Abstract
TNF is central to inflammation and may play a role in the pathogenesis of asthma. The 3'-untranslated region of the TNF transcript contains AU-rich elements (AREs) that are targeted by the RNA-binding protein, tristetraprolin (also known as zinc finger protein 36 (ZFP36)), which is itself up-regulated by inflammatory stimuli, to promote mRNA degradation. Using primary human bronchial epithelial and pulmonary epithelial A549 cells, we confirm that interleukin-1β (IL1B) induces expression of dual-specificity phosphatase 1 (DUSP1), ZFP36, and TNF. Whereas IL1B-induced DUSP1 is involved in feedback control of MAPK pathways, ZFP36 exerts negative (incoherent) feed-forward control of TNF mRNA and protein expression. DUSP1 silencing increased IL1B-induced ZFP36 expression at 2 h and profoundly repressed TNF mRNA at 6 h. This was partly due to increased TNF mRNA degradation, an effect that was reduced by ZFP36 silencing. This confirms a regulatory network, whereby DUSP1-dependent negative feedback control reduces feed-forward control by ZFP36. Conversely, whereas DUSP1 overexpression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6 h, an effect that was predominantly due to elevated transcription. This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and -independent mechanisms. In terms of repression by dexamethasone, neither silencing of DUSP1, silencing of ZFP36, nor silencing of both together prevented the repression of IL1B-induced TNF expression, thereby demonstrating the need for further repressive mechanisms by anti-inflammatory glucocorticoids. In summary, these data illustrate why understanding the competing effects of feedback and feed-forward control is relevant to the development of novel anti-inflammatory therapies.
Highlights
Characterization of TNF Expression in the Presence of IL1B and Dexamethasone in A549 Cells—As a prelude to analyzing the regulation of TNF expression by dual-specificity phosphatase 1 (DUSP1) and ZFP36, we characterized the expression of TNF following IL1B treatment in the absence and presence of the synthetic glucocorticoid, 112 JOURNAL OF BIOLOGICAL CHEMISTRY
RNA was extracted for real-time PCR analysis of TNF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
E, A549 cells were either not treated or stimulated with IL1B (1 ng/ml) for 1, 2, or 6 h, or A549 cells were either not treated or stimulated with IL1B (1 ng/ml) or a combination of IL1B and dexamethasone (1 M) for 2 h, and total protein was prepared for Western blot analysis of uncleaved (ϳ25 kDa) TNF and GAPDH
Summary
Whereas DUSP1 overexpression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6 h, an effect that was predominantly due to elevated transcription This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and -independent mechanisms. Phosphorylation is suggested to enhance ZFP36 stability and to promote targeting of ARE-containing transcripts [19] Such a MAPKdependent negative feed-forward regulatory loop suggests that MAPK activation may act to reduce the expression of AREcontaining genes via increased ZFP36 activity (Fig. 1). Given interest in therapeutically targeting MAPK pathways in inflammatory disease and the fact that glucocorticoids induce DUSP1 to reduce MAPK activity, we have used TNF as a model ZFP36 target gene to explore the relationship(s) between the regulation of MAPK activation by DUSP1 with the expression of ZFP36 and effects on downstream gene expression
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