Functional Analysis of KSRP Interaction with the AU-Rich Element of Interleukin-8 and Identification of Inflammatory mRNA Targets

Abstract

Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.Rapid degradation controls the levels of many mRNAs that are translated into transiently expressed proteins. These include cytokines, growth factors, proto-oncogene products, and other proteins participating in acute reactions. The short half-lives of their mRNAs depend on regulatory RNA sequences, the most widely distributed being AU-rich elements (AREs) located in their 3Ј untranslated regions (UTRs) (8,39). AREs have been divided into three classes, differing in the sequences and modes of degradation imposed by them (8). Class I AREs contain one to three scattered AUUUA motifs, and class II AREs contain multiple overlapping AUUUA motifs. Class III AREs are less well defined and lack an AUUUA motif. With the search pattern WWWU(AUUUA)UUUW, 4,000 human mRNAs have been reported to contain AREs and grouped into the ARED database (1), where the class II AREs are further subdivided into different groups, depending on the number of AUUUA motifs present in an ARE.Interleukin-8 (IL-8) is a member of the CXC chemokine family, released from different types of cells in response to direct cell stress, pathogens, or the proinflammatory cytokines tumor necrosis factor (TNF) and IL-1 (reference 25 and references therein). It attracts and activates leukocytes and also plays a role in angiogenesis. Studying its induction in response to IL-1, we previously observed that in addition to transcriptional activation of the IL-8 gene, its mRNA is stabilized (26,46). The latter response involves the activation of p38 mitogenactivated protein (MAP) kinase and its substrate kinase MK2. Stabilization of IL-8 mRNA can contribute to enhanced IL-8 expression, e.g., in viral infection (22). Our recent studies showed that the IL-8 mRNA contains an ARE which consists of two functionally distinct domains. They cooperate for maximal destabilization and interaction with cytoplasmic proteins in vitro (44).Control of mRNA degradation by AREs involves the function of protei...

Interleukin-1 Activates Synthesis of Interleukin-6 by Interfering with a KH-type Splicing Regulatory Protein (KSRP)-dependent Translational Silencing Mechanism

“…The 3Ј-UTR of IL-1␣ or the 3Ј-UTR of ␤-globin was cloned immediately downstream of the IL-1␣ coding region to generate pUHD10/ IL1-IL1-IL1 or pUHD10/IL1-IL1-B, respectively. Plasmids for expression of Strep-tagged GFP and KSRP have been described previously (15).…”

“…Cells and Materials-HeLa cells were cultured and transfected with plasmids (see above) or with siRNAs specific for GFP or KSRP mRNAs (Qiagen) by the calcium phosphate method as described (15). Recombinant human IL-1␣ was obtained from Promocell.…”

“…Microarray Analysis-RNA of pooled sucrose gradient fractions was subjected to quality control and analyzed using whole human genome oligonucleotide microarrays (G4112F, identification number 014850, Agilent Technologies) in essence as described (15). Data were filtered according to a stringent multistep approach that accounted for 1) the quality of the measurements (hybridization performance), 2) the consistency among replicate assays, 3) the intensity range, and 4) -fold change values (see also the legend to Table 1).…”

“…Radiolabeled RNA probes spanning the IL-6 3Ј-UTR with or without nt 828 -1002 were in vitro transcribed with the T7 Megascript kit (Ambion) using 32 P-labeled UTP. Purification of the labeled RNA probes, incubation with cytoplasmic extracts or purified Strep-tagged proteins, and separation of the samples on non-denaturing gels were performed as described (15).…”

“…10 -13). The proinflammatory cytokine IL-1 can induce stabilization of KSRP target mRNAs, including that of IL-6 (14,15), by reducing interaction of KSRP with the ARE presumably as a result of KSRP phosphorylation by p38 MAP kinase (10).…”