The International Journal of Biochemistry & Cell Biology 41 (2009) 1157–1164 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel The alpha-1-antitrypsin gene promoter in human A549 lung derived cells, and a novel transcription initiation site Kevin Morgan 1 , Sally Chappell 1 , Tamar Guetta-Baranés, Stephen Morley, Noor Kalsheker ∗ School of Molecular Medical Sciences, Institute of Genetics, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom article info Article history: Received 25 April 2008 Received in revised form 17 October 2008 Accepted 22 October 2008 Available online 28 October 2008 Keywords: SerpinA1 Cytokine Promoter Lung Gene regulation abstract Alpha-1-antitrypsin (AAT), also called serine proteinase inhibitor A1 (Serpin A1), is the most abundant serpin in human plasma. A major physiological role of AAT is to protect the lung from the destructive effects of excess uninhibited neutrophil elastase. During inflammation, circulating levels of AAT may increase twofold-to-threefold as part of the acute-phase response. The liver is the main contributor to this increase. However, local synthesis may provide an important mechanism for controlling neutrophil elastase activity at sites of inflammation, and previous studies have shown a marked increase in production after cytokine stimulation. In the current study we report a distinct transcription initiation site for AAT expression in the lung alveolar epithelial cell line A549, which is located nine bases upstream of the previously mapped full- length monocyte transcription start-site, and show using site-directed mutagenesis that two Sp1 sites and a putative TATA box are functional. EMSA experiments provide evidence for Sp1 and Sp3 binding to these two Sp1 sites. We have also mapped the minimal promoter region and a cell-specific element essential for expression in A549 cells, both of which reside in an 865bp fragment upstream of the transcription start- site. Understanding the mechanisms of AAT gene regulation in a lung-derived cell line has important implications for understanding the control of localised lung tissue damage which occurs as a result of excess proteolytic activity. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction Alpha-1-antitrypsin (AAT) is a major inhibitor of serine pro- teinases in human plasma, and the archetype of the serpin superfamily of proteins. Although AAT has broad specificity, its main target proteinase is neutrophil elastase (NE), an enzyme that degrades the extracellular matrix. The elastic tissues of the lung alveoli are particularly vulnerable to damage by NE and deficiency states of AAT are associated with chronic lung disease resulting from destruction of connective tissue components by uninhibited NE (Crystal, 1990). The major site of AAT synthesis is the liver. Other cell types, including monocytes, macrophages (Mornex et al., 1986), bronchial epithelial cells and lung alveolar epithelial cells make it in substantially lesser amounts (Sallenave et al., 1997) particularly under basal conditions. However, in the extracellular matrix, local ∗ Corresponding author at: Clinical Chemistry, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom. Tel.: +44 115 8230720; fax: +44 115 8230722. E-mail addresses: Kevin.morgan@nottingham.ac.uk (K. Morgan), Sally.chappell@nottingham.ac.uk (S. Chappell), Tamar.Guetta-Baranes@nottingham.ac.uk (T. Guetta-Baranés), noor.kalsheker@nottingham.ac.uk (N. Kalsheker). 1 These authors contributed equally to this paper. production may be an important mechanism for controlling NE activity at physiologically relevant tissue sites (Cichy et al., 1997). It has been proposed that AAT production increases up to 110- fold in lung epithelial cells after treatment with combinations of oncostatin-M (OSM), transforming growth factor  (TGF) and dex- amethasone (Sallenave et al., 1997; Boutten et al., 1998) suggesting that this dramatic increase in production after cytokine stimulation is likely to be physiologically relevant in the lung. The estimated fold-increase in AAT production may have been exaggerated due to very low levels present under basal conditions. Other anti-elastases are also produced locally by the lung, e.g. SLPI and elafin, but greater than 90% of the anti-NE protection in the alveolar walls is provided by AAT. The elements of the gene required for control of tissue- specific expression in the lung are as yet undefined. The AAT gene consists of seven exons, including three untrans- lated exons (1A, 1B and 1C) and exons 2–5 that code for the protein. Distinct promoters and transcription start-sites have been identified for hepatocytes and extra-hepatic tissues such as monocytes/macrophages and the cornea. There are three possible upstream transcriptional start-sites in monoctyes designated M1, M2 and M3, which generate transcripts of 2, 1.95 and 1.8 kb respec- tively (Perlino et al., 1987; Hafeez et al., 1992) in addition to the 1.6kb hepatocyte transcript (Long et al., 1984). A distinct corneal start-site which generates a 1.9 kb transcript has also been proposed 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.10.020