Proteins binding A ؉ U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1␤ and tumor necrosis factor ␣. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that posttranslational modifications of the major cytoplasmic isoform, p40 AUF1 , are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40 AUF1 recovered from polysomes was phosphorylated on Ser 83 and Ser 87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40 AUF1 .In eukaryotes, cytoplasmic mRNA stability is an important checkpoint in the control of gene expression. Many mRNAs encoding regulatory proteins like cytokines, inflammatory mediators, and oncoproteins are constitutively unstable. This ensures that the steady-state levels of these mRNAs, and hence their potential for translation, remain low but also that new steady-state levels are approached quickly following changes in the rate of mRNA synthesis (reviewed in Ref. 1). In mammals, a common feature of many unstable mRNAs is the presence of an A ϩ U-rich element (ARE) 1 within the 3Ј-untranslated region (3Ј-UTR). These elements range from 40 to 150 nucleotides in length and exhibit significant variability in sequence composition, but they usually include one or more AUUUA motifs within a U-rich context (2). In general, mRNA turnover mediated by AREs consists of rapid 3Ј 3 5Ј shortening of the poly(A) tail, followed by decay of the mRNA body (3, 4).The regulation of mRNA decay kinetics by AREs involves their association with any of a number of cellular ARE-binding factors (reviewed in Ref. 5). One such factor, AUF1 (also referred to as heterogeneous nuclear ribonucleoprotein D), is expressed as a family of four protein isoforms resulting from alternative splicing of a common pre-mRNA (6). The larger isoforms, designated by their apparent molecular weights as p42 AUF1 and p45 AUF1 , are largely nuclear (7), probably due to the presence of a binding determinant for components of the nuclear scaffold (8). By contrast, p37 AUF1 and p40 AUF1 lack this seq...

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Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3 untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1-and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (AREmRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.Many mRNAs encoding proteins transiently required for inflammatory responses, cell proliferation, and intracellular signaling are labile due to AU-rich elements (AREs) in their 3Ј untranslated regions (UTRs) (14,21,57). ARE association by ELAV-like (embryonic lethal, abnormal vision) proteins, such as HuR, blocks ARE-mediated mRNA decay (AMD) (31). By contrast, association of proteins such as AUF1, tristetraprolin (TTP), BRF1 (butyrate-responsive factor-1), K-homology splicing regulatory protein (KSRP), ring finger K-homology domain 1 (RKHD1), polymyositisscleroderma 75-kDa antigen (PM-Scl75), or microRNA miR16 or miR289 with an ARE promotes AMD (6,8,12,18,24,34,43). The phosphorylation state of TTP, BRF1, and AUF1 affects AMD efficiency (3,37,51,56), indicating that signal transduction networks regulate this pathway.AUF1 has four protein isoforms-p37, p40, p42, and p45-generated by alternative pre-mRNA splicing (50). Based upon extensive biochemical studies of AUF1, we proposed an integrated, three-step model for induction of AMD by AUF1 via assembly of a trans-acting complex that targets the mRNA for degradation (52). The first step is dynamic AUF1 dimer binding to an ARE and formation of an oligomeric AUF1 complex (7, 52). Stabilizing ARE-binding proteins (AUBPs) may compete with AUF1 for binding to the ARE during this step, thus preventing AUF1 oligomerization and subsequent factor recruitment (25). Binding of AUF1 to an ARE then permits the second step involving recruitment of additional trans-acting factors including eukaryotic translation initiation factor eIF4G, poly(A)-binding protein, dual-functional heat shock/AUBPs Hsp/Hsc70 (27), and additional unknown proteins, forming a multisubunit AUF1-and signal tra...

Resveratrol, a trihydroxystilbene found in grapes and other plants, has been shown to be active in inhibiting multistage carcinogenesis. Using resveratrol as a prototype, we have synthesized a number of polyhydroxy- and polymethoxy-stilbenes and tested their anti-proliferative effect in normal and transformed human cells. Here we show that one of the resveratrol analogs, 3,4,5,4'-tetrahydroxystilbene (R-4), specifically inhibited the growth of SV40 virally transformed WI38 cells (WI38VA) at 10 microM, but had no effect on normal WI38 cells at even higher concentrations. R-4 also prominently induced apoptosis in WI38VA cells, but not in WI38 cells. RNase protection assay showed that R-4 significantly induced the expression of p53, GADD45 and Bax genes and concomitantly suppressed the expression of bcl-2 gene in WI38VA, but not in WI38 cells. A large increase in p53 DNA binding activity and the presence of p53 in the Bax promoter binding complex suggested that p53 was responsible for the Bax gene expression induced by R-4 in transformed cells. Within 4 h of treatment with R-4, the Bax to bcl-2 protein ratio in WI38 and WI38VA cells was, respectively, 0.1 and 105, a difference of three orders of magnitude. While R-4 prominently induced the p53/Bax pro-apoptotic genes, it also concomitantly suppressed the expression of Cox-2 in WI38VA cells. Taken together, our study suggests that the induction of p53 gene by R-4 in transformed cells may play a key role in the differential growth inhibition and apoptosis of transformed cells.

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