloned a fragment of the CTGF promoter between -805 and -23 upstream of the minimal, non-TGF-beta -responsive herpes simplex virus TK promoter (21). Confirming previous results (21), we found that, in fibroblasts, the segment of the CTGF promoter lying between -805 and -23 conferred TGF-beta responsiveness to the TK promoter (Fig. 4A). However, this promoter construct did not confer TGF-beta responsiveness to the TK promoter in MvLu epithelial cells (Fig. 4A). Thus, the fragment of the CTGF promoter that lies between nucleotides -805 and -23 is sufficient to confer fibroblast-specific TGF-beta responsiveness to a heterologous promoter.


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Fig. 4.   Delineation of the CTGF promoter elements necessary and sufficient to confer TGF-beta responsiveness in fibroblasts to a heterologous (herpes simplex virus minimal TK) promoter. A, elements between nucleotides -244 and -143 and -126 and -77 are necessary for the TGF-beta response (p < 0.05). Various CTGF promoter fragments were subcloned into pTK-SEAP (Clontech) upstream of the minimal TK promoter. The resultant constructs were transfected into cells as indicated along with a CMV-beta -galactosidase control plasmid. After a serum starvation step of 18 h, cells were incubated with and without 25 ng/ml TGF-beta 2 for 24 h. Previously the region of the CTGF promoter between -244 and -143 was analyzed for its role in TGF-beta induction of CTGF promoter activity and was found to consist of a functional Smad element (20). B, mapping of the TGF-beta -responsive element between nucleotides -126 and -77 of the CTGF promoter. The TK/SEAP promoter/reporter construct driven by nucleotides -351 to -77 of the CTGF promoter was mutated between nucleotides -126 and -77 as indicated. Five linker scanning CTGF promoter mutants were generated (m1-m5). The resultant constructs were transfected into NIH 3T3 fibroblasts and incubated with or without TGF-beta as in part A. Mutation of nucleotides -97 to -92 or -92 to -83 (m3 or m4) abolished the ability of the CTGF promoter fragment to confer TGF-beta responsiveness in fibroblasts to the TK promoter (p < 0.05). Values expressed are averages of adjusted SEAP expression ± S.D. (n = 6). ND, not done; HFF, human foreskin fibroblasts.

To precisely identify the CTGF promoter elements required for fibroblast-selective TGF-beta induction, we introduced a progressive series of 5' and 3' deletions in the CTGF promoter and assayed the ability of these fragments to confer TGF-beta responsiveness to the TK promoter. CTGF/TK promoter constructs that lacked either nucleotides -244 to -143 or -126 to -77 of the CTGF promoter no longer responded to TGF-beta in fibroblasts (Fig. 4A). The element in the CTGF promoter between nucleotides -244 and -143 has been extensively analyzed. This CTGF promoter segment consists of two elements involved with CTGF gene expression: the BCE-1 site necessary for basal CTGF promoter activity in fibroblasts (20) and a Smad site necessary for the TGF-beta response (Ref. 20 and Fig. 5). Accordingly, for further study, we focused on analyzing the contribution of the region of the CTGF promoter between -126 and -77 to the induction of CTGF by TGF-beta 2.


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Fig. 5.   The sequence 5'-GAGGAATG-3' is necessary for the TGF-beta induction of CTGF in fibroblasts. A full-length CTGF promoter (spanning nucleotides -805 to +17)/SEAP reporter construct (-805) or otherwise identical constructs possessing either a scrambled 5'-GAGGAATG motif (5'), a scrambled 3'-GAGGAATG motif (3'), both GAGGAATG motifs scrambled (BOTH), or a mutated Smad motif (SMAD; Ref. 20) were transfected into NIH 3T3 cells. After an 18-h incubation in serum-free media, TGF-beta 2 (25 ng/ml) was added for a further 24 h. All experiments were performed in six-well plates. Cells were cotransfected with a CMV promoter-driven vector encoding beta -galactosidase (0.25 µg) to control for differences in transfection efficiency. Values expressed are averages of adjusted SEAP expression ± S.D. (n = 6). **, statistically significant difference in expression values (p < 0.05) relative to controls.

A Repeat of the Sequence 5'-GAGGAATG-3', Located between -126 and -77 of the CTGF Promoter, Is Necessary for the Fibroblast-specific Induction of CTGF by TGF-beta -- Nucleotides -351 to -77 of the CTGF promoter conferred TGF-beta responsiveness in fibroblasts to the minimal TK promoter (Fig. 4A). However, nucleotides -351 to -126 were not able to confer TGF-beta inducibility to the TK promoter (Fig. 4A). To further characterize the contribution of nucleotides -126 to -77 to TGF-beta induction of the CTGF promoter, we decided to mutate the -351 to -77 CTGF promoter fragment between nucleotides -126 and -77 and assess the ability of the resultant mutant CTGF promoter fragments to confer TGF-beta responsiveness to the TK promoter. The sequence between nucleotides -126 and -77 of the resultant CTGF promoter mutants is shown in Fig. 4B. We found that although three of the new mutant CTGF/TK promoter constructs could still respond to TGF-beta , constructs bearing mutations that disrupted either nucleotides -97 to -92 or -92 to -83 no longer responded to TGF-beta treatment (Fig. 4B, m3 and m4). Interestingly these regions of the CTGF promoter each contain a single copy of the nucleotides 5'-GAGGAATG-3' (Fig. 5, underlined). Mutation of either the 5' or the 3' repeat or both repeats (Fig. 5, 5', 3', and BOTH) in the context of a full-length CTGF promoter (-805 to +17)/SEAP reporter construct abolished the ability of the CTGF promoter to respond to TGF-beta in fibroblasts (Fig. 5). This result was similar to the effect of mutating the Smad element in the context of the -805 construct (Fig. 5, SMAD). Thus both the Smad and the GAGGAATG motifs are necessary for the fibroblast-selective TGF-beta induction of the CTGF promoter.

Nuclear Factors Greatly Enriched in Fibroblasts, Relative to Epithelial Cells, Bind the GAGGAATG TGF-beta Response Element of the CTGF Promoter-- To determine whether proteins from fibroblast nuclear extracts specifically bound the GAGGAATG motifs in the CTGF promoter, we subjected a radiolabeled double-stranded oligomer spanning nucleotides -126 to -77 of the CTGF promoter to gel shift analysis with nuclear extracts prepared from dermal fibroblasts. One protein-DNA complex formed when we combined probe and fibroblast nuclear protein (Fig. 6). This complex was specific as formation of this complex was inhibited by adding a 100-fold molar excess of unlabeled wild-type probe (Fig. 6, wt) but not by otherwise identical oligomers containing mutations in either the 5' or 3' GAGGAATG repeat (Fig. 6, 5' and 3'). Conversely protein from nuclear extracts of MvLu epithelial cells did not appreciably bind our -126 to -77 CTGF promoter probe (Fig. 6). Thus, binding of fibroblast-enriched nuclear factors to the GAGGAATG element of the CTGF promoter seemed to be in part responsible for the fibroblast-specific induction of the CTGF promoter by TGF-beta 2.


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Fig. 6.   Gel shift analysis of the CTGF promoter between nucleotides -126 and -77 reveal greatly enriched binding of fibroblast, relative to epithelia, nuclear proteins. A double-stranded DNA oligomer spanning nucleotides -126 to -77 of the CTGF promoter was end-labeled and subjected to gel shift analysis with nuclear extracts from human foreskin fibroblast (hff) or MvLu cells as indicated (5 µg/lane). Competitors used (200-fold molar excess) were cold wild-type probe (wt) or oligomers identical to the probe but possessing mutations in either the 5'- or 3'-GAGGAATG motifs as indicated.

The precise identity of these fibroblast factors binding the GAGGAATG TGF-beta response element of the CTGF promoter is under investigation; however, it did not escape our notice that the sequence GAGGAATG is a perfect consensus binding motif for the TEF/TEAD family of transcription factors (35-39). To assess whether members of this family could modify the TGF-beta induction of CTGF in fibroblasts, we cotransfected into fibroblasts empty expression vector (EMPTY) or expression vector encoding the ubiquitous, prototypical member of the TEF/TEAD family, TEF-1, along with the full-length (-805 to +17) CTGF promoter/SEAP reporter construct. Overexpression of TEF-1 suppressed basal and TGF-beta -induced CTGF promoter activity (Fig. 7, CTGF-SEAP). Conversely overexpression of TEF-1 had no effect on the TGF-beta induction of SBE-lux (Fig. 7, SBE-lux). It is possible that overexpression of TEF-1 competed for the binding to the CTGF promoter of a TEF/TEAD family member that is required for basal and TGF-beta -induced gene expression. Interestingly this phenomenon of squelching by the overexpression of TEF-1 is a general characteristic of TEF-regulated genes; that is, overexpression of TEF-1 generally suppresses TEF-responsive promoters (38). In the literature, these results have been interpreted by hypothesizing that overexpression of TEF-1 results in the sequestering of a limiting, common cofactor necessary for the ability of TEF family members to induce expression of target promoters (38, 39). Confirming this notion, overexpression of the TEF family cofactor YAP65 (40) markedly attenuated the ability of transfected TEF-1 to suppress the induction of the CTGF promoter by TGF-beta (Fig. 8). Furthermore, overexpression of YAP65 increased basal and TGF-beta -induced CTGF promoter activity, suggesting that the expression of CTGF might be dependent on the YAP65 transcriptional coactivator.


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Fig. 7.   Transfection of expression vector encoding TEF-1 reduces basal and TGF-beta -induced CTGF expression. NIH 3T3 cells were transfected with a full-length CTGF promoter (-805 to +17)/SEAP reporter plasmid or SBE-lux (0.5 µg/well) along with either empty expression vector or expression vector encoding TEF-1 as indicated (1.0 µg/well). After an 18-h incubation in serum-free media, TGF-beta 2 (25 ng/ml) was added for a further 24 h. All experiments were performed in six-well plates. Cells were cotransfected with a CMV promoter-driven vector encoding beta -galactosidase (0.25 µg) to control for differences in transfection efficiency. Values expressed are averages of adjusted SEAP expression ± S.D. (n = 6). **, statistically significant difference in expression values (p < 0.05) relative to controls.


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Fig. 8.   The TEF coactivator YAP65 activates basal and TGF-beta -induced CTGF promoter activity. NIH 3T3 cells were transfected with a full-length CTGF promoter (-805 to +17)/SEAP reporter plasmid (0.5 µg/well) along with either empty expression vector or expression vectors encoding TEF-1 or YAP65 as indicated (0.5 µg each/well). After an 18-h incubation in serum-free media, TGF-beta 2 (25 ng/ml) was added for a further 24 h. All experiments were performed in six-well plates. Cells were cotransfected with a CMV promoter-driven vector encoding beta  galactosidase (0.25 µg) to control for differences in transfection efficiency. Values expressed are averages of adjusted SEAP expression ± S.D. (n = 6). **, statistically significant difference in expression values (p < 0.05) relative to controls.

That overexpression of TEF-1 reduced basal CTGF promoter activity and blocked the TGF-beta induction of CTGF and that factor binding to the consensus TEF binding element, GGAATGG, of the CTGF promoter was greatly enriched in fibroblasts relative to epithelial cells suggests that a fibroblast-enriched member of the TEF/TEAD family of transcription factors, or at least factors that compete with binding of TEF/TEAD family members to this element, may act with the Ras/MEK/ERK cascade and Smads to permit the fibroblast-specific induction of CTGF by TGF-beta 2.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our interest in examining CTGF expression arises out of a desire to block the expression or activity of a downstream mediator of the profibrotic effects of TGF-beta but to leave other effects of TGF-beta intact. CTGF is induced by TGF-beta in fibroblasts and not in keratinocytes (17) and acts with TGF-beta to promote sustained fibrosis (12). Thus, CTGF is likely to be a fibroblast-selective effector of the profibrotic effects of TGF-beta . We believe that understanding the regulation of CTGF in fibroblasts cells should therefore provide novel methods of developing antifibrotic therapeutics and that these therapies would be perhaps more selective than generally blocking TGF-beta signaling (16). Intriguingly a small molecule inhibitor of CTGF expression, namely Iloprost, has recently been shown to alleviate fibrosis in scleroderma patients (14, 15).

In addition to the general Smad pathway, TGF-beta activates other signaling pathways, the nature of which depends on the cell type or target of interest (41). The ability of TGF-beta to specifically activate target genes is considered to be due in part to the interaction of the general, ubiquitous TGF-beta -stimulated Smad signaling pathway with these other signaling pathways. For example, in addition to the effects on Smad phosphorylation, TGF-beta can signal via Ras and Rac proteins and activate certain MAP kinases including the extracellular signal-regulated kinases ERK 1 and 2 and JNK (42-44). In this report, we have found that Ras/MEK/ERK potentiates but that JNK suppresses TGF-beta -induced CTGF gene expression in fibroblasts. Targeting the TGF-beta induction of Ras/MEK/ERK in fibroblasts may be useful in developing novel antifibrotic therapeutics.

The antagonistic nature of Ras/MEK/ERK and JNK in the induction of CTGF is intriguing. The TGF-beta induction of JNK seems to be ubiquitous (45-47). Similarly the TGF-beta induction of Ras/MEK/ERK appears to be ubiquitous, for example, occurring in cells lines known to induce CTGF in response to TGF-beta treatment, including fibroblast and mesangial cells (15, 48), and in cells that do not induce CTGF such as epithelial cells (41). However, Ras/MEK/ERK seems to modulate TGF-beta -dependent transcription in a promoter-specific fashion as blocking this cascade did not affect the ability of TGF-beta to induce expression of the generic Smad-responsive promoter SBE-lux (this report). This interpretation that Ras/MEK/ERK seems not to be directly involved with Smad action is consistent with our recent observations that antagonizing Ras/MEK/ERK does not significantly affect Smad phosphorylation or the ability of Smad 3/4 to activate expression of target promoters such as CTGF (15, 22).

In physiologically relevant promoters, Smads act with other transcription factors, the identity of which depends on the gene of interest, to induce promoter activity. In some promoters, Smads act with Ap-1/c-Jun to induce gene expression; and in these cases, activating JNK/c-Jun enhances expression of target genes (49-53). However, in promoters whose induction does not involve Ap-1, overexpressing c-Jun or JNK or activating JNK inhibits TGF-beta -induced expression (Refs. 54 and 55 and this report). These opposing effects may be due to competition for limiting amounts of Smads (54, 55) as the effect of overexpressing c-Jun on the TGF-beta induction of CTGF can be rescued by overexpressing Smad 3/4 (this report). Thus, the TGF-beta induction of Ras/MEK/ERK seems to be profibrotic, that is, necessary for TGF-beta -induced fibrosis in vivo (15). Conversely activation of JNK would be expected to be antifibrotic as this pathway suppresses the induction of both collagen and CTGF by TGF-beta (Refs. 54 and 55 and this report). Thus which MAP kinase pathway is predominantly active in fibroblasts at a given juncture is likely to have a profound influence on the successful termination of wound healing or on the perpetuation of the fibrotic response. Therefore, pharmacological alteration of MAP kinase pathways in fibroblasts may be useful in developing novel antifibrotic therapies.

In physiologically relevant promoters, Smads are thought not themselves to activate gene expression but are believed to act with basal transcription factors, which vary depending on the gene of interest, to activate gene expression. The finding that a consensus TEF/TEAD binding element was essential for the TGF-beta induction of CTGF is intriguing because to our knowledge these elements have not been implicated in the control of TGF-beta -induced gene expression. The TEF/TEAD family possesses at least four members that are expressed in a tissue- and developmental stage-specific fashion and have been shown to be involved with activation of tissue-specific gene expression (35-39, 56-59). However, recently an additional TEF family member was discovered that was induced in fibroblasts by fibroblast growth factor and serum (60). In addition, splice variants of known TEF family members have been found to have different sequence binding or activation specificity (60, 61), and there may be cell type-specific methods of activating these transcription factors (59). Taken together with our observation that factors present in fibroblast, but not epithelial, nuclear extracts bound a region of the CTGF promoter that was required for the fibroblast-specific induction of CTGF by TGF-beta , these observations raise the intriguing possibility that novel factors of the TEF family might yet be found that are involved with the activation of CTGF expression in fibroblasts.

In conclusion, as far as we are aware this is the first report characterizing a TGF-beta -responsive promoter element that acts in fibroblasts but not in epithelial cells. We have identified a central role for protein kinase C, Ras/MEK/ERK, and a consensus TEF/TEAD binding motif in the fibroblast-selective and promoter-specific induction of CTGF by TGF-beta . Specifically how these components interact to activate CTGF expression awaits further characterization and cloning of the factor binding to the consensus TEF motif in the CTGF promoter. Given the role of CTGF in sustained fibrosis, identifying signaling pathways, trans-acting factors, and cis-acting sequences involved with the TGF-beta -specific induction of CTGF in fibroblasts should continue to suggest new targets around which to develop new, selective antifibrotic strategies.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant AR45879, the Arthritis Research Campaign (UK), the Raynaud's and Scleroderma Association Trust, and the Nightingale Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Fibrogen, Inc., 225 Gateway Blvd., South San Francisco, CA 94080. Tel.: 650-866-7336; Fax: 650-866-7207.

Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M210366200

    ABBREVIATIONS

The abbreviations used are: TGF, transforming growth factor; CTGF, connective tissue growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; JNK, c-Jun NH2-terminal kinase; TEF, transcription enhancer factor; MKK, MAP kinase kinase; TK, thymidine kinase; lux, luciferase; PKC, protein kinase C; CMV, cytomegalovirus; SEAP, secreted enhanced alkaline phosphatase; SBE, Smad binding element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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