Biol. Chem., Vol. 385, pp. 649–653, July 2004 • Copyright  by Walter de Gruyter • Berlin • New York 2004/403090 SMAD-signaling in chronic obstructive pulmonary disease: transcriptional down-regulation of inhibitory SMAD 6 and 7 by cigarette smoke Jochen Springer 1,2 , Frank R. Scholz 3 , Christian Peiser 1 , David A. Groneberg 1, * and Axel Fischer 1 1 Pneumology and Immunology, Otto-Heubner-Centre, Charite ´ School of Medicine, Campus Buch, Charite ´, Free University and Humboldt-University, D-13353 Berlin, Germany 2 Department of Medicine/Cachexia Research, Charite ´ School of Medicine, Campus Buch, Free University and Humboldt-University, D-13353 Berlin, Germany 3 Department of Hematology and Oncology, Charite ´ School of Medicine, Free University and Humboldt University, D-13353 Berlin, Germany * Corresponding author e-mail: david.groneberg@charite.de Abstract Transforming growth factor-b1 is a potent mediator of fibrosis stimulating the secretion of extracellular matrix proteins and is involved in airway remodeling in chronic obstructive pulmonary disease (COPD). Signals from the TGF superfamily are mediated by the SMAD group of transcription factors. Here, the expression of the regula- tory SMAD2, 3, the co-SMAD4 and the inhibitory SMAD6 and 7 was assessed in bronchial biopsies of COPD patients and controls by quantitative RT-PCR. While SMAD2 was not expressed and SMAD3 and 4 displayed no change, the inhibitory SMAD6 and 7 were significantly down-regulated in COPD. To reveal the molecular basis of tobacco smoke-induced airway remodeling and to test whether it may interfere with intracellular SMAD signal- ing, the airway epithelial cell line A549 was incubated with cigarette smoke extract (1% and 10%) for 48 hours, which led to down-regulation of SMAD6 and 7 at both concentrations tested. It can be concluded that TGF-b-mediated effects in COPD are influenced by a disturbed intracellular feed- back mechanism of inhibitory SMADs. Also, the effects of non-volatile components in tobacco smoke may partly be regulated via a smoke-induced down-regulation of inhibitory SMADs. Keywords: airways; chronic obstructive airway disease; cigarette smoke; COPD; lung; SMAD; TGF-b. Introduction Chronic obstructive pulmonary disease (COPD) is cur- rently estimated to be the 3 rd most common cause of death in 2020 (Lopez and Murray, 1998). The disease is characterized by an irreversible and progressive devel- opment of airflow limitation featuring cough, mucus hypersecretion, inflammatory changes and remodeling of the airway wall (Madison and Irwin, 1998; Barnes, 2000). A common feature in the remodeling process is the fibrosis of the airway wall, which contributes to the observed severe airway obstruction (Jeffery, 2001). This fibrosis is associated with a deposition of collagen I, col- lagen III and fibronectin (Massague, 1990). Transforming growth factor-b1 (TGF-b1) potently stimulates the release of these proteins, while it inhibits the synthesis of enzymes that degrade the extra-cellular matrix pro- teins, such as matrix metallopoteinases (Overall et al., 1989; Duvernelle et al., 2003). Furthermore, TGF-b1 increases the expression of inhibitors of these matrix- degrading enzymes (Murphy and Docherty, 1992). TGF- b1-expression itself has been shown to be induced in smokers and COPD-patients (Takizawa et al., 2001). Therefore, TGF-b1 is regarded as a major contributor to the alteration in the extracellular matrix such as the sub- basement membrane thickening in asthma (Vignola et al., 1998) and the bronchial wall fibrosis in COPD (Fabbri et al., 2003). The downstream signaling of TGF-receptors is medi- ated by the SMAD group of transcription factors (Gro- neberg et al., 2004). The term SMAD is derived from the Drosophila homolog MAD and the C. elegans homolog SMA (Roberts et al., 2000). The vertebrate SMAD family includes stimulatory, receptor-activated SMADs (1, 2, 3, 5, 8 and 9; Massague, 1998), a common mediator SMAD4 (Liu et al., 1997) and inhibitory SMADs (6 and 7; Imamura et al., 1997; Nakao et al., 1997). The receptor- activated SMADs hetero-oligomerize with the common mediator SMAD4 to translocate into the nucleus, where the complex may either bind directly to the DNA or par- ticipate in other transcriptional complexes (Shi et al., 1997). The inhibitory SMADs function as a negative feed- back mechanism in TGF-b signaling by either binding stably to TGF-b receptors or by competing for the com- mon mediator SMAD4, which lead to a reduction of sig- nal strength or termination of the signal (Roberts et al., 2000). Therefore, alterations in inhibitory SMAD expres- sion may have a strong influence on TGF-b-signaling and subsequent effects in COPD pathophysiology such as fibrosis as suggested earlier (Groneberg et al., 2004). Here, the expression of TGF-b1-signaling relevant SMADs (2, 3, 4, 6 and 7) was investigated at the tran- scriptional level using a recently published approach of real-time quantitative RT-PCR (Huang and Lai, 2003) in bronchial biopsies taken from COPD patients. Quantifi- cation with the highly sensitive real-time RT-PCR was chosen as protein contents in the biopsies were not suf- ficient for Western blotting studies. The deleterious Brought to you by | Purdue University Libraries Authenticated Download Date | 5/24/15 8:23 AM