Browsing by Author "Rider, Christopher Francis"

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    Modulation of glucocorticoid-inducible gene expression: Effects of inflammatory stimuli and long-acting β2-adrenoceptor agonists
    (2014-09-15) Rider, Christopher Francis; Newton, Robert; Giembycz, Mark Anthony
    Glucocorticoids, acting on the glucocorticoid receptor (GR; NR3C1), are the most effective medication for controlling inflammation in the majority of asthmatics. However, some asthmatics, particularly those with severe disease, have reduced responses to glucocorticoids, a condition known as glucocorticoid resistance. Likewise, glucocorticoids have reduced effectiveness in asthmatics who smoke and during exacerbations, which are frequently induced by human rhinovirus (HRV) infection. Glucocorticoids reduce inflammatory protein production by directly inhibiting inflammatory transcription factors, including nuclear factor-kappa B (NF-κB) and by enhancing the expression of potentially anti-inflammatory genes (transactivation). The importance of transactivation in glucocorticoid activity is increasingly apparent, but the impact of inflammatory mediators on anti-inflammatory gene expression is understudied. Human bronchial epithelial, BEAS-2B, cells stably transfected with a 2×glucocorticoid response element (GRE) reporter system, which models glucocorticoid-inducible gene expression, demonstrate concentration-dependent activation by glucocorticoids. However, dexamethasone-induced 2×GRE activation was time-dependently reduced by pre-treatment with inflammatory mediators, including tumor necrosis factor (TNF), interleukin-1β (IL1B) and cigarette smoke extract (CSE). Furthermore, TNF pre-treatment decreased dexamethasone-induced mRNA expression of genes with potentially anti-inflammatory activity, including cyclin dependent kinase inhibitor 1C (CDKN1C) and TSC22 domain family protein 3 (TSC22D3/GILZ), in bronchial epithelial and airway smooth muscle cells. Likewise, pre-incubations with HRV or the synthetic double-stranded viral RNA mimetic polyinosinic:polycytidylic acid (poly(I:C)) reduced dexamethasone-induced 2×GRE activation. Poly(I:C) also reduced dexamethasone-induced CDKN1C expression. Approaches to reverse TNF-induced glucocorticoid hyporesponsiveness were evaluated, including addition of long-acting β2-adrenoceptor agonists (LABAs), use of novel GR agonists and inflammatory signalling pathway inhibition. LABAs, such as formoterol, potentiated 2×GRE reporter activation and CDKN1C expression through a time- and PKA-dependent mechanism that did not enhance GR expression, agonist affinity or translocation, but instead allows for gene specific control. Therefore, LABA addition functionally reversed glucocorticoid hyporesponsiveness induced by TNF or poly(I:C). However, novel GR agonist-induced 2×GRE activation was repressed by TNF or poly(I:C) treatment, with the degree of repression correlating with agonist efficacy. Finally, inhibition of the NF-κB and c-Jun N-terminal kinase (JNK) mitogen activated protein kinase pathways partially reversed TNF-induced glucocorticoid hyporesponsiveness. These results may contribute to the development of improved treatments for combating glucocorticoid hyporesponsiveness during exacerbations and in severe asthma.