Exposure of macrophages to LPS elicits the production of proinflammatory cytokines, such as TNF-α, through complex signaling mechanisms. Mitogen-activated protein (MAP) kinases play a critical role in this process. In the present study, we have addressed the role of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory cytokine production using RAW264.7 macrophages. Analysis of MAP kinase activity revealed a transient activation of c-Jun N-terminal kinase (JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38, whereas blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-α and IL-6. Induction of MKP-1 by LPS was found to be extracellular signal-regulated kinase dependent and involved enhanced gene expression and increased protein stability. Finally, MKP-1 expression was also induced by glucocorticoids as well as cholera toxin B subunit, an agent capable of preventing autoimmune diseases in animal models. These findings highlight MKP-1 as a critical negative regulator of the macrophage inflammatory response, underscoring its premise as a potential target for developing novel anti-inflammatory drugs.

Nuclear import of HuR, a shuttling RNA-binding protein, is associated with reduced stability of its target mRNAs. Increased function of the AMP-activated protein kinase (AMPK), an enzyme involved in responding to metabolic stress, was recently shown to reduce the cytoplasmic levels of HuR. Here, we provide evidence that importin ␣1, an adaptor protein involved in nuclear import, contributes to the nuclear import of HuR through two AMPK-modulated mechanisms. First, AMPK triggered the acetylation of importin ␣1 on Lys 22 , a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin ␣1 on Ser 105 . Accordingly, expression of importin ␣1 proteins bearing K22R or S105A mutations failed to mediate the nuclear import of HuR in intact cells. Our results point to importin ␣1 as a critical downstream target of AMPK and key mediator of AMPK-triggered HuR nuclear import.

MKP-2 is a member of the mitogen-activated protein (MAP) kinase phosphatase family which has been suggested to play an important role in the feedback control of MAP kinase-mediated gene expression. Although MKP-2 preferentially inactivates extracellular signalregulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK) MAP kinase subfamilies, the mechanisms underlying its own regulation remain unclear. In this report, we have examined the MKP-2 interaction with and catalytic activation by distinct MAP kinase subfamilies. We found that the catalytic activity of MKP-2 was enhanced dramatically by ERK and JNK but was affected only minimally by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, whereas JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the NH 2 -terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised the catalytic activation of MKP-2 by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 whereas MKP-2 mutants incapable of binding to ERK/ p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for the substrate preference of MKP-2 and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function.

Wu X, Guo R, Chen P, Wang Q, Cunningham PN. TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho-and myosin light chain kinase-dependent mechanism.

Arsenic trioxide (As 2 O 3 ) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As 2 O 3 is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As 2 O 3 in acute promyelocytic leukemia NB4 cells. CASPASE-10 expression was found to be potently induced by As 2 O 3 . Accordingly, caspase-10 activity also substantially increased in response to As 2 O 3 treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As 2 O 3 -triggered apoptosis. Interestingly, the treatment of NB4 cells with As 2 O 3 markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As 2 O 3 potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that the effect of As 2 O 3 on histone H3 phosphoacetylation at the CASPASE-10 gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.Acute promyelocytic leukemia (APL) 1 accounts for ϳ10 -15% of adult myeloid leukemias with 3,500 -5,500 new cases diagnosed annually (1, 2). The vast majority of APL patients harbor the chromosomal translocation t(15,17)(q22;q21) involving the retinoic acid receptor ␣ (RAR␣) gene on chromosome 15 and the promyelocytic leukemia (PML) gene on chromosome 17, generally giving rise to two fusion genes, PML-RAR␣ and RAR␣-PML (1, 3). Studies using transgenic mice have demonstrated that the protein product of the PML-RAR␣ fusion gene is primarily responsible for the leukemogenic property of this characteristic translocation (4, 5). All-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, can induce complete remission in most APL patients associated with an enhancement of differentiation pathways (6). Recent studies (7-9) have provided strong evidence that the induction of leukemia by the PML-RAR␣ protein relies on its ability to repress gene transcription by recruiting transcription repressor complexes. Pharmacological doses of ATRA stimulate the release of the transcription repressor complexes from PML-RAR␣, thereby activating the transcription of genes critical for normal granulocytic differentiation. However, ATRA is not curative, and resistance rapidly develops usually within 10 months of therapy (2, 6). Therefore, alternative therapies are necessary.Recently, As 2 O 3 was identified as a potent anti-leukemic agent for treating not only newly diagnosed but also relapsed APL patients (2, 10 -13), and it is remarkably effective in ATRA-refractory patients (2, 12). However, the mechanisms underlying its therapeutic effects are not well understood (2,...

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Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is the archetypal member of the dual-specificity protein phosphatase family, the expression of which can be rapidly induced by a variety of growth factors and cellular stress. Since MKP-1 protein localizes in the nucleus, it has been suggested to play an important role in the feedback control of MAP kinase-regulated gene transcription. Recently it has been demonstrated that the interaction of several cytosolic MAP kinase phosphatases with MAP kinases can trigger the catalytic activation of the phosphatases. It is unclear whether such a regulatory mechanism can apply to nuclear MAP kinase phosphatases and serve as an additional apparatus for the feedback control of MAP kinase-mediated gene expression. Here we have shown that MKP-1 associates directly with p38 MAP kinase both in vivo and in vitro, and that this interaction enhances the catalytic activity of MKP-1. The point mutation Asp-316-->Asn in the C-terminus of p38, analogous to the ERK2 (extracellular-signal-regulated kinase 2) sevenmaker mutation, dramatically decreases its binding to MKP-1 and substantially compromises its stimulatory effect on the catalytic activity of this phosphatase. Consistent with its defective interaction with MKP-1, this p38 mutant also displays greater resistance to dephosphorylation by the phosphatase. Our studies provide the first example of catalytic activation of a nuclear MAP kinase phosphatase through direct binding to a MAP kinase, suggesting that such a regulatory mechanism may play an important role in the feedback control of MAP kinase signalling in the nuclear compartment.

SummaryConstitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukaemogenesis. We have examined, by cDNA microarray analysis, the changes in gene expression induced by FLT3/ITD or constitutively activated wild type FLT3 signalling. A limited set of genes was consistently affected by FLT3 inhibition. In confirmation of their FLT3 dependence, these genes returned toward pretreatment levels of expression after reversal of FLT3 inhibition. Several of the most significantly affected genes are involved in the RAS/mitogen-activated protein kinase, Janus kinase/signal transducer and activator of transcription and phosphatidylinositol 3 kinase (PI3K)/AKT pathways. These data suggest that constitutively activated FLT3 works through multiple signal transduction pathways. PIM1, MYC and CCND3 were chosen from this gene set to explore their biological roles. Knock-down of these genes by small interfering RNA showed that these genes play important roles in constitutively activated FLT3 expressing cells. The alterations of the gene expression profiles in these cells help to further elucidate the mechanisms of FLT3-mediated leukaemogenesis.Keywords: Fms-like tyrosine kinase 3, Fms-like tyrosine kinase 3/internal tandem duplication, acute myeloid leukaemia, gene expression profile. research paper ª