Glucocorticoid receptor activation by long acting steroids and its modification by inflammation

Abstract

s: Glucocorticoids have an effective anti-inflammatory, anti-proliferative and immunomodulatory activity. Therefore this class of drugs is used worldwide for the treatment of inflammatory diseases and to prevent rejection following organ transplantation. Inhaled glucocorticoids are the cornerstone treatment of asthma and advanced chronic obstructive pulmonary disease (COPD). The new long acting glucocorticoids mometasone and Ciclesonide have recently been introduced for the treatment of asthma. The aim of these studies is to assess the kinetics and molecular pathways of glucocorticoid receptor (GR) activation and traffic in human lung fibroblasts and bronchial smooth muscle cells. Furthermore the effects of new long acting glucocorticoids on GR activation, complex formation with the transcription factor C/EBP and its isoforms proliferation will be evaluated. Glucocorticoids enter the cytosol of cells by diffusion and bind to an intracellular specific receptor, the glucocorticoid receptor (GR), inducing a conformational change and leading thereby to its activation. The active GR translocates into the nucleus, where it binds to its cognate DNA recognition sequence, the glucocorticoid response element (GRE). Often the activation of the GR is associated with a parallel activation of the transcription factor C/EBP-, followed by de novo synthesis of p21(waf1/cip1), which in turn is mediating the GR dependent inhibition of the cell cycle. Both, GR and C/EBP- are necessary to induce and activate p21(waf1/cip1) and to arrest the cell cycle at the transition from the G1- to the S-phase. Fibroblasts and bronchial smooth muscle cells are centrally involved in airway remodeling of patients with asthma and COPD. Therefore three cell types were chosen to assess the effect of the classical and new glucocorticoids on GR activation, cell proliferation and extracellular matrix deposition under different conditions. Four cell culture conditions were chosen to mimic different stages of inflammation: confluent cells without serum representing intact, not damaged tissue; confluent cells with 5% fetal calf serum to mimic early stages of inflammation; sub-confluent cells without serum to represent damaged tissue with no active inflammatory process; sub-confluent cells with 5% serum to represented tissue with active inflammation-induced lung tissue remodeling. The results showed that cell density and inflammation alter the localization and function of the GR. In sub-confluent cells dexamethasone activated the nuclear accumulation and DNA binding of the GR persistently, while in confluent cells its activity declined. In sub-confluent cells, GR interacted with a 42 kDa C/EBP-α isoprotein, which resulted in an up-regulation of p21(Waf1/Cip1) expression and suppression of proliferation. In confluent cells glucocorticoids induced p27(Kip1) expression via p38 MAP kinase and a 52 kDa C/EBP-β isoprotein. Furthermore, p27(Kip1) did not mediate the anti-proliferative effect of glucocorticoids, but simultaneous inhibition of p21(Waf1/Cip1) and p27(Kip1) unlocked contact inhibition in confluent cell cultures. The studies further showed that in lung fibroblasts the long acting steroid mometasone in contrast to ciclesonide or the short acting steroids dexamethasone, budesonide or fluticasone did not induce tachyphylaxis and had a preferable effect on airway remodeling. Mometasone maintained the increased GR activity significantly over 24 hours, compared to that induced by dexamethasone which peaked at 6 hours and declined thereafter. Similarly drug specific effects were observed for the expression of C/EBP- and p21(Waf1/Cip1). The anti-proliferative effect of mometasone was significantly stronger and long lasting compared to all other steroids. Similar anti-proliferative effects were observed in bronchial smooth muscle cells. Regarding airway remodeling we observed that mometasone, in contrast to all other steroids, did not further stimulate serum dependent synthesis and deposition of extracellular matrix and did not affect matrix metalloproteinase-2 and -9 expression or activity. Therefore this novel steroid will have a lower modulatory effect of tissue remodeling compared to the other steroids. Interestingly the long duration effects of the new glucocorticoid ciclesonide originated from the metabolism of the epithelial cells. In conclusion, our findings suggest that the action of glucocorticoids differs under non-inflammatory and inflammatory conditions in human lung fibroblasts and bronchial smooth muscle cells. Different doses of inhaled steroids might therefore be applied in patient with asthma and COPD dependent on the current status of inflammation in order to reduce side effects. The long acting glucocorticoid mometasone might help to reduce overall steroid dosage and might thereby reduce the risk of tachyphylaxis, enhanced tissue remodeling and other typical steroid side effects. Ciclosonide is a new long acting steroid, but its action on mesenchymal cells depends on the function of epithelial cells. These studies not only improve the understanding of the effect of different glucocorticoid on mesenchymal lung cells but might also help to find optional treatment doses.