Genetic Determinants of Functional Impairment in ...

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Correspondence and requests for reprints should be addressed to Jack A. Elias, ... Becky Mercer, Constance Brinckerhoff, and Jeanine. D'Armiento. Institute of ...
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the level of pulmonary artery pressure is a good indicator of prognosis in patients with COPD. The pathogenesis of PH in patients with advanced COPD is incompletely understood. Chronic hypoxemia, morphologic changes in lung parenchyma (2, 3), and inflammation (4) are potential contributing factors. Here, we investigated whether inflammatory cytokines were related to the PH process in COPD. In 50 patients with COPD (age, 62 ⫾ 3 yr), pulmonary artery pressure (Pap) was measured during right heart catheterization, and blood samples were collected for determination of genotypes and serum levels of interleukin (IL)-1␤, IL-6, and monocyte chemoattractant protein-1 (MCP-1). As compared with a control group of 50 smokers, patients with COPD had elevated serum levels of IL-6 (p ⬍ 0.001) and MCP-1 (p ⬍ 0.01) but unchanged levels of IL-1␤. Pap was positively correlated with serum IL-6 levels but not with serum IL-1␤ and MCP-1 levels. The IL-6 G/G polymorphism (–174G/C) correlated with a higher level of IL-6 among patients with COPD but not among control subjects. Patients carrying the IL6 GG genotype showed higher serum levels of IL-6 and higher Pap than those carrying the CC or CG genotype. These results indicate that the inflammatory cytokine IL-6 may contribute to PH in COPD and that IL-6 gene polymorphism confers susceptibility to PH in patients with COPD. Since we previously reported that the serotonin transporter (5-HTT) gene polymorphism was associated with PH in COPD, the combined actions of the allelic variants of these two genes are being examined in a larger population of patients with COPD. Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

References 1. Pauwels RA, Rabe KF. Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 2004;364:613–620. 2. Eddahibi S, Fabre V, Boni C, Martres MP, Raffestin B, Hamon M, Adnot S. Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ Res 1999;84:329–336. 3. Eddahibi S, Chaouat A, Morrell N, Fadel E, Fuhrman C, Bugnet AS, Dartevelle P, Housset B, Hamon M, Weitzenblum E, et al. Polymorphism of the serotonin transporter gene and pulmonary hypertension in chronic obstructive pulmonary disease. Circulation 2003;108:1839–1844. 4. Humbert M, Monti G, Brenot F, Sitbon O, Portier A, Grangeot-Keros L, Duroux P, Galanaud P, Simonneau G, Emilie D. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med 1995;151:1628–1631.

PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY

VOL 3 2006

Channing Laboratory and Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Rationale: Patients with severe chronic obstructive pulmonary disease (COPD) may have varying levels of disability despite similar levels of lung function. This variation may reflect different COPD subtypes, which may have different genetic predispositions. Methods: In 304 subjects from the National Emphysema Treatment Trial, we genotyped 80 markers in 22 positional and/ or biologically plausible candidate genes. Regression models were used to test for association, using a test-replication approach to guard against false-positive results. For significant associations, effect estimates were recalculated using the entire cohort. Positive associations with dyspnea were confirmed in families from the Boston Early-Onset COPD Study. Results: The test-replication approach identified four genes—microsomal epoxide hydrolase (EPHX1), latent transforming growth factor ␤ binding protein-4 (LTBP4), surfactant protein B (SFTPB), and transforming growth factor ␤-1 (TGFB1)—that were associated with COPD-related phenotypes. In all subjects, single nucleotide polymorphisms (SNPs) in EPHX1 (p ⭐ 0.03) and in LTBP4 (p ⭐ 0.03) were associated with maximal output on cardiopulmonary exercise testing. Markers in LTBP4 (p ⭐ 0.05) and SFTPB (p ⫽ 0.005) were associated with six-minute walk test distance. SNPs in EPHX1 were associated with carbon monoxide diffusing capacity (p ⭐ 0.04). Three SNPs in TGFB1 were associated with dyspnea (p ⭐ 0.002), one of which replicated in the family study (p ⫽ 0.02). Conclusions: Polymorphisms in several genes appear to be associated with COPD-related traits other than forced expiratory volume in 1 second. These associations may identify genes in pathways important for COPD pathogenesis. Conflict of Interest Statement : C.P.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.L.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.J.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.K.S. received grant support, consulting fees, and honoraria from GlaxoSmithKline for studies of COPD genetics. He also received a speaker fee from Wyeth for a talk on COPD genetics and also received honoraria from Bayer.

(Received in original form March 16, 2006; accepted in final form April 11, 2006 ) (Received in original form March 16, 2006; accepted in final form March 21, 2006 ) Correspondence and requests for reprints should be addressed to Serge Adnot, M.D., Professeur de Physiologie, Service de Physiologie–Explorations Fonctionnelles, Ho ˆ pital Henri Mondor, 94010 Creteil, France. E-mail: serge.adnot@creteil. inserm.fr Proc Am Thorac Soc Vol 3, 2006 DOI: 10.1513/pats.200603-038MS Internet address: www.atsjournals.org

Genetic Determinants of Functional Impairment in Chronic Obstructive Pulmonary Disease Craig P. Hersh, Dawn L. DeMeo, Ross Lazarus, Juan C. Celedo´n, Benjamin A. Raby, John J. Reilly, and Edwin K. Silverman

Supported by NIH grants HL71393 (Edwin Silverman), HL075478 (Edwin Silverman), N01HR76102 (John Reilly), HL080242 (Craig Hersh), and by an American Lung Association Career Investigator Award (Edwin Silverman). Correspondence and requests for reprints should be addressed to Craig P. Hersh, M.D., M.P.H., Channing Laboratory, Brigham and Women’s Hospital, 181 Longwood Avenue, Boston, MA 02115. E-mail: [email protected] Proc Am Thorac Soc Vol 3, 2006 DOI: 10.1513/pats.200603-039MS Internet address: www.atsjournals.org

Genetic Control of Transforming Growth Factor-␤1–induced Emphysema and Fibrosis in the Murine Lung Chun Geun Lee, Soojung Cho, Robert J. Homer, and Jack A. Elias

Cookson: Genetics of COPD

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Section of Pulmonary and Critical Care Medicine and Department of Pathology, Yale University School of Medicine, New Haven, Connecticut

Institute of Human Nutrition, and Department of Medicine, Columbia University, New York, New York; and Department of Biochemistry, Dartmouth Medical School, Lebanon, New Hampshire

Transforming growth factor-␤1 (TGF-␤1) is a multifunctional cytokine that plays a critical role in tissue injury, inflammation, and repair, and is overexpressed in tissues from patients with chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. However, its role in the pathogenesis of these diseases has been controversial. This is particularly true in COPD where emphysema has been associated with TGF-␤1 excess and with interventions that block TGF-␤1 activation or signaling. To begin to define the processes that control the respiratory effector responses of TGF-␤1, we generated transgenic mice in which TGF-␤1 was selectively overexpressed in the lung. Interestingly, transgenic TGF-␤1 caused emphysema with minimal fibrosis in Balb/c mice and prominent fibrosis and honeycombing in C57BL/6 mice. The emphysema in Balb/c mice was associated with heightened apoptosis and matrix metalloproteinase-12 expression. In contrast, the fibrosis in C57BL/6 mice was associated with enhanced expression of lysyl oxidase and cysteine rich protein (cyr 61). These studies demonstrate that TGF-␤1 is a multifunctional cytokine that simultaneously induces tissue injury and fibrosis and can generate emphysematous and/or fibrotic phenotypes. They also demonstrated that the tissue effects of TGF-␤1 depend, in part, on the balance of apoptosis, proteolysis, and fibrosis. Last, they demonstrate that genetic influences regulate the effector responses of TGF-␤1 in the lung. These genetic controls may regulate the degree to which TGF-␤1–induced tissue remodeling responses result in emphysema and/or fibrosis in the injured lung.

Rationale: Differences in matrix metalloproteinase (MMP)-1 promoter sequence influence MMP-1 expression. We have shown that cigarette smoke exposure induces MMP-1 gene transcription in human small airway epithelial cells (SAECs) and that this activation requires extracellularly regulated kinase (ERK) 1/2 mitogen-activated protein (MAP) kinase. No evidence exists, however, regarding the response of the 1G and 2G MMP-1 promoter sequences to cigarette smoke. There is evidence from cancer cell lines supporting a role for the 2G polymorphism in enhancing MMP-1 transcription. The objective of this study was to determine whether the 2G insertion polymorphism plays a role in the induction of MMP-1 by cigarette smoke, and whether MAPK signaling is directly involved in MMP-1 promoter activation in lung epithelial cells. Methods: SAECs were transfected with plasmid containing the firefly luciferase gene driven by the full-length MMP-1 promoter containing either the 1G or 2G polymorphic sequence at ⫺1607 bp. In addition, constructs in which increasing portions of the distal promoter were deleted were used to determine the specific regions of the promoter activated by smoke. Renilla luciferase plasmid was cotransfected to control for transfection efficiency. After 24 hours’ recovery, cells were treated with media containing 5% cigarette smoke extract and incubated for another 24 hours. The cells were lysed in Dual Luciferase Buffer (Promega, Madison, WI) and MMP-1 promoter activity was read as relative light units (RLU) in a 96-well plate luminometer. Results: Cigarette smoke extract induced MMP-1 promoter activity. However, the response of the 2G insertion polymorphism was at least threefold greater than the 1G sequence (p ⬍ 0.05), demonstrating the enhanced responsiveness of this polymorphism to cigarette smoke. Importantly, treatment of cells with the ERK inhibitor PD98059 blocked induction of the MMP-1 promoter, demonstrating that the MMP-1 promoter is a direct target of ERK signaling. The various deletion constructs did not respond to smoke, suggesting that the distal region of the promoter is required for MMP-1 induction by smoke. Conclusions: The ⫺1607 2G insertion polymorphism of the human MMP-1 promoter exhibits enhanced responsiveness to cigarette smoke, compared with the 1G sequence. Importantly, ERK1/2 MAPK is required for MMP-1 promoter activity in SAECs. Individuals with the 2G insertion polymorphism who smoke may be predisposed to enhanced MMP-1 expression in the lung, contributing to the development of chronic obstructive pulmonary disease.

Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form March 16, 2006; accepted in final form April 12, 2006 ) Correspondence and requests for reprints should be addressed to Jack A. Elias, M.D., Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 300 Cedar Street, S441 TAC, New Haven, CT 06519. E-mail: jack. [email protected] Proc Am Thorac Soc Vol 3, 2006 DOI: 10.1513/pats.200603-040MS Internet address: www.atsjournals.org

Activation of the MMP-1 Promoter by Cigarette Smoke in Human Small Airway Epithelial Cells Requires ERK MAP Kinase Signaling

Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. (Received in original form March 16, 2006; accepted in final form March 20, 2006 ) Supported by the Alpha-1 Anti-trypsin Foundation.

Differential Response of the 1G and 2G Promoter Sequences

Correspondence and requests for reprints should be addressed to Jeanine D’Armiento, M.D., Ph.D., Assistant Professor, Department of Medicine, Division of Molecular Medicine, Columbia University, College of Physicians & Surgeons, New York, NY 10032. E-mail: [email protected]

Becky Mercer, Constance Brinckerhoff, and Jeanine D’Armiento

Proc Am Thorac Soc Vol 3, 2006 DOI: 10.1513/pats.200603-041MS Internet address: www.atsjournals.org