Gooptu, B. | Search bioRxiv

bioRxiv2023

Defining the mechanism of galectin-3-mediated TGF-β1 activation and its role in lung fibrosis.

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-{beta}1 (TGF-{beta}1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF individuals, although its mechanism of action is not precisely defined. We hypothesised that galectin-3 potentiates TGF-{beta}1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-{beta}1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L--lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-{beta}1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to the v integrins, v{beta}1, v{beta}S and v{beta}6 and also to the TGF{beta}RII subunit in a glycosylation-dependent manner. This galectin-3 binding is heterogeneous and not a 1:1 binding stoichiometry. These binding interactions were blocked by small molecule inhibitors of galectin-3 which target the carbohydrate recognition domain. Binding of galectin-3 to the {beta}1 integrin was validated in vitro by co-immunoprecipitation (Co-IP) in HLFs. In addition, proximity ligation assay (PLA) data indicates that galectin-3 and the {beta}1 integrin colocalize closely (40 nm) on the cell surface of HLFs, that colocalization is increased by TGF-{beta}1 treatment and galectin-3 inhibitors prevented this colocalization. In the absence of TGF-{beta}1 stimulation, such colocalization was detectable only in HLFs isolated from IPF patients suggesting that the proteins are inherently more closely associated in the disease state. Taken together, this data suggests that galectin-3 promotes TGF-{beta}1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-{beta}1 signaling cascade. Declaration of InterestsRGJ reports grants or contracts from AstraZeneca, Biogen, Galecto Biotech, GlaxoSmithKline, Nordic Biosciences, RedX, Plaint, consulting fees from Bristol Myers Squibb, Chiesi, Daewoong Veracyre, Resolution Therapeutics and Pliant, honoraria from Boehringer Ingelheim, Chiesi, Roche, PatientMPower, AstraZeneca, advisory roles with Boehringer Ingelheim, Galapagos and Vicore, non-financial support from NuMedii, and is a Trustee for Action for Pulmonary Fibrosis. AEJ reports grant funding from Galecto Biotech. BG reports CASE student project partnership with Galecto Biotech, honoraria from GlaxoSmithKline and Vertex and grants and fellowships from UKRI MRC and BBSRC, Alpha-1 Foundation, BLF/A+LUK, Wellcome Trust. RJS, ACM, FRZ, JFC are Galecto employees with shares/options in the company. NRP is a Roche employee. GH, RML, PS, SBC, DJS declare no competing interests.

molecular biology↗

bioRxiv2022

CD98 is critical for a conserved inflammatory response to diverse injury stimuli relevant to IPF exacerbations and COVID pneumonitis

Progressive fibrosing interstitial lung diseases (PFILDs) cause substantial morbidity and mortality. Antifibrotic agents slow progression, but most of the clinical need remains unmet. The archetypal PFILD is idiopathic pulmonary fibrosis (IPF). Chronic progression is driven by transforming growth factor (TGF-){beta}1 signalling. It is punctuated by inflammatory flares known as acute exacerbations (AE-IPF), which are associated with accelerated decline and high mortality. We hypothesized that acute injury responses underlying exacerbations and the mechanisms of chronic fibrosis overlap at the molecular level, via a cell surface assembly nucleated by galectin-3 that we term the gal-3-fibrosome. We focused upon a putative pro-inflammatory galectin-3 ligand, the CD98:integrin complex. Our data indicate CD98 and {beta}1-integrin co-localise with galectin-3 within epithelial cells in IPF lung tissue, and within 40 nm in human lung tissue treated with TGF-{beta}1 compared to controls. CD98 is required for interleukin (IL-)6 and IL-8 responses to biochemical and biophysical conditions mimicking stimuli of AE-IPF in vivo, ex vivo and in cells, and for an interstitial neutrophilic response in a mouse model. We demonstrate this pathway progresses via intracellular influx of Ca2+ mediated by TRPV4, and NF-{kappa}B activation, operating in positive feedback. Lastly we show the CD98- and galectin-3-dependence of IL-6 and IL-8 responses to the SARS-CoV-2 spike protein receptor binding domain and the conservation of this response pattern between lung epithelial cells and monocyte-derived macrophages. Taken together our findings identify CD98 as a key mediator of both pro-fibrotic and acute inflammatory responses in the lung with relevance to AE- and chronic progression of IPF, and the priming of fibrotic lungs for acute inflammatory responses. They similarly implicate CD98 and galectin-3 as mediators of COVID pneumonitis and worse outcomes in ILD patients with COVID.

cell biology↗