MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSHbeads or anti-Myc linked to magnetic beads MK21 mediates several p38␣,␤ MAP kinase-dependent processes (for a review, see Ref. 1), demonstrated most clearly by results from targeted disruption of the MK2 gene in mice (2). MK2 (Ϫ,Ϫ) mice have suppressed stress responses. Cellular studies show deficits in motility, chemotaxis, and cytokine production. Macrophages taken from MK2 (Ϫ,Ϫ) mice exhibit normal TNF mRNA induction in response to endotoxin but do not release TNF protein. Cellular TNF protein was markedly decreased in MK2 (Ϫ,Ϫ) macrophages, suggesting a block in production of TNF from TNF mRNA (2). TNF expression is regulated both via mRNA stability and translation (3, 4) but is not completely understood.The p38␣,␤ MAP kinase pathway regulates stability of mRNAs that contain AU-rich elements in their 3Ј-untranslated regions. Examples include TNF, COX-2, interleukin-6, and interleukin-1␤ (4 -6). Evidence chiefly comes from mRNA stabilization caused by transfection of mutationally activated MEK3/MEK6 or by the addition of agents that activate p38 MAPK and conversely from destabilization caused by the addition of a p38␣,␤ MAP kinase inhibitor. Since p38␣,␤ is required to activate MK2, these experiments do not dissect contributions from MK2. Studies in MK2 (Ϫ,Ϫ) cells suggest that MK2 regulates stability of some cytokine mRNAs (2, 4). Lasa et al. (5) first reported that expression of a mutant of MK2 with constitutive activity stabilized COX-2 mRNA in the presence of SB2035780 and that expression of a kinase-defective MK2 blocked the stabilization induced by activated MEK6, arguing that MK2 is necessary and sufficient to induce stabilization of at least the COX-2 mRNA (5). How MK2 regulates cytokine production post-transcriptionally is unknown. Mahtani et al. (7) reported that tristetraprolin (TTP) is an in vitro substrate for MK2, motivating the detailed studies we describe.TTP (for a review, see Ref. 8) destabilizes class II AU-rich elements and is the prototype for a non-zinc finger class of nucleic acid-binding proteins. Destabilization requires integrity of the TTP tandem Cys 3 His RNA binding domains that coordinate zinc in a disklike structure (9 -11). TTP-null mice exhibit many defects including inflammatory arthritis and systemic lupus erythematosis-like symptoms attributed to increased production of TNF (12).TTP is phosphorylated in cells treated with growth factors or cytokines. Phosphorylation occurs at more than one site evident by the appearance of two distinct slower migrating forms of TTP on gels after stimulation that are reversed by phosphatase treatment (7, 13). TTP under...

Oxidative stress broadly impacts cells, initiating regulatory pathways as well as apoptosis and necrosis. A key molecular event is protein S-glutathionylation, and thioltransferase (glutaredoxin) is a specific and efficient catalyst of protein-SSG reduction. In this study 30-min exposure of H9 and Jurkat cells to cadmium inhibited intracellular protein-SSG reduction, and this correlated with inhibition of the thioltransferase system, consistent with thioltransferase being the primary intracellular catalyst of deglutathionylation. The thioredoxin system contributed very little to total deglutathionylase activity. Thioltransferase and GSSG reductase in situ displayed similar dose-response curves (50% inhibition near 10 M cadmium in extracellular buffer). Acute cadmium exposure also initiated apoptosis, with H9 cells being more sensitive than Jurkat. Moreover, transfection with antisense thioltransferase cDNA was incompatible with cell survival. Collectively, these data suggest that thioltransferase has a vital role in sulfhydryl homeostasis and cell survival. In separate experiments, cadmium inhibited the isolated component enzymes of the thioltransferase and thioredoxin systems, consistent with the vicinal dithiol nature of their active sites: thioltransferase (IC 50 Ϸ 1 M), GSSG reductase (IC 50 Ϸ 1 M), thioredoxin (IC 50 Ϸ 8 M), thioredoxin reductase (IC 50 Ϸ 0.2 M). Disruption of the vicinal dithiol on thioltransferase (via oxidation to C22-SS-C25; or C25S mutation) protected against cadmium, consistent with a dithiol chelation mechanism of inactivation.

Fig. 2. Confocal imaging of cell penetration. BHK cells were treated for 1 h with DyLight 550 fluorescently labeled cargo proteins β-Gal (A), HRP (B) and myoglobin (C) (rendered as white in left panels, red in center and right panels), in either the absence or presence of TAT-CaM, washed and imaged live. Center images are optical sections set at a similar depth of the nucleus (NucBlue staining, white, center and right panels), as determined by position within the Z-stack. Orthogonal projections are shown at the right (boxed in red) and top (boxed in green) sides of each panel. Cytoplasmic compartments in live cells were visualized using CellTracker Green CMFDA dye (green in right panels). Comparison of TAT-CaM-treated versus untreated cells indicates that cargo proteins are entering the cell, and are localized primarily to the cytoplasm. Scale bars in all panels, 20 μm. Each experiment was replicated at least twice with the same results. 2474 ABSTRACTThe use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.

We have shown that chronic elevated glucose (25 mM) increases monocyte adhesion to human aortic endothelial cells (EC). This increased adhesion is mediated primarily through induction of interleukin (IL)-8 via activation of the transcription factor AP-1 (Srinivasan, S., Yeh, M., Danziger, E. C., Hatley, M. E., Riggan, A. E., Leitinger, N., Berliner, J. A., and Hedrick, C. C. (2003) Circ. Res. 92, 371-377). In the current study, we identified the elements in the AP-1 transcriptional complex that are activated by glucose. These elements include c-Jun, c-Fos, and Fra-1. AP-1 is activated by cellular oxidative stress, and we have reported significant production of ROS by high glucose-cultured cells. We examined signaling pathways upstream of AP-1 in EC that lead to AP-1 activation by HG. EC cultured in 25 mM glucose had a 2-fold increase in p38 phosphorylation compared with control normal glucose-cultured EC. Inhibition of the p38 pathway using 5 M SB203580 significantly reduced glucose-mediated IL-8 mRNA production by 60%. Furthermore, blocking p38 pathway activation using a dominant-negative p38 construct significantly reduced glucose-mediated monocyte adhesion by 50%. Thus, glucose-stimulated monocyte adhesion is primarily regulated through phosphorylation of p38 with subsequent activation of AP-1, leading to IL-8 production. To study this pathway in the setting of diabetes, we used the db/db mouse. P38 phosphorylation was increased in diabetic db/db mice compared with control mice. We found a dramatic elevation in plasma levels of KC, the mouse ortholog of IL-8 in diabetic db/db mice (1800 ؎ 100 pg/ml KC in db/db versus 300 ؎ 75 pg/ml in C57BL/6J control mice, p < 0.0001). Inhibition of the p38 pathway in diabetic db/db mice significantly reduced monocyte adhesion by 50%. Taken together, these data indicate that chronic elevated glucose in diabetes activates the p38 MAP kinase pathway to increase inflammatory IL-8 gene induction and monocyte/endothelial adhesion.