Regulator of G-Protein Signaling 5 (RGS5) Proteins in Human Optic Nerve Head (ONH) Astrocytes in Response to Hydrostatic Pressure

Abstract

Purpose:: Elevated intraocular pressure (IOP) is one of the major risk factors involved in pathogenesis of glaucomatous optic neuropathy. RGS5 is highly expressed in human normal and glaucomatous ciliary body and trabecular meshwork where it inhibits the activity of G-protein signaling. We detected abundant RGS5 mRNA expression in ONH astrocytes. In this study, we identified the cellular localization and functional changes of RGS5 in human (ONH) astrocytes after exposure to hydrostatic pressure (HP).

Methods:: Primary cultures of human ONH astrocytes were generated from normal donors. After purification and characterization, astrocytes were transferred to HP chamber filled with a mixture of 92%air and 8% CO₂. Cells exposed to sustained 60mmHg HP for 3, 6, 24 and 48 hours were compared with the cells exposed to control ambient pressure (CP). Astrocytes on coverslips were fixed and processed for standard indirect immunofluorescence staining. Cytoplasmic and nuclear proteins were harvested from astrocytes at different time points after exposure to HP. Protein expression levels were detected and compared by Western blot.

Results:: RGS5 was detected in cytosolic fraction and at low levels in the nuclear fraction by Western blot. After exposure to HP, nuclear protein levels of RGS5 dramatically increased at 3h and 6h and returned to control levels at 24h and 48h. Cytoplasmic protein levels of RGS5 did not change during exposure to HP. Immunofluorescence staining showed that RGS5 was abundant in human ONH astrocytes and located predominantly in the cytoplasm, and also confirmed nuclear translocation from cytoplasm to nucleus after 3h and 6h exposure to HP.

Conclusions:: Our results indicate that HP induced transient translocation of RGS5 from cytoplasm into nucleus. RGS5 shuttling between nucleus and cytoplasm suggest that RGS5 has additional functions in G-protein receptor signaling. Nuclear localization of RGS5 in response to pressure may be related to modulation of nuclear G-protein coupled receptors such as prostaglandin EP3 and EP4 which affect the transcription of genes such as NOS2. This study may help us understand the cellular mechanism by which astrocytes respond to elevated IOP.

Keywords: astrocytes: optic nerve head • signal transduction