Background—Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor β1 (TGF-β1), interleukin 10 (IL-10), and tumour necrosis factor α (TNF-α), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions.
Aim—To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression.
Materials/Methods—Seventy seven human DNA samples were sequenced—45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)—using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit.
Results—A C to G substitution was identified at position −132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position −447. These substitutions at −132 and −447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position −43 (G), −47 (C), −71 (G) and a C to T substitution at position −198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites.
Conclusion—Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.
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The gene encoding connective tissue growth factor (ccn2; ctgf) is an immediate early gene that is crucially involved in various aspects of tissue response to injury, including extracellular matrix production, (myo)fibroblast activation, and proliferation. An imbalance in the regulation of these processes may lead to excessive sclerosis and fibrosis, with subsequent loss of function of the injured organ or tissue. The ccn2 (ctgf) gene is consistently overexpressed in fibrotic disorders.1–10 The CCN2 (CTGF) protein synergises with transforming growth factor β (TGF-β) to induce persistent fibrosis in vivo.11, 12 Thus, dysregulation of ccn2 (ctgf) expression might contribute to the fibrotic phenotype.
TGF-β is one of the most important inducers of ccn2 (ctgf), but other regulatory factors, such as lipophosphatidic acid (LPA), high glucose, tissue factor, thrombin, dexamethasone, and tumour necrosis factor α (TNF-α) have also been identified.13–19 TGF-β induction of ccn2 (ctgf) and its overexpression in the fibrotic disorder scleroderma are mediated through sequences located in the ccn2 (ctgf) promoter.20, 21 Several general transcription factor binding sites have been predicted in the promoter region of the ccn2 (ctgf) gene (such as AP-1, SP-1, and TATA box binding sites). However, the only motifs specifically shown to be involved with ccn2 (ctgf) gene regulation are a unique TGF-β response element and a SMAD binding element.20, 21
For various growth factors and cytokines (for example, TGF-β1, interleukin 10 (IL-10), and TNF-α), polymorphisms within 5′ upstream regulatory sequences have a pronounced effect on transcription, by altering the structure of transcription factor binding sites.22–24 Several of these genotypes have been found to be associated with specific pathological conditions, and thus might provide clues about gene regulation, disease susceptibility, disease severity, and clinical outcome.25
Ischaemic heart disease (IHD) is strongly associated with atherosclerosis. ccn2 (ctgf) is one of the most prominent genes overexpressed in advanced atherosclerotic lesions.26
Here, we report on the DNA analysis of a panel of 32 patients suffering from IHD and 45 healthy controls in search of polymorphic sequences within the 5′ untranslated region of the human ccn2 (ctgf) gene and the relation of the polymorphisms to predicted transcription factor binding sites.
Materials and methods
HUMAN DNA SAMPLES
DNA samples of 45 previously healthy white cardiac transplant donors were obtained by homogenisation of spleen tissue in RPMI-1640 medium (Gibco BRL, Life Technologies, Breda, the Netherlands) and subsequent isolation of mononuclear cells by Ficoll-Paque (Pharmacia, Upsaala, Sweden) differential density centrifugation. Genomic DNA from cell pellets was isolated after proteinase K digestion and ethanol precipitation using the conventional salting out method. DNA was dissolved in 0.1 × TE (10 mM Tris/HCl (pH 7.5), 1 mM EDTA (pH 8.0)).
DNA samples of 32 individuals suffering from ischaemic heart failure were obtained from heparinised blood. Peripheral mononuclear cells were isolated by Ficoll-Paque differential density centrifugation. Genomic DNA was isolated from cell pellets using the salting out method, as described above.
CCN2 (CTGF) PROMOTER POLYMORPHISM ANALYSIS
To assess the occurrence of polymorphic sequences in the ccn2 (ctgf) promoter, −21M13 labelled forward and M13 labelled reverse primers elongated with templates (shown in lower case) for universal sequencing were selected for the amplification of a 600 bp fragment upstream of the transcription start site (−559 to +39). Primer sequences were as follows: −21M13 forward primer, 5′-tgtaa aacgacggccagtCAGGTAGGCATCTTGAG-3′; M13 reverse primer, 5′-caggaaacagctatgacc CACTGGCTGTCTCCTC-3′. Amplification was performed using a PE9600 PCR machine. The PCR conditions were: 94°C for three minutes; then 35 cycles of 94°C for 20 seconds, 56°C for 30 seconds, and 72°C for 10 minutes. PCR products were sequenced using an M13 forward and reverse dye primer cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, CS+ (Perkin Elmer, Nieuwekerk a/d Yssel, The Netherlands). Sequences were compared with the originally published sequence in Genbank (accession number, X9251120).
TRANSFAC DATABASE SEARCH
To identify possible transcription factor binding motifs within the 600 bp ccn2 (ctgf) promoter fragment, we used the software program Matinspector Professional 4.3.27
CCN2 (CTGF) PROMOTER POLYMORPHISMS
We analysed the DNA from a panel of 32 patients suffering from IHD and 45 healthy controls.
A new single nucleotide substitution polymorphism (G to C) was found at position −447. This new −447C allele was found in six of the 45 healthy controls and in five of the 32 patients with IHD. Table 1 shows the calculated genotype frequencies for both populations. The occurrence of the GC genotype in patients with IHD was not significantly different from the controls, as determined by Fisher's exact test (p = 0.367). Polymorphic sequences were submitted to Genbank with the accession number AF316368.
We found a C to G substitution at position −132 in one of the 32 patients with IHD. This patient had the −447G genotype. This C to G substitution at position –132 was not seen in the 45 healthy controls. Because the −132G genotype was only identified in one individual (one of 72), it remains to be determined whether this substitution represents a true new polymorphism or an incidental mutation. This sequence has been submitted to Genbank with the accession number AF316366.
CCN2 (CTGF) PROMOTER SEQUENCE CORRECTIONS ON GENBANK X92511
In all 77 human DNA samples (32 patients with IHD, 45 healthy controls), single nucleotide insertions were found at positions −43 (G insertion), −47 (C insertion), and −71 (G insertion) compared with the original ccn2 (ctgf) promoter sequence. In addition, a C to T substitution was found at position −198 in all DNA samples analysed (fig 1). These corrections have been submitted to Genbank with the accession number AF316367.
TRANSFAC ANALYSIS OF THE CCN2 (CTGF) PROMOTER
TRANSFAC analysis identified several additional (compared with the original description of the ccn2 (ctgf) promoter) potential binding sites for known transcription factors in the 600 bp fragment upstream of the transcription start site (fig 1; table 2).
The previously identified TGF-β responsive element (TβRE; position −156 to −144) and SMAD binding element (SBE; position −174 to −167), both involved in ccn2 (ctgf) gene regulation, are not included in the TRANSFAC database, and were added to the promoter map according to the literature.20, 21
As shown in fig 1, the corrections on the Genbank sequence X92511 at position −43, −47, −71, and −198 are all located outside of the predicted transcription factor binding sites. Therefore, these corrections would not be expected to influence the transcription regulation of the ccn2 (ctgf) gene.
However, the polymorphism found at position −447 (G to C) is close to a 100% consensus myeloid zinc finger protein (MZF1) binding site and the substitution at position −132 (C to G) lies adjacent to a 100% consensus Pbx-1 homeo domain box, which might suggest a potential effect on ccn2 (ctgf) transcription regulation.
The coordinated actions of growth factors and cytokines in the tissue response to injury may lead to optimal repair of the injured organ or tissue. However, an imbalance in the repair process can lead to excessive matrix production and fibroblast proliferation, resulting in progressive sclerosis and fibrosis, and subsequent loss of function. Therefore, differential regulation of the expression of factors involved in the repair process might be crucially important for the outcome of the repair process.
CCN2 (CTGF) is a newly identified factor that is thought to play an important role in repair and fibrosis.1–10 The ccn2 (ctgf) gene is overexpressed in a variety of fibrotic disorders, such as advanced atherosclerotic lesions,26 and synergises with TGF-β to produce prolonged fibrosis in vivo.11, 12 Thus, the regulation of ccn2 (ctgf) expression might be crucially important for optimal regulation of tissue response to injury.
Polymorphisms in the promoter region of genes might have a pronounced effect on the transcription and expression of the corresponding gene. Different genotypes may result in interindividual variation in transcription and expression. In this way, genetic variations may have a considerable impact on the susceptibility, severity, and clinical outcome of disease.25 We investigated the occurrence of polymorphic sites in the promoter region of ccn2 (ctgf) and their frequency distribution in healthy controls and patients with IHD.
A new polymorphism was found at position −447 in six of 45 healthy controls and in five of 32 patients with IHD. This polymorphism (G to C) is close to a binding site for MZF1, which acts as a bifunctional transcription regulator depending on the cellular environment. MZF1 is essential for human granulopoiesis and has been shown to increase the growth rate of NIH3T3 cells, when transfected into these fibroblasts. Moreover, these MZF1 transfected cells were able to develop into fibrosarcomas when injected into athymic mice.28 In vitro, MZF1 transfected NIH3T3 cells lost contact inhibition and were capable of anchorage independent growth. In normal rat kidney fibroblasts (NRK), ccn2 (ctgf) expression is essential for anchorage independent growth induced by TGF-β.29 In addition, the epidermal growth factor induced proliferation of NIH3T3 fibroblasts was significantly increased by costimulation with CCN2 (CTGF). When injected into wound chambers in vivo, CCN2 (CTGF) stimulated fibroblast proliferation and granulation tissue formation.30 These observations would be compatible with a role for MZF1 in ccn2 (ctgf) gene regulation. Therefore, polymorphisms located in close proximity to the MZF1 site might greatly influence MZF1 regulated ccn2 (ctgf) expression. According to TRANSFAC analysis, the observed G to C substitution results in a slight reduction of matrix similarity from 0.987 to 0.980. Whether MZF1 is involved in ccn2 (ctgf) regulation and whether the polymorphism at position −447 has an effect on the transcriptional response is currently under investigation.
The C to G substitution at position -132 in the ccn2 (ctgf) promoter, identified in one of the patients with IHD, lies adjacent to a Pbx-1 homeo domain box. Heterodimers between Pbx and Hox genes regulate the expression of genes involved in anterior–posterior patterning and development.31 The ccn2 (ctgf) gene is expressed in embryonic development.32 It is distantly related to the twisted gastrulation gene, which is required for the specification of dorsal midline fates.33 There are no studies to date that link ccn2 (ctgf) regulation with Hox gene expression. The role of the putative Pbx-1 site and the possible relevance of a substitution mutation at position −132 (resulting in a slight reduction in matrix similarity from 0.821 to 0.819) in ccn2 (ctgf) expression and development remain to be investigated.
To date, the only transcription factors known to play a role in ccn2 (ctgf) gene expression are a unique TGF-β cis regulatory element and a binding site for SMAD3 and SMAD4, general factors known to be necessary for the TGF-β mediated induction of genes.20, 21 To date, no polymorphisms have been identified that are related to these sequences.
In addition to the polymorphisms at positions −447 and −132, we have identified insertions of single nucleotides at positions −43, −47, and −71 and a substitution at position −198 in all 77 human DNA samples tested. These insertions and substitution are not located near to predicted transcription factor binding sites, and thus are not expected to be relevant for regulation of ccn2 (ctgf) transcription.
In conclusion, we have corrected the published sequence of the promoter of the ccn2 (ctgf) gene and identified polymorphisms in close proximity to the MZF1 and Pbx-1 binding sites. It remains to be investigated whether these polymorphisms influence transcription regulation. Preliminary screening of patients with IHD did not reveal a significant association with any of the genotypes. Future in vitro studies and analysis of other patient populations should reveal whether ccn2 (ctgf) promoter polymorphisms might contribute to disease susceptibility and clinical outcome.
Thanks to A Leask (FibroGen Inc) for helpful discussion and critical reading of the manuscript and B Westland for technical assistance. This project was sponsored by a grant from the Dutch Kidney Foundation, nr 96–1545.
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