# p16INK4a+ fibroblasts regulate epithelial regeneration after injury in lung alveoli through the SASP

> **NIH NIH K99** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2023 · $177,984

## Abstract

PROJECT SUMMARY ABSTRACT
Cellular Senescence is as an acquired state cells enter in response to environmental stressors or tumor
evasion. Senescent cells cease their proliferative capacity and enhance their ability to respond and regulate
the microenvironment through the senescence-associated secretory phonotype (SASP). Another shared
characteristic of cellular senescence is the upregulation of the tumor suppressor cyclin inhibitor p16INK4a. With
age there’s accumulation of senescence cells in tissues along with the upregulation of p16INK4a expression.
While removal of p16INK4a expressing cells using genetic mouse tools slows down aging, it also adversely
impacts wound healing during injury repair, suggesting contradictory roles of p16INK4a during homeostasis and
injury response. We generated an ultra-sensitive p16INK4a reporter mouse line name, named INK4A H2B-GFP
Reporter-In-Tandem, or INKBRITE to further understand in vivo p16INK4+ cells. In INKBRITE adult lungs,
p16INK4+ cells are predominantly within immune and fibroblasts populations. p16INK4+ fibroblasts express
features of senescence including polyploidy, increase in cell size, low proliferation capacity and ability to
promote airway epithelial cell growth after injury. My main goal is to determine if the capacity to promote
regeneration is restricted to airway p16INK4+ and epithelium or other regionally specific p16INK4 expressing
fibroblast can support epithelial growth. Whether the capacity to promote epithelial regeneration is restricted to
airway p16INK4+ fibroblasts or other spatially defined fibroblast subpopulations such as alveolar fibroblast can
promote epithelial growth through SASP, remains unknown. To fill this knowledge gap, I will isolate alveolar
p16INK4a+ fibroblasts and determine their capacity to promote epithelial growth after acute epithelial injury by 1)
ex vivo 3D co-culture assay, 2) identify the transcribed SASP through RNA sequencing, and 3) in vivo using
known senolytics Dasatinib and Quercetin (D&Q). Our p16INK4a induction and knockdown studies showed the
requirement of p16INK4a for expression of known SASP factors such as IL6, Ereg, Ccl8. Another aspect that has
been largely underexplored in the identity and dependence p16INK4 expression of SASP factors in vivo. For the
R00 phase of my proposed work, I will further explore how the expression of p16INK4 is able to reprogram the
SASP to support tissue repair, specifically epithelial regeneration. I will with our tools to functionally induce and
remove p16INK4 in fibroblasts and 1) asses epithelial growth, 2) transcriptome analysis, and 3) proteomics to
capture secreted proteins to identify p16INK4a-dependent SASP factors. I have extensive knowledge on working
with INKBRITE and the tools to manipulate p16 INK4a expression which will allow me to pursue the proposed
work with ease. With additional training from Drs. Peng and Sheppard I will expand my current knowledge of
lung biology and cellular senescence while e...

## Key facts

- **NIH application ID:** 10643269
- **Project number:** 1K99HL168365-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Nabora Soledad Reyes de Barboza
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $177,984
- **Award type:** 1
- **Project period:** 2023-05-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10643269

## Citation

> US National Institutes of Health, RePORTER application 10643269, p16INK4a+ fibroblasts regulate epithelial regeneration after injury in lung alveoli through the SASP (1K99HL168365-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10643269. Licensed CC0.

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