Dual-specificity phosphatases: therapeutic targets in cancer therapy resistance.

PURPOSE: Therapy resistance is the principal obstacle to achieving cures in cancer patients and its successful tackling requires a deep understanding of the resistance mediators. Increasing evidence indicates that tumor phosphatases are novel and druggable targets in translational oncology and their modulation may hinder tumor growth and motility and potentiate therapeutic sensitivity in various neoplasms via regulation of various signal transduction pathways. Dual-specificity phosphatases (DUSPs) are key players of cell growth, survival and death and have essential roles in tumor initiation, malignant progression and therapy resistance through regulation of the MAPK signaling pathway. In this review, different aspects of DUSPs are discussed. METHODS: A comprehensive literature review was performed using various websites including PubMed. RESULTS: We provide mechanistic insights into the roles of well-known DUSPs in resistance to a wide range of cancer therapeutic approaches including chemotherapy, radiation and molecular targeted therapy in human malignancies. Moreover, we discuss the development of DUSP modulators, with a focus on DUSP1 and 6 inhibitors. Ultimately, the preclinical investigations of small molecule inhibitors of DUSP1 and 6 are outlined. CONCLUSION: Emerging evidence indicates that the DUSP family is aberrantly expressed in human malignancies and plays critical roles in determining sensitivity to a wide range of cancer therapeutic strategies through regulation of the MAPK signaling pathways. Consequently, targeting DUSPs and their downstream molecules can pave the way for more effective cancer therapies.

Bexarotent Attenuated Chronic Constriction Injury-Induced Spinal Neuroinflammation and Neuropathic Pain by Targeting Mitogen-Activated Protein Kinase Phosphatase-1.

It is widely accepted that neuroinflammation in the spinal cord contributes to the development of central sensitization in neuropathic pain. Mitogen-activated protein kinase (MAPK) activation plays a vital role in the development of neuroinflammation in the spinal cord. In this study, we investigated the effect of bexarotene (bex), a retinoid X receptor agonist, on MAPKs activation in chronic constriction injury (CCI)-induced neuropathic pain. The data showed that daily treatment with bex 50 mg/kg significantly alleviated CCI-induced nociceptive hypersensitivity in rats. Bex 50 mg/kg/day inhibited CCI-induced MAPKs (p38MAPK, ERK1/2, and JNK) activation and upregulation of proinflammatory factors (IL-1ß, tumor necrosis factor-α and IL-6). Bex also reversed CCI-induced microglia activation in the ipsilateral spinal cord. Furthermore, bex treatment significantly upregulated MKP-1 in the spinal cord. These effects were completely abrogated by MKP-1 inhibitor BCI. These results indicated that bex relieved CCI-induced neuroinflammation and neuropathic pain by targeting MKP-1. Therefore, bex might be a potential agent for the treatment of neuropathic pain. PERSPECTIVE: Bex could relieve neuropathic pain behaviors in animals by reversing MKP-1 downregulation and MAPKs activation in the spinal cord. Therapeutic applications of bex may be extended beyond cutaneous T-cell lymphoma.

Dual-specificity phosphatase 1 regulates cell cycle progression and apoptosis in cumulus cells by affecting mitochondrial function, oxidative stress, and autophagy.

Dual-specificity phosphatase 1 (DUSP1) is differentially expressed in cumulus cells of different physiological states, but its specific function and mechanism of action remain unclear. In this study, we explored the effects of DUSP1 expression inhibition on cell cycle progression, proliferation, apoptosis, and lactate and cholesterol levels in cumulus cells and examined reactive oxygen species levels, mitochondrial function, autophagy, and the expression of key cytokine genes. The results showed that inhibition of DUSP1 in cumulus cells caused abnormal cell cycle progression, increased cell proliferation, decreased apoptosis rates, increased cholesterol synthesis and lactic acid content, and increased cell expansion. The main reason for these effects was that inhibition of DUSP1 reduced ROS accumulation, increased glutathione level and mitochondrial membrane potential, and reduced autophagy levels in cells. These results indicate that DUSP1 limits the biological function of bovine cumulus cells under normal physiological conditions and will greatly contribute to further explorations of the physiological functions of cumulus cells and the interactions of the cumulus-oocyte complex.

Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition.

Structure based virtual screening attempts to discover DUSP1 inhibitors have yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its analogues were synthesized to explore structure activity relationship (SAR) of DUSP1 inhibition.

Targeted Inhibition of the Dual Specificity Phosphatases DUSP1 and DUSP6 Suppress MPNST Growth via JNK.

PURPOSE: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors. EXPERIMENTAL DESIGN: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. RESULTS: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. CONCLUSIONS: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.

MAPK Reliance via Acquired CDK4/6 Inhibitor Resistance in Cancer.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1-S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor-resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201-14. ©2018 AACR.

A Potential Mechanism for Immune Suppression by Beta-Adrenergic Receptor Stimulation following Traumatic Injury.

BACKGROUND: ß-Adrenergic agents suppress inflammation and may play an important role in posttraumatic infections. Mechanisms may include inhibition of MAP kinase signaling. We sought to determine whether MKP-1 contributed to catecholamine suppression of innate immunity and also wanted to know whether early catecholamine treatment after traumatic injury increases the risk of later nosocomial infection. METHODS: We performed experiments using THP-1 cells and peripheral blood mononuclear cells from healthy individuals. We exposed cells to epinephrine and/or LPS and measured inflammatory gene transcription and MAP kinase activation. We inhibited MKP-1 activity to determine its role in catecholamine-induced immune suppression. Finally, we studied injured subjects to determine whether early catecholamine treatment was associated with nosocomial infection. RESULTS: Epinephrine increases MKP-1 transcripts and protein and decreases LPS-induced p38 and JNK phosphorylation and TNF-α gene transcription. RNAi inhibition of MKP-1 at least partially restores LPS-induced TNF-α gene expression (p = 0.024). In the clinical cohort, subjects treated with ß-adrenergic agents had an increased risk of ventilator-associated pneumonia (aOR = 1.9; 95% CI = 1.3-2.6) and bacteremia (aOR = 1.5; 95% CI = 1.1-2.3). CONCLUSIONS: MKP-1 may have a role in catecholamine-induced suppression of innate immunity, and exogenous catecholamines might contribute to nosocomial infection risk.

MKP-1 suppresses PARP-1 degradation to mediate cisplatin resistance.

Understanding the mechanisms of platinum compound resistance, including cisplatin resistance, has important implications for improving cancer treatments. Previous studies identified a potential role for mitogen-activated protein kinase phosphatase-1 (MKP-1) in cisplatin resistance. This work focuses on the regulation of poly(ADP-ribose) polymerase-1 (PARP-1) expression by MKP-1. We found that MKP-1 overexpression stimulates PARP-1 and poly(ADP-ribose) (PAR) protein expression and cisplatin resistance while its downregulation suppresses PARP-1 and PAR protein expression and cisplatin resistance. Silencing MKP-1 promoted PARP-1 ubiquitination, which decreased PARP-1 protein levels. We also found that silencing c-Jun N-terminal kinase 1/2 (JNK1/2) decreased PARP-1 ubiquitination while increasing total PARP-1 protein levels. Furthermore, we showed that acquired cisplatin-resistant ovarian cancer cells expressed high levels of MKP-1 and PARP-1 proteins, and that silencing MKP-1 or PARP-1 increased cisplatin sensitivity in resistant cells. Notably, the pharmacologic inhibition of PARP activity restored cisplatin sensitivity in MKP-1 overexpressing cells. Thus, this work indicates that suppression of JNK1/2 activity by MKP-1 maintains PARP-1 levels and suggests that MKP-1-mediated cisplatin resistance can be bypassed by PARP-1 inhibition.

MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in response to DNA damage.

Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.

Plumbagin induces apoptosis in lymphoma cells via oxidative stress mediated glutathionylation and inhibition of mitogen-activated protein kinase phosphatases (MKP1/2).

Maintaining cellular redox homeostasis is imperative for the survival and normal functioning of cells. This study describes the role and regulation of MAPKinases in oxidative stress mediated apoptosis. Plumbagin, a vitamin K3 analog and a pro-oxidant, was employed and it induced apoptosis in both mouse and human T-cell lymphoma cell lines via increased oxidative stress, caspase activity and loss of mitochondrial membrane potential. The pro-oxidant and cytotoxic effects of plumbagin were sensitive to antioxidants indicating a decisive role of cellular redox balance. Plumbagin induced persistent activation of JNK and pharmacological inhibition as well as shRNA-mediated JNK knock-down rescued cells from plumbagin-induced apoptosis. Further, plumbagin induced cytochrome c release, FasL expression and Bax levels via activation of JNK pathway. Exposure of lymphoma cells to plumbagin led to inhibition of total and specific phosphatase activity, increased total protein S-glutathionylation and induced glutathionylation of dual specific phosphatase- 1 and 4 (MKP-1 and MKP-2). The in vivo anti-tumor efficacy of plumbagin was demonstrated using a mouse model. In conclusion, oxidative stress mediated tumor cytotoxicity operates through sustained JNK activation via a novel redox-mediated regulation of MKP-1 and MKP-2.

CaMKII protects MKP-1 from proteasome degradation in endothelial cells.

CaMKs are a widely distributed family of kinases with multiple and often cell specific effects on intracellular signal transduction pathway. In endothelial cells, it has been recognized a role for CamKII in several pathways such as eNOS activation and nitric oxide production. It is not clear though, whether CaMKII interfere with other endothelial cell functions such as ERK activation and cell proliferation. We explored this issue in primary cultured rat endothelial cells and we evaluated the effect on endothelial cell proliferation and DNA synthesis. CaMKII inhibition through Cantide, conducted into the cell through Antoennapedia (ANT-CN), showed positive effects on proliferation and H(3)-thimdine incorporation similar to insulin stimulation. Accordingly, both CaMKII pharmacological inhibition and silencing through shRNA produced activation of the p44/42 MAPK. These observations leaded to the hypothesis that CamKII could regulate p44/p42 by interfering with specific ERK phosphatases. Indeed, we found that CaMKII interacts and protect the dual specific phosphatase MKP-1 from proteasome mediated degradation while this complex is disrupted by CaMKII inhibitors. This study reveals that CaMKII, besides phosphorylation through the known ras-raf-mek pathway, can regulate also dephosphorylation of p44/p42 by modulation of MKP-1 level. This novel finding opens to a novel scenario in regulation of endothelial cell functions.

Mitogen-activated protein kinase phosphatase 1 negatively regulates MAPK signaling in mouse hypothalamus.

Mitogen-activated protein kinase phosphatase 1 (MKP-1) is shown to negatively regulate MAPK signaling in various peripheral tissues as well as the central nervous system such as cortex, striatum and hippocampus. In this study, we examined whether MKP-1 regulates MAPK signaling in the mouse hypothalamus. Intraperitoneal injection of TNFα significantly increased MKP-1 mRNA expression in paraventricular and arcuate nuclei in the hypothalamus. TNFα treatment induced increases in MKP-1 expression at both mRNA and protein levels, accompanied by the inactivation of MAPK signaling in mouse hypothalamic explants. Inhibition of MKP-1 by its inhibitor or siRNA increased MAPK activity in the explants. Our data indicate that MKP-1 negatively regulates MAPK signaling in the mouse hypothalamus.

TGF-ß1 and hypoxia-dependent expression of MKP-1 leads tumor resistance to death receptor-mediated cell death.

Sporadic occurrence of transformed tumor cells is under the surveillance of the host immune system and such cells are effectively eliminated by immune-mediated cell death. During tumor progression, the antitumor effects of the tumor microenvironment are suppressed by diverse immunosuppressive mechanisms. In this research, we suggest novel immune evasion strategy of tumor cells through a transforming growth factor (TGF)-ß1- and hypoxia-dependent mechanism. Experimental results showed that TGF-ß1 and hypoxia induced mitogen-activated protein kinase phosphatase (MKP)-1 expression within 1 h, resulting in attenuation of c-Jun N-terminal kinase (JNK) phosphorylation and subsequent death receptor-mediated cell death. In addition, analysis of microarray data and immunostaining of MKP-1 in hepatocellular carcinoma (HCC) patient samples revealed that expression of MKP-1 is notably higher in tumors than in normal tissues, implying that MKP-1-dependent suppression of immune-mediated cell death takes place only in the tumor. To prove that MKP-1 can act as a mediator of immune escape by tumors, we determined whether chemo-resistance against several anticancer drugs could be overcome by knockdown of MKP-1. Cytotoxic assays showed that chemotherapy with siRNA targeting MKP-1 was significantly more effective than chemotherapy in the presence of MKP-1. Thus, we conclude that TGF-ß1 and hypoxia ensure tumor cell survival and growth through expression of MKP-1.

Kaposi's sarcoma-associated herpesvirus suppression of DUSP1 facilitates cellular pathogenesis following de novo infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), and KSHV activation of mitogen-activated protein kinases (MAPKs) initiates a number of key pathogenic determinants of KS. Direct inhibition of signal transduction as a therapeutic approach presents several challenges, and a better understanding of KSHV-induced mechanisms regulating MAPK activation may facilitate the development of new treatment or prevention strategies for KS. MAPK phosphatases, including dual-specificity phosphatase-1 (DUSP1), negatively regulate signal transduction and cytokine activation through MAPK dephosphorylation or interference with effector molecule binding to MAPKs, including the extracellular signal-regulated kinase (ERK). We found that ERK-dependent latent viral gene expression, the induction of promigratory factors, and cell invasiveness following de novo infection of primary human endothelial cells are in part dependent on KSHV suppression of DUSP1 expression during de novo infection. KSHV-encoded miR-K12-11 upregulates the expression of xCT (an amino acid transporter and KSHV fusion/entry receptor), and existing data indicate a role for xCT in the regulation of 14-3-3ß, a transcriptional repressor of DUSP1. We found that miR-K12-11 induces endothelial cell secretion of promigratory factors and cell invasiveness through upregulation of xCT-dependent, 14-3-3ß-mediated suppression of DUSP1. Finally, proof-of-principle experiments revealed that pharmacologic upregulation of DUSP1 inhibits the induction of promigratory factors and cell invasiveness during de novo KSHV infection. These data reveal an indirect role for miR-K12-11 in the regulation of DUSP1 and downstream pathogenesis.

MAPK signaling drives inflammation in LPS-stimulated cardiomyocytes: the route of crosstalk to G-protein-coupled receptors.

Profound cardiovascular dysfunction is an important cause of mortality from septic shock. The molecular underpinnings of cardiac dysfunction during the inflammatory surge of early sepsis are not fully understood. MAPKs are important signal transducers mediating inflammation whereas G-protein signaling pathways modulate the cardiac response to stress. Using H9c2 cardiomyocytes, we investigated the interaction of MAPK and G-protein signaling in a sepsis model to test the hypothesis that the cardiomyocyte inflammatory response is controlled by MAPKs via G-protein-mediated events. We found that LPS stimulated proinflammatory cytokine production was markedly exacerbated by siRNA knockdown of the MAPK negative regulator Mkp-1. Cytokine production was blunted when cells were treated with p38 inhibitor. Two important cellular signaling molecules typically regulated by G-protein-coupled receptors, cAMP and PKC activity, were also stimulated by LPS and inflammatory cytokines TNF-α and IL-6, through a process regulated by Mkp-1 and p38. Interestingly, neutralizing antibodies against Gα(s) and Gα(q) blocked the increase in cellular cAMP and PKC activation, respectively, in response to inflammatory stimuli, indicating a critical role of G-protein coupled receptors in this process. LPS stimulation increased COX-2 in H9c2 cells, which also express prostaglandin receptors. Blockade of G-protein-coupled EP4 prostaglandin receptor by AH 23848 prevented LPS-induced cAMP increase. These data implicate MAPKs and G-proteins in the cardiomyocyte inflammatory response to LPS as well as crosstalk via COX-2-generated PGE(2). These data add to our understanding of the pathogenesis of septic shock and have the potential to guide the selection of future therapeutics.

MKP-1: a critical phosphatase in the biology of macrophages controlling the switch between proliferation and activation.

Macrophages play a central role in the immune response. These cells either proliferate in response to, for example, growth factors or become activated in response to, for example, LPS and develop functional activities. Experiments carried out in mice showed that macrophage proliferation requires a short period of ERK phosphorylation, while an extended period is required for macrophage activation. The length of phosphorylation is controlled by the MAPK phosphatase-1 (MKP-1), a nuclear-localized dual-specificity phosphatase that dephosphorylates the MAPKs ERK, p38, and c-Jun NH(2) -terminal kinase (JNK). MKP-1 is induced in macrophages by growth factors, as well as by activators such as LPS, but with different kinetics; to achieve the different functional outcomes (proliferation versus activation), the inhibition of MKP-1 by cytokines such as IFN-γ blocks macrophage proliferation and induces activation. The data presented in this review show that this phosphatase is the switch between macrophage proliferation and activation.

Targeting mitogen-activated protein kinase phosphatase-1 (MKP-1): structure-based design of MKP-1 inhibitors and upregulators.

Mitogen-activated protein kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate both phospho-tyrosine and phospho-threonine residues on mitogen-activated protein kinases (MAPKs). Because the MAPK family of signalling molecules (phospho-p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)) play essential roles in cell signalling pathways that regulate cell growth and inflammation, controlling MAPK-mediated pathways is a therapeutically attractive strategy. While small molecule MAPK inhibitors have utility, in this review we will focus on exploring the potential of targeting the endogenous MAPK deactivator--MKP-1. Importantly, there is a strong justification for developing both inhibitors and upregulators of MKP-1 because of the diverse roles played by MAPKs in disease: for example, in cancer, MKP-1 inhibitors may prove beneficial, as MKP-1 is overexpressed and is considered responsible for the failure of JNK-driven apoptotic pathways induced by chemotherapeutics; conversely, in inflammatory diseases such as asthma and arthritis, MKP-1 reduces MAPK-mediated signalling and developing novel ligands to upregulate MKP-1 levels would be a therapeutically attractive anti-inflammatory strategy. Thus, in this review we utilise MKP-1 homology modeling to highlight the structural features of MKP-1 inhibitors that permit potent and selective inhibition, and to provide insights into the structural requirements for selective MKP-1 upregulators.

Interleukin-17A stimulates cardiac fibroblast proliferation and migration via negative regulation of the dual-specificity phosphatase MKP-1/DUSP-1.

The dual-specificity mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) inactivates MAP kinases by dephosphorylation. Here we show that the proinflammatory cytokine interleukin (IL)-17A induces adult mouse primary cardiac fibroblast (CF) proliferation and migration via IL-17 receptor A//IL-17 receptor C-dependent MKP-1 suppression, and activation of p38 MAPK and ERK1/2. IL-17A mediated p38 MAPK and ERK1/2 activation is inhibited by MKP-1 overexpression, but prolonged by MKP-1 knockdown. IL-17A induced miR-101 expression via PI3K/Akt, and miR-101 inhibitor reversed MKP-1 down regulation. Importantly, MKP-1 knockdown, pharmacological inhibition of p38 MAPK and ERK1/2, or overexpression of dominant negative MEK1, each markedly attenuated IL-17A-mediated CF proliferation and migration. Similarly, IL-17F and IL-17A/F heterodimer that also signal via IL-17RA/IL-17RC, stimulated CF proliferation and migration. These results indicate that IL-17A stimulates CF proliferation and migration via Akt/miR-101/MKP-1-dependent p38 MAPK and ERK1/2 activation. These studies support a potential role for IL-17 in cardiac fibrosis and adverse myocardial remodeling.

MKP-1: a negative feedback effector that represses MAPK-mediated pro-inflammatory signaling pathways and cytokine secretion in human airway smooth muscle cells.

Airway smooth muscle (ASM) plays an important immunomodulatory role in airway inflammation in asthma. In our previous in vitro studies in ASM cells delineating the pro-inflammatory mitogen-activated protein kinase (MAPK) signaling pathways activated by tumor necrosis factor α (TNFα), we observed that TNFα concomitantly induces the rapid, but transient, upregulation of the anti-inflammatory protein-mitogen-activated protein kinase phosphatase 1 (MKP-1). As this was suggestive of a negative feedback loop, the aim of this study was to investigate the molecular mechanisms of MKP-1 upregulation by TNFα and to determine whether MKP-1 is a negative feedback effector that represses MAPK-mediated pro-inflammatory signaling pathways and cytokine secretion in ASM cells. Herein, we show that TNFα increases MKP-1 mRNA expression and protein upregulation in a p38 MAPK-dependent manner. TNFα does not increase MKP-1 transcription (measured by MKP-1 promoter activity); rather, we found that TNFα-induced MKP-1 mRNA stability is regulated by the p38 MAPK pathway. Inhibiting MKP-1 upregulation (with triptolide) demonstrated the precise temporal control exerted on MAPK signaling by MKP-1. In the absence of MKP-1, downstream phosphoprotein targets of MAPKs (such as MSK-1 and histone H3) are not turned off at the right time, allowing pro-inflammatory pathways to continue in an unrestrained manner. This is confirmed by knocking-down MKP-1 by siRNA where enhanced secretion of the neutrophil chemoattractant cytokine-interleukin 8 was detected in the absence of MKP-1. Thus, by activating p38 MAP kinase, TNFα concomitantly upregulates the MAPK deactivator MKP-1 to serve as an important negative feedback effector, limiting the extent and duration of pro-inflammatory MAPK signaling and cytokine secretion in ASM cells.