Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells
Vitaly Ievlev, … , John F. Engelhardt, Kalpaj R. Parekh
Vitaly Ievlev, … , John F. Engelhardt, Kalpaj R. Parekh
Published December 13, 2022
Citation Information: JCI Insight. 2023;8(2):e162041. https://doi.org/10.1172/jci.insight.162041.
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Research Article Pulmonology Stem cells

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

Abstract

Keratin expression dynamically changes in airway basal cells (BCs) after acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In bronchiolitis obliterans (BO) after lung transplantation, BC clonogenicity declines, which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated these keratins’ roles using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs did not differentiate into club and ciliated cells but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo after injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, whereas dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts with Krt14-KO phenotype and resembles the phenotype in BO with decreased clonogenicity, increased Krt14, and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior, which is relevant in chronic disease states like BO.

Authors

Vitaly Ievlev, Thomas J. Lynch, Kyle W. Freischlag, Caitlyn B. Gries, Anit Shah, Albert C. Pai, Bethany A. Ahlers, Soo Park, John F. Engelhardt, Kalpaj R. Parekh

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Figure 7

Krt14-KO airway BCs lose Sfn and upregulate p63, whereas Krt15-KO cells downregulate p63 early in differentiation.

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Krt14-KO airway BCs lose Sfn and upregulate p63, whereas Krt15-KO cells ...
(A–C) Immunofluorescence staining of Krt14 and Sfn in Krt14-WT (A), Krt14-gRNA–treated cells (B) and Krt15-gRNA–treated primary airway BCs (C). (D) Quantification of immunofluorescence staining for Sfn from panels A–C. (E) Proximity ligation assay (PLA) of the interaction between Krt14 or Krt15 and Sfn or negative control (NC) in passage 2 (p2) healthy human SAE and quantification of the PLA signal. (F and G) Western blot analysis of the cytosolic (F) and nuclear (nucl) (G) fractions collected from Krt14-WT and Krt14-KO airway BCs on ALI day 5. (H) Normalized quantification of Western blot band intensity. (I) qPCR analysis of Krt14-KO and Krt14-WT differentiating cultures on ALI day 5. (J) Western blot analysis of the nuclear fraction collected from Krt15-KO airway BCs on ALI day 5. (K) qPCR analysis of Krt15-KO and Krt15-WT differentiating cultures on ALI day 5. (L) Quantification of CFE of dNp63-KO compared with Scrambled-KO primary mouse SAE cells. Crispr-KO and CFE assays were performed similarly to Krt15-KO in Figure 5. (E, H, I, and L) Graphs show mean data ± SEM from 3 independent pools of primary cells. (K) Graph mean data ± SEM of technical replicates from a cell pool isolated from 3 independent Krt15-WT or KO mice. (F, G, and J) Lanes were run on the same gel but were noncontiguous. Statistical significance in was determined by 2-way ANOVA, Sidak multiple comparison test (H, I, and K); by 1-way ANOVA, Tukey multiple comparison test (D and E); or by 2-tailed t test in L; ****P < 0.0001). Scale bars: 20 μm.