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. K E Y W O R D S diabetic retinopathy, galectin-1, glucocorticoid, HIF-1α, hypoxia, Müller glia, transactivation, TSC22D3

Hypoxia Induces Galectin-1 Expression Via Autoinduction of Placental Growth Factor in Retinal Pigment Epithelium Cells

“… 17 We and others demonstrated the hypoxic stimulation of galectin-1/ LGALS1 expression in various cell species. 8 , 10 , 12 , 13 Consistent with previous reports, RPE cells demonstrated time-dependent responsiveness to hypoxia in LGALS1 transcripts ( Fig. 1 A) and products ( Figs.…”

“…RPE cells were transfected with the pGL4 vector containing one of three fragments spanning nucleotides –500 bp to +67 bp from the LGALS1 transcription start site (promoter region; pGal), +450 bp to +1750 bp (enhancer region; pGal+AP-1), and AP-1 site (TGACTCA)-mutated enhancer region (pGalΔAP-1), as previously described. 14 , 15 Mutation in two HRE sites in the LGALS1 promoter region (CA CG C to CA AA C, positions at –441 bp to –437 bp and –427 bp to –423 bp) 8 , 12 was synthesized and sequenced by Integrated DNA Technologies (Coralville, IA, USA), and subcloned into the pGL4 vector (pGalΔHRE). The pRL-CMV Renilla luciferase plasmid (Promega) was used as internal control.…”

“…Assays were performed using the SimpleChIP Enzymatic Immunoprecipitation Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's protocols. After chromatin was immunoprecipitated with antibodies, immunoprecipitates were evaluated by real-time qPCR using the primers specific for the previously described HRE sites in the LGALS1 promoter region 8 , 12 and AP-1-binding site in the LGALS1 enhancer region, 14 – 16 together with 2% input DNA as reference samples. All primers are listed in Supplementary Table S1 .…”

“…Real-time qPCR was performed as described previously. 12 ChIP-qPCR signals were calculated as percentage of input.…”

“… 10 – 13 Moreover, we have recently shown the significant role of hypoxia-responsive elements (HREs) in the LGALS1 promoter region in hypoxia-induced galectin-1/ LGALS1 expression in Müller glial cells. 12 In addition to hypoxia, our previous studies revealed that IL-1β induced galectin-1 expression in Müller glial cells via activator protein 1 (AP-1) activation following diabetes-associated inflammatory cascades including extracellular signal-regulated kinase (ERK) 1 and 2 and phosphatidylinositol-3 kinase (PI3K)/AKT pathways. 13 , 14 In this study, we explored a novel mechanism for the upregulation of galectin-1 expression in RPE cells, which proved to be dependent on the autoinduction of hypoxia-induced placental growth factor (PlGF)/ PGF .…”