This program focuses on the function and regulation of MAP kinase phosphatases. Specifically, we are interested in the role of MAP kinase phosphatase-1 (MKP-1) in the regulation of pro-inflammatory cytokine production by macrophages. Macrophages serve as the first-line defense against pathogenic microbial insult. Among their vast anti-microbial arsenal, macrophages produce potent pro-inflammatory cytokines such as TNF-alpha, IL-1, IL-6 and IL-8, which induce inflammation and recruit other immune cells; e.g., neutrophils and T-lymphocytes. Although these pro-inflammatory cytokines are beneficial to the host defense, they can also trigger pathological conditions when expressed in excess. For example, massive stimulation of macrophages following a severe gram-negative bacterial infection leads to excessive production of pro-inflammatory cytokines, including TNF-alpha and IL-1, and the development of fatal septic shock syndrome, characterized by fever, disseminated intravascular coagulation, and multiple organ failure. In addition, higher levels of pro-inflammatory cytokines are also implicated in a variety of chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and Crohn's disease. Moreover, recent studies suggest that macrophage hyperactivity and the over-production of TNF-alpha contribute to autologous cell destruction and T cell-mediated autoimmune diseases. In macrophages stimulated by bacterial endotoxin lipopolysaccharides (LPS), the biosynthesis of cytokines, especially TNF-alpha, is regulated at multiple levels and involves a multitude of signal transduction pathways. Both the MAP kinases and the transcription factor NF-kappaB play a critical role in mediating TNF-alpha biosynthesis. Particularly important in the regulation of TNF-alpha expression is an AU-rich element (ARE) residing in the 3' untranslated region of the TNF-alpha mRNA that represses TNF-alpha expression post-transcriptionally. MAP kinases, including ERK, JNK, and p38, have been shown to target this ARE to increase TNF-alpha biosynthesis in response to LPS stimulation. The critical role of p38 in TNF-alpha biosynthesis has been well established. Selective inhibition of p38 using specific imidazole compounds such as SB2305080 substantially decreases the translation of TNF-alpha in LPS-stimulated macrophages. Furthermore, inactivation of the gene for MAPKAP kinase-2 (MAPKAPK-2) , a downstream target of p38, abolishes LPS-triggered TNF-alpha production and renders mice resistant to endotoxin-induced septic shock. Moreover, deletion of the TNF-alpha ARE bypasses the requirement of p38/MAPKAPK-2 for LPS-induced TNF-alpha production. JNK also appears to play a role in relieving the ARE-mediated translational silencing of TNF-alpha mRNA, as glucocorticoids have been shown to inhibit LPS-induced JNK activation and reduce TNF-alpha production by macrophages. More recently, using transgenic mice, Dumitru et al. demonstrated that LPS-triggered ERK activation is required for the nucleocytoplasmic transport of TNF-alpha mRNA via a mechanism that involves the TNF-alpha ARE. MKP-1 is the archetype of the MAP kinase phosphatase family that shows substrate preferences toward JNK and p38 MAP kinases. MKP-1 is highly induced by a variety of extracellular stimuli while the signaling pathways controlling its transcription are not well understood. During the past year, we have examined the role of MKP-1 in the macrophage response to inflammatory stimuli using RAW264.7 cells and LPS and a model. Analysis of MAP kinase activity revealed a transient activation of JNK and p38 following LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38; while blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 at a modest level accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-alpha and IL-6. Induction of MKP-1 by LPS was found to involve both enhanced gene expression and increased protein stability. Through using the pharmacological inhibitors of the MAP kinase pathways, we found that the transcriptional induction of MKP-1 in response to LPS is primarily regulated by the ERK pathway with p38 playing only a minor role. LPS stimulation also results in a 3-4-fold increase in MKP-1 half-life. This increase is substantially diminished in the presence of the MEK1/2 inhibitor or by deleting the C-terminal domain of MKP-1, suggesting that MKP-1 stabilization in response to LPS is primarily controlled through ERK-mediated phosphorylation. Finally, we found that MKP-1 expression was 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. Our studies also suggest that the infectious pathogen Vibrio cholerae may exploit the MAP kinase phosphatase to circumvent the host innate immunity. Another area we are investigating is the therapeutic mechanism of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia. As2O3 is highly effective for the treatment of acute promyelocytic leukemia, even in patients unresponsive to all-trans retinoic acid therapy. As2O3 is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. Using cDNA arrays, we have examined the changes of gene expression profiles triggered by clinically achievable doses of As2O3 in acute promyelocytic leukemia NB4 cells. CASPASE-10 expression was found to be potently induced by As2O3. Accordingly, Caspase-10 activity also substantially increased in response to As2O3 treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, markedly blocked caspase-3 activation and significantly attenuated As2O3-triggered apoptosis. Interestingly, treatment of NB4 cells with As2O3 markedly increased histone H3 phosphorylation at Serine-10, an event that is associated with acetylation of the Lysine-14 residue. Chromatin immunoprecipitation (ChIP) assays revealed that As2O3 potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that the effect of As2O3 on chromatin remodeling at the CASPASE-10 gene may play an important role in the induction of apoptosis and contribute to its therapeutic mechanism for acute promyelocytic leukemia.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
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