Substrate Stiffness Modulates TGF-β Activation and ECM-Associated Gene Expression in Fibroblasts

Area of Science:

• Biotechnology
• Cell Biology
• Biomaterials Science

Background:

• Transforming growth factor-β (TGF-β) is a key cytokine regulating extracellular matrix (ECM) gene expression during tissue injury and fibrosis.
• Fibrosis development is influenced by both biochemical and mechanical factors in the tissue microenvironment.
• The specific role of substrate stiffness in modulating TGF-β activity and ECM gene expression in fibroblasts remains underexplored.

Purpose of the Study:

• To investigate the effect of varying substrate stiffness on TGF-β protein activation in HMF3S fibroblasts.
• To analyze the impact of substrate stiffness on the expression of ECM-associated genes and matrix metalloproteinases (MMPs).
• To understand the relationship between substrate stiffness and fibroblast morphology, proliferation, and cell cycle progression.

Main Methods:

• Fabrication of polydimethylsiloxane (PDMS) substrates with defined stiffness (40 kPa, 300 kPa, 1.5 MPa).
• Culture of HMF3S fibroblasts on substrates of varying stiffness.
• Quantification of TGF-β protein secretion using a reporter assay.
• Analysis of ECM gene expression (Collagen I, Collagen III, integrin α5) and MMP activity (MMP-2, MMP-9) via RT-PCR, Western blotting, and gelatin zymography.
• Assessment of cell and nuclear morphology, tubulin expression, proliferation, and cell cycle status.

Main Results:

• TGF-β protein activation was significantly reduced on softer substrates (40 kPa and 300 kPa).
• Integrin α5 expression decreased on softer substrates, correlating with impaired TGF-β activation.
• Expression of Collagen I, Collagen III, and MMP-2 was lower on softer substrates; MMP-9 activity was minimal across all stiffnesses.
• Fibroblasts exhibited rounded cell and nuclear morphologies with increased tubulin expression on compliant substrates.
• Cell proliferation was higher on stiffer substrates, while softer substrates induced cell cycle arrest.

Conclusions:

• Substrate stiffness plays a critical role in regulating fibroblast-mediated ECM production and TGF-β signaling.
• Mechanical cues from the microenvironment significantly influence fibroblast behavior, impacting fibrotic processes.
• These findings highlight feedback mechanisms between substrate stiffness and ECM regulation, relevant to understanding and potentially treating fibrosis.