Glucocorticoid receptor inhibits Müller glial galectin‐1 expression via DUSP1‐dependent and ‐independent deactivation of AP‐1 signalling

Abstract

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.

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

“…Specific siRNAs against TSC22D3 (hs.Ri.TSC22D3.13.1), DUSP1 (hs.Ri.DUSP1.13.3) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L . Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.…”

“…Total RNA isolation was performed from cells using SuperPrep II Cell Lysis & RT Kit for qPCR (TOYOBO) and from tissue samples using TRI reagent (Molecular research centre), as previously described . The following primers were used: human LGALS1 (forward 5′‐CGC TAA GAG CTT CGT GCT GAA C‐3′, reverse 5′‐CAC ACC TCT GCA ACA CTT CCA G‐3′), human HIF1A (HIF‐1α; forward 5′‐TGC TCA TCA GTT GCC ACT TC‐3′, reverse 5′‐TCC TCA CAC GCA AAT AGC TG‐3′), human VEGFA (forward 5′‐CAG ATT ATG CGG ATC AAA CCT CA‐3′; reverse 5′‐CAA GGC CCA CAG GGA TTT TC‐3′), human TSC22D3 (forward 5′‐ATC TGC AAC CGC AAC ATC GAC C‐3′, reverse 5′‐GCA TAC ATC AGA TGA TTC TTC ACC‐3′), human DUSP1 (forward 5′‐CTG CCT TGA TCA ACG TCT CA‐3′, reverse 5′‐CTG TGC CTT GTG GTT GTC CT‐3′), human ACTB (β‐actin; forward 5′‐CTG GAA CGG TGA AGG TGA CA‐3′, reverse 5′‐ AAG GGA CTT CCT GTA ACA ATG CA‐3′), mouse Lgals1 (forward 5′‐GTC TCA GGA ATC TCT TCG CTT C‐3′, reverse 5′‐TCC CCG AAC TTT GAG ACA TTC‐3′, probe 5′‐TTC AAT CAT GGC CTG TGG TCT GGT‐3′), mouse Tsc22d3 (forward 5′‐TCA ATG AGG GCA TCT GCA ACC G‐3′, reverse 5′‐CAT CAG GTG GTT CTT CAC GAG G‐3′), and mouse Actb (forward 5′‐CAT CCG TAA AGA CCT CTA TGC CAA C‐3′, reverse 5′‐ATG GAG CCA CCG ATC CAC A‐3′).…”

“…Human LGALS1 promoter (−500 bp to +67 bp from the LGALS1 transcription start site) was synthesized and sequenced by Integrated DNA Technologies and subcloned into the pGL4 vector (Promega). Luciferase reporter construct containing 3 consensus hypoxia‐responsive elements (HREs) was obtained from Addgene.…”

“…All animal experiments were conducted in accordance with the ARVO (Association of Research in Vision and Ophthalmology) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Review Committee for Animal Experimentation of Hokkaido University. Procedures for murine model of streptozotocin‐induced diabetes were described in our previous reports . At 2 months after induction of diabetes, dexamethasone and triamcinolone acetonide (50 pmol/eye for each) were injected into the vitreous cavity.…”

“…In addition to the hypoxic induction of galectin‐1, we revealed an interleukin (IL)‐1β‐driven inflammatory pathway to produce galectin‐1 in human Müller glial cells as well as in the retina of diabetic mice . Activator protein (AP)‐1 is the key transcription factor for the hypoxia‐unrelated LGALS1 expression in the downstream of IL‐1β receptor‐mediated phosphorylation of phosphatidylinositol‐3 kinase (PI3K)/AKT and extracellular signal‐regulated kinase (ERK)1/2 . Recently, we have shown that glucocorticoids inhibit IL‐1β‐induced galectin‐1 expression via dual‐specificity phosphatase (DUSP)1‐dependent and DUSP1‐independent deactivation of AP‐1 signalling ( ie transactivation and transrepression, respectively) in Müller glial cells …”