D19-a - Mechanical Signaling-Mediated PKM2 and Hsp27 Phosphorylation Regulate Fibroblast Fibrotic Phenotype

Saturday, October 11, 2025

10:00 AM - 12:00 PM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

VW

Vincent Wollner

Research Intern
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital., United States

Co-Author(s)

CB

Casey Baxter

Researcher
Baylor College of Medicine, United States
AK

Arin Kumar

Research Intern
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital., United States
JT

Jaime Tellez

Researcher
Baylor College of Medicine, United States
Mary Elizabeth Guerra, MD

Research Assistant
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital., United States
TL

Taylor Lee

Researcher
Baylor College of Medicine, United States
Varun Akondy, n/a

Research Intern
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children's Hospital.
Cypress, Texas, United States
SK

Sonya Keswani, n/a

Researcher
Baylor College of Medicine, United States
ZC

Zara Chandrasekhar

Research Intern
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital., United States
ML

Mary Hui Li

Research Scientist
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital., United States
Ling Yu, PhD

Research Scientist
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital., United States
Sundeep Govind Keswani, MD, MBA, FACS, FAAP

Researcher
Baylor College of Medicine
Houston, Texas, United States

Primary Investigator(s)

Swathi Balaji, PhD

Associate Professor
Laboratory for Regenerative Tissue Repair, Department of Pediatric Surgery, Baylor college of Medicine and Texas Children’s Hospital.
Houston, Texas, United States

Introduction: : Fibroblasts (FB) respond to changes in mechanical forces in the wound milieu by reorganizing the actin cytoskeleton. Heat shock protein (Hsp27) phosphorylation is known to govern actin dynamics and contractility. Oxidative phosphorylation and glycolysis regulate lactate and ATP during this process, with pyruvate kinase (PK) being the rate-limiting enzyme in glycolysis. The role of PKM1/M2 isoforms and Hsp27 in fibroblast responses during wound healing is unknown. We hypothesize that fibroblasts regulate their metabolism and fibrotic phenotype in response to mechanical signaling via phosphorylation changes in PKM2 and Hsp27-actin cytoskeleton dynamics.

Materials and

Methods: : FB were isolated from 8wk C57BL/6J mice and cultured on either flexible silicone membranes (flex – 930kPa Young's modulus) or tissue culture plastic dishes (TCP - 1x107kPa) with +/- TGF-b (10ng/ml, 24h). Total and phosphorylated PKM1/M2 and Hsp25 (the mouse homolog of Hsp27) were analyzed using immunoblotting. Hsp25 intracellular expression and alignment with actin were assessed by immunofluorescence. a-SMA levels (qRT-pcr; immunoblotting) were analyzed. Lastly, the effects of TEPP (100uM), which regulates p-PKM2 and glycolysis, and J2 (10uM), an Hsp25 inhibitor, were tested. n=3/group.

Results, Conclusions, and Discussions:: PKM2 (1.5-fold) and a-SMA (10-fold) expression was higher in cells on TCP compared to cells on flex at baseline. Hsp27 staining was more perinuclear in cells on flex but co-localized with a-SMA in cells on TCP. TGF-b treatment induced increases in total PKM2 (1.7-fold), total Hsp-25 (2.2-fold), and total p-Hsp25 (4-fold) in cells on TCP. TGF-b treatment on flex induced more modest increases in total PKM2 (1.4-fold) and p-Hsp25 (1.8-fold). Total Hsp25 did not show increased expression after TGF-b treatment on flex. PKM1 expression was constant across stiffness conditions and TGF-b treatment. p-PKM2 expression increased after treatment with TGF-b in cells on TCP and flex. TEPP treatment was effective in reducing the effect of TGF-b on PKM2, Hsp25, and p-Hsp25 on both TCP and flex, with expression levels returning closer to baseline. J2 treatment in cells on TCP was effective in reducing the effect of TGF-b on Hsp25 and p-Hsp25 but was less effective in reducing p-Hsp25 levels on flex. In contrast, J2 treatment was more effective in reducing PKM2 levels in cells on flex compared to TCP. TGF-b induced more a-SMA in cells on flex compared to TCP (2.3 vs 1.6-fold). TEPP treatment reduced a-SMA in cells on both TCP and flex, while J2 treatment was more effective in cells on TCP.

Acknowledgements and/or References (Optional): : Our data shows that fibroblasts display distinct PKM2 and Hsp27 activation in different microenvironmental stiffness conditions, with increases in glycolysis and a-SMA expression in stiffer environments. We further show that fibroblasts are amenable to treatment with TEPP to regulate glycolysis and fibrosis markers in varying stiffness conditions, while J2 is most effective in higher stiffness environments.