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.

Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) has been shown to play a critical role in mediating the feedback control of MAP kinase cascades in a variety of cellular processes, including proliferation and stress responsiveness. Although MKP-1 expression is induced by a broad array of extracellular stimuli, the mechanisms mediating its induction remain poorly understood. Here we show that MKP-1 mRNA was potently induced by arsenite and ultraviolet light and modestly increased by heat shock and hydrogen peroxide. Interestingly, arsenite also dramatically induces phosphorylation-acetylation of histone H3 at a global level which precedes the induction of MKP-1 mRNA. The transcriptional induction of MKP-1, histone H3 modification, and elevation in MKP-1 mRNA in response to arsenite are all partially prevented by the p38 MAP kinase inhibitor SB203580, suggesting that the p38 pathway is involved in these processes. Finally, analysis of the DNA brought down by chromatin immunoprecipitation (ChIP) reveals that arsenite induces phosphorylation-acetylation of histone H3 associated with the MKP-1 gene and enhances binding of RNA polymerase II to MKP-1 chromatin. ChIP assays following exposure to other stress agents reveal various degrees of histone H3 modification at the MKP-1 chromatin. The differential contribution of p38 and ERK MAP kinases in mediating MKP-1 induction by different stress agents further illustrates the complexity and versatility of stress-induced MKP-1 expression. Our results strongly suggest that chromatin remodeling after stress contributes to the transcriptional induction of MKP-1.The mitogen-activated protein (MAP) kinases play a central role in orchestrating many short-and long-term changes in the cell in response to extracellular stimuli (50). To date, three major MAP kinase subfamilies have been well characterized in mammalian cells: the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK), and p38 (11,40,50). Thus far, numerous proteins with a wide spectrum of biological functions have been identified as the targets of MAP kinase cascades, including protein kinases, cytoskeletal components, phospholipase A2, stalhmin, and the Na ϩ /H ϩ antipump NHE1 (6,23,42). In addition to the proteins that function on the membrane or in the cytoplasm, MAP kinases also play a crucial role in regulating gene transcription. Upon activation, MAP kinases translocate from the cytoplasm to the nucleus, where they phosphorylate and activate a multitude of transcription factors, including c-Myc, c-Jun, c-Fos, Elk-1, and ATF-2, ultimately resulting in enhanced gene transcription (8,24,58). The fact that a broad variety of extracellular signals conscript MAP kinase cascades to convey their specific messages suggests that MAP kinase cascades serve a myriad of purposes and the cascades need to be tightly controlled.The activities of all MAP kinases are regulated through reversible phosphorylation of two different amino acid residues (threonine ...

Background: Oxidative stress-induced DNA damage is repaired by proteins in the base excision pathway. Results: We identified a novel interaction between two DNA repair proteins, OGG1 and PARP-1. Conclusion: OGG1-PARP-1 binding has both a functional and biological consequence. Significance: These results provide insight into the factors that regulate DNA repair under normal and oxidative stress conditions.

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.

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,...

Although epidemiological studies have long established that inorganic arsenic is a potent human carcinogen, the underlying mechanisms are still poorly understood. Recent studies suggest that inorganic arsenic may act as a tumor promoter by perturbing key signaling transduction pathways. We have shown previously that arsenite can potently activate the mitogen-activated protein kinase cascades and induce the expression of proliferation-associated genes, including protooncogenes c-jun and c-fos. In order to elucidate further the molecular mechanisms underlying its tumor-promoting properties, we investigated the signaling events involved in arsenite-mediated induction of c-fos and cjun. We found that induction of both c-fos and c-jun by arsenite can be substantially inhibited by the MEKselective inhibitor U0126, suggesting that the ERK pathway is critically involved in their up-regulation. Interestingly, arsenite dramatically induced the phosphorylation and acetylation of histone H3 preceding the induction of mRNAs encoding c-fos and c-jun. Finally, chromatin immunoprecipitation assays revealed that arsenite treatment markedly induced the phosphorylation/acetylation of histone H3 associated with the c-fos and c-jun genes through an ERK-dependent pathway. Our results strongly suggest that arsenic-triggered alterations in chromatin structure perturb specific gene transcription, including that of proto-oncogenes c-jun and c-fos, and may thereby contribute to the carcinogenic process.

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