Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2005) 9 P133

BES2005 Poster Presentations Steroids (17 abstracts)

Modulation of glucocorticoid sensitivity by retroviral gene transfer

P Le Rouzic , H Gillingham , A Berry , HJ Garside , A White & DW Ray

Endocrine Sciences Research Group, University of Manchester, Manchester, UK.

Glucocorticoid sensitivity is dependent on the intracellular concentration of the glucocorticoid receptor (GR). GR expression is regulated in a complex, cell-type specific manner, but in most cell types the GR is subject to negative autoregulation by glucocorticoid exposure. Many human diseases are associated with reduced tissue sensitivity to glucocorticoids, including inflammatory arthritis, asthma, and chronic obstructive pulmonary disease. Therefore there is interest in approaches to increase glucocorticoid sensitivity.

We have evaluated a retroviral gene transfer method for conferring glucocorticoid sensitivity to GR deficient cell lines. Retroviral vectors allow up to 8kB of insert to be packaged, so allowing both the full length GR, and also an antibiotic selection cassette to be included. We also made a GR-EYFP fluorescent fusion protein, to allow easy tracking of infection, and also intracellular trafficking of the transduced GR.

All retroviruses were generated in HEK packaging cells, and the conditioned medium used fresh to infect recipient cells, either COS 7, or fresh HEK cells, neither of which express functional GR. Infected cells were analysed by fluorescent microscopy, and also by FACS. Infection rate approaching 70% were achieved, and stable expression was obtained by antibiotic selection. The GR-EYFP fusion was found in the cytoplasm of quiescent cells, and in the nucleus after dexamethasone treatment, confirming appropriate intracellular localisation. The GR-EYFP has authentic glucocorticoid receptor activity in COS 7 cells and interacts with SRC-1 and NFkB in the presence of dexamethasone and NCOR in the presence of RU486 using BRET analysis.

These data show that retroviral infection affords an efficient way to permanently alter cellular GR expression, with implications for anti-inflammatory therapeutics.

Volume 9

24th Joint Meeting of the British Endocrine Societies

British Endocrine Societies 

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