The role of senescence marker P16INK4A in alveolar epithelial stem cells during injury-triggered differentiation

The National Heart, Lung, and Blood Institute (NHLBI) awarded The Regents of the University of California, San Francisco (UCSF) $297,806 on September 30, 2025, under the Cardiovascular Diseases Research program (CFDA 93.837) to investigate the role of the senescence marker P16INK4A in alveolar epithelial stem cells during injury-triggered differentiation.

The research examines how alveolar type 2 cells (AT2s), which serve as lung stem cells, differentiate into alveolar type 1 cells and how the P16 protein drives pathological changes in idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis. The project uses multi-transgenic mouse models and conditional knockout approaches to determine the fate of P16-expressing AT2s in vivo and in vitro and to test whether AT2-specific loss of P16 ameliorates the fibrotic phenotype. The research tests the hypothesis that P16 is required for AT2s to enter a profibrotic state during injury-triggered differentiation and that removing P16 from AT2s will reduce fibrosis progression.

Work is performed in San Francisco, California. The period of performance runs through November 29, 2026. This is a Project Grant, the standard assistance type for individual research projects under the NHLBI portfolio.

Abstract

The delicate lung alveolus relies on endogenous progenitor cells to maintain its function and regenerate after repeated insults. The alveolar epithelium contains alveolar type 2 cells (AT2S), which act as stem cells capable of self-renewal and differentiation into alveolar type 1 cells (AT1S), which cover the alveolus to facilitate gas exchange. AT2 to AT1 differentiation is a highly regulated process dictated by internal and external cues. Single- cell RNA sequencing has identified a "transitional AT2" on this differentiation trajectory, marked by keratin 8 and senescence proteins like P16. In models of resolving lung injury, transitional AT2S gradually disappear as they differentiate into AT1S. However, in idiopathic pulmonary fibrosis (IPF) or severe bleomycin (BLM)-induced pulmonary fibrosis, transitional AT2S persist and are thought to drive the disease. IPF is a devastating progressive age-related lung disease with no cure, and anti-fibrotic medications do not reverse fibrosis, suggesting another mechanism underlies IPF pathogenesis. Using a highly sensitive validated mouse P16 reporter developed in our laboratory, preliminary data show that P16 is rarely expressed in AT2S at steady state but increases in a subset of AT2S and their derivatives after BLM-induced lung injury. I will use a multi-transgenic mouse model of pulmonary fibrosis to determine the fate of P16-expressing AT2S in vivo and in vitro. Using a conditional knock-out of P16 in AT2S, i will test the functional role of P16 in AT2S during injury and repair. My hypothesis is that P16 has no effect on AT2S during homeostasis, but P16 is required for AT2S to enter a profibrotic state and during injury-triggered differentiation. Furthermore, i predict that AT2-specific loss of P16 will ameliorate the fibrotic phenotype in a BLM-induced mouse model. This research will demonstrate how manipulating senescence pathways affects AT2 stem cell function and could lead to new therapeutic targets to reverse pulmonary fibrosis. This proposal is part of a comprehensive training plan built with my mentors to develop the skills and knowledge needed to become a successful independent investigator in lung regeneration with an emphasis on chronic and age-related lung diseases. I have assembled a career development committee composed of physician-scientists and basic scientists from pulmonology and aging who will serve as both scientific and career advisors. Upon completing this training plan, with support from my team and ucsf's strong research environment, i will be well-positioned to launch my career in regenerative medicine and contribute to understanding chronic lung diseases.