(438) Krt14 and Krt15 Influence Airway Basal Cell Proliferative Capacity and Fate

Background: Airway basal cells (BCs) are important in maintaining airway epithelial homeostasis integrity and are marked by expression of Keratin5 (Krt5) - an abundant cytoskeletal protein that can heterodimerize with Keratin14 (Krt14) or Keratin15 (Krt15) to form intermediate filaments. Krt15 is expressed in BCs at homeostasis in vivo, while Krt14 is expressed in BCs during injury- or diseases-associated regeneration of the surface airway epithelium (SAE). This altered Krt14-expressing BC state has been associated with a decline in stem cell function, but the causality of this association and the mechanisms by which Krt14 and Krt15 impact BC behavior have not been evaluated.

Methods: In this study we used mouse genetic knockout approaches in vitro and in vivo to study the functions of Krt14 and Krt15 in BCs. We additionally present there a novel ex vivo ferret tracheal injury model that provides new insights into the mechanics of airway epithelial wound repair.

Results: After evaluating the keratin profile of cultured mouse tracheal BCs we found that it resembles that of BCs in vivo following injury, with enhanced Krt14 expression and reduced Krt15 expression after several passages. CRISPR/Cas9-mediated knockout of Krt14 and Krt15 in primary BCs leads to opposite phenotypes: loss of Krt15 impairs colony formation efficiency (CFE) on irradiated fibroblasts, while the loss of Krt14 enhances CFE. Knockout of Krt14 also leads to re-activation of Krt15 expression in advanced-passage BCs. We generated in vivo murine knockout models of Krt14 and Krt15 and observed that loss of Krt14 leads to reduced size of submucosal glands (SMGs), where Krt14 is expressed at homeostasis in glandular myoepithelial and ductal basal cells. Krt14-KO mice also contained reduced club cells numbers in the trachea. Additionally, the nuclei of Krt14-KO BCs appeared enlarged and elongated during air-liquid interface differentiation and produced fewer club cells. Krt14-KO cultures had reduced levels of tumor suppressor Sfn and increased oncogene p63 early in differentiation. Krt15-KO mice had fewer label retaining basal cells 21 days after injury, suggesting a role for this keratin in stem cell maintenance. However, Krt15 appears to be dispensable for club and ciliated cell differentiation in vivo and in vitro.

Conclusions: Regeneration by basal stem cells occurs in most lung diseases that lead to injury of the airway epithelium, including cystic fibrosis. The mechanisms that drive commitment of basal stem cells to regenerate an injured airway are not completely understood. In this work, we demonstrate that regenerative changes in BC Krt15 and Krt14 expression have a direct impact on BC behavior by affecting structure, function and fate. Overall, this work suggests that keratins may be actively involved in regulation of BC fate decisions (see the graphical abstract for details).

Acknowledgements:

References: