Dissertation
Airway stem cell fate decisions during regeneration are influenced by Sox9, Krt14 and Krt15
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2022
DOI: 10.25820/etd.007524
Abstract
Respiratory diseases are an enormous burden on the society being the third most common leading cause of death in the United States. Airways and alveoli are the two major epithelial compartments that get damaged and need to be repaired in lung diseases and injuries. In this work we describe a novel ex vivo ferret tracheal injury model and the mechanisms that govern regeneration of proximal airways. In particular, we investigate how the interactions between Sox9 and Wnt signaling as well as the balance between keratins 14 and 15 influence stem cell commitment to the surface epithelial repair. Through a combination of knockout and lineage tracing experiments we found that Sox9 transiently marks a glandular stem cell compartment capable of surface epithelial regeneration in Wnt dependent manner. Sox9 has been previously demonstrated to be a negative feedback mediator that represses Wnt signals. We further demonstrate that Wnt signaling promotes proliferation of airway glandular progenitors while Sox9 is necessary for their efficient migration to the airway surface. Transit-amplifying progenitors and slow-cycling basal stem cells can be distinguished based on their keratin expression: Krt14 expression is upregulated after injury in cycling progenitors, while Krt15 is present at homeostasis in quiescent basal stem cells. We demonstrate that these keratins not only mark the fate transition of airway basal progenitors, but also affect their proliferative capacity and differentiation. Specifically, Krt14 knockout primary airway basal cells have increased proliferative capacity, but impaired club cell differentiation, while the opposite is true for Krt15 knockout. One of the key differences between Krt14 and Krt15 that can explain this outcome is the presence of a conserved Cysteine 373 residue in Krt14 that is capable of forming interfilamentous S-S bonds and recruiting Sfn, which is a tumor suppressor protein known to stabilize nuclear p53 and target dNp63α for degradation. We observed increased dNp63α expression in Krt14-KO and a decreased dNp63α expression in Krt15-KO, which can mechanistically explain how the balance between Krt14 and Krt15 can affect airway stem cell fate decisions. Overall, understanding the circuits that govern stem cell injury responses can inform therapies for respiratory diseases that cause damage to the airway epithelium.
Details
- Title: Subtitle
- Airway stem cell fate decisions during regeneration are influenced by Sox9, Krt14 and Krt15
- Creators
- Vitaly Ievlev
- Contributors
- John Engelhardt (Advisor)Kalpaj Parekh (Advisor)Charles Yeaman (Committee Member)Botond Banfi (Committee Member)Brad Amendt (Committee Member)Amy Ryan (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Cell and Developmental Biology)
- Date degree season
- Autumn 2022
- DOI
- 10.25820/etd.007524
- Publisher
- University of Iowa
- Number of pages
- xvii, 156 pages
- Copyright
- Copyright 2022 Vitaly Ievlev
- Grant note
- This work was supported by grants from the National Institutes of Health P30 DK054759, P01 HL152960, R01 DK047967 to JFE, and a grant from the Cystic Fibrosis Foundation to JFE. RNA-seq data presented herein were obtained at the Genomics Division of the Iowa Institute of Human Genetics, which is supported, in part, by the University of Iowa Carver College of Medicine. Flow cytometry data presented herein were obtained at the University of Iowa Flow Cytometry Facility, which is supported by the University of Iowa Carver College of Medicine. (79) We would like to thank the members of Parekh and Engelhardt labs for discussion of the data used in this paper. This work was funded by the following grants: R01 HL136370 (to KRP); P01 HL152960 (to JFE), NHLBI Contract 75N92019R0014 (to JFE); P30 DK054759 (to JFE), R01 HL165404 (to JFE); T32 HL007638 (to TJL), K99HL155843 (to TJL). Transgenic mice were generated at the University of Iowa Genome Editing Core Facility directed by William Paradee, PhD and supported in part by grants from the NIH and from the Roy J. and Lucille A. Carver College of Medicine. We wish to thank Norma Sinclair, Rongbin Guan and Joanne Schwarting for their technical expertise in generating transgenic mice. (133)
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Date submitted
- 11/21/2022
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 147-156).
- Public Abstract (ETD)
Respiratory diseases are an enormous burden to society being the third most common leading cause of death in the United States. COPD, asthma, COVID-19 and other lung diseases damage airway surface, which is later repaired by stem cells. These dedicated progenitor cells are capable of multiplying and later specializing into different cell types that are essential for antimicrobial defense. To improve the efficiency of airway repair, it is essential to understand what regulates stem cell commitment to regeneration.
In this work we developed a novel airway injury model and used it along with existing models to define the roles of several proteins that can affect stem cell fate decisions. These proteins are: keratin14 (Krt14), keratin15 (Krt15) and Sox9. Krt14 appears on the surface of injured airways and gradually displaces Krt15 over time, while Sox9 marks the stem cells that are normally hidden in the airway glands and migrate onto the surface only after injury.
By removing Krt14 and Krt15 from cells in culture and in mice, we demonstrated that Krt15 is essential for basal stem cell self-renewal while Krt14 is required to specialize generic stem cells into secretory cells that produce mucus to keep the airways hydrated and protected from microbes. The reason why these two structurally similar keratins (Krt14 and Krt15) have such different functions can be explained by a small difference in their molecular composition (amino acid Cys373), which allows Krt14 to cross-link cellular skeleton and make it more rigid. This can recruit other proteins that limit replication and allow stem cells to specialize into differentiated progeny.
We also demonstrate that airway surface repair by Sox9+ gland stem cells cannot occur without Lef1 – a key component of Wnt signaling that stimulates cell division. Interestingly, we show that Sox9 and Lef1 regulate different processes. Specifically, Wnt promotes progenitor cell expansion and activates Sox9, while Sox9 then represses Wnt and promotes cell migration and long-term survival.
Overall, these findings help us better understand the importance of the above-mentioned proteins in airway repair, which can inform treatments for various lung diseases in the future.
- Academic Unit
- Anatomy and Cell Biology; Craniofacial Anomalies Research Center
- Record Identifier
- 9984647456602771
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