Abstract The goal of this application is to determine the role of estrogen and its receptors - estrogen receptor 1 (ESR1) and G protein coupled estrogen receptor (GPER1) – in regulating intraocular pressure (IOP) through trabecular meshwork (TM) and Schlemm's canal (SC) endothelial cells. IOP is the primary and only modifiable risk factor for patients affected with primary open-angle glaucoma (POAG) in the clinic. TM and SC modulate majority of aqueous humor outflow resistance in the conventional pathway. Despite the significant research progress in TM/SC outflow dynamics, limited therapeutic targets are available for modulating the conventional outflow. It is necessary to identify novel targets to lower IOP more efficiently in glaucoma patients with progressive visual field loss. Several recent genome-wide association studies have identified >150 IOP- associated genomic loci, which is too many to follow up functionally. Our comprehensive bioinformatics analyses of these IOP-associated genomic loci indicate the enrichment of ESR1-related gene networks among these IOP genes. Factors related with menarche, menopause, and oophorectomy have been associated with POAG. Genetic associations have been identified between sequence variants in estrogen receptor pathway genes and POAG. Estrogen and ESR1-related pathways including aromatase may affect the aqueous humor outflow facility and regulate IOP levels. The presence of estradiol in aqueous humor and ESR1 protein in the TM/SC region further supports the potential role of estrogen and ESR1-related pathways in IOP regulation. Based on our preliminary data on the elevated IOP levels from mouse models with the loss of Esr1 or Gper1 as well as their interaction with Nos3, we hypothesize that activation of estrogen signaling via ESR1 and GPER1 decreases IOP and POAG risk by modulating the turnover of extracellular matrix in the TM and the NO signaling in the SC. We propose to determine the in vivo effects of loss of estrogen signaling via Gper1 (Aim 1) or Esr1 (Aim 2) in murine eyes using tissue-specific knockout mice, and to determine the underlying mechanisms of Gper1 and Esr1-mediated IOP regulation using in vitro primary cell culture models (Aim3). Successful completion of this project will help reveal the critical role of estrogen signaling in aqueous outflow pathway and identify novel therapeutic targets to reduce IOP more effectively for this sight-threatening disease.