DUSP1 promotes senescence of retinoblastoma cell line SO-Rb5 cells by activating AKT signaling pathway.

OBJECTIVE: Retinoblastoma seriously threats to human health and life. Molecular targeted therapy of retinoblastoma supplies the direction of research in the future. This study aims to investigate the impact of DUSP1 on human retinoblastoma SO-Rb5 cell senescence. MATERIALS AND METHODS: Angiotensin II (AGII) was used to induce human retinoblastoma SO-Rb5 cell senescence model. DUSP1 over-expression plasmid and small interfere RNA (siRNA) were transfected into SO-Rb5 cells by Lipofectamine. Dual specificity phosphatase 1 (DUSP1), p53, p16, and protein kinase B (Akt) signaling expressions were detected with Western blot assay. SH-6 was applied to inhibit Akt signaling in SO-Rb5 cells. Cell senescence was evaluated by using ß-galactosidase test. RESULTS: DUSP1 level increased in SO-Rb5 cells induced by AGII. Senescence protein p53 and p16 significantly upregulated in SO-Rb5 cell senescence model, together with ß-galactosidase staining. DUSP1 plasmid transfection significantly enhanced DUSP expression, triggered SO-Rb5 cell senescence, and inhibited Akt signaling activation. DUSP1 siRNA exhibited the opposite effects. SH-6 significantly increased SO-Rb5 cell senescence induced by AGII through inhibiting Akt signaling. CONCLUSIONS: DUSP1 facilitated human retinoblastoma SO-Rb5 cell senescence induced by AGII through inhibiting Akt signaling pathway.

A cannabinoid receptor 2 agonist reduces blood-brain barrier damage via induction of MKP-1 after intracerebral hemorrhage in rats.

BACKGROUND AND PURPOSE: The blood-brain barrier (BBB) disruption and the following development of brain edema, is the most life-threatening secondary injury after intracerebral hemorrhage (ICH). This study is to investigate a potential role and mechanism of JWH133, a selected cannabinoid receptor type2 (CB2R) agonist, on protecting blood-brain barrier integrity after ICH. METHODS: 192 adult male Sprague-Dawley (SD) rats were randomly divided into Sham; ICH + Vehicle; ICH + JWH 1.0 mg/kg, ICH + JWH 1.5 mg/kg and ICH + JWH 2.0 mg/kg; ICH + SR + JWH respectively. Animals were euthanized at 24 h following western blots and immunofluorescence staining, we also examined the effect of JWH133 on the brain water contents, neurobehavioral deficits and blood brain barrier (BBB) permeability, meanwhile reassessed the inflammatory cytokines concentrations around the hematoma by enzyme-linked immunosorbent assay (ELISA) in each group. RESULTS: JWH133 (1.5 mg/kg) administration ameliorated brain edema, neurological deficits and blood-brain barrier damage, as well as microglia activation. The expression of pro-inflammatory mediators interleukin 1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metallopeptidase-2/9 (MMP2/9) were attenuated, but not monocyte chemoattractant protein-1 (MCP-1). Additionally, decreases in zonula occludens-1 (ZO-1) and claudin-5 expression were partially recovered by JWH133. Furthermore, JWH133 upregulated the expression level of MKP-1, which leads to the inhibition of MAPKs signaling pathway activation, especially for ERK and P38. However, these effects were reversed by pretreatment with a selective CB2R antagonist, SR144528. CONCLUSIONS: CB2R agonist alleviated neuroinflammation and protected blood-brain barrier permeability in a rat ICH model. Further molecular mechanisms revealed which is probably mediated by enhancing the expression of MKP-1, then inhibited MAPKs signal transduction.

miR-424 protects PC-12 cells from OGD-induced injury by negatively regulating MKP-1.

OBJECTIVE: It's of great significance to investigate the novel targets of drugs for the treatment of stroke. In this study, we explored the neuroprotective role of miR-424 in oxygen glucose deprivation (OGD)-induced injuries in PC-12 cells. MATERIALS AND METHODS: PC-12 cells were subjected to OGD stimulation to mimic ischemic injury. The expressions of miR-424 and mitogen-activated protein kinase phosphatase-1 (MKP-1) were altered by transient transfection with miR-424 mimic, miR-424 inhibitor, pEX-MKP-1, or sh-MKP-1. Cell counting kit-8 (CCK-8) assay, flow cytometry, and quantitative reverse transcription polymerase chain reaction (qRT-PCR), were conducted to respectively detect cell viability, apoptotic cells, and the expression of miR-424 and MKP-1. The protein expressions of several factors were determined by Western blot. Meanwhile, relative luciferase activity assay was done to verify the predicted targets association. RESULTS: OGD induced injury in PC-12 cells by suppressing cell viability and inducing apoptosis. OGD also induced the expression of miR-424 in PC-12 cells. Overexpression of miR-424 protected PC-12 cells from OGD-induced injury by increasing cell viability and decreasing apoptosis. MKP-1 was a direct target of miR-424, and its expression was negatively regulated by miR-424. Up-regulation of expression of MKP-1 aggravated OGD-induced cell injury by inhibiting the expression of hypoxia-inducible factor 1α (HIF-1α), thus inhibiting the PI3K/AKT/mTOR pathways. CONCLUSIONS: miR-424 protected PC-12 cells from OGD-induced injury through direct suppression of MKP-1 expression, as MKP-1 promoted OGD-induced cell injury by inhibiting the expression of HIF-1α and PI3K/AKT/mTOR pathways.

Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB.

Eucalyptus oil (EO) used in traditional medicine continues to prove useful for aroma therapy in respiratory ailments; however, there is a paucity of information on its mechanism of action and active components. In this direction, we investigated EO and its dominant constituent 1,8-cineole (eucalyptol) using the murine lung alveolar macrophage (AM) cell line MH-S. In an LPS-induced AM inflammation model, pre-treatment with EO significantly reduced (P ≤0.01or 0.05) the pro-inflammatory mediators TNF-α, IL-1 (α and ß), and NO, albeit at a variable rate and extent; 1,8-cineole diminished IL-1 and IL-6. In a mycobacterial-infection AM model, EO pre-treatment or post-treatment significantly enhanced (P ≤0.01) the phagocytic activity and pathogen clearance. 1,8-cineole also significantly enhanced the pathogen clearance though the phagocytic activity was not significantly altered. EO or 1,8-cineole pre-treatment attenuated LPS-induced inflammatory signaling pathways at various levels accompanied by diminished inflammatory response. Among the pattern recognition receptors (PRRs) involved in LPS signaling, the TREM pathway surface receptor (TREM-1) was significantly downregulated. Importantly, the pre-treatments significantly downregulated (P ≤0.01) the intracellular PRR receptor NLRP3 of the inflammasome, which is consistent with the decrease in IL-1ß secretion. Of the shared downstream signaling cascade for these PRR pathways, there was significant attenuation of phosphorylation of the transcription factor NF-κB and p38 (but increased phosphorylation of the other two MAP kinases, ERK1/2 and JNK1/2). 1,8-cineole showed a similar general trend except for an opposite effect on NF-κB and JNK1/2. In this context, either pre-treatment caused a significant downregulation of MKP-1 phosphatase, a negative regulator of MAPKs. Collectively, our results demonstrate that the anti-inflammatory activity of EO and 1,8-cineole is modulated via selective downregulation of the PRR pathways, including PRR receptors (TREM-1 and NLRP3) and common downstream signaling cascade partners (NF-κB, MAPKs, MKP-1). To our knowledge, this is the first report on the modulatory role of TREM-1 and NLRP3 inflammasome pathways and the MAPK negative regulator MKP-1 in context of the anti-inflammatory potential of EO and its constituent 1,8-cineole.

p38α MAPK inhibits stretch-induced JNK activation in cardiac myocytes through MKP-1.

Mechanical stretch is a major determinant that leads to heart failure, which is associated with a steady increase in myocardial angiotensinogen (Aogen) expression and formation of the biological peptide angiotensin II (Ang II). c-jun NH2-terminal kinase (JNK) and p38α have been found to have opposing roles on stretch-induced Aogen gene expression in neonatal rat ventricular myocytes (NRVM). JNK negatively regulated Aogen expression in NRVM following acute stretch, whereas with prolonged stretch, JNK phosphorylation was downregulated and p38α was found responsible for upregulation of Aogen expression. However, the mechanisms responsible for regulation of these kinases, especially the cross-talk between p38 and JNK1/2, remain to be determined. In this study, a combination of pharmacologic and molecular approaches (adenovirus-mediated gene transfer) were used to examine the mechanisms by which p38 regulates JNK phosphorylation in NRVM under stretch and non-stretch conditions. Pharmacologic inhibition of p38 significantly increased JNK phosphorylation in NRVM at 15 min, whereas overexpression of wild-type p38α significantly decreased JNK phosphorylation. While p38α overexpression prevented stretch-induced JNK phosphorylation, pharmacologic p38 inhibition abolished the JNK dephosphorylation during 15-60 min of stretch. Expression of constitutively-active MKK3 (MKK3CA), the upstream activator of p38, abolished JNK phosphorylation in both basal and stretched NRVM. Pharmacologic inhibition of MAP kinase phosphatase-1 (MKP-1) or protein phosphatase-1 (PP1) increased JNK phosphorylation in NRVM, suggesting the involvement of these phosphatases on reversing stretch-induced JNK activation. Inhibition of MKP-1, but not PP1, reduced JNK phosphorylation in NRVM overexpressing MKK3CA under basal conditions (no-stretch). Inhibition of MKP-1 also enhanced stretch-induced JNK phosphorylation in NRVM at 15 to 60 min. In summary, these results indicate that MKP-1 inhibits JNK phosphorylation in stretched NRVM through p38 dependent and independent mechanisms, whereas PP1 regulates JNK through a p38-independent mechanism.

Atrial overexpression of angiotensin-converting enzyme 2 improves the canine rapid atrial pacing-induced structural and electrical remodeling. Fan, ACE2 improves atrial substrate remodeling.

The purpose of this study was to investigate whether atrial overexpression of angiotensin-converting enzyme 2 (ACE2) by homogeneous transmural atrial gene transfer can reverse atrial remodeling and its mechanisms in a canine atrial-pacing model. Twenty-eight mongrel dogs were randomly divided into four groups: Sham-operated, AF-control, gene therapy with adenovirus-enhanced green fluorescent protein (Ad-EGFP) and gene therapy with Ad-ACE2 (Ad-ACE2) (n = 7 per subgroup). AF was induced in all dogs except the Sham-operated group by rapid atrial pacing at 450 beats/min for 2 weeks. Ad-EGFP and Ad-ACE2 group then received epicardial gene painting. Three weeks after gene transfer, all animals except the Sham group underwent rapid atrial pacing for another 3 weeks and then invasive electrophysiological, histological and molecular studies. The Ad-ACE2 group showed an increased ACE2 and Angiotensin-(1-7) expression, and decreased Angiotensin II expression in comparison with Ad-EGFP and AF-control group. ACE2 overexpression attenuated rapid atrial pacing-induced increase in activated extracellular signal-regulated kinases and mitogen-activated protein kinases (MAPKs) levels, and decrease in MAPK phosphatase 1(MKP-1) level, resulting in attenuation of atrial fibrosis collagen protein markers and transforming growth factor-ß1. Additionally, ACE2 overexpression also modulated the tachypacing-induced up-regulation of connexin 40, down-regulation of connexin 43 and Kv4.2, and significantly decreased the inducibility and duration of AF. ACE2 overexpression could shift the renin-angiotensin system balance towards the protective axis, attenuate cardiac fibrosis remodeling associated with up-regulation of MKP-1 and reduction of MAPKs activities, modulate tachypacing-induced ion channels and connexin remodeling, and subsequently reduce the inducibility and duration of AF.

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.

Mutant PIK3CA controls DUSP1-dependent ERK 1/2 activity to confer response to AKT target therapy.

BACKGROUND: Alterations in the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway are frequent in urothelial bladder cancer (BLCA) and thus provide a potential target for novel therapeutic strategies. We investigated the efficacy of the AKT inhibitor MK-2206 in BLCA and the molecular determinants that predict therapy response. METHODS: Biochemical and functional effects of the AKT inhibitor MK-2206 were analysed on a panel of 11 BLCA cell lines possessing different genetic alterations. Cell viability (CellTiter-Blue, cell counts), apoptosis (caspase 3/7 activity) and cell cycle progression (EdU incorporation) were analysed to determine effects on cell growth and proliferation. cDNA or siRNA transfections were used to manipulate the expression of specific proteins such as wild-type or mutant PIK3CA, DUSP1 or CREB. For in vivo analysis, the chicken chorioallantoic membrane model was utilised and tumours were characterised by weight and biochemically for the expression of Ki-67 and AKT phosphorylation. RESULTS: Treatment with MK-2206 suppressed AKT and S6K1 but not 4E-BP1 phosphorylation in all cell lines. Functionally, only cell lines bearing mutations in the hotspot helical domain of PIK3CA were sensitive to the drug, independent of other genetic alterations in the PI3K or MAPK signalling pathway. Following MK-2206 treatment, the presence of mutant PIK3CA resulted in an increase in DUSP1 expression that induced a decrease in ERK 1/2 phosphorylation. Manipulating the expression of mutant or wild-type PIK3CA or DUSP1 confirmed that this mechanism is responsible for the induction of apoptosis and the inhibition of tumour proliferation in vitro and in vivo, to sensitise cells to AKT target therapy.Conclusion or interpretation:PIK3CA mutations confer sensitivity to AKT target therapy in BLCA by regulating DUSP1 expression and subsequent ERK1/2 dephosphorylation and can potentially serve as a stratifying biomarker for treatment.

Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase-1.

BACKGROUND AND PURPOSE: 3',5'-Cyclic nucleotide PDE4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5'AMP, down-regulating cAMP signalling in cells. MAPK phosphatase-1 (MKP-1) is an endogenous p38 MAPK signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and whether MKP-1 is involved in the anti-inflammatory effects of rolipram. EXPERIMENTAL APPROACH: The effect of rolipram on TNF production was investigated in J774 mouse macrophage cell line and in primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(-/-) mice. We also investigated the effect of rolipram on carrageenan-induced paw inflammation in WT and MKP-1(-/-) mice. KEY RESULTS: MKP-1 expression was enhanced by rolipram, by a non-selective PDE inhibitor IBMX and by a cAMP analogue 8-Br-cAMP in J774 cells and in PM. Enhanced MKP-1 mRNA expression by rolipram was reversed by a PKA inhibitor. Rolipram, IBMX and 8-Br-cAMP also inhibited TNF production in activated macrophages. Accordingly, rolipram inhibited TNF production in PMs from WT mice but, interestingly, not in PMs from MKP-1(-/-) mice. Furthermore, rolipram attenuated carrageenan-induced paw inflammation in WT but not in MKP-1(-/-) mice. CONCLUSIONS AND IMPLICATIONS: PDE4 inhibitor rolipram was found to enhance the expression of MKP-1, and MKP-1 mediated, at least partly, the anti-inflammatory effects of PDE4 inhibition. The results suggest that compounds that enhance MKP-1 expression and/or MKP-1 activity hold potential as novel anti-inflammatory drugs.

Impaired Notch-MKP-1 signalling in hidradenitis suppurativa: an approach to pathogenesis by evidence from translational biology.

Recent findings in familial hidradenitis suppurativa (HS) demonstrated loss-of-function mutations of components of the γ-secretase (GS) complex leading to decreased protease cleaving activity, which may compromise canonical Notch signalling. Appropriate Notch signalling is of pivotal importance for maintaining the inner and outer root sheath of the hair follicle and skin appendages. This viewpoint on the pathogenesis of HS is primarily supported by circumstantial evidence derived from translational biology. Impaired Notch signalling is proposed to be the major pathogenic mechanism of HS. Deficient Notch signalling switches the fate of outer root sheath cells, resulting in conversion of hair follicles to keratin-enriched epidermal cysts. Impaired Notch signalling may compromise apocrine gland homoeostasis as well. Damage-associated molecular pattern molecules released by either ruptured epidermal cysts exposing keratin fibres or altered structural components of less maintained apocrine glands may both stimulate TLR-mediated innate immunity. All aggravating factors of HS, that is, smoking, obesity, skin occlusion, androgens and progesterone, may further promote inflammation by release of proinflammatory cytokines derived from activated monocyte/macrophages. Inappropriate Notch signalling may not only initiate inflammation in HS but may lead to insufficient feedback inhibition of overstimulated innate immunity. Regular Notch signalling via induction of MAPK phosphatase-1 (MKP-1) terminates TLR-MAPK-signalling in macrophages and IL-23 secreting DCs, the key players for Th17 cell polarization. Thus, impaired Notch signalling links HS to other Th17-driven comorbidities. All major therapeutic interventions in HS appear to attenuate increased MAPK activation of innate immune cells due to impaired Notch-mediated feedback regulation of innate immunity.

MKP-1 is essential for canonical vitamin D-induced signaling through nuclear import and regulates RANKL expression and function.

Vitamin D(3,) and its most active form, 1,25(OH)(2)D(3), are well known to stimulate osteoclastogenesis through stromal cell induction of the receptor activator of nuclear factor-κB ligand (RANKL). MAPK phosphatase-1 (MKP-1) is a phosphatase classically known to negatively regulate the innate immune response through dephosphorylation of p38, ERK, and c-Jun N-terminal kinase activity. This paper describes a new function of MKP-1 in permitting genomic 1,25(OH)(2)D(3) signaling and downstream osteoclastogenesis through RANKL. Initially, quantitative RT-PCR (qRT-PCR) and immunoblot analysis comparing bone marrow stromal cells (BMSC) revealed that 1,25(OH)(2)D(3)-induced vitamin D receptor (VDR), cytochrome P 45024a1, and RANKL mRNA expression and protein were significantly attenuated or absent in MKP-1(-/-) BMSC. Immunoblot analysis from cellular fractions of wild type and MKP-1(-/-) BMSC stimulated with 10(-7) m 1,25(OH)(2)D(3) revealed retinoid X receptor (RXR)α nuclear import was impaired in MKP-1(-/-) BMSC, whereas VDR import was not. Proximity ligation assays revealed that baseline VDR-RXRα heterodimer translocation was unchanged, yet 1,25(OH)(2)D(3)-induced nuclear translocation of VDR-RXRα heterodimers was reduced in MKP-1(-/-) BMSC. A functional consequence was observed as BMSC from MKP-1(-/-) mice treated with 1,25(OH)(2)D(3) and cocultured with RAW 264.7 cells had a 91% decrease in osteoclastogenesis and a 94.5% decrease in mineralized matrix resorption compared with wild-type cocultures (P < 0.01). These results reveal an unexpected, permissive role for MKP-1 in canonical 1,25(OH)(2)D(3) signaling via VDR-RXRα heterodimer nuclear import and downstream osteoclastogenesis through stromal cell RANKL expression.

[Research progress on MKP-1 in tumor drug resistance].

The main obstacle for chemotherapy is tumor drug resistance. Studying the mechanisms of drug resistance and reversing drug resistance is the key to improve the effectiveness of chemotherapy. It has been reported that MKP-1 plays an important role in tumor drug resistance. MKP-1, as a negative regulator of MAPKs, is involved in the MAPKs mediated drug resistance and is regulated by ERK and p38 signaling pathways.However, the relationship between MKP-1 and other drug resistance-related signaling pathways is not clear and requires further investigation.

S-Nitrosylation of mitogen activated protein kinase phosphatase-1 suppresses radiation-induced apoptosis.

Radiotherapy is a key modality for head and neck cancer (HNC) treatment. Mitogen activated protein kinase phosphatase-1 (MKP-1) protein levels are elevated in various tumors and are negatively correlated with efficacy of chemo- or radio-therapy. However, the mechanisms underlying the moderate radiosensitivity of HNC and the increased MKP-1 protein levels are still dismal. Here we show that S-nitrosylation of MKP-1 on Cysteine 258 enhances MKP-1 protein stability, phosphatase activity, and MKP-1-mediated anti-apoptotic effect on HNC radiotherapy. Co-culturing MKP-1 transfected HNC cell lines with activated macrophages for mimicking the microenvironment of the irradiated cancer cells further confirms that S-nitrosylation-mediated increase of MKP-1 activity correlates with decrease of HNC radiosensitivity. Therefore, S-nitrosylation of MKP-1 presents a novel mechanism underlying the enhanced MKP-1 expression levels and MKP-1-mediated radio-resistance in head and neck cancer.

p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair.

Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma.

OBJECTIVE: Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers. This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK(1/2)), as the key regulatory mechanism of the MAPKs, in lung squamous cell carcinoma (SCC). METHODS: We assessed the expressions of MKP-1 and p-ERK(1/2) in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma, and it was negatively correlated with tumor differentiation (P<0.01). However, the expression of p-ERK(1/2) or ERK(1/2) was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma, and it was positively correlated with tumor differentiation (P<0.01). CONCLUSIONS: Our data indicates the relevance of MKP-1 and p-ERK(1/2) in SCC as a potential positive and negative prognostic factor. The imbalanced expression of MKP-1 and p-ERK(1/2) may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

ICER evokes Dusp1-p38 pathway enhancing chemotherapy sensitivity in myeloid leukemia.

PURPOSE: The inducible cyclic adenosine monophosphate (cAMP) early repressor (ICER) is found downregulated in acute myeloid leukemia (AML), failing to control cAMP response element binding protein (CREB) transcriptional activity, recently demonstrated to mediate AML progression. We aimed to characterize ICER's role in drug sensitivity by treating myeloid cell lines and primary AML with chemotherapics. EXPERIMENTAL DESIGN: The effects on CREB target genes induced by ICER restoration and drug treatment were studied by quantitative real-time PCR (qRT-PCR) and western blot. Cell cycle and apoptosis analysis were performed. Possible ICER-evoked pathways were investigated in vitro. The mechanism involved in enhanced drug sensitivity was described in primary AML cultures by silencing ICER main target genes. RESULTS: AML cell lines reduced cell growth and enhanced apoptotic behavior after chemotherapy treatment if ICER was expressed. A significantly lowered expression of CREB target genes involved in cell cycle control (CyA1, B1, D1), and in the mitogen-activated protein kinase signaling pathway (ERK, AKT, DUSP1/4), was found after Etoposide treatment. The dual-specificity phosphatases DUSP1 and DUSP4, directly repressed by ICER, activated the p38 pathway, which triggered enhanced caspase-dependent apoptosis. The silencing of DUSP1/4 in HL60 confirmed the same enhanced drug sensitivity induced by ICER. Primary AML cultures, silenced for DUSP1 as well as restored of ICER expression, showed DUSP1 downregulation and p38 activation. CONCLUSION: ICER mediates chemotherapy anticancer activity through DUSP1-p38 pathway activation and drives the cell program from survival to apoptosis. ICER restoration or DUSP1 inhibition might be possible strategies to sensitize AML cancer cells to conventional chemotherapy and to inhibit tumor growth.

Mitogen-activated protein kinase phosphatase-1 is a key regulator of hypoxia-induced vascular endothelial growth factor expression and vessel density in lung.

Although mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key deactivator of MAP kinases, known effectors of lung vessel formation, whether it plays a role in the expression of proangiogenic vascular endothelial growth factor (VEGF) in hypoxic lung is unknown. We therefore hypothesized that MKP-1 is a crucial modulator of hypoxia-stimulated vessel development by regulating lung VEGF levels. Wild-type MKP-1(+/+), heterozygous MKP-1(+/-), and deficient MKP-1(-/-) mice were exposed to sea level (SL), Denver altitude (DA) (1609 m [5280 feet]), and severe high altitude (HYP) (∼5182 m [∼17,000 feet]) for 6 weeks. Hypoxia enhanced phosphorylation of p38 MAP kinase, a substrate of MKP-1, as well as α smooth muscle actin (αSMA) expression in vessels, respiratory epithelium, and interstitium of phosphatase-deficient lung. αSMA-positive vessel (<50 µm outside diameter) densities were markedly reduced, whereas vessel wall thickness was increased in hypoxic MKP-1(-/-) lung. Mouse embryonic fibroblasts (MEFs) of all three genotypes were isolated to pinpoint the mechanism involved in hypoxia-induced vascular abnormalities of MKP-1(-/-) lung. Sustained phosphorylation of p38 MAP kinase was observed in MKP-1-null MEFs in response to hypoxia exposure. Although hypoxia up-regulated VEGF levels in MKP-1(+/+) MEFs eightfold, only a 70% increase in VEGF expression was observed in MKP-1-deficient cells. Therefore, our data strongly suggest that MKP-1 might be the key regulator of vascular densities through the regulation of VEGF levels in hypoxic lung.

Anti-inflammatory properties of the PI3K pathway are mediated by IL-10/DUSP regulation.

Resolution of inflammation is an important hallmark in the course of infectious diseases. Dysregulated inflammatory responses may have detrimental consequences for the affected organism. Therefore, tight regulation of inflammation is indispensable. Among numerous modulatory signaling pathways, the PI3K/PTEN signaling pathway has been proposed recently to be involved in the regulation of innate immune reactions. Here, we attempted to elucidate molecular mechanisms that contribute to the modulatory properties of the PI3K signaling pathway in inflammation. PTEN-deficient macrophages, which harbor constitutively active PI3Ks, were analyzed in response to gram-negative bacteria and PAMPs such as LPS. PTEN-deficient cells showed reduced inflammatory cytokine production, which was accompanied by reduced MAPK signaling activation in early- as well as late-phase activation. Simultaneously, we found increased levels of the MKP DUSP1, as well as the anti-inflammatory cytokine IL-10. Our data suggest that differential DUSP1 regulation coupled with enhanced IL-10 production contributes to the anti-inflammatory properties of the PI3K pathway.

Modulation of gonadotropin-releasing hormone-induced extracellular signal-regulated kinase activation by dual-specificity protein phosphatase 1 in LbetaT2 gonadotropes.

As the regulator of pituitary reproductive hormone synthesis, the hypothalamic neuropeptide GnRH is the central regulator of reproduction. A hallmark of GnRH action is the differential control of gene expression in pituitary gonadotropes through varied pulsatile stimulation. Among other signaling events, GnRH activation of the ERK family of MAPKs plays a significant role in the transcriptional regulation of the luteinizing hormone ß-subunit gene and regulation of cap-dependent translation. We evaluated the ERK response to different GnRH pulse amplitudes in the gonadotrope cell line LßT2. We found that low-amplitude stimulation with GnRH invokes a rapid and transient ERK activation, whereas high-amplitude stimulation invokes a prolonged activation specifically in the cytoplasm fraction of LßT2 cells. Nuclear and cytoplasmic targets of ERK, Ets-like gene 1, and eukaryotic initiation factor 4E, respectively, are similarly activated. Feedback control of ERK activation occurs mainly through the dual-specificity protein phosphatases (DUSPs). DUSP1 is localized to the nucleus in LßT2 cells but DUSP4, another member implicated in GnRH feedback, exists in both the nucleus and cytoplasm. Manipulation of nuclear DUSP activity through overexpression or knockdown of Dusp1 modulates the ERK response to low and high GnRH pulse amplitudes and activation of the Lhb promoter. Dusp1 overexpression abolishes sustained ERK activation and inhibits Lhb promoter activity induced by high amplitude pulses. Conversely, Dusp1 knockdown enhances ERK activation by low-amplitude stimulation and increases stimulation of Lhb promoter activity. We conclude that DUSP1 feedback activity modulates ERK activation and the transcriptional response to GnRH.

MAPK-directed phosphatases preferentially regulate pro- and anti-inflammatory cytokines in experimental visceral leishmaniasis: involvement of distinct protein kinase C isoforms.

The role of phosphatases in the impairment of MAPK signaling, which is directly responsible for Leishmania-induced macrophage dysfunction, is still poorly understood. Gene expression profiling revealed that Leishmania donovani infection markedly up-regulated the expression of three phosphatases: MKP1, MKP3, and PP2A. Inhibition of these phosphatases prior to infection points toward preferential induction of the Th2 response through deactivation of p38 by MKP1. On the other hand, MKP3 and PP2A might play significant roles in the inhibition of iNOS expression through deactivation of ERK1/2. Among various PKC isoforms, PKCzeta was associated with induction of MKP3 and PP2A in infected macrophages, whereas PKCepsilon was correlated with MKP1 induction. Inhibition of phosphatases in L. donovani-infected BALB/c mice shifted the cytokine balance in favor of the host by inducing TNF-alpha and iNOS expression. This was validated by cystatin, an immunomodulator and curing agent for experimental visceral leishmaniasis, which showed that inhibition of MKPs and PP2A activity may be necessary for a favorable T cell response and suppression of organ parasite burden. This study, for the first time, suggests the possibility of the involvement of MAPK-directed phosphatases in the establishment of L. donovani infection.