Abstract
Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.
Highlights
Inflammatory gene expression is reduced by anti-inflammatory glucocorticoids
Effect of Dexamethasone on mitogen-activated protein kinase (MAPK) Activation by IL1B—Activation of the p38 and extracellular regulated kinase (ERK) MAPK pathways by IL1B was previously found to be repressed by dexamethasone, in A549 cells [28, 29]
We show that IL1B robustly activated, as assessed by TXY motif phosphorylation, the p38, ERK, and JUN N-terminal kinase (JNK) MAPK cascades and that dexamethasone co-treatment repressed all three pathways at 1, 2, and 6 h post-treatment (Fig. 1A)
Summary
Inflammatory gene expression is reduced by anti-inflammatory glucocorticoids. Results: With an inflammatory stimulus, glucocorticoids enhance expression of the phosphatase, DUSP1. Repression of cyclooxygenase-2 (PTGS2) expression by dexamethasone is prevented by transcriptional blockers in human fetal lung IMR-90 fibroblasts and A549 type II epithelial cells [12, 13] Such data imply that glucocorticoid-dependent gene expression plays a role in repression [9, 11]. As the dexamethasone-dependent repression of these mRNAs was attenuated by both a NR3C1 receptor antagonist and siRNA-mediated knock-down of NR3C1 [30], these genes represent prima facie targets for glucocorticoid-inducible anti-inflammatory genes. This model is used to explore possible repressive roles of DUSP1 induced by IL1B and IL1B plus the synthetic glucocorticoid, dexamethasone
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