Homeostatic Role for GPR158 in Modulating Intraocular Pressure (IOP)

Abstract

Purpose : GPR158 is a glutamate family GPCR with unconventional activities, identified in screens for proteins interacting with RGS7 (PMID: 22689652) and genes associated with steroid-induced ocular hypertension (PMID: 27666015). GPR158 is expressed in trabecular meshwork (TBM) cells and expression is induced by glucocorticoid stress hormones (PMID: 23451275). Here we investigated possible stress-associated effects of GPR158 on IOP.

Methods : GPR158 was placed under control of the tet-on system in a lentivirus expression vector (pSLIK), then the construct was stably infected into immortalized human TBM cells (TM-1). The transgene was induced by doxycycline (100 ng/ml). A competitive ELISA was used for cAMP quantification (Thermo Fisher Scientific, MA). GPR158 knockout (KO) mice (KO Mouse Project repository, Davis, CA) were bred onto the C57Bl/6J background. Mice were anesthetized with isoflurane, 5 min before IOP measurement by Tonolab (Icare, NC). Epinephrine at 109 μM was applied topically (2 μL) to the right eye of GPR158 KO or wild type mice, and then IOP was measured at 30, 60 and 90 min and compared to the untreated eye.

Results : TM-1 cell treatment with epinephrine (adrenergic receptor ligand) at 250 μM induced a spike in the cAMP level. Epinephrine administration with GPR158 transgene induction enhanced the cAMP level by 25.2% over controls ) after 30 min (p<0.025, n=3, ANOVA), 17.6% after 60 min (p<0.04, n=3, ANOVA) and 18.6% after 180 min (p<0.01, n=3, ANOVA). GPR158 transgene induction alone had no effect on cAMP. Epinephrine treatment decreased IOP by 4.19 mmHg in wild type mice at 60 min (p<0.02; n=3, paired t-test). In contrast, epinephrine treatment had little or no effect on IOP in GPR158 KO mice. No difference in IOP was apparent between wildtype and KO mice at 3-months of age (GPR158^+/+ = 12.1 mmHg, GPR158^-/- = 11.5 mmHg; p=0.58, n=3, t-test) and 9-month of age (GPR158^+/+ = 15.8 mmHg, GPR158^-/- = 18.5 mmHg; p=0.18, n=3, t-test). However, by 6-months of age, IOP was significantly higher in wild type than KO mice (GPR158^+/+ = 14.15 mmHg, n=3; GPR158^-/- = 11.78 mmHg, p<0.008, n=3, t-test).

Conclusions : GPR158 cooperates with adrenergic receptors to enhance cAMP production in TM-1 cells in response to stress. KO mice are insensitive to epinephrine’s IOP-lowering effect. Finding GPR158 ligands might enhance current therapeutics for reducing IOP of glaucoma patient.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.