Chronic inflammation and mucous cell metaplasia are common pathologic features of the majority of pulmonary diseases, including allergic asthma, pneumonia, and chronic bronchitis. The number of metaplastic mucous cells is regulated by Bcl-2 family-mediated cell death, specifically Bcl-2 and Bcl-2 interacting killer (Bik), in airway epithelia. However, it is not known whether Bcl-2 and Bik regulate inflammation, or the mechanism by which this regulation occurs. Preliminary results demonstrated that cigarette smoke -induced inflammation is significantly increased in bik-/- compared to bik+/+ mice, shown by increased numbers of macrophages and lymphocytes in the lung. Additionally, transgenic expression of Bik in airway epithelia significantly attenuated allergen-induced eosinophilia in comparison to controls, demonstrating an anti- inflammatory role for Bik. In order to determine of Bik modulates inflammation through inhibition of the pro- inflammatory transcription factor, Nuclear Factor-kappaB (NF-?B), its activation was assessed after adenoviral expression of GFP, wild type Bik, mutant Bik (BH3mutBik, Leucine 61to alanine substitution renders Bik unable to induce cell death), or a peptide containing the mutant BH3 domain of Bik . Interestingly, expression of both Bik and BH3mutBik equally in an airway cell line suppressed NF-?B activation in response to TNF-? stimulation. Inhibition of NF-?B in the presence of BH3mutBik occurred in conjunction with NF-?B's association with Bcl-2, a known binding partner of Bik. Because Bik and Bcl-2 associate via their BH3 domains, the effect on NF-?B activation was examined after blocking of the BH3 domain in Bcl-2 using the BH3 mimetic, ABT-263. ABT-263 reduced activation of NF-?B and enhanced its association to Bcl-2 in comparison to controls. Collectively, these data suggest that binding of the BH3 domain of Bcl-2, either by Bik or a BH3 mimetic, blocks nuclear translocation of NF-?B and inflammation.
Specific aims of this proposal will (1) demonstrate the molecular mechanism by which Bik and Bcl-2 regulate NF-?B activation and inflammation and (2) test the role of Bik and Bcl-2 following House Dust Mite (HDM)-induced inflammation in vivo. Expression of Bik and Bcl-2 will be modulated in in vitro airway epithelial models, and Bik/Bcl-2 peptides and truncation mutants will be generated in order to test their impact on NF-?B activation. Additionally, adenoviral constructs of Bik and BH3mutBik, and ABT-263, will be administered in the mouse lung during HDM exposure to evaluate their impact on HDM-induced inflammation. According to the hypothesis, it is anticipated that both overexpression of BH3mutBik and inhibition of Bcl-2 will dampen HDM-induced inflammation in the lung. Based on preliminary data demonstrating that the BH3mutBik dampens NF-?B signaling independent of cell death, a novel role for Bcl-2 proteins will be established. Completion of these aims will not only delineate a novel mechanism for Bcl- 2 proteins and NF-?B signaling, but will also result in highly relevant translational research for inflammatory diseases of the lung.
Chronic inflammation in the lung contributes to and exacerbates the vast majority of pulmonary diseases. Current therapeutics can alleviate inflammation; however, they are not effective in every patient, nor are they effective consistently, which can lead to serious health complications. The present application aims to elucidate a novel role/mechanism for the Bcl-2 family proteins, Bcl-2 and Bik, in regulating inflammation in the lung.
