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Published ahead of print on July 13, 2006, doi:10.1164/rccm.200512-1839OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 915-922, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200512-1839OC

Original Article

Functional Prostaglandin-Endoperoxide Synthase 2 Polymorphism Predicts Poor Outcome in Sarcoidosis

Michael R. Hill, Anastasia Papafili, Helen Booth, Phillippa Lawson, Marianne Hubner, Huw Beynon, Catherine Read, Gisela Lindahl, Richard P. Marshall, Robin J. McAnulty* and Geoffrey J. Laurent*

Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, The Rayne Institute; Department of Rheumatology, Royal Free Hospital, London, United Kingdom; and Wilhelminenspital, Vienna, Austria

Correspondence and requests for reprints should be addressed to Michael R. Hill, D.Phil., Centre for Respiratory Research, Department of Medicine, Royal Free and University College Medical School, The Rayne Institute, 5 University Street, London WC1E 6JJ, UK. E-mail: michael.hill{at}ucl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: The majority of patients with sarcoidosis resolve their condition; however 5–10% of patients with sarcoidosis develop pulmonary fibrosis with poor prognosis. Prostaglandin-endoperoxide synthase 2 (PTGS2) is a key regulatory enzyme in the synthesis of the antifibrotic agent prostaglandin E2 and is reduced in sarcoidosis lung. A promoter polymorphism in PTGS2, –765G>C, is reported to reduce its expression.

Objectives: To investigate if –765G>C is associated with susceptibility to, and poorer outcome within, sarcoidosis and to examine a possible mechanism by which –765G>C reduces PTGS2 expression.

Methods: We used a case-control design study and genotyped –765G>C in a white British population of 198 patients with sarcoidosis and 166 control subjects. Patients with sarcoidosis were classified before genotyping as having persistent or nonpersistent disease using clinical criteria that included chest radiography staging, need for treatment, lung function, and longitudinal follow-up. Electrophoretic mobility shift assays were used to identify changes in transcription factor binding caused by the –765G>C polymorphism.

Results: Carriage of the –765C allele was strongly associated with susceptibility to sarcoidosis (odds ratio, 2.50; 95% confidence interval, 1.51–4.13; p = 0.006) and, within this disease, with poorer outcome (odds ratio, 3.11; 95% confidence interval, 1.35–7.13; p = 0.008). The association with sarcoidosis was replicated in a second Austrian population. Electrophoretic mobility shift assays revealed that the –765C allele causes a loss of Sp1/Sp3 transcription factor binding and an increase in Egr-1 binding to the region.

Conclusion: Our data suggest that the –765G>C polymorphism identifies individuals who are susceptible to sarcoidosis and, more importantly, at risk of pulmonary fibrotic disease. An altered Sp1/Sp3 binding to the –765 region may contribute to the mechanism by which –765G>C reduces PTGS2 expression.

Key Words: cyclooxygenase-2 • gene regulation • PTGS2 • sarcoidosis

Sarcoidosis is a multisystem disease characterized by chronic inflammation and granuloma formation. Sarcoidosis affects up to 40 per 100,000 individuals each year, with lung involvement in almost all cases (1–4). The cause and mechanisms behind sarcoidosis remain unclear; however, genetic susceptibility is recognized as a major factor (5). The extent, functional impact, and prognosis of lung involvement is variable, ranging from a self-limiting infiltrate that resolves spontaneously or persists to chronic progressive fibrosis, which occurs in 5–10% of patients. There are no means to identify individuals at greater risk of poorer outcome; however, emerging evidence suggests that a reduced expression of prostaglandin-endoperoxide synthase 2 (PTGS2) or cyclooxygenase-2 in fibrotic lung diseases, including sarcoidosis, removes an important inhibitory control that may limit progressive fibrotic lung disease (6–9).

PTGS2 is an inducible isoform of prostaglandin-endoperoxide synthase (also known as cyclooxygenase) involved in the enzymatic conversion of arachidonic acid to prostanoids. These prostanoids provide an important homeostatic control in normal tissues and regulate inflammation (10). Prostaglandin E2 (PGE2) is the predominant prostanoid produced in the normal lung after up-regulation of PTGS2 with concentrations up to 50 times greater than those of other PTGS products (11). It is also the main prostanoid produced by lung fibroblasts and exerts bronchoprotective effects by inhibiting their proliferation, differentiation into myofibroblasts, and collagen synthesis (6, 12–14). However, in pulmonary fibrosis, PGE2 levels are reduced by as much as 50% in the lung (15), despite increased levels of mediators known to induce its synthesis. We and others have shown that fibroblasts derived from the lungs of patients with pulmonary fibrosis fail to up-regulate their production of PGE2 in response to inflammatory and profibrotic mediators such as interleukin (IL)-1, tumor necrosis factor–{alpha}, and transforming growth factor (TGF)-beta (6–8). This is due to a decreased capacity to induce PTGS2 expression (6, 7) and not to differences in PTGS1 expression, altered release of the substrate arachidonic acid, or steps in the prostanoids' pathway downstream of PTGS2 (6, 7). These cells exhibit normal response to exogenous PGE2, suggesting that they have normal PGE2 receptor expression and signaling (6, 7). PTGS2-deficient mice exhibit an enhanced fibrotic response in the lung in response to fibrotic agents, including bleomycin (6, 16). Furthermore, the severity of injury in these mice seems to be dependent on reduced PGE2 production (17).

We considered that dysregulation of PTGS2 expression may be due to polymorphisms within the gene and have recently described a novel promoter polymorphism, –765G>C (18). The –765C allele has 30% lower promoter activity compared with the –765G allele in reporter gene assays, and, in a systemic inflammatory response, the magnitude of rise in levels of C-reactive protein (CRP) after coronary artery bypass surgery was strongly genotype dependent, with carriers of the –765C allele having significantly lower CRP levels (18). Furthermore, it has recently been shown that the –765C allele is protective against myocardial infarction and stroke (19), suggesting that –765G>C has an important role in chronic inflammation. Based on these studies, we hypothesized that the –765C allele would be involved with fibrotic lung disease in sarcoidosis. In this study, we have investigated whether the –765G>C polymorphism is involved with susceptibility and poorer outcome in sarcoidosis. A possible mechanism by which –765G>C reduces PTGS2 expression is also reported. Some of the results of these studies have been previously reported in the form of abstracts (20, 21).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
All the United Kingdom subjects in this study were white British, and all the Austrian subjects were white Austrian. Sarcoidosis was diagnosed using guidelines from the consensus statement on sarcoidosis, including the patient's history and radiologic and clinical features, which were consistent with the disease (1). United Kingdom patients with pulmonary sarcoidosis (n = 198; 119 female, 79 male; mean age, 39.5 ± 11.5 yr) were recruited from the University College London Hospitals, Royal Free, and St. Georges Hospitals (London, UK). Healthy United Kingdom control subjects (n = 166; 77 female, 89 male; mean age 41.4 ± 10.9 yr) were obtained through the blood transfusion service based in London (National Blood Service, London, UK). Austrian patients with pulmonary sarcoidosis (n = 76; 46 female, 30 male; mean age 43.5 ± 11.9 yr) were recruited from the Wilhelminenspital, Vienna, Austria. Healthy Austrian control subjects (n = 130; 75 female, 55 male; mean age 44.3 ± 18.9 yr) were obtained from the blood transfusion service Wilhelminenspital, Vienna. None of the patients with sarcoidosis participating in this study were receiving prescribed nonsteroidal antiinflammatory medication. The studies had hospital ethics committee approval, and written informed consent was obtained from all participants.

An easily applied classification system was developed at the initiation of the project to allow patients to be separated into those with nonpersistent pulmonary sarcoidosis and those with persistent pulmonary sarcoidosis (Table 1). Chest radiographs were assessed blind to the genotype data by a consultant radiologist, and standard radiographic staging was applied (1). Patients with stage IV appearances with volume loss, lung distortion, and/or fibrocystic disease were placed in the persistent disease group. The remainder were assessed at least 2 yr after diagnosis. Those with grade II or III appearances who had required immunosuppressant therapy for symptomatic lung disease and had abnormal lung function as defined by FVC and/or total lung capacity (TLC) and/or transfer factor (diffusing capacity of carbon monoxide [DLCO]) < 80% of the predicted values were also classified as having persistent pulmonary sarcoidosis. Subjects with stage II or III appearances that did not fulfill these requirements and subjects with stage 0 or I appearances at least 2 yr from diagnosis were classified as having nonpersistent pulmonary sarcoidosis.


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  		TABLE 1. CRITERIA USED TO SUBCLASSIFY PATIENTS WITH PULMONARY SARCOIDOSIS INTO PERSISTENT AND NONPERSISTENT DISEASE CATEGORIES

 
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes by standard phenol/chloroform techniques (22). The –765G>C polymorphism was genotyped as previously described (18).

Functional Studies
The methods used for measuring PGE2 synthesis in lung fibroblast cell lines (6) and the electrophoretic mobility shift assays (EMSA) studies have been reported (23, 24). Additional detail is provided in the online supplement.

Statistical Analysis
Statistical analysis was performed using SPSS version 11.5 (SPSS, Inc., Chicago, IL), and p <= 0.05 was considered as significant. {chi}2 Tables were used to compare the observed numbers of each genotype with those expected for a population in Hardy-Weinberg equilibrium and to compare genotype frequencies between the patient and the control groups. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from binary logistic regression analysis and adjusted for age and sex when comparing patient and control groups or for age alone when the sexes were considered separately. When comparing nonpersistent and persistent disease within sarcoidosis, the ORs were adjusted for duration of disease, corticosteroid use, and smoking status. Geometric means ± SD have been quoted where age, lung function, and duration of disease parameters are shown. Student's t test was used to compare the means of age between control subjects and patients with sarcoidosis and the mean duration of disease between United Kingdom patients with sarcoidosis and Austrian patients. {chi}2 Tables were used to compare chest X-ray (CXR) staging, gender, smoking history, and use of corticosteroids across categories. Lung function parameters were compared by univariate linear regression analysis and adjusted for age, sex, duration of disease, and corticosteroid use and also for smoking status within the United Kingdom population.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
–765G>C in Patients with Sarcoidosis and Control Subjects
The rare allele frequencies in the United Kingdom control subjects (OR, 0.10; 95% CI, 0.07–0.13) are similar to those we previously reported (18), and these differ significantly from the individuals with pulmonary sarcoidosis (OR, 0.20; 95% CI, 0.16–0.24; p = 0.001). The demographic data for all the subjects are shown in Table 2. The United Kingdom populations were suitably matched for age (41.4 ± 10.9 yr compared with 39.5 ± 11.5 yr; p = 0.11) but differed in their sex distribution (control subjects, 46.4% female; patients with sarcoidosis, 60.1% female; p = 0.009). There was no evidence for an interaction between sex and genotype (p = 0.392); however, the effect of the –765G>C polymorphism was clearly strongest in female subjects (Table 3). Overall, the rare –765C allele was associated with increased susceptibility to sarcoidosis in this population (OR, 2.50; 95% CI, 1.51–4.13) for the GC and CC genotypes combined; p = 0.006). Within the female sex, the OR was 3.17 (95% CI, 1.53–6.56; p = 0.002) (Table 3). There also seemed to be a gene–dose effect. Overall, GC subjects had an OR risk of 2.40 (95% CI, 1.42–4.06; p = 0.001), and CC subjects had an OR risk of 3.40 (95% CI, 0.89–13.05; p = 0.075).


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  		TABLE 2. DEMOGRAPHIC DATA FOR THE UNITED KINGDOM AND AUSTRIAN POPULATIONS*

 

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  		TABLE 3. GENOTYPE DISTRIBUTION OF THE –765G>C VARIANT BY SEX IN UNITED KINGDOM AND AUSTRIAN WHITE CONTROL SUBJECTS AND SUBJECTS WITH PULMONARY SARCOIDOSIS

 
The United Kingdom results were replicated in a second smaller population of Austrian subjects (Table 3). The control subjects and patients with sarcoidosis were well matched for age (44.3 ± 18.9 yr compared with 43.5 ± 11.9 yr; p = 0.76) and sex (control subjects, 57.7% female; patients with sarcoidosis, 60.5% female; p = 0.69; Table 2). The mean length of time patients were with disease was significantly less in the Austrian population compared with the United Kingdom population (2.4 ± 2.0 yr compared with 8.9 ± 8.2 yr, respectively; p = < 0.001). The majority (93.5%) of the Austrian patients with sarcoidosis were CXR stage II/III compared with the United Kingdom patients, of whom 50.8% were CXR stage 0/I, 24.3% CXR stage II/III, and 24.9% CXR stage IV (Table 2). The United Kingdom and Austrian patient populations had a similar history of corticosteroid use and lung function for TLC and DLCO % predicted; however, the FEV1 and FVC % predicted differed between these two groups (Table 2). In the Austrian population, carriage of the –765C allele was associated with susceptibility to sarcoidosis (OR, 1.91; 95% CI, 1.04–3.49; p = 0.036). Similar to the United Kingdom population, the effect of the –765G>C was strongest in female subjects (within this group, carriage of the –765C allele gave an OR risk of 3.35 (95% CI, 1.47–7.64; p = 0.004).

Relationship of –765G>C with Persistent and Nonpersistent Disease in Sarcoidosis
Patients with sarcoidosis were classified as having persistent or nonpersistent pulmonary disease based on the criteria specified (see Table 1). Twenty patients remain unclassified because they had no CXR score available 2 yr after diagnosis (n = 19) or because they were less than 2 yr from diagnosis (n = 1). Of the remaining subjects, 76 were classified with persistent disease and 102 with nonpersistent disease. The demographic data for these two groups of patients are shown in Table 4. The two groups did not differ by age (p = 0.17), sex (p = 0.19), corticosteroid use (p = 0.47), or smoking status (p = 0.22), but they differed significantly with respect to CXR staging, lung function, and duration of disease (Table 4). The high-resolution computed tomography (HRCT) data supported the presence of fibrosis. Of the patients with an HRCT, 23 had evidence of fibrosis. Ten of these subjects were CXR stage IV, six were CXR stage II, and seven were CXR stage III. However, all these subjects could be categorized with persistent disease without the HRCT information. The rare allele frequencies of the –765G>C polymorphism differ significantly between the persistent (OR, 0.26; 95% CI, 0.19–0.33) and nonpersistent disease (OR, 0.13; 95% CI, 0.08–0.17) groups (p = 0.005). The rare allele frequencies of the nonpersistent subjects were similar to the frequencies found in the healthy control subjects (OR, 0.10; 95% CI, 0.07–0.13), suggesting that the association of the –765C allele with susceptibility to sarcoidosis seems to be explained by the presence of the persistent disease subjects. Within the sarcoidosis population, carriage of the rare –765C allele was associated with poorer outcome (OR, 3.11; 95% CI, 1.35–7.13; p = 0.008). The effect of the –765G>C polymorphism was strongest in the female subjects. Carriage of the –765C allele gave an OR risk of 5.49 (95% CI, 1.82–16.59; p = 0.003; see Table 3). As secondary outcomes, the effect of genotype on CXR staging and lung function at least 2 yr from diagnosis was considered (Table 5). There was no significant effect of genotype on these secondary outcomes; however, a trend for TLC and DLCO % predicted to be reduced in patients carrying the –765C allele was observed (Table 5).


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  		TABLE 4. DEMOGRAPHIC DATA FOR PERSISTENT AND NONPERSISTENT PULMONARY SARCOIDOSIS AT 2 YEARS AFTER DIAGNOSIS

 

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  		TABLE 5. COMPARISON OF CHEST RADIOGRAPH STAGING AND LUNG FUNCTION AT LEAST 2 YEARS AFTER DIAGNOSIS BY PROSTAGLANDIN-ENDOPEROXIDE SYNTHASE 2 –765G>C GENOTYPE

 
Several of the Austrian subjects with sarcoidosis did not have clinical data available 2 yr after diagnosis. Furthermore, because this group was much smaller than the group of United Kingdom patients with sarcoidosis, it is not feasible to investigate whether the –765G>C genotype has an effect on poorer outcome in the Austrian population.

Effect of –765G>C Genotype on Lung Fibroblast PGE2 Synthesis
The effect of –765G>C genotype on PGE2 synthesis was examined in lung fibroblast cell lines that had been stimulated with transforming growth factor (TGF)-beta1. Nine cell lines had been established; seven were homozygous for the common –765G allele (GG), and two were heterozygous for the –765G>C polymorphism (GC). The GC cell lines produced little or no PGE2 basally and were not stimulated further by TGF-beta1 (Figure 1). By comparison, PGE2 synthesis by the GG cell lines ranged basally from 31 to 2720 pg/105 cells (median 670 pg/105 cells), and this production was enhanced up to 14-fold when the cells were in the presence of TGF-beta1 (range, 55–3200 pg/105 cells; median, 1,084 pg/105 cells) (Figure 1).


Figure 1
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  		Figure 1. Effect of –765G>C genotype on transforming growth factor (TGF)-beta1–induced lung fibroblast prostaglandin E2 (PGE2) production. Fibroblasts were grown to confluence and incubated for 24 h in media alone or in media with TGF-beta1. Results are expressed in picograms of PGE2 per 105 cells. Each point represents the mean PGE2 production from six replicate cultures of an individual cell line. Cell lines homozygous for the –765G allele (GG) are shown with a bold line; heterozygote (GC) cell lines are shown with a dashed line.

 
Nuclear Protein Binding to the –765 PTGS2 Promoter Region
The EMSA experiments revealed that the –765G>C polymorphism altered transcription factor binding to the –765 region. An initial experiment with hrecSp1 confirmed that Sp1 could bind to the region containing –765G; however, this binding is disrupted in the presence of –765C (Figure 2A). When nuclear extracts from human lung fibroblasts were used in EMSA, a number of complexes were formed (Figure 2B). Antibody supershift and blocking experiments showed that Sp1, Sp3, and Egr-1 are involved in binding to this region but that these proteins bind differentially to the –765G and –765C alleles (Figure 2B). A set of smaller, unidentified protein complexes are present that are unaffected by these antibodies. A supershifted complex (SS1, Figure 2B) was obtained with anti-Sp1 antibody in the presence of –765G but not –765C, whereas anti-Sp3 antibody prevents the formation of a complex (complex II, Figure 2B) that is observed only with –765G. Antibodies to Sp2 and Sp4 had no apparent effect. Antibody specific for Egr-1 gave a supershift (SS2, Figure 2B) in the presence of –765C, and a reduction in the intensity of Complex I formed with –765G (Figure 2B).


Figure 2
Figure 2
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  		Figure 2. Binding of human recombinant (hrec) Sp1 protein and nuclear extract derived from human lung fibroblasts to oligonucleotide probes spanning –780 to –751 of the human prostaglandin-endoperoxide synthase 2 promoter region. In the presence of a G base at –765 (G probe), (A) six complexes (1–6) were formed with hrecSp1; however, the presence of a C base at –765 (C probe) considerably reduced the ability of hrecSp1 to bind this region. (B) Several complexes are formed with nuclear extract (NE) derived from lung fibroblasts; however, there is a clear difference in the binding profile to the G probe compared with the C probe (Lanes 3 and 4). Preincubation with an anti-Sp1 antibody results in the formation of a supershifted complex (SS1) in the presence of the G probe (Lane 5) but not with the C probe (Lane 6). Preincubation with an anti-Sp3 antibody prevents complex II formation with the G probe (Lane 9), but this is not seen with the C probe (Lane 10). Sp2 and Sp4 antibodies do not seem to have any obvious effects (Lanes 7 and 8 and Lanes 11 and 12, respectively). Preincubation with an anti–Egr-1 antibody results in formation of a supershift with the C probe (SS2, Lane 14) and reduction of the intensity of complex I with the G probe (Lane 13). IgG antibody was used a negative control (Lanes 15 and 16). Both experiments (A and B) are representative of three experiments, with probe alone reactions loaded in Lanes 1 and 2.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The –765G>C Polymorphism Is Associated with Sarcoidosis and Predicts Poor Outcome
This study is the first report of PTGS2 genotypes associated with sarcoidosis. Carriage of the rare –765C allele of the –765G>C polymorphism confers susceptibility to sarcoidosis in a United Kingdom population sample. These results were confirmed in a second group of subjects. The association of the –765C allele with sarcoidosis in the United Kingdom seems to be due to the presence of subjects with persistent disease (47% of subjects with persistent sarcoidosis disease carried the –765C allele, compared with 18% of healthy subjects and 23% of subjects with nonpersistent sarcoidosis), suggesting the potential for using –765G>C as a prognostic marker in this disease to predict increased risk of poor outcome.

Sarcoidosis affects the lungs in almost all patients. Pulmonary manifestations may resolve spontaneously or progress to pulmonary fibrosis, respiratory failure, and death in 5–10% of cases. Identifying individuals likely to have poor prognosis is difficult. At the outset of this study, we devised a classification system that could be easily applied to population studies to separate patients into those with persistent/fibrotic pulmonary sarcoidosis and those with nonpersistent/nonfibrotic pulmonary sarcoidosis. This was based on a combination of chest radiograph staging, lung function, need for treatment with immunosuppressants, and duration of disease. Chest radiograph staging is well established as a prognostic guide in pulmonary sarcoidosis (25). The HRCT data supported the presence of fibrosis and revealed evidence of fibrosis in subjects who did not have CXR stage IV disease. This is not surprising because HRCT is considerably more sensitive than CXR; however, all the subjects with CXR stage II or III disease that had evidence of fibrosis by HRCT were categorized as having persistent disease using our classification system. This supports the need for several disease-relevant criteria to be assessed to accurately determine outcome in sarcoidosis. Lung function abnormalities in sarcoidosis are complex; however, FVC and DLCO have been shown to be inversely correlated with the degree of reticular pattern changes on CT scanning (26). The need for immunosuppressant therapy was included in our classification system to reflect patients with progressive symptomatic pulmonary disease as recommended in international guidelines (1). The most commonly used immunosuppressants are corticosteroids; however, their use for pulmonary involvement is controversial (27, 28). Duration of disease was also considered important because spontaneous resolution of chest radiograph stage II or greater is unlikely 2 yr after diagnosis (1), and CT abnormalities are less likely to resolve after this time (29). Using these criteria, we found a strong association of the –765C allele with persistent/fibrotic disease in sarcoidosis that is consistent with reduced PTGS2 expression having a role in the fibrotic reaction associated with fibroproliferative lung disease (6, 7). There was no significant effect of genotype on CXR staging and lung function when these were considered as independent outcome measures.

Role of –765G>C in Other Inflammatory and Respiratory Conditions
Since our initial report describing the –765G>C polymorphism (18), several studies have investigated this polymorphism (19, 30, 31). We previously reported that the –765C allele may be protective in a systemic inflammatory response by virtue of its association with decreased levels of CRP. CRP is a known predictor of poorer outcome in bypass surgery and in cardiovascular disease. Cipollone and colleagues have recently confirmed our initial observation of reduced plasma CRP levels in carriers of –765C in a prospective study of cardiovascular disease (19). However, it has also been reported that female carriers of the –765CC genotype are susceptible to type 2 diabetes and bronchial asthma (30, 31). The fact that the –765C allele seems to be protective in cardiovascular disease while conferring susceptibility in other diseases, including fibrotic lung disease, is consistent with PTGS2 having proinflammatory, antiinflammatory, and antifibrotic roles (6, 17, 32).

–765G>C Is Functional and May Exert Transcriptional Control through Altered Binding of Sp1/Sp3 and Egr-1
Using reporter gene assays in lung fibroblasts, we previously showed that the –765G>C polymorphism is functional, with the –765C allele having lower promoter activity than the –765G allele (18). We considered that a functional outcome of the –765C allele in lung fibroblasts may be reduced PGE2 synthesis. Preliminary data in this study seem to support this; however, a larger study needs to confirm these findings. Several other studies have correlated –765G>C genotypes with biomarkers of disease, including CRP, metalloproteinase, and prostaglandins; however, there is some disagreement among these studies (18, 19, 31).

The –765G>C polymorphism is located within a putative Sp1 transcription factor binding motif and may explain its functional effect. We now confirm this as a possible mechanism. Using nuclear extracts derived from lung fibroblasts and EMSAs, supershift and blocking antibody reactions revealed that Sp1 and Sp3 are displaced from binding to the region when the –765C allele is present. However, in the presence of the –765C allele, there is enhanced binding of Egr-1. In most promoters, Sp1 and Sp3 recognize the classical Sp1 consensus element 5'GGGGCGGGG3' with comparable affinity and specificity (33). It is therefore not surprising that displacement of Sp1 binding to the –765 region is also accompanied by the displacement of Sp3 in the presence of the –765C allele. The Sp1/Sp3 ratio is often critical for gene activation. Sp1 is typically a transcriptional activator that is required for the expression of a variety of genes (34). Sp3, however, seems to be a bifunctional regulator that acts as a repressor or activator whose activity depends on the context of DNA binding sites in a promoter (34, 35). Egr-1 also binds to GC-rich DNA sequences (5'-GCGGGGGCG-3') and is able to displace Sp1 from a large number of promoters. Egr-1 acts primarily as a transcriptional inducer of gene expression, with potent effects on profibrotic genes, such as platelet-derived growth factor, TGF-beta, and tumor necrosis factor-{alpha}. However, Egr-1 displacement of Sp1 can also result in transcription repression (36, 37). Further studies are needed to determine whether Egr-1 acts as a repressor or as a less potent activator of PTGS2 transcription in lung fibroblasts and to examine whether the Sp1/Sp3 and Egr-1 relationship is affected by cell type. This might help explain the disparity reported among studies investigating functional outcomes of the –765G>C polymorphism (18, 31).

Transcriptional regulation in the –765 region may involve other transcription factors. The numbers of complexes formed in our EMSA studies suggest that nuclear proteins other than Sp1, Sp3, and Egr-1 binding can bind to this region. Further studies are required to confirm whether reduced PTGS2 expression by altered Sp1/Sp3 binding to the –765 region has a role in other fibrotic and inflammatory diseases.

Clinical Implications
The results of this study indicate that the PTGS2 –765C allele is a promising candidate for a simple genotype analysis that may help identify patients with sarcoidosis who are at greater risk of progressive fibrotic disease and who could be targeted for more aggressive or specific antifibrotic therapy. The effect of the –765G>C polymorphism seems to be strongest in women. Female gender is a known risk factor for sarcoidosis and modifies the effect of the –765G>C polymorphism in our populations. It is important to investigate –765G>C in other ethnic groups. African Americans are reported to have a higher prevalence and a more severe form of sarcoidosis than white subjects, and the frequency of –765G>C is reported to be much higher in African Americans (38). Current therapy for sarcoidosis centers on the use of corticosteroids. However, their effectiveness in the later fibrotic stages of sarcoidosis is limited, and this may relate to current concepts of the pathogenesis of pulmonary fibrosis, indicating that paracrine interactions between local cells rather than chronic inflammation drive progressive fibrosis (39, 40). Therefore, identifying patients at greater risk of fibrosis, such as those carrying the PTGS2 –765C allele, may provide the option for more aggressive immunosuppressive therapy in these individuals in the early stages of disease when it may be more effective or for the future use of other novel antifibrotic therapies that are currently under development and in clinical trials (41–43).


   Acknowledgments
 
The authors thank the participating patients and the clinical units providing access to patients and controls for these studies: Helen Booth (University College London Hospitals), Huw Beynon and Paul Dilworth (Royal Free Hospital, London), Charlotte Rayner (St George's Hospital, London), Norman Johnson (Whittington Hospital, London), Himender Makker (North Middlesex Hospital, London), Marianne Hubner (Wilhelminenspital, Vienna), and Kevin Hart (National Blood Service, London).


   FOOTNOTES
 
* These authors contributed equally to the study. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200512-1839OC on July 13, 2006

Conflict of Interest of Statement: M.R.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.P.M. is currently an employee of GlaxoSmithKline (GSK) and holds stock options within GSK. He was working at University College London when contributing to this work. His position at GSK concerns drug development in asthma and allergy and does not relate to the diseases or genes that constitute this manuscript. GSK currently has cyclooxygenase-2 inhibitors in development. However, such development at present is not for respiratory disease and he has no connection with these efforts. R.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.J.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 2, 2005; accepted in final form July 12, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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