Q11 - Fibroblast mechanoperception instructs pulmonary developmental and pattern specification gene expression programs

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

RB

Rishi Bhogaraju

Undergraduate Researcher
University of Virginia, United States

Primary Investigator(s)

AM

Andrew Miller

Visiting Scholar
University of Virginia, United States

Co-Author(s)

TB

Thomas H. Barker, PhD

Professor
University of Virginia
Charlottesville, Virginia, United States

Last Author(s)

TB

Thomas H. Barker, PhD

Professor
University of Virginia
Charlottesville, Virginia, United States

Co-Author(s)

MC

Mete Civelek

Professor
University of California, Los Angeles, United States
Daniel Abebayehu, PhD

Assistant Professor
University of Virginia, United States
RH

Riley Hannan

Postdoctoral Fellow
University of Virginia, United States
PH

Ping Hu

Director of Research
Vasarya Therapeutics, United States

Introduction: : HOXA5 is a developmental transcription factor responsible for lung patterning and respiratory development in the embryo. Homozygous knockout of HOXA5 is embryonic lethal due to alveolar simplification and tracheal malformation, making HOXA5 requisite for complete airway maturation [1,2]. We have previously shown that loss of Thy-1 (CD90) impairs cellular mechanoperception, the cell’s ability to appropriately respond to ECM stiffness cues, in pulmonary fibroblasts by disrupting the stoichiometry of Fn-binding integrins on the fibroblast cell surface. Loss of Thy-1 leads to a myofibroblast morphology on healthy (soft) tissue stiffnesses and has been previously tied to idiopathic pulmonary fibrosis progression and incomplete fibrosis resolution in bleomycin mouse models [3, 4]. Past studies have shown Thy-1’s role in weakly inhibiting ⍺vβ3 integrin avidity, suggesting that Thy-1 loss impairs the fibroblast’s ability to properly sense ECM mechanics by disrupting the balance of active ⍺vβ3: ⍺5β1 integrin [3, 5]. Multi-omic (ATAC-seq and RNA-seq) interrogation of Thy-1KO fibroblasts revealed epigenetic and transcriptional silencing of HOXA5 across varying substrate stiffness conditions, yet the connection between integrin signaling and HOXA5 expression remains unknown. This study aims to address the hypothesis that HOXA5 is (i) a mechanoresponsive transcription factor as well as (ii) sensitive to integrin binding stoichiometry and downstream focal adhesion signaling on the cell surface.

Materials and

Methods: : Immortalized human lung fibroblasts were transfected with a CRISPR/Cas9 knockout for Thy-1. For genome and chromatin architecture analysis, wild type (WT) and Thy-1 knockouts (Thy-1KO) were cultured on 4 kPa and 25 kPa hydrogels as well as tissue culture plastic (TCP; ~1GPa) for either 24 or 72 hours. RNA and DNA were harvested for RNA-seq and ATAC-seq after the appropriate culture time for library generation and analysis. WT and Thy-1KO fibroblasts were cultured on 5 kPa, 100 kPa, and 1 GPa substrates, and lysed for western blot analysis of HOXA5 expression in response to substrate stiffness. To capture the interplay of integrin stoichiometry and HOXA5 expression, WT and Thy-1KO cells were cultured on 5 kPa, 100 kPa, and 1 GPa substrates coated with the engineered fibronectin fragments Fn4G (favoring ⍺vβ3 binding), Fn9*10 (favoring ⍺5β1binding), or full fibronectin (Fnfull). These samples were lysed and analyzed via western blot to determine the impact of substrate stiffness and integrin utilization on HOXA5 expression. To assess the independent roles of cytoskeletal regulation and focal adhesion signaling on HOXA5 expression, both WT and Thy-1KO cells were again cultured on 5 kPa, 100 kPa, and 1 GPa substrates and stimulated with cytoskeletal inhibitors Y-27632 (Rho associated protein kinase inhibitor) or Latrunculin A (depolymerizes actin), and c-SRC or SRC Family Kinase inhibitors KB SRC4 and PP2, respectively. The inhibition studies concluded with western blot analysis of HOXA5 expression, serving to analyze HOXA5 expression after disrupting known cytoskeletal and focal adhesion signaling pathways.

Results, Conclusions, and Discussions:: Analysis of ATAC-seq and RNA-seq data showed an overall decrease in HOXA5 transcription as well as reduced chromatin accessibility at the HOXA5 promoter site in Thy-1KO fibroblasts (Figure 1A). Further validation via western blot analysis showed decreased HOXA5 expression in Thy-1KO cells across all substrate conditions (5kPa, 100kPa, and 1 GPa) compared to WT cells. Strikingly, HOXA5 expression in WT cells was negatively correlated with substrate stiffness, with expression on 1 GPa substrates matching Thy-1KO levels, suggesting a role for cytoskeletal and/or integrin signaling pathways in HOXA5 suppression (Figure 1B, C). Use of cytoskeletal inhibitors Latrunculin A and Y-27632 showed an increase in HOXA5 expression, indicating cytoskeletal contractility as an active negative regulator of HOXA5 (Figure 1D, E). However, only Latrunculin A induced a statistically significant increase in HOXA5 expression in WT cultures while only Y-27632 increased expression in Thy-1KO cultures. Stimulation with KB SRC4 and PP2 lead to significant increases in HOXA5 expression in both WT and Thy-1KO cells cultured on TCP, underscoring HOXA5 as a target in mechanically sensitive focal adhesion pathways in stiff environments (Figure 2A). When cultured on plates coated with Fn4G, both WT and Thy-1KO cells showed a significant decrease in HOXA5 expression on soft substrates. Thus, regardless of Thy-1 status, increased ⍺vβ3 avidity forces a decrease in HOXA5 expression, highlighting a direct connection between integrin stoichiometry and HOXA5 activation. In total, these results suggest HOXA5 is a novel mechanoresponsive developmental factor, whose expression dynamics are mediated by the stoichiometry of the Fn-binding integrins ⍺vβ3 and ⍺5β1. This study presents a novel link between integrin stoichiometry, focal adhesion signaling, and the regulation of developmental transcription factors, specifically those essential to the development of the embryonic lung.

Acknowledgements and/or References (Optional): : [1] Aubin, J., Lemieux, M., Tremblay, M., Bérard, J. & Jeannotte, L. (1997) Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects. Dev Biol 192(2), 432-445 https://doi.org/10.1006/dbio.1997.8746
[2] Kinkead, R., Leblanc, M., Gulemetova, R., Lalancette-Hebert, M., Lemiux, M, Mandeville, I., Jeannotte, L. (2004). Respiratory adaptations to lung morphological defects in adult mice lacking Hoxa5 gene function. Pediatric Research 56, 553-562. https://doi.org/10.1203/01.PDR.0000139427.26083.3D
[3] Fiore, V.F. Wong, S.S, Tran, C., Tan, C., Xu, W., Sulchek, T., White, E.S., Hagood, J.S., Barker, T.H. (2018). αvβ3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis. JCI Insight 3(20). https://doi.org/10.1172/jci.insight.97597
[4] Hagood, J.S., Prabhakaran, P., Kumbla, P., Salazar, L., MacEwen, M.W., Barker, T.H., Ortiz, L.A., Schoeb, T., Siegal, G.P., Alexander, C.B., Pardo, A., Selman, M. (2005) Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis. The American Journal of Pathology 167(2), 365-379. https://doi.org/10.1016/S0002-9440(10)62982-3
[5] Fiore, V.F., Strane, P.W., Bryskin, A.V., White, E.S., Hagood, J.S., Barker, T.H. (2015). Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction. J Cell Biol 211(1), 173-190. https://doi.org/10.1083/jcb.201505007