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Calver, J.F. (2023) Defining the mechanism of galectin-3-mediated TGF-β1 activation and its role in lung fibrosis. PhD thesis, University of Nottingham.

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Abstract

Introduction: Traction-dependent activation of the pro-fibrotic mediator transforming growth factor-β1 (TGF-β1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 potentiates TGF-β1 signaling to promote fibrogenesis and the efficacy of galectin inhibition in IPF patients is currently under investigation. Small molecule galectin-3 inhibitors block lysophosphatidic acid (LPA)-induced and TGF-β1-induced suppressor of mothers against decapentaplegic (SMAD) signaling in human lung fibroblasts (HLFs). However, the exact mechanism by which galectin-3 promotes fibrogenesis has not yet been defined. It was hypothesised that galectin-3 promotes TGF-β1 SMAD signaling and induces fibrogenesis by interacting directly with components of this signaling cascade in fibroblasts.

Methods: Full length galectin-3 protein was expressed and purified in-house (bacterial expression system). Surface plasmon resonance (SPR) was used to determine whether galectin-3 physically interacts with αv integrins, proteins constituting the large latent TGF-β1 complex (latency-associated peptide (LAP) and latent TGF-β binding protein 1 (LTBP1)) or the TGF-β1 receptor subunits (TGFβRI/ TGFβRII). SPR data was analysed in GraphPad Prism and on-off rate maps plotted in EVILFIT. The effects of protein deglycosylation or small molecule galectin inhibitors on binding was investigated and solution competition binding curves plotted in GraphPad Prism. Key binding data was validated in vitro by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA) in HLFs.

Results: By SPR galectin-3 bound to the αv integrins and TGFβRII subunit in a glycosylation-dependent manner, with enzymatic removal of glycans blocking the binding events. This galectin-3 binding was heterogeneous and not a 1:1 binding stoichiometry. Small molecule galectin-3 inhibitors blocked these binding interactions via the carbohydrate-recognition domain (CRD). No galectin-3 binding was detected to LAP, LTBP1 or the TGFβRI subunit. The binding of galectin-3 to the β1 integrin was validated in HLFs by Co-IP and PLA. Galectin-3 successfully co-immunoprecipitated with β1 integrin pulldown and inversely. Galectin-3 and the β1 integrin were also colocalised within 40 nm in IPF HLFs and galectin-3 inhibition prevented this colocalisation.

Conclusions: This work has encompassed cell, molecular and biophysical methods to define the mechanism of galectin-3-mediated TGF-β1 activation in fibroblasts and its potential role in IPF development. Galectin-3 bound to the αvβ1 integrin and TGFβRII subunit and this binding was blocked by galectin-3 inhibitors. Future work is required to validate the galectin-3-TGFβRII SPR binding data in vitro. It is hypothesised that upon binding, galectin-3 self-associates to form a lattice between the αvβ1 integrin and TGF-β1 receptor on adjacent cells to facilitate receptor clustering. Subsequently, when TGF-β1 is activated (traction-dependent), it is in close enough proximity to the TGF-β1 receptor to bind and signal, causing fibrogenesis. Understanding the precise role of galectin-3 in IPF pathogenesis may be critical for the continued development of more effective and selective treatments for IPF patients.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	John, A.E. Jenkins, R.G. Scott, D.J. Tatler, A.L.
Keywords:	Pulmonary fibrosis, fibroblast, transforming growth factor beta (TGF‐β), integrin, galectin
Subjects:	W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	76234
Depositing User:	Calver, Jessica
Date Deposited:	13 Dec 2023 04:40
Last Modified:	13 Dec 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/76234

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