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 ∼40- and a ∼45-kDa phosphorylated version of TTP protein were rapidly induced in response to interleukin (IL)-1β. Analysis with IκBαΔN, a dominant version of inhibitor of κBα (IκBα), as well as dominant-negative and small-molecule IκB kinase (IKK) inhibitors demonstrated that IL-1β-induced TTP is nuclear factor-κB (NF-κB)-dependent. Likewise, TTP expression and formation of the ∼45-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 ∼40-kDa form of TTP. In the context of IL-1β, 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-1β, this repressive effect may be explained by repression of the p38 MAPK pathway by dexamethasone. Knockdown of TTP protein by siRNA elevated IL-1β-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-1β plus dexamethasone, suggesting that feedback control by TTP also occurs in the context of IL-1β plus dexamethasone. Taken together, our data demonstrate that NF-κB and p38 MAPK are critical to the induction of TTP by IL-1β and that TTP induction provides feedback control of the ARE-containing genes GM-CSF and IL-8.
Footnotes
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This study was supported by a start-up grant from the Alberta Heritage Foundation for Medical Research (to R.N.); the Canadian Institutes of Health Research [Grants 128534, 171818] (to R.N.); a Lung Association of Alberta and the North West Territories studentship (to E.M.K.); and an Izaak Walton Killam postdoctoral fellowship (to N.S.H.). Real-time PCR was performed by virtue of an equipment and infrastructure grant from the Canadian Fund for Innovation and the Alberta Science and Research Authority. Work in the laboratory of R.N. was also supported by grants from AstraZeneca, GlaxoSmithKline, and Nycomed.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.151423.
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ABBREVIATIONS: IL, interleukin; TNF, tumor necrosis factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; NF-κB, nuclear factor-κB; ARE, adenine- and uridine-rich element; MAPK, mitogen-activated protein kinase; TTP, tristetraprolin; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; PD098059, 2′-amino-3′-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PS-1145, N-(6-chloro-9H-pyrido[3,4-b]indol-8-yl)-3-pyridinecarboxamide dihydrochloride; ML120B, N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2-methylnicotinamide; DMSO, dimethyl sulfoxide; MOI, multiplicity of infection; Ad, adenovirus; IκBα, inhibitor of κBα; IKK, IκB kinase; siRNA, short interfering RNA; GFP, green fluorescent protein; HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; CIAP, calf alkaline intestinal phosphatase; ANOVA, analysis of variance; MEK, mitogen-activated protein kinase kinase.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. - Received January 25, 2009.
- Accepted May 11, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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