Canonical and Truncated Transglutaminase-2 Drive TGF-β1 Autocrine Loop to Induce Fibrosis in Graves’ Orbitopathy

To evaluate the role of transglutaminase 2 (TGM2) in fibroblasts of Graves' orbitopathy (GO) and explore its potential mechanisms in orbital fibrosis. A key gene selection model for GO was established through bioinformatics and machine learning. Orbital fibroblasts (OFs) were cultured from orbital connective tissue samples. Subsequently, three-dimensional spheroid models were developed. Lentiviral transduction was used to establish TGM2 overexpression and knockdown models. Fibrosis levels were assessed using Western blot, PCR, and collagen contraction assays. TGM2 activity was evaluated by FITC-cadaverine staining and the colorimetric assay. The canonical and truncated TGM2 isoforms were selectively introduced to restore expression. TGF-β1 levels in cell culture supernatants were measured by ELISA. The results of bioinformatics and machine learning indicate that TGM2 is a key characteristic gene in GO. Knockdown of TGM2 markedly suppresses the expression of fibrosis-related genes and reduces the proliferation, migration, and adhesion capabilities of fibroblasts. TGF-β1 can upregulate TGM2 expression and induces the production of both the canonical and truncated TGM2 forms. Similar results were observed in GO tissues. Restoration of TGM2_V2 expression following knockdown can prevent the inhibitory effects on guanosine triphosphate (GTP). Additionally, TGM2 is involved in the autocrine loop of TGF-β1. TGM2 inhibitors significantly reverse the fibrotic response induced by TGF-β1 in OFs. TGM2 is highly expressed in GO fibroblasts and plays a key role in the TGF-β1 autocrine feedback loop. TGF-β1 induces more truncated TGM2 variants that bypass GTP inhibition, exacerbating fibrosis. Inhibiting TGM2 activity significantly reduces fibrosis markers.