A73-05 Bik in Airway Epithelium Increases Expression of Uchl1 in Human and Mouse Airway Cells

Introduction While the Bcl-2-interacting killer (Bik) is central in inducing cell death in airway epithelial cells (AECs), we have shown that Bik-deficiency leads to emphysema. A single nucleotide polymorphism (SNP) in the BIK gene affects its expression and thereby modulates low-level inflammation in humans and enhances the decline in lung function. Therefore, beyond its apoptotic and anti-inflammatory function, BIK has been implicated in the regulation of neuroendocrine cells (NECs) within the airway epithelium as genetic induction of BIK in airway club cells increases NEC numbers. Here, we investigated the anatomical distribution of BIK in the lung and tested whether BIK induces UCHL1, a marker for NECs in adults, in human airway epithelial cells. Methods In Bik-/- mice, the Bik gene was disrupted by replacing its first two coding exons, including the BH3 domain, with a E. coli β-galactosidase reporter cassette. Harvested lungs were immunostained with anti-β-galactosidase, anti-acetylated α-tubulin, anti-CC10, and anti-UCHL1 antibodies to assess BIK transcriptional activity and localization. Human bronchial epithelial cells (16HBE) were transduced with an adenoviral BIK expression vector and analyzed for UCHL1 expression. In parallel, 16HBE cells were treated with lipidcoated, TAT-linked BIK or BIK-MUT (BH3-only) peptides for 24 h, and UCHL1 levels were assessed by Western blotting. Mouse tracheal epithelial stem cells (MTEC) from Bik-/- mice were differentiated at an air-liquid interface (ALI) for 28 days and treated with BIK or BIK-MUT peptides for 72 h before UCHL1 analysis. Adenovirus and peptides were applied at 10 MOI and 10 μM, respectively, in all experiments. Results Because BIK protein undergoes rapid proteasomal degradation, β-galactosidase expression from the reporter cassette in Bik-/- mice was used as a surrogate marker of Bik transcriptional activity and anatomical distribution. Lung tissues from the Bik-/- mice when immunostained for β-galactosidase showed that BIK transcriptional activity was predominantly localized to airway and alveolar epithelial cells. In human bronchial epithelial cells, adenoviral expression of BIK increased UCHL1 expression. Likewise, differentiated MTEC at the ALI treated with lipid-coated BIK or BIK-MUT peptides exhibited elevated UCHL1 expression. Conclusions BIK transcriptional activity is enriched in the airway. The BIK-induced expression of UCHL1 axis was conserved in human and mouse bronchial epithelial cells. Expression of UCHL1 was also efficient in well-differentiated cultures that were treated with lipidcoated BIK peptides. These findings highlight a potential therapeutic pathway, with future studies aimed at defining how BIK-UCHL1 signaling contributes to regeneration and repair of the lung parenchyma. This abstract is funded by: RO1HL068111 and R01HL16699