Glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor alpha inflammatory signaling.

During inflammation, activated lymphocytes or macrophages secrete inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) or interleukin 1β (IL-1β). These cytokines in turn not only activate components of the inflammatory system but also exert profound excitatory effects on the hypothalamic-pituitary-adrenal axis, resulting in the synthesis and secretion of glucocorticoids. These adrenal steroids subsequently exert anti-inflammatory effects on many cell types, including T cells, macrophages, eosinophils, neutrophils, mast cells, and endothelial and epithelial cells, thereby creating a classical endocrine feedback loop (12). Glucocorticoids accomplish these actions in a glucocorticoid receptor (GR)-dependent manner through repression of proinflammatory signaling pathways, such as those activated by nuclear factor κB (NF-κB) or the mitogen-activated protein kinase (MAPK) pathway. GR-mediated abrogation of these proinflammatory signaling pathways leads to repression of the production of a number of cytokines, chemokines, and inflammatory enzymes that are relevant to inflammatory diseases, including TNF-α, granulocyte/macrophage colony-stimulating factor (GM-CSF), ILs (IL-1β, IL-2, IL-3, IL-6, IL-8, and IL-11), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (1), by poorly understood mechanisms. Because of these profound anti-inflammatory effects, exogenous synthetic glucocorticoids are often prescribed for several immune and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and asthma (4). Transient inflammatory responses are often tightly controlled by both transcriptional and posttranscriptional mechanisms regulating proinflammatory gene expression. Posttranscriptional control, via regulation of mRNA turnover, is known to be conferred by adenylate-uridylate-rich elements (AREs) located in the 3′ untranslated regions (UTRs) of transcripts encoding various cytokines, chemokines, and other inflammatory proteins (3). These AREs, composed of multiple overlapping AUUUA motifs, promote the deadenylation of the cytokine mRNA and its subsequent degradation by the exosome (11). Therefore, once an adequate inflammatory response has been achieved, the ARE-dependent mRNA decay and subsequent inhibition of protein synthesis is vital for returning cytokines to basal levels. Prolonged expression of proinflammatory cytokines can lead to a variety of tissue-destructive pathologies. For example, transgenic mice expressing ARE-deleted TNF-α transcripts develop both chronic inflammatory arthritis and Crohn's disease-like inflammatory bowel disease as a result of prolonged overexpression of TNF-α (20). Therefore, the mRNA instability conferred by the AU-rich UTR is an important mechanism by which inflammatory responses are kept restrained. The mechanism by which AREs regulate mRNA instability involves trans-acting factors which bind to the ARE and promote mRNA deadenylation and subsequent degradation. One such RNA binding protein is tristetraprolin (TTP) (23), also known as TIS11 (39), Nup 475 (14), or GOS24 (39). TTP is the prototype of a small family of tandem CCCH zinc finger proteins which bind to AREs through specific interactions with zinc finger motifs (21), promoting deadenylation through recruitment of a poly(A) RNase (22). TTP was initially described as being encoded by an immediate-early response gene (ZFP-36) which is rapidly and transiently induced in fibroblasts in response to insulin, phorbol esters, and serum (14, 23, 39). Since then, transient induction of TTP has been reported in T cells stimulated with transforming growth factor β (TGF-β) (30) and in macrophages treated with either TNF-α or lipopolysaccharide (LPS) (10). Importantly, in studies with myeloid cell types, TTP was found to be a component of a negative feedback loop which interferes with TNF-α production by destabilizing its mRNA. Model systems that characterize the effect of TTP on the TNF-α 3′ UTR have recently been developed. Human TTP reduces TNF-α 3′ UTR-dependent luciferase expression when cotransfected into HEK293 cells. Furthermore, mice deficient in TTP display increased TNF-α mRNA stability, overexpression of the cytokine, and a chronic inflammatory disease characterized by severe arthritis, autoimmunity, and myeloid hyperplasia (37). Overexpression of GM-CSF and COX-2 in cells derived from TTP-deficient mice has also been reported (9, 32), and less-direct evidence suggests that other targets of TTP include IL-2 (31, 33), IL-3 (35), and IL-8 (5). While GR repression of the proinflammatory transcription factors NF-κB and AP-1 is widely known as an important mechanism by which glucocorticoids exert their anti-inflammatory effects (13), glucocorticoids can also posttranscriptionally regulate genes of the inflammatory response. For example, they have been found to destabilize the mRNAs of some cytokines and inflammatory mediators (15, 17, 29); however, the mechanisms underlying these actions are unclear. Recently, GR-mediated induction of MAPK phosphatase 1 (MKP-1) and subsequent interruption of p38 MAPK signaling has been proposed as a new anti-inflammatory mechanism, and glucocorticoids have been shown to destabilize COX-2 mRNA through this mechanism (24, 25). However, the possibility that glucocorticoids may exert anti-inflammatory actions through other mechanisms clearly exists. We show here for the first time that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. This induction appears to be through a direct, GR-mediated transcriptional mechanism, with downstream effects on TNF-α. These studies thus represent a novel signaling pathway by which glucocorticoids exert their anti-inflammatory effects.