Pulmonary infections are responsible for significant morbidity and mortality, particularly in individuals with chronic illness and acute lung injury. The specific host defenses that are compromised in these clinical settings are incompletely defined at the molecular level. Recently, numerous classes of antimicrobial peptides have been isolated and characterized, unveiling a previously unrecognized component of mammalian host defense. Tracheal Antimicrobial Peptide (TAP) is an abundant peptide isolated from bovine tracheal mucosa which exhibits potent broad-spectrum antimicrobial activity in vitro. TAP is a product of epithelial cells lining the large airways, and may participate in the antimicrobial defense of respiratory tissue. Furthermore, the expression of TAP in cultured airway cells is modulated by bacteria and lipopolysaccharide (LPS). The long range goal of this research is to understand the role that these antimicrobial peptides of the respiratory tract play in host defense. The focus of this proposal is to define mechanisms of regulation of expression of TAP at the molecular level. It is hypothesized that this regulation might occur via the binding of transcriptional activating proteins to sequences flanking the TAP gene. The following experiments are proposed: l. The level of control of expression of TAP will be characterized. 2. The regions containing the cis-acting DNA sequences responsible for the tissue-specific, developmental and regulated expression of TAP will be defined; 3. The protein binding sites within these regions will be identified. Preliminary data suggest that TAP expression is controlled at the level of transcription. To determine whether this is indeed the case we will perform northern blot and nuclear run-on analysis. A series of plasmids containing segments of the 5' flanking control region linked to a reporter gene will be constructed. These plasmids will be transfected into bovine tracheal epithelial cells. The expression of these plasmids in response to LPS and other factors will then be examined in order to identify the regions responsible for regulated expression of TAP. The specific sequences which are bound by transcription factors will be identified by gel mobility shift assays and nuclease protection assays. This analysis will also be extended to tissue-specific and developmental control. One of the many important physiological functions of the mammalian respiratory tract is to maintain defense against potential pathogenic agents present in inspired air. A more complete definition of this function at the molecular level will foster our understanding of the pathophysiology of airway disease. Defining the mechanisms regulating expression of antimicrobial peptides in the lung may ultimately lead to therapeutic modulation of endogenous peptide expression, and may lead to the development of novel therapeutics designed to work in the respiratory tract.
| Ryan, L K; Rhodes, J; Bhat, M et al. (1998) Expression of beta-defensin genes in bovine alveolar macrophages. Infect Immun 66:878-81 |
