GENOTYPIC VARIATION IN THE TRANSFORMING GROWTH FACTOR-β1... : Transplantation (original) (raw)
* Abbreviations: CF, cystic fibrosis; PCR, polymerase chain reaction; SSCP, single-stranded conformational polymorphism; TGF, transforming growth factor.
Transforming growth factor (TGF*)-β1 is a multifunctional cytokine that controls the proliferation and differentiation of many cell types (1-4). This cytokine is also involved in the production and degradation of the extracellular matrix in three ways. TGF-β1 activates gene transcription (5), thereby increasing synthesis and secretion of collagen and other matrix proteins (6). Furthermore, it decreases the synthesis of proteolytic enzymes such as collagenase (7), which degrade matrix proteins, and increases the synthesis of protease inhibitors such as plasminogen activator inhibitor, which block the activity of the proteolytic enzymes (8). TGF-β1 also increases transcription, translation, and processing of cellular receptors for matrix proteins (9). Consequently, this cytokine is thought to play a critical role in fibrotic conditions and in the process of graft damage, particularly in the transplanted lung (10).
The active form of TGF-β1 is a 25-kDa disulfide-linked dimer of two identical chains of 112 amino acids, synthesized in a latent form as a protein containing 390 amino acids (11,12). Many cells (including T lymphocytes, monocytes, endothelial cells, fibroblasts, and others) secrete this cytokine, but the main source in normal circumstances is platelets, from which it is released in α-granules (13,14). The human gene encoding TGF-β1 is located on chromosome 19q13 (15), and the promoter region of this cytokine has been characterized by Kim et al. (16). All positions are defined relative to the first major transcription start site (position +1). The first +840 bases are a nontranslated region and codon one begins at position +841 (16).
We have observed interindividual variation in TGF-β1 production in vitro. Our hypothesis is that these differences in the induced expression and production of TGF-β1 are a consequence of genetic sequence polymorphism in the regulatory regions of the TGF-β1 gene.
In this study, our aim was to identify polymorphisms in the promoter region of the TGF-β1 gene and in the 5′ end of the signaling sequence, which might result in observed quantitative variations in the TGF-β1 production. Further, we have attempted to assess whether these polymorphisms are associated with the severity of fibrosis in patients secondary to lung diseases predisposing to lung transplantation, such as cystic fibrosis (CF), cryptogenic fibrosing alveolitis, bronchiectasis, and obliterative bronchiolitis, and with the development of graft fibrosis (as defined by biopsy) after lung transplantation.
MATERIALS AND METHODS
Study subjects. Anticoagulated venous blood samples (5 ml) were collected from two groups using the Vacutainer system (Becton Dickinson, Mountain View, CA) and wide bore needles according to NBA recommendations. One group was a healthy control group of 107 individuals with no recent infection or recent history of treatment with immunosuppressive drugs. The experimental group was a cohort of 95 patients from the Thoracic Transplant Unit at Wythenshawe Hospital who had undergone lung transplantation between January 1990 and August 1996.
DNA extraction and amplification. DNA was prepared from whole blood using a phenol precipitation method (17) after digestion with proteinase K (Boehringer Mannheim, Mannheim, Germany). Specific oligonucleotide primers were designed based on the published TGF-β1 sequence (16,18). Using specifically designed pairs of primers, overlapping fragments that covered a segment between position -1321 upstream of the first major transcription starting point and +966 (codon 42) in the first exon were amplified using the polymerase chain reaction (PCR). A Perkin Elmer 9600 thermal cycler (Applied Biosystems, Foster City, CA) was used for PCR amplification in 30-µl reaction mixtures containing: 50 mM KCl; 10 mM Tris-HCl; 0.1% Triton X100; 200 µM each dATP, dGTP, dTTP, and dCTP (Life Technologies, Gaithersburg, MD); 1.5 mM MgCl2; 0.4 M Betaine (Sigma Chemical Co., Poole, UK); 0.5 µM each primer; DNA; and 1 U of Taq polymerase (Life Technologies).
Detection and characterization of TGF-β1 gene polymorphism. Initially, 30 samples were screened using a single-stranded conformational polymorphism (SSCP) method to detect mutations (19). Specific restriction enzyme digestion was performed to cleave PCR products that were fragments larger than 300 bp. The resulting products were placed on ice after heat denaturation of the DNA at 95°C for 5 min with loading buffer (95% formamide, EDTA, bromophenol blue, xylene cyanol). Samples were loaded onto a nondenaturing 8% (49:1) acrylamide:bisacrylamide gel using a model SA-32 vertical gel system (Life Technologies). Bands were visualized using a standard silver staining technique. An ABI PRISM Dye Deoxy Terminator Cycle Sequencing Kit and an ABI 373 A DNA Sequencer were used to sequence complete DNA fragment, which existent polymorphisms detected by SSCP.
Screening of TGF-β1 gene polymorphism. Two biotinylated oligonucleotide probes (Table 1) were designed to detect each polymorphism by a dot-blot hybridization technique, sequence-specific oligonucleotide probing. Specifically amplified PCR products that encompassed the polymorphisms were blotted onto Hybond N+ nylon membranes (Amersham, Buckinghamshire, UK). The membranes were placed in denaturing solution (0.5 M NaOH and 1.5 M NaCl) for 5 min and then in neutralizing solution (1.5 M NaCl and 0.5 M Tris) for 1 min. After rapid drying at 80°C for 10 min, the DNA samples were immobilized onto the nylon surface by cross-linking with ultraviolet light using a Stratagene Stratalinker (Stratagene, La Jolla, CA). The membranes were incubated with hybridization buffer for 30 min at 42.5°C, and then 200 ng of the specific probe (Table 1) was added and allowed by hybridize for 2 hr at 42.5°C. The membranes were washed twice at room temperature in 5× SSC (0.75 M NaCl and 0.075 M sodium citrate) with 0.1% sodium dodecyl sulfate for 5 min. To remove nonspecifically bound probes, the stringency washing was performed twice for 15 min using 1× SSC containing 0.1% sodium dodecyl sulfate. Finally, to visualize the hybridized probes, membranes were treated with 0.5% blocking solution (Marvel, Premier), incubated with streptavidin-conjugated horseradish peroxidase (Amersham), and detected using a commercial chemiluminescent method (ECL system; Amersham). The membranes were then placed on x-ray films (Fuji), which were subsequently developed, and the resulting blots were analyzed.
Primers and probes used for analysis of the TGF-β1 gene
Cell culture. To examine TGF-β1 production in vitro, peripheral blood leukocytes from healthy individuals (n=34) were isolated by centrifugation over Histopaque 1083 (Sigma) and suspended in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal calf serum (Sigma), 1% nonessential amino acids, 1% sodium pyruvate, 1% L-glutamine, 1% HEPES buffer solution, and 1% penicillin-streptomycin mixture (all from Life Technologies). Leukocytes were cultured at 106 cells/ml and stimulated with a mixture of two mitogens of 2 µg/ml phytohemagglutinin-P and 50 ng/ml phorbol myristate acetate, at 37°C with 5% CO2. TGF-β1 is detectable from day 2 onward. Culture supernatants were harvested on day 5, when TGF-β1 levels were maximal in all cultures, and stored at -20°C. Total TGF-β1 concentrations were measured using a specific ELISA (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The results were calculated by reference to a standard curve and expressed as picogram per milliliter. The production of TGF-β1 by cells from different individuals was reproducible when tested on more than one occasion.
Histology. Ninety-five sections from explanted lungs were reviewed to verify the pretransplant histology. Seven hundred and eighty transbronchial biopsies from all patients were studied subsequently. Paraffin-embedded sections of lung, stained with hematoxylin and eosin, were systematically scanned in a microscope using a ×10 objective. Slides were examined by two independent observers. Each successive field was individually assessed for severity of interstitial fibrosis and allotted a score between 0 and 8 using a predetermined scale of severity according to the method of Ashcroft (20). After examining the whole section, a mean score of all the fields was taken as the fibrosis score. For the present analysis, specimens were divided into those that did not show fibrosis at all (fibrosis score=0) and those that did (fibrosis score >0).
Statistical analysis. Students' t test was used to analyze the difference in the TGF-β1 production. The probability value of differences between controls and clinical groups was calculated by the chi-square test.
RESULTS
Detection of polymorphism. Five polymorphisms were identified in the TGF-β1 gene between position -1321 and +966 (codon 42) relative to the first major transcription start site (Fig. 1). Analysis of the DNA sequence from 34 randomly selected healthy individuals revealed five different patterns by SSCP. These variants were sequenced to ascertain the position and nature of the putative polymorphisms. The following polymorphisms were identified (Fig. 1): two of the polymorphisms were single base substitutions located in the upstream region of the gene at positions -800 (G→ A) and -509 (C→ T) from the first transcribed nucleotide; an insertion of a cytosine base was revealed in the nontranslated region at position +72; and two single base substitutions were found in the first exon at positions +869 (T→C) and +915 (G→ C). In these latter polymorphisms, changes in nucleotide bases affected the amino acid coding sequence, which resulted in changes in amino acid residues. The polymorphism at position +869 represents a change from the amino acid leucine to proline in codon 10, whereas the polymorphism at position +915 causes an arginine to proline substitution in codon 25.
Position of polymorphisms in the TGF-β1 gene. The TGF-β1 gene was sequenced from position -1321 through +966, relative to the first major transcription start point (TSP1). Allelic substitutions were found at positions -800 (G/A), -509 (C/T), +869 (codon 10, T/C), and +915 (codon 25, G/C). An allelic insertion was found at position +72. The shaded region of exon 1 encodes the signal sequence (codons 1 to 29) of the translated TGF-β1 protein.
Genotype distribution and frequency. Four putative haplotypes could be deduced from the normal control data. These and their frequencies are presented in Table 2. Insertion of a cytosine at position +72 segregates with a proline residue at codon 25. In addition, cytosine at position -509 is strongly associated with leucine at codon 10, except where there is an insertion at position +72 as observed in two individuals having a haplotype herein named haplotype 4 (Table 2). Polymorphisms at position -800 seem to segregate independently of the other polymorphisms.
Frequencies of four putative haplotypes in 104 controls
The allele distribution and frequencies of these polymorphisms, studied using single-specific oligonucleotide probe analysis in a control group and patient populations, are shown in Tables 3 and 4, whereas the patterns of genotype inheritance (homozygosity and heterozygosity) are shown in Table 5.
Distribution of TGF-β1 alleles in pretransplant lung fibrosis in pulmonary transplant recipients_a_
Distribution of TGF-β1 alleles in lung allograft fibrosis and no allograft fibrosis_a_
Distribution of TGF-β1 genotypes in controls and in patients with or without fibrotic disease, and in lung transplant recipients who developed fibrosis in their transplants
TGF-β1 genetic polymorphisms and TGF-β1 production. The TGF-β1 gene polymorphism at codon 25 is significantly associated (P<0.02) with TGF-β1 production in vitro. TGF-β1 production by peripheral blood leukocytes from 34 healthy individuals was stimulated in vitro with phytohemagglutinin and phorbol myristate acetate, and TGF-β1 protein production was measured by ELISA. Individuals with the arginine/arginine (codon 25) homozygous genotype (n=25) had a mean production of 10037±745 pg/ml, whereas the arginine/proline genotype (n=9) gave a mean value of 6729±883 pg/ml. Proline/proline homozygous genotypes are rare and were not tested. An alternative interpretation, given the complete linkage disequilibrium between the cytosine insertion at position +72 and arginine at codon 25, is that low TGF-β1 production is equally significantly associated with the polymorphism at position +72. There were no significant relationships between any of the other identified TGF-β1 polymorphisms and TGF-β1 production as defined in our in vitro assay.
TGF-β1 gene polymorphisms are associated with pretransplant lung fibrosis. Patients were analyzed in two groups, those who had histologically proven fibrosis in their lungs before transplantation and those who did not. All patients with pretransplant fibrosis carried the high TGF-β1 producing allele, codon 25 arginine (+72 C-) allele, compared with controls (P<0.02) (Table 3); only one patient was not homozygous (Table 5). Furthermore, the difference in genotype at codon 25 (+72) between patients with and without pretransplant fibrosis was more marked (P<0.004; Table 3). In addition, the genotype encoding leucine at codon 10 was associated with pretransplant fibrosis (P<0.005; Table 3). In the CF patients with lung fibrosis, all were homozygous for arginine at codon 25 (P<0.01; Table 3), and the frequency of leucine at codon 10 was increased (P<0.01) compared with controls (Table 3). Also, all CF patients were homozygous for the allele having a G at position -800 (P<0.01) compared with controls (Table 3).
TGF-β1 gene polymorphisms are associated with development of fibrosis in the transplanted lungs. After lung transplantation, some grafts undergo fibrosis. The development of fibrosis in the transplanted lung is associated (P<0.03; Table 4) with the codon 25 arginine (+72 C-) and with homozygosity for this allele (P<0.03; Table 4). This difference is more marked when patients with fibrotic and nonfibrotic grafts are compared (P<0.01; Table 4). On the other hand, the G allele at position -800 is associated both with patients who developed fibrosis (P<0.03) and those who do not develop fibrosis (P<0.04) when they are compared with the control group.
DISCUSSION
The TGF-β1 gene is polymorphic. This study has identified polymorphisms in the TGF-β1 gene that influence TGF-β1 production in vitro and are associated with lung fibrosis as an indication for lung transplantation and the development of lung allograft fibrosis. We have analyzed a region of the TGF-β1 gene from position -1321 to +966 (codon 42) in the first exon. Our study independently confirms, using a different methodological approach, the presence of polymorphisms in the TGF-β1 gene as reported recently by Cambien et al. (21).
In previous studies, we have elucidated and analyzed polymorphisms in the promoter regions of inflammatory and anti-inflammatory cytokine genes and have shown or confirmed the correlation between differences in gene sequence and phenotypic expression (22,23). In addition, we have shown associations between these cytokine genotypes, in vitro phenotype, and clinical presentation in solid organ transplantation. In this article, we sought similar associations with TGF-β1 phenotype and genotype in lung transplant recipients.
Of the five polymorphic sites identified in the TGF-β1 gene (Fig. 1), two are within the 29-amino acid signal sequence. Signal sequences regulate posttranslational protein synthesis and are usually 15-25 amino acids long comprising three regions: a short positively charged N terminal; a central strongly hydrophobic core typically 8-12 residues long; and a more polar region, defining the cleavage site (24,25). The hydrophobic core is responsible for transmembrane transport of the protein.
TGF-β1 gene polymorphism is associated with TGF-β1 production. The polymorphism at codon 25 was significantly correlated (P<0.02) with TGF-β1 production in vitro. This polymorphism occurs within the peptide signal sequence that is cleaved from the active TGF-β1 protein at codon 29 (26). The proline/arginine substitution at codon 25 corresponds to an exchange of a small, neutral residue for a charged residue that could have a direct effect on the adjacent cleavage site. Protein transcription and/or translation might also be affected as a result of the predicted change in folding of the amino acid chain. Alternatively, the proline/arginine substitution at codon 25 is in linkage with an insertion (+C) at position +72, a site that may form a stem loop configuration in the DNA and that may regulate gene activity (27).
The polymorphism at codon 10, within the hydrophobic α-helix of the signal sequence, involves the replacement of a hydrophobic leucine residue with a small, neutral proline residue. Such a change would alter the overall hydrophobicity of the core transport sequence and disrupt the α-helical structure of the region, thereby altering its ability to direct protein transport across the endoplasmic reticulum.
TGF-β1 gene polymorphisms are associated with fibrotic lung pathology. In this study, we observed significant associations between TGF-β1 genotype and the development of fibrotic lung pathology. Because TGF-β1 is implicated in fibrosis, the five polymorphisms identified were tested as potential genetic markers for the observed intraindividual differences in fibrotic lung pathology in 95 lung allograft recipients after transplantation. The presence of allograft fibrosis was defined as a histological score in transbronchial biopsy. In the posttransplant group (Table 4), a significant correlation (P<0.01) was observed between the presence of arginine at codon 25/insertion (C) at position +72 and the development of fibrosis. Hence, the high TGF-β1 genotype is associated with lung fibrosis, as predicted by our hypothesis.
Another significant association was seen when we analyzed the presence of fibrosis in recipients with different primary diseases leading to the requirement for transplant (Tables 3 and 5). In patients with a fibrotic pathology predisposing to lung transplantation (including those with a primary diagnosis of CF, cryptogenic fibrosing alveolitis, obliterative bronchiolitis, and bronchiectasis who went on to develop fibrosis in the native lung) (n=45), there was a significant association with the presence of a leucine residue at codon 10 compared with normal controls (P<0.02) or with patients without lung fibrosis (P<0.004).
Significance of TGF-β1 genotypes. The precise role of TGF-β1 polymorphisms and their significance in the progression of primary lung disease toward fibrosis and in the development of speculation. In the posttransplant situation, individuals with a genetically determined propensity to produce greater amounts of the free TGF-β1 protein might be more prone to fibrotic complications due to the net increased concentration of this fibrogenic cytokine. In those fibrotic diseases resulting in a chronic decline in lung function, the requirement for a transplant might be exacerbated by an enhanced chronic release of TGF-β1. In this scenario, those individuals with a leucine residue at codon 10 could be identified as a group in which disease progression (at least in CF), and consequently the need for a lung transplant, may be accelerated. Indeed, we have direct evidence for an association between leucine at codon 10, elevated circulation levels of TGF-β1 and reduced lung function, and forced expiratory volume 1 in patients with CF. The TGF-β1 genotype in unselected CF patients shows a normal distribution, and yet all of the CF patients requiring lung transplantation are of the high-producer TGF-β1 genotype (Abdalla HM, Webb AK, and Hutchinson IV, unpublished data). Recently, Suthanthiran's laboratory has shown that TGF-β1 plays a role in progressive fibrosis in kidney patients (28).
We conclude that knowledge of TGF-β1 polymorphisms may have prognostic significance in a wider range of fibrotic/sclerotic conditions and may lead to the development of new treatment strategies.
REFERENCES
1. Sporn M, Roberts A, Wakefield M, Assoian R. Transforming growth factor-β biological function and chemical structure. Science 1986; 233: 532.
2. Anzano M, Roberts A, Sporn M. Anchorage-independent growth of primary rat embryo cells is induced by platelet-derived growth factor and inhibited by type-beta transforming growth factor. J Cell Physiol 1986; 126: 321.
3. Kehrl J, Wakfield K, Roberts A, et al. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986; 163: 1037.
4. Kehrl J, Taylor A, Delsing G. Further studies of the role of TGF-β in human B cell function. J Immunol 1989; 143: 1868.
5. Ignotz R, Endo T, Massague J. Regulation of fibronectin and type I collagen mRNA level by transforming growth factor-β. J Biol Chem 1987; 262: 6443.
6. Ignotz R, Massague J. Transforming growth factor-β stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 1986; 261: 4337.
7. Edwards D, Murphy G, Reynolds J, et al. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987; 6: 1899.
8. Laiho M, Saksela O, Keski-Oja J. Transforming growth factor-β induction of type-1 plasminogen activator inhibitor. J Biol Chem 1987; 262: 17467.
9. Sporn M, Roberts A. The transforming growth factor-βs. In: Sporn MB, Roberts AB, eds. Peptide growth factors and their receptors I. New York: Springer-Verlag, 1991: 419.
10. Vaillant P, Menard O, Vignaud J-M, Martinet N, Martinet Y. The role of cytokines in human lung fibrosis. Monaldi Arch Chest Dis 1996; 51: 145.
11. Nicola NA. Transforming growth factor-β1. In: Gitelman SE, Derynck R, eds. Guidebook to cytokines and their receptors. Oxford: University Press, 1995: 223.
12. Kanzaki T, Olofesson A, Moren A, Wernstedt C, Hellman U. TGFβ1 binding protein: a component of the latent complex of TGFβ1 with multiple repeat sequences. Cell 1990; 61: 1051.
13. Assoian R, Komoriya A, Meyers C, Miller D, Sporn M. Transforming growth factor β in human platelets. J Biol Chem 1983; 258: 7155.
14. Meager A. Assays for transforming growth factor β. J Immunol Med 1991; 141: 1.
15. Fujii D, Brissenden J, Derynck R, Franke U. Transforming growth factor-β gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat Cell Mol Genet 1986; 12: 281.
16. Kim S, Glick A, Sporn M, Roberts A. Characterization of the promoter region of the human transforming growth factor-β1 Gene. J Biol Chem 1989; 264: 402.
17. Kunle L, Smith K, Boyer S, et al. Analysis of Y chromosome specific reiterated DNA in chromosomal variants. Proc Natl Acad Sci USA 1977; 74: 1245.
18. Derynck R, Rhee L, Chen EY, Tilburg AV. Intron-exon structure of the human transforming growth factor-β precursor gene. Nucleic Acids Res 1987; 15: 3188.
19. Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5: 874.
20. Ashcroft T, Simpson J, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988; 41: 467.
21. Cambien F, Ricard S, Troesch A, et al. Polymorphisms of the transforming growth factor-β1 gene in relation to myocardial infarction and blood pressure. Hypertension 1996; 28: 881.
22. Turner D, Williams D, Sankaran D, Lazarus M, Sinnot PJ, Hutchinson IV. An investigation of polymorphism in the interleukin-10 gene promoter. Euro J Immunogenet 1997; 24: 1.
23. Wilson A, Symons J, McDowell T, McDevitt H, Duff G. Effects of a polymorphism in the human tumor necrosis factor α promoter on transcriptional activation. Proc Natl Acad Sci USA 1997; 49: 3195.
24. Heijne GV. Structural and thermodynamic aspects of the transfer of proteins into and across membranes. In: Current topic in membranes and transport, Vol. 24. San Diego, CA: Academic Press, 1985: 151.
25. Walter P, Johnson A. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 1994; 10: 87.
26. Miyazono K, Hellman U, Wernstedt C, Heldin C-H. Latent high molecular weight complex of transforming growth factor-β. J Biol Chem 1988; 263: 6407.
27. Kim SJ, Park K, Rudkin BB, Dey BR, Sporn MB, Roberts AB. Nerve growth factor induces transcription of transforming growth factor-β1 through a specific promoter element in PC12 cells. J Biol Chem 1994; 269: 3739.
28. Suthanthiran M. Clinical application of molecular biology: a study of allograft rejection. Am J Med Sci 1997; 313: 264.
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