Regulation of Tristetraprolin Expression by Interleukin-1β and Dexamethasone in Human Pulmonary Epithelial Cells: Roles for Nuclear Factor-κB and p38 Mitogen-Activated Protein Kinase

Abstract

The mRNA-destabilizing protein tristetraprolin (TTP) negatively regulates adenine-and uridine-rich element (ARE)-containing mRNAs. In A549 pulmonary cells, TTP mRNA and both a ϳ40-and a ϳ45-kDa phosphorylated version of TTP protein were rapidly induced in response to interleukin (IL)-1␤. Analysis with IB␣⌬N, a dominant version of inhibitor of B␣ (IB␣), as well as dominant-negative and small-molecule IB kinase (IKK) inhibitors demonstrated that IL-1␤-induced TTP is nuclear factor-B (NF-B)-dependent. Likewise, TTP expression and formation of the ϳ45-kDa phosphorylated form of TTP are blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580). By contrast, and despite a 3-to 4-fold induction of TTP mRNA, the anti-inflammatory glucocorticoid dexamethasone only modestly induced expression of the ϳ40-kDa form of TTP. In the context of IL-1␤, dexamethasone exerted a marginal repressive effect on TTP mRNA expression and more considerably reduced TTP protein. Given a requirement for p38 MAPK in the induction of TTP by IL-1␤, this repressive effect may be explained by repression of the p38 MAPK pathway by dexamethasone. Knockdown of TTP protein by siRNA elevated IL-1␤-induced expression of granulocyte macrophage-colony-stimulating factor (GM-CSF) and IL-8, demonstrating a role for TTP in feedback control. Likewise, knockdown of TTP increased GM-CSF expression in the presence of IL-1␤ plus dexamethasone, suggesting that feedback control by TTP also occurs in the context of IL-1␤ plus dexamethasone. Taken together, our data demonstrate that NF-B and p38 MAPK are critical to the induction of TTP by IL-1␤ and that TTP induction provides feedback control of the ARE-containing genes GM-CSF and IL-8.Recruitment of inflammatory cells, including neutrophils, monocyte/macrophage, eosinophils, and T cells, is central to inflammation and is driven by increases in the expression of cytokines, chemokines, adhesion molecules, and other inflammatory proteins. For example, activation of structural cells in the airways by inflammatory insult or cytokines, such as interleukin (IL)-1␤ or TNF-␣, leads to the expression and production of IL-8 (CXCL8) and granulocyte macrophagecolony-stimulating factor (GM-CSF) (Laberge and El Bassam, 2004). Because IL-8 is primarily a neutrophil chemoattractant (Mukaida, 2003), whereas GM-CSF stimulates myeloid-derived cells, including basophils, eosinophils, neutrophils, monocyte/ macrophages, as well as subsets of dendritic cells, these two proteins differentially contribute to inflammatory cell influx (Martinez-Moczygemba and Huston, 2003). ABBREVIATIONS: IL, interleukin; TNF, tumor necrosis factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; NF-B, nuclear factor-B; ARE, adenine-and uridine-rich element; MAPK, mitogen-activated protein kinase; TTP, tristetraprolin; ERK, extracellular signalregulated kinase; DMEM, Dulbecco's modified Eagle's medium; PD098059, 2Ј-amino-3Ј-methoxyflavone; U0126, 1...

Negative Feed-forward Control of Tumor Necrosis Factor (TNF) by Tristetraprolin (ZFP36) Is Limited by the Mitogen-activated Protein Kinase Phosphatase, Dual-specificity Phosphatase 1 (DUSP1)

“…11B). This is clearly observed at 1 and 2 h following IL1B treatment and has been shown previously in A549 cells and in LPS-treated macrophages (15,16). Contrary to this, a number of groups report ZFP36 to be induced by glucocorticoids (16,31,32,52).…”

“…This is clearly observed at 1 and 2 h following IL1B treatment and has been shown previously in A549 cells and in LPS-treated macrophages (15,16). Contrary to this, a number of groups report ZFP36 to be induced by glucocorticoids (16,31,32,52). Although this glucocorticoid-inducibility seems relatively minor in A549 cells, the response may contribute toward restoring or maintaining ZFP36 expression at longer times following IL1B plus dexamethasone treatment (Fig.…”

“…More surprisingly, loss of DUSP1 reduced, relative to control, the mRNA expression for a number of inflammatory genes, including CCL2, CXCL3, and PTGS2, at longer (6 h) times (24). Given that 1) such mRNAs contain AREs, 2) ZFP36 can destabilize ARE-containing mRNAs, and 3) ZFP36 is induced in A549 cells in a p38 MAPK-dependent manner, ZFP36 represents a candidate to explain this enhanced loss of inflammatory mRNAs following DUSP1 knockdown (16,(41)(42)(43). We confirm, in A549 cells, that ZFP36 is induced by IL1B to reduce TNF mRNA stability and exerts feed-forward inhibition of TNF mRNA and protein expression.…”

“…However, in addition to DUSP1, glucocorticoids induce expression of multiple effector genes, and this may lead to redundant actions (30). Indeed, ZFP36 is modestly up-regulated by glucocorticoids in the human airway epithelial cells as well as in pulmonary A549 and bronchial BEAS-2B epithelial cells and in the airways following glucocorticoid inhalation (16,31,32). 3 Furthermore, a role for ZFP36 in the repression of inflammatory gene expression is indicated (31,33).…”

“…Whereas increasing ZFP36 reduces the expression of inflammatory genes, ZFP36 protein expression is itself highly dependent on mitogen-activated protein kinase (MAPK) activation (16,18). Following pro-inflammatory stimulation, ZFP36 protein appears initially as a ϳ40-kDa protein, which becomes phosphorylated and migrates at ϳ45 kDa on SDS-PAGE (16,19). Phosphorylation is suggested to enhance ZFP36 stability and to promote targeting of ARE-containing transcripts (19).…”