Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease
Abstract
To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up- or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor α, IL-1β, and IFN-γ) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (+/+) mice but not detected in that of EGR1 null (-/-) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.
Chronic obstructive pulmonary disease (COPD) is a slowly progressive and irreversible disorder characterized by the functional abnormality of airway obstruction, which is a significant cause of morbidity, mortality, and health care costs. COPD is a collective term describing two separate chronic lung diseases: emphysema and chronic bronchitis, which are caused largely by a common agent, cigarettes (1, 2). Cigarette smoke has been generally accepted as the most important of many risk factors for the development of COPD, which accounts for ≈80-90% of COPD cases in the United States (3). However, only 15-20% of heavy smokers develop clinically significant airflow obstruction, which suggests a genetic susceptibility to the development of the disease (4). The genes that determine this genetic susceptibility to cigarette smoking and disease progression to COPD are poorly understood.
To better understand the candidate genes involved in the development of COPD in smokers, we performed serial analysis of gene expression (SAGE) and microarray analysis as a complementary approach to analyze the global gene expression profiles of lung tissue from smokers who are at risk (GOLD-0) and who have developed moderate (GOLD-2) COPD (5). SAGE (6), based on the 10-bp tag for sufficient individual gene identification and the concatenation of SAGE tags for high-efficient gene identification, has the additional advantage of allowing unbiased and comprehensive analysis of a large number of differentially expressed genes without prior knowledge of the genes when applied to any particular cell system in different conditions (7, 8). This tag-based approach has the potential to identify new genes expressed at lower levels (9).
In this study, we compared the tags present in the GOLD-2 smoker samples with GOLD-0 smoker controls and generated a comprehensive profile of gene expression patterns in these lung tissues. We selected genes that were consistently differentially expressed in GOLD-2 smokers versus GOLD-0 smoker controls by both SAGE and microarray analysis, with confirmation of expression in the lung tissue of GOLD-2 smokers and fibroblasts from emphysema patients. We demonstrate that gene expression profiling analysis represents a powerful approach to provide insights to novel pathways involved in the pathogenesis of COPD.
Pkyr, packs per year; FVC, forced vital capacity.
Codelink microarray analysis was performed on independent individual lung tissue samples from GOLD-2 and GOLD-0 smokers.
Click here to view.Acknowledgments
We thank Emeka Ifedigbo for his enthusiasm for all aspects of this work, and Liqiang Xi and Paul R. Reynolds for assistance with the QRT-PCR technique. This work was supported by National Institutes of Health Grants R01-HL60234, R01-AI42365, and R01-HL55330 (to A.M.K.C.).
Notes
Author contributions: A.M.K.C. designed research; W.N., C.-J.L., S.M.A., S.L.O., and S.C.W. performed research; C.A.F.-B., Y.P.D., R.S., S.H., Z.Z., D.J.P., J.M.P., and J.C.H. contributed new reagents/analytic tools; W.N., C.-J.L., N.K., S.H., D.G.P., J.C.H., and A.M.K.C. analyzed data; W.N., C.-J.L., D.G.P., and A.M.K.C. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: COPD, chronic obstructive pulmonary disease; SAGE, serial analysis of gene expression; QRT-PCR, real-time quantitative RT-PCR; CSE, cigarette smoke extract; MMP, matrix metalloproteinase; FEV1, forced expiratory volume in 1 sec; TNF-α, tumor necrosis factor α; GO, gene ontology.
References
- 1. Voelkel, N. F. & MacNee, W. (2002) in Chronic Obstructive Lung Diseases (BC Decker, Hamilton, ON, Canada), pp. 90-113.
- 2. Petty, T. L. (2002) Chest121, 116s-120s. [[PubMed]
- 3. Sethi, J. M. & Rochester C. L. (2000) Clin. Chest Med.21, 1-26. [PubMed]
- 4. Mayer, A. S. & Mewman, L. S. (2001) Respir. Physiol.128, 3-11. [[PubMed]
- 5. Pauwels, R. A., Buist, A. S., Calverley, P. M. A., Jenkins, C. R. & Jurd, S. S. (2001) Am. J. Respir. Crit. Care Med.163, 1256-1276. [[PubMed]
- 6. Velculescu, V. E., Zhnag, L., Vogelstein, B. & Kinzler, K. W. (1995) Science270, 484-487. [[PubMed]
- 7. Kagnoff, M. F. & Eckmann, L. (2001) Curr. Opin. Microbiol.4, 246-250. [[PubMed]
- 8. Nacht, M., Dracheva, T., Gao, Y., Fujii, T., Chen, Y., Player, A., Akmaev, V., Cook, B., Dufault, M., Zhang, M., et al. (2001) Proc. Natl. Acad. Sci. USA98, 15203-15208.
- 9. Boheler, K. R. & Stern, M. D. (2003) Trends Biotechnol.21, 55-57. [[PubMed]
- 10. Ning, W., Chu, T. J., Li, C. J., Choi, A. M. & Peters, D. G. (2004) Physiol. Genomics18, 70-78. [[PubMed]
- 11. Mirnics, K., Middleton, F. A., Marquez, A., Lewis, D. A. & Levitt, P. (2000) Neuron28, 53-67. [[PubMed]
- 12. Kaminski, N. & Friedman, N. (2002) Am. J. Respir. Cell Mol. Biol.27, 1-8. [[PubMed]
- 13. Benjamini, Y. & Hochberg, Y. (1995) J. R. Stat. Soc. B57, 289-300. [PubMed]
- 14. Godfrey, T. E., Kim, S. H., Chavira, M., Ruff, D. W., Warren, R. S., Gray, J. W. & Jensen, R. H. (2000) J. Mol. Diagn.2, 84-91.
- 15. Zhou. X, Tan, F. K., Xiong, M., Milewicz, D. M., Feghali, C. A., Fritzler, M. J., Reveille, J. D. & Arnett, F. C. (2001) J. Immunol.167, 7126-7133. [[PubMed]
- 16. Clark, R. S. B., Kochanek, P. M., Watkins, S. C., Chen, M., Dixon, C. E., Seidberg, N. A., Melick, J., Loeffert, J. E., Nathaniel, P. D., Jin, K. L., et al. (2000) J. Neurochem.74, 740-753. [[PubMed]
- 17. Ishii, T., Matsuse, T., Igarashi, H., Masuda, M., Teramoto, S. & Ouchi, Y. (2001) Am. J. Physiol.280, L1189-L1195. [[PubMed]
- 18. Ning, W., Song, R., Li, C., Park, E., Mohsenin, A., Choi, A. M. & Choi, M. E. (2002) Am. J. Physiol.283, L1094-L1102. [[PubMed]
- 19. Kleiner, D. E. & Stetler-Stevenson, W. G. (1994) Anal. Biochem.218, 325-329. [[PubMed]
- 20. Hastie, N. D. & Bishop, J. O. (1976) Cell9, 761-744. [[PubMed]
- 21. de Waard, V., van den Berg, B. M., Veken, J., Schultz-Heienbrok, R., Pannekoek, H. & van Zonneveld, A. J. (1999) Gene226, 1-8. [[PubMed]
- 22. Draghici, S., Khatri, P., Bhavsar, P., Shah, A., Krawetz, S. A. & Tainsky, M. A. (2003) Nucleic Acids Res.31, 3775-3781.
- 23. Al-Shahrour, F., Diaz-Uriarte, R. & Dopazo, J. (2004) Bioinformatics20, 578-580. [[PubMed]
- 24. Zhang, W., Yan, S. D., Zhu, A., Zou, Y. S., Williams, M., Godman, G. C., Thomashow, B. M., Ginsburg, M. E., Stern, D. M. & Yan, S. F. (2000) Am. J. Pathol.157, 1311-1320.
- 25. DiCamillo, S. J., Carreras, I., Panchenko, M. V., Stone, P. J., Nugent, M. A., Foster, J. A. & Panchenko, M. P. (2002) J. Biol. Chem.277, 18938-18946. [[PubMed]
- 26. Takizawa, H., Tanaka, M., Takami, K., Ohtoshi, T., Ito, K., Satoh, M., Okada, Y., Yamasawa, F., Nakahara, K. & Umeda, A. (2001) Am. J. Respir. Crit. Care Med.163, 1476-1483. [[PubMed]
- 27. Knight, D(2001) Immunol. Cell Biol.79, 160-164. [[PubMed][Google Scholar]
- 28. Zhu, Y. K., Liu, X., Ertl, R. F., Kohyama, T., Wen, F. Q., Wang, H., Spurzem, J. R., Romberger, D. J. & Rennard, S. I. (2001) Am. J. Respir. Cell Mol. Biol.25, 620-627. [[PubMed]
- 29. Churg, A., Zay, K., Shay, S., Xie, C., Shapiro, S. D., Hendricks, R. & Wright, J. L. (2002) Am. J. Respir. Cell Mol. Biol.27, 368-374. [[PubMed]
- 30. Shi, L., Kishore, R., McMullen, M. R. & Nagy, L. E. (2002) Am. J. Physiol.282, C1205-C1211. [[PubMed]
- 31. Sho, E., Sho, M., Singh, T. M., Nanjo, H., Komatsu, M., Xu, C., Masuda, H. & Zarins, C. K. (2002) Exp. Mol. Pathol.73, 142-153. [[PubMed]
- 32. Chen, J., Rowley, J. D. & Wang, S. M. (2000) Proc. Natl. Acad. Sci. USA97, 349-353.
- 33. Lee, S. L. Sadovsky, Y., Swirnoff, A. H., Polish, J. A., Goda, P., Gavrilina, G. & Milbrandt, J. (1996) Science273, 1219-1221. [[PubMed]
- 34. Yan, S. F., Pinsky D. J., Mackman, N. & Stern, D. M. (2000) J. Clin. Invest.105, 553-554.
- 35. Haas, T. L., Stitelman, D., Davis, S. J., Apte, S. S. & Madri, J. A. (1999) J. Biol. Chem.274, 22679-22685. [[PubMed]
- 36. Yang, G., Nguyen, X., Ou, J., Rekulapelli, P., Stevenson, D. K. & Dennery, P. A. (2001) Blood97, 1306-1313. [[PubMed]
- 37. Senior, R. M. (2000) Chest117, 320s-323s. [[PubMed]
- 38. MacNee, W(2001) Eur. J. Pharmacol.429, 195-207. [[PubMed][Google Scholar]
- 39. Shin, H. J., Park, K. K., Lee, B. H., Moon, C. K. & Lee, M. O. (2003) Toxicology191, 121-131. [[PubMed]