June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Overexpression of GPR158, an Orphan Member of G Protein-Coupled Receptor Family C, Mimics Effects of Glucocorticoids on Trabecular Meshwork Cells
Author Affiliations & Notes
  • Nitin Patel
    Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, CA
  • Tatsuo Itakura
    Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, CA
  • M Elizabeth Fini
    Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, CA
    Cell & Neurobiology, Keck School of Medicine of USC, Los Angeles, CA
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3565. doi:
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      Nitin Patel, Tatsuo Itakura, M Elizabeth Fini; Overexpression of GPR158, an Orphan Member of G Protein-Coupled Receptor Family C, Mimics Effects of Glucocorticoids on Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3565.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Administration of glucocorticoids (GCs) causes spiking of intraocular pressure in a subset of patients due to a reduction in aqueous outflow facility. GPR158 is expressed in trabecular meshwork (TM) cells of the conventional outflow pathway and transcription is stimulated by GCs (ARVO, Patel et al., 2011). We investigated the possible role of GPR158 in controlling TM cellular activities known to be affected by GCs.

Methods: We used the immortalized TM-1 cell line and primary TM cells isolated from discarded human eye tissue. GPR158 cDNA was cloned into two mammalian expression vectors: pcDNA 3.1(+) and pEGFP-C1, the latter generates C-terminus GFP fusion with GPR158. TM barrier function was quantified using the In Vitro vascular permeability assay , which measures permeability of a confluent cell monolayer to FITC-dextran.

Results: Similar to treatment with GCs, transient overexpression of GPR158 in TM cells increased the rate of cell proliferation (4-fold), and significantly enhanced TM barrier function. Conversely, siRNA knockdown of endogenous GPR158 inhibited cell proliferation (barrier function studies in progress). GPR158 overexpression was accompanied by elevated expression of cyclin D1 and the intercellular tight junction proteins, ZO-1 and occludin. Both endogenous and overexpressed GPR158 protein localized to punctate structures in the nucleus of TM cells. Significantly, overexpressed GPR158 protein was shifted entirely to the plasma membrane when TM cells were treated with inhibitors of clathrin-mediated endocytosis (concanavalin A or chlorpromazine). Mutation of a nuclear localization signal (NLS) in the 8th helix of GPR158 also shifted the protein out of the nucleus, but not to the plasma membrane, instead found within discrete vesicles under the plasma membrane. These results suggest that newly synthesized GPR158 traffics to the plasma membrane of TM cells, where it is rapidly internalized in endocytic vesicles for translocation to the nucleus. Importantly, GPR158 stimulation of TM cell proliferation was completely abrogated by failure to translocate to the nucleus (barrier function studies in progress).

Conclusions: TM cell overexpression of nuclear localized GPR158 mimics the effects of GCs. Pharmaceutical-assisted retention of GPR158 at the plasma membrane may be a novel strategy to treat GCs-induced ocular hypertension.

Keywords: 735 trabecular meshwork • 633 outflow: trabecular meshwork • 487 corticosteroids  
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