Glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced Müller glial galectin-1 expression via HIF-1α destabilization

Abstract

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.

Keywords: HIF-1α; Müller glia; TSC22D3; diabetic retinopathy; galectin-1; glucocorticoid; hypoxia; transactivation.

Figure 1

Glucocorticoid‐mediated suppression of hypoxia‐induced galectin‐1/LGALS1 expression in human Müller glial cells. (A) Müller glial cells were pretreated with aldosterone (Ald, 1 μmol/L), dexamethasone (Dex, 1 μmol/L) or triamcinolone acetonide (TA, 1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h, and LGALS1 gene expression levels were analysed. (B‐D) Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h, and galectin‐1 protein expression levels in culture medium (B) and cell lysate (C, D) were analysed by ELISA (B, C) and immunoblot analysis (D). (E) Müller glial cells were pretreated with the glucocorticoid receptor antagonist RU486 (1 μmol/L) for 30 min before culture with Dex (1 μmol/L) and TA (1 μmol/L) in hypoxia (1% O₂) for 24 h, and LGALS1 gene expression levels were analysed. *P < .05, **P < .01, n = 6 per group

Figure 2

Glucocorticoid‐mediated reduction of HIF‐1α protein and DNA‐binding activity with no impact on LGALS1 mRNA stability. (A) After Müller glial cells were cultured in hypoxia (1% O₂) for 24 h, the transcription inhibitor actinomycin D (2.5 μg/mL) with or without dexamethasone (Dex, 1 μmol/L) or triamcinolone acetonide (TA, 1 μmol/L) was added and cells were harvested at the indicated times. RNA was extracted for real‐time qPCR analysis of LGALS1. (B) Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h, and HIF‐1α protein expression levels were analysed. (C, D) Müller glial cells were transfected with the control reporter pRL‐CMV, together with consensus HRE‐luciferase reporter (C) or human LGALS1 promoter‐luciferase reporter (D). Transfected Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h and assayed for luciferase activities. (E) Müller glial cells were cultured in hypoxia (1% O₂) for 1 h before harvest of samples. Binding of HIF‐1α to HREs in the LGALS1 promoter region was analysed by ChIP‐qPCR. (F) Müller glial cells were pretreated with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h, and HIF1A gene expression levels were analysed. *P < .05, **P < .01, n = 4‐6 per group

Figure 3

Glucocorticoid‐transactivated TSC22D3 interaction with and ubiquitination of HIF‐1α leading to suppression of hypoxia‐induced galectin‐1/LGALS1 expression. (A) Müller glial cells were pretreated with the glucocorticoid receptor antagonist (RU486, 1 μmol/L) for 30 min before culture with dexamethasone (Dex, 1 μmol/L) or triamcinolone acetonide (TA, 1 μmol/L) in hypoxia (1% O₂) for 24 h, and TSC22D3 gene expression levels were analysed. (B) After hypoxic stimulation (1% O₂) for 24 h, co‐IP of human Müller glial cell extracts using anti‐TSC22D3 and anti‐HIF‐1α antibodies was performed, followed by immunoblot analyses for HIF‐1α and TSC22D3. (C) Müller glial cells were incubated in hypoxia (1% O₂) with or without TA in the presence of the proteasome inhibitor MG132 (10 μmol/L) for 24 h. After co‐IP of cell extracts with anti‐HIF‐1α antibody, ubiquitinated HIF‐1α was detected using anti‐ubiquitin (Ub) antibody in TA‐treated cell extracts. (D, E) TSC22D3 (D) and LGALS1 (E) mRNA expression levels in human Müller glial cells exposed to control‐ or TSC22D3‐siRNA combined with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h. (F) Galectin‐1, HIF‐1α and TSC22D3 protein expression levels in human Müller glial cells exposed to control‐ or TSC22D3‐siRNA combined with Dex (1 μmol/L) or TA (1 μmol/L) for 30 min before culture in hypoxia (1% O₂) for 24 h. (G) Müller glial cells transfected with control‐ or TSC22D3‐siRNA were incubated in hypoxia (1% O₂) with or without TA in the presence of MG132 (10 μmol/L) for 24 h. After co‐IP of cell extracts with anti‐HIF‐1α antibody, ubiquitinated HIF‐1α was detected using anti‐Ub antibody. *P < .05, **P < .01, n = 6 per group

Figure 4

Glucocorticoid‐mediated inhibition of diabetes‐induced retinal galectin‐1 and HIF‐1α together with transactivation of TSC22D3 in mice. (A‐C) Retinal Lgals1 (A) and Tsc22d3 (B) expression in mice with streptozotocin (STZ)‐induced diabetes at 2 mo. Dexamethasone (Dex, 50 pmol/eye) or triamcinolone acetonide (TA, 50 pmol/eye) were injected intravitreally to STZ mice, followed by mRNA (A, B) and protein (C) expression analyses after 24 h. (C) Immunoblot analyses for galectin‐1, TSC22D3 and HIF‐1α in the retina of diabetic mice treated with Dex or TA. *P < .05, **P < .01, n = 6‐8 per group

Figure 5

Tissue co‐localization of HIF‐1α and galectin‐1 in glial cells in the epiretinal fibrovascular tissue excised from eyes of patients with PDR. (A‐C) Double labelling of HIF‐1α (green) and GFAP (red) with DAPI (blue) counterstaining in PDR fibrovascular tissues. (D‐F) Double labelling of HIF‐1α (green) and galctin‐1 (red) with DAPI (blue) counterstaining. (G‐I) Double labelling of HIF‐1α (green) and TSD22D3 (red) with DAPI (blue) counterstaining. Scale bar = 20 μm

Figure 6

A schema showing that glucocorticoid‐transactivated TSC22D3 suppresses hypoxia‐ and diabetes‐induced galectin‐1 expression through HIF‐1α destabilization. Glucocorticoid‐bound glucocorticoid receptor (GR) transactivates TSC22D3 via glucocorticoid response element (GRE), causing ubiquitin‐proteasome system (UPS)‐mediated degradation of HIF‐1α, which is otherwise stabilized by hypoxia and diabetes for the induction of Müller glial galectin‐1 expression