Heat Shock Protein 90 Is An Essential Molecular Chaperone for Nuclear Transport of Glucocorticoid Receptor Beta

Abstract

Abstract: : Purpose:In human, there are two glucocorticoid receptor isoforms termed GRα and GRß. The actions of exogenous glucocorticoids in the treatment of a wide variety of diseases are achieved by binding to the GRα receptors. In contrast, the function of GRß has only been recently appreciated as GRß has been implicated in glucocorticoid sensitivity in asthma and glaucoma. GRα undergoes hormone dependent translocation from the cytoplasm to the nucleus by associating with heat shock protein 90 (Hsp90) multi–protein heterocomplex. Currently we are investigating the potential role of Hsp90 in regulation of nuclear transport and stability of GRß in TM ells. Methods:Confocal immunofluorescence microscopy was performed to detect the correlation of subcellular expression of Hsp90 with GRß in primary cultured normal and glaucomatous TM cell lines. A GRß expression vector was constructed and overexpression of GRß was performed in a transformed HTM–5 cell line. Both confocal microscopy and Western Blot were used to measure the overexpression and nuclear accumulation of GRß as well as the expression of Hsp90 in the nucleus in these GRß transfected HTM–5 cells. 17–AAG, a Hsp90 inhibitor, was used to study the chaperone function of Hsp90 in nuclear transport and stability of GRß. Lactacystin, a specific proteasome inhibitor, was applied to identify the degradation pathway of GRß. Furthermore, co–immunoprecipitation and Western Blot were used to investigate the formation of a GRß–Hsp90 complex. Results:In the normal TM cell line, which had a high level of GRß in the nucleus, Hsp90 was also concentrated in the nuclear region, while in the glaucomatous TM cell line, GRß and Hsp90 were both present at lower concentrations in the nucleus. Transfection with a GRß expression construct in HTM–5 cells caused the overexpression and accumulation of GRß protein in the nucleus and also lead to an increase in Hsp90 in the nucleus. Most importantly, 17–AAG completely blocked the accumulation of GRß in the nucleus and further lead to the degradation of GRß in the cytoplasm through a proteasome–mediated pathway.In addition, co–immunoprecipitation and a subsequent Western Blot demonstrated that GRß was able to physically associate with Hsp90. Conclusions:This is the first report in which Hsp90 was identified as essential molecular chaperone for transport of GRß from the cytoplasm to the nucleus. This finding also suggests that regulation of the GRß levels in the nucleus could influence subsequent responses to glucocorticoids often seen clinically such as glucocorticoid resistance in asthma or glucocorticoid sensitivity in glaucoma.

Keywords: receptors • chaperones • pharmacology