The epithelial-mesenchymal transition (EMT) is a key process in fibrogenic diseases where transdifferentiated myofibroblasts produce excessive amounts of extracellular matrix, resulting in organ dysfunction. Idiopathic epiretinal membrane (iERM) is a vision-threatening disorder characterized by fibrocellular proliferation and contraction on the central retina. Müller glial cells, which regulate retinal physiology and structure, are the major cellular components in the iERM tissue; however, the pathological role of this cell type remains incompletely understood. Here we revealed the involvement of Müller glial-mesenchymal transition (GMT), as an alternative to EMT, in the pathogenesis of iERM lacking epithelial contribution in nature. Of various pro-fibrotic cytokines, transforming growth factor (TGF)-β1 stimulation to human Müller glial cells exclusively increased mRNA and protein levels of several EMT-related molecular markers, together with the transcription factor SNAIL but not SLUG or TWIST. TGF-β1-stimulated Müller cells also exhibited EMT-related cell motility, while reducing the expression of glutamine synthetase (GS), a Müller glial marker. Notably, all of these TGF-β-induced EMT features were reversed by SNAI1 knockdown in Müller cells. iERM patient specimens demonstrated co-immunolocalization of SNAIL with TGF-β1, GS, and smooth muscle protein 22. Our data implicated a critical role of the TGF-β-SNAIL axis in Müller GMT to promote iERM formation.

Galectin‐1/LGALS1 is a hypoxia‐induced angiogenic factor associated with diabetic retinopathy (DR). Recently, we elucidated a hypoxia‐independent pathway to produce galectin‐1 in Müller glial cells stimulated by interleukin (IL)‐1β. Here we revealed glucocorticoid receptor (GR)‐mediated inhibitory mechanisms for Müller glial galectin‐1/LGALS1 expression. Activator protein (AP)‐1 site in the LGALS1 enhancer region, to which activating transcription factor2, c‐Fos and c‐Jun bind, was shown to be essential for IL‐1β‐induced galectin‐1/LGALS1 expression in Müller cells. Ligand (dexamethasone or triamcinolone acetonide)‐activated GR induced dual specificity phosphatase (DUSP)1 expression via the glucocorticoid response element and attenuated IL‐1β‐induced galectin‐1/LGALS1 expression by reducing phosphorylation of these AP‐1 subunits following AKT and extracellular signal‐regulated kinase (ERK)1/2 deactivation. Moreover, activated GR also caused DUSP1‐independent down‐regulation of IL‐1β‐induced LGALS1 expression via its binding to AP‐1. Administration of glucocorticoids to mice attenuated diabetes‐induced retinal galectin‐1/Lgals1 expression together with AKT/AP‐1 and ERK/AP‐1 pathways. Supporting these in vitro and in vivo findings, immunofluorescence analyses showed co‐localization of galectin‐1 with GR and phosphorylated AP‐1 in DUSP1‐positive glial cells in fibrovascular tissues from patients with DR. Our present data demonstrated the inhibitory effects of glucocorticoids on glial galectin‐1 expression via DUSP1‐dependent and ‐independent deactivation of AP‐1 signalling (transactivation and transrepression), highlighting therapeutic implications for DR.

(Pro)renin receptor [(P)RR], a new component of the tissue renin-angiotensin system (RAS), plays a crucial role in inflammation and angiogenesis in the eye, thus contributing to the development of proliferative diabetic retinopathy (PDR). In this study, we investigated systemic factors related to plasma levels of soluble form of (P)RR [s(P)RR] in patients with PDR. Twenty type II diabetic patients with PDR and 20 age-matched, non-diabetic patients with idiopathic macular diseases were enrolled, and plasma levels of various molecules were measured by enzyme-linked immunosorbent assays. Human retinal microvascular endothelial cells were stimulated with several diabetes-related conditions to evaluate changes in gene expression using real-time quantitative PCR. Of various systemic parameters examined, the PDR patients had significantly higher blood sugar and serum creatinine levels than non-diabetic controls. Protein levels of s(P)RR, prorenin, tumor necrosis factor (TNF)-α, complement factor D (CFD), and leucine-rich α-2-glycoprotein 1 (LRG1) significantly increased in the plasma of PDR subjects as compared to non-diabetes, with positive correlations detected between s(P)RR and these inflammatory molecules but not prorenin. Estimated glomerular filtration rate and serum creatinine were also correlated with plasma s(P)RR, but not prorenin, levels. Among the inflammatory molecules correlated with s(P)RR in the plasma, TNF-α, but not CFD or LRG1, application to retinal endothelial cells upregulated the mRNA expression of (P)RR but not prorenin, while stimulation with high glucose enhanced both (P)RR and prorenin expression. These findings suggested close relationships between plasma s(P)RR and diabetes-induced factors including chronic inflammation, renal dysfunction, and hyperglycemia in patients with PDR.

Galectin-1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin-1β-driven inflammatory pathway for galectin-1 expression in vitro and in vivo. Here, we show glucocorticoid-mediated inhibitory mechanism against hypoxia-inducible factor (HIF)-1α-involved galectin-1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia-induced increases in galectin-1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia-stimulated cells decreased HIF-1α protein, but not mRNA, together with its DNA-binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co-immunoprecipitation revealed that glucocorticoid-transactivated TSC22D3 interacted with HIF-1α, leading to degradation of hypoxia-stabilized HIF-1α via the ubiquitin-proteasome pathway. Silencing TSC22D3 reversed glucocorticoid-mediated ubiquitination of HIF-1α and subsequent down-regulation of HIF-1α and galectin-1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes-induced retinal HIF-1α and galectin-1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co-localization of galectin-1 and HIF-1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid-transactivated TSC22D3 attenuates hypoxia-and diabetes-induced retinal glial galectin-1/LGALS1 expression via HIF-1α destabilization, highlighting therapeutic implications for DR in the era of anti-vascular endothelial growth factor treatment. K E Y W O R D S diabetic retinopathy, galectin-1, glucocorticoid, HIF-1α, hypoxia, Müller glia, transactivation, TSC22D3