Knock-Down of Glucocorticoid Receptor Beta (GRβ) Enhances the Ocular Hypertensive Response to Dexamethasone in C57BL/6J Mice

Topical ocular treatment with glucocorticoids (GCs) yields an increase in intraocular pressure (IOP) in a subset (~35–40%) of the human population, known as steroid responders. If treatment is continued, this ocular hypertension (OHT) can develop into GC-induced glaucoma. In mice, the GC-mediated OHT response is modulated by the GC receptor, which exists as two major splice variants, GRα and GRβ, in the TM cell cytosol. GRα contains a GC-binding domain. Following GC binding, the GRα-GC complex enters the TM cell nucleus and transactivates genes associated with increased synthesis/export of extracellular matrix (ECM), causing OHT. GRβ has no ligand-binding domain but appears to inhibit gene transactivation by the GRα-GC complex. We previously described the effects of over-expression of GRβ on the GC-induced OHT response on IOP in mice. Here, we investigated the effect of the GC Dexamethasone (DEX) on the OHT response in C57BL/6J mice, under conditions of GRβ knockdown. A total of 20 × C57BL/6J mice (♀), aged 3–4½ months were used. Animals were weighed. Baseline (BL) conscious IOP measurements (TonoLab) and direct ophthalmoscopy (Keeler Instruments Specialist Model Ophthalmoscope) assessments were made. Animals were divided into 4 cohorts (n = 5/cohort), and anesthetized (isoflurane (2.5%) and O2(g) (0.8 L/min)). Cohorts 1 and 2 (n = 10 animals) were given an intravitreal (IVT) injection in both eyes (OU) of Lenti.shRNA.Scramble (2.2 × 109 TU/mL; 1 μL bolus injection (total 2.2 × 106 TU/eye)). Cohorts 3 and 4 (n = 10 animals) were given an IVT injection OU of Lenti.shRNA.GRβ (1.09 × 109 TU/mL; 2 μL bolus injection (total 2.18 × 106 TU/eye)). Each subsequent week, for 8 weeks, conscious IOPs were measured (OU), and direct ophthalmoscopy was performed. Animals were then anesthetized and given a periocular injection OU of a 20 μL bolus (per eye) of either vehicle solution for DEX (DEX-Veh) (Cohorts 1 (n = 5 animals) and 3 (n = 5 animals), or micronized DEX suspension (Cohorts 2 (n = 5 animals) and 4 (n = 5 animals)). At the conclusion of the study, animals were euthanized and TM tissues were collected (OD and OS pooled per animal) for assay of GRβ mRNA expression using QPCR. GRβ mRNA expression in the TM was significantly reduced by greater than 95% in the Lenti.shRNA.GRβ group vs. the Lenti.shRNA.Scramble group (p < 0.0001). Injection of Lenti.shRNA.Scramble or Lenti.shRNA.GRβ had no effect on baseline IOP. Subsequent injections of DEX over the following 8 weeks led to a significant increase in mean IOP in both groups (baseline 12.5 mmHg; 23 mmHg in Lenti.shRNA.Scramble cohort (p < 0.0001); and 28 mmHg in Lenti.shRNA.GRβ cohort (p < 0.0001)). DEX-induced IOP was significantly higher in the shRNA.GRβ cohort compared to the shRNA.Scramble cohort (p < 0.0001). Animals given vehicle control did not show an increase in IOP. Eyes that received DEX had very mild lenticular cataracts at Week 8, but there was no significant reduction in body weight. GRβ plays a role in modulating the IOP response to DEX in C57BL/6J mice. Knockdown of endogenous mRNA encoding GRβ results in an enhanced IOP response to DEX.