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

Verma, BK and Chatterjee, A and Kondaiah, P and Gundiah, N (2023) Substrate Stiffness Modulates TGF-β Activation and ECM-Associated Gene Expression in Fibroblasts. In: Bioengineering, 10 (9).

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Official URL: https://doi.org/10.3390/bioengineering10090998


Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates the expression of ECM-associated genes during early injury. Tissue fibrosis development is driven by synergistic cues between the evolving biochemical and mechanical milieu. Few studies have addressed the role of substrate stiffness on TGF-β activity and extracellular matrix (ECM)-associated genes. We used a commercial formulation of polydimethylsiloxane (PDMS) to fabricate substrates of 40 kPa, 300 kPa, and 1.5 MPa stiffness, and cultured the HMF3S fibroblasts on substrates. We quantified TGF-β protein secreted by HMF3S cells on different substrates using a TGF-β responsive promoter reporter assay. We also tested for variations in gene expression levels on the substrates using RT-PCR and Western blotting and determined the MMP-2 and MMP-9 activities with gelatin zymography. The results showed that TGF-β protein activation was significantly compromised at lower stiffnesses. The expression of integrin α5 decreased on lower stiffness substrates and correlated with inefficient TGF-β protein activation. Collagen I, collagen III, and MMP-2 expression levels were lower on softer substrates; there was little MMP-9 activity on all substrates. Cell and nuclear morphologies were more rounded on compliant substrates, correlating with increased tubulin expression. Proliferations were higher on stiffer substrates, whereas cells on softer substrates showed cell cycle arrest. These results demonstrated critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts, relevant in fibrosis. © 2023 by the authors.

Item Type: Journal Article
Publication: Bioengineering
Publisher: Multidisciplinary Digital Publishing Institute (MDPI)
Additional Information: The copyright for this article belongs to the Authors.
Keywords: cell–matrix interactions; collagen; MMP; polydimethylsiloxane; TGF-β, substrate stiffness
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Division of Electrical Sciences > Electronic Systems Engineering (Formerly Centre for Electronic Design & Technology)
Division of Mechanical Sciences > Mechanical Engineering
Date Deposited: 17 Dec 2023 08:28
Last Modified: 17 Dec 2023 08:28
URI: https://eprints.iisc.ac.in/id/eprint/83442

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