Abstract
Purpose: :
An alternative splicing variant of human glucocorticoid receptor gene, termed GRß, has dominant negative activity on the glucocorticoid receptor GRα and has been implicated in a variety of steroid–resistant diseases. Previously, we reported that GRß blocks the cellular response to dexamethasone (DEX) in the regulation of the expression of glaucomatous markers in trabecular meshwork (TM) cells. Currently we investigated the effect of GRß on the regulation of phagocytosis and the action of DEX treatment in normal and glaucomatous TM cell lines.
Methods: :
Transformed human normal NTM–5 cell line, which expresses relatively high amount of GRß, and glaucomatous GTM–3 cell line, which has lower GRß expression, was used. Alexa Fluor 488 conjugated Straphylococcus aureus bioparticles were used to track the abilities of the cells to phagocytose. Cells were treated with 100 nM DEX for 24 hours then incubated with bioparticles opsonized with rabbit IgG for one hour, followed by fixation and incubated with Alexa Fluor 633 conjugated goat anti–rabbit IgG to differentiate intracellular from extracellular bioparticles. DAPI nuclear staining was used to calculate cell numbers. A confocal microscope was used to visualize cells and bioparticles. Overexpression of GRß by transfection of the GRß expression plasmid was performed to study the inhibition of DEX–induced decrease in phagocytotic activity of NTM–5 cells.
Results: :
Normal NTM–5 cells ingested more bioparticles than GTM3 cells. DEX treatment significantly decreased the phagocytosis of bioparticles in NTM–5 and GTM–3 cells, while GTM3 cells were more sensitive to DEX, compared to NTM–5 cells. Transient transfection of pCMX–hGRß plasmid increased the expression of GRß and consequently retained the phagocytotic activity of NTM–5 cells in the presence of the challenge of DEX.
Conclusions: :
Our data demonstrate that the expression level of GRß in TM cells can regulate the sensitivity to DEX in terms of the change of phagocytotic activity by steroid treatment. The lower expression of GRß in glaucomatous TM cells could contribute to the loss of function of TM cells, and contribute to the reduced aqueous humor outflow that is seen in the presence of steroids.
Keywords: corticosteroids • receptors • trabecular meshwork