EP2 receptor agonist protects against trabecular meshwork apoptosis induced by ER stress

Abstract

Purpose: Glaucoma is the second leading cause of blindness in the world characterized by the degeneration of retinal ganglion cells (RGCs) and optic nerve atrophy. Our aim is to provide insight into the potential involvement of mitochondrial impairment in the apparition and progression of the glaucomatous phenotype in DBA/2J mice.

Methods: Primary human TM cells were pretreated with vehicle control or butaprost acid or dibutyril cyclic AMP (dbcAMP) and then incubated with tunicamicyin 1 µg/ml. After 24h, TM cell viability was quantified by total ATP content (Celltiter-Glo) and DAPI stained nucleus count. Caspase 3/7 and 9 activations were analysed (caspase 3/7 and caspase 9-Glo) at 6h and 24h. Expression level of Bcl-2, Bax and phospho-Bad were analysed by western blot. Reactive oxygen species (ROS) accumulation were measured at 24h (ROS-Glo).

Results: Butaprost protected TM cell death induced by tunicamycin in a concentration dependent manner as analysed by increased ATP content and nucleus number at 24h. Furthermore, the prevention against cell death was due to inhibition of mitochondrial-dependent apoptosis as both executioner caspase 3/7 and caspase 9 activations were decreased by butaprost. These effects seems linked to increased cAMP as dbcAMP reproduced the same protection. Butaprost did not modify Bcl-2 and Bax level but increased phosphorylation of Bad. Finally, butaprost decreased ROS accumulation induced by tunicamycin.

Conclusions: Butaprost prevents TM cell from apoptosis induced by ER stress and increases anti-apoptotic phosphorylation of Bad. This protection is accompanied by a decrease in ROS accumulation. These results give insight in the function of EP2 signalling in the TM and are of importance in the development of prostaglandin analogues to lower the intraocular pressure.