Expression Of Dual-specificity Phosphatase Research Articles

The expression of dual-specificity phosphatase 1 mRNA is downregulated in lesional psoriatic skin

The p38 mitogen-activated protein kinase (MAPK) plays an important role in inflammatory processes and displays increased activity in psoriasis. Dual-specificity phosphatase 1 (DUSP1) is an important negative regulator of p38 MAPK activity. To study mRNA expression of DUSP1 in normal human epidermal keratinocytes (NHEKs) stimulated with proinflammatory cytokines and to investigate DUSP1 in psoriatic skin. NHEKs were cultured in vitro and punch biopsies were obtained from the skin of patients with psoriasis vulgaris and atopic dermatitis. mRNA expression was analysed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In NHEKs, interleukin (IL)-1β induced DUSP1 mRNA expression in a rapid and time-dependent manner through the p38 MAPK/mitogen- and stress-activated kinase (MSK) signalling pathway. DUSP1 mRNA expression was demonstrated to be significantly downregulated in psoriatic skin lesions compared with paired samples of nonlesional psoriatic skin. This was in contrast to atopic dermatitis. The downregulation of DUSP1 mRNA in lesional psoriatic skin was not explained by the difference in the mRNA expression of the potential DUSP1 transcript stability-affecting proteins Hu antigen R or tristetraprolin. Furthermore, DUSP1 mRNA expression was shown not to increase during the early course of treatment with the antitumour necrosis factor-α antibody adalimumab. In lesional psoriatic skin, the p38 MAPK negative feedback mechanism provided by DUSP1 seems to be inhibited. Downregulation of DUSP1 may contribute to the sustained inflammatory response seen in psoriasis.

Identification of androgen-regulated genes in human prostate

Androgens are essential for the development of the prostate and prostate cancer. We examined androgen-regulated gene expression in the human prostate. Samples from benign and malignant prostate tissue and samples containing prostate tissue obtained from prostate cancer patients three days after surgical castration were further processed as probes for a GeneChip array. The comparison of gene expression profiles in castrated samples and in benign or malignant prostate tissue samples revealed androgen-regulated genes. We further evaluated the genes which were differentially expressed in benign and malignant prostate samples. The androgen-regulated expression of dual specificity phosphatase 1 (DUSP1) was confirmed in the LNCaP prostate cancer cell line, as the expression of DUSP1 increased with androgen treatment over the course of time. The expression of the genes CRISP3, PCA3, OR51E2, HOXC6, AGR3, AMACR and SLC14A1 was affected by castration in addition to differential expression in the benign and malignant prostate. These sample results require further investigation for the role of AGR3 and SLC14A1 in prostate cancer as these associations have not been reported previously.

AMP-activated Protein Kinase Antagonizes Pro-apoptotic Extracellular Signal-regulated Kinase Activation by Inducing Dual-specificity Protein Phosphatases in Response to Glucose Deprivation in HCT116 Carcinoma

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant-negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

Induction of Dual Specificity Phosphatase 1 (DUSP1) by Gonadotropin-Releasing Hormone (GnRH) and the Role for Gonadotropin Subunit Gene Expression in Mouse Pituitary Gonadotroph LbetaT2 Cells1

We examined the expression of dual specificity phosphatase 1 (DUSP1) by gonadotropin-releasing hormone (GnRH) stimulation and investigated the role of DUSP1 on gonadotropin gene expression using LbetaT2 gonadotroph cell line. DUSP1 expression was markedly increased 60 min after GnRH stimulation, and mitogen-activated protein kinase 3/1 (MAPK3/1) activation was gradually decreased after 60 min. GnRH-induced MAPK3/1 activation was completely inhibited by U0126, a MEK inhibitor, whereas GnRH-induced DUSP1 expression was partially inhibited by U0126. GnRH-induced DUSP1 induction was inhibited by triptolide, a diterpenoid triepoxide. In contrast, this compound potentiated MAPK3/1 activation. U0126 prevented GnRH-stimulated gonadotropin subunit promoter activation dose dependently, and 10 muM of U0126 reduced the effects of GnRH on the Lhb and Fshb promoters to 79.15% and 55.66%, respectively. GnRH-stimulated activation of Lhb and Fshb promoters as well as serum response factor (Srf) promoters were almost completely inhibited by triptolide, suggesting that this component had a nonspecific effect to the cells. Dusp1 siRNA reduced the expression of DUSP1 and augmented MAPK3/1 phosphorylation, but it did not increase of gonadotropin promoters. By overexpression of DUSP1, both GnRH-stimulated Lhb and Fshb promoters were significantly reduced. We have previously shown that insulin-like growth factor 1 (IGF1) increases MAPK3/1 but does not activate gonadotropin subunit promoters. IGF1 failed to induce DUSP1 expression. In addition, under pulsatile GnRH stimulation, DUSP1 expression was observed following high-frequency GnRH pulses but not following low-frequency pulses. Our study demonstrated that DUSP1, induced by GnRH, functions not only as an MAPK3/1-inactivating phosphatase but also as an important mediator in gonadotropin subunit gene expression regulation.

Spatiotemporal Regulation of ERK2 by Dual Specificity Phosphatases

Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.

Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase 1

Glucocorticoids (GCs), which are used in the treatment of immune-mediated inflammatory diseases, inhibit the expression of many inflammatory mediators. They can also induce the expression of dual specificity phosphatase 1 (DUSP1; otherwise known as mitogen-activated protein kinase [MAPK] phosphatase 1), which dephosphorylates and inactivates MAPKs. We investigated the role of DUSP1 in the antiinflammatory action of the GC dexamethasone (Dex). Dex-mediated inhibition of c-Jun N-terminal kinase and p38 MAPK was abrogated in DUSP1−/− mouse macrophages. Dex-mediated suppression of several proinflammatory genes (including tumor necrosis factor, cyclooxygenase 2, and interleukin 1α and 1β) was impaired in DUSP1−/− mouse macrophages, whereas other proinflammatory genes were inhibited by Dex in a DUSP1-independent manner. In vivo antiinflammatory effects of Dex on zymosan-induced inflammation were impaired in DUSP1−/− mice. Therefore, the expression of DUSP1 is required for the inhibition of proinflammatory signaling pathways by Dex in mouse macrophages. Furthermore, DUSP1 contributes to the antiinflammatory effects of Dex in vitro and in vivo.

Synergistic induction of tissue factor by coagulation factor Xa and TNF: Evidence for involvement of negative regulatory signaling cascades

Enzymes of the blood coagulation pathway enhance the inflammatory response leading to endothelial dysfunction, accounting, in part, for the vascular complications occurring in sepsis and cardiovascular disease. The responses of endothelial cell activation include induction of the expression of tissue factor (TF), a membrane glycoprotein that promotes thrombosis, and of E-selectin, a cell adhesion molecule that promotes inflammation. In this report, we demonstrate synergistic interactions between the coagulation factor Xa (fXa) and the proinflammatory cytokines TNF, IL-1beta, and CD40L, leading to enhanced expression of TF and E-selectin in endothelial cells. A detailed analysis of the molecular pathways that could account for this activity of fXa showed that fXa inhibited the cytokine-induced expression of dual specificity phosphatases, MAP kinase phosphatase-L, -4, -5, and -7, blocking a negative regulatory effect on c-Jun N-terminal kinase. The synergistic interaction between fXa and TNF was also involved in the inhibition of A20 and IkappaBalpha expression in the IkappaB kinase-NF-kappaB pathway. The data indicate that inhibition of negative regulatory signaling accounts for the amplification of cytokine-induced endothelial cell activation by fXa.

The dual specificity phosphatase transcriptome of the murine thymus

Properly regulated mitogen-activated protein (MAP) kinase activity is critical for normal thymocyte development. MAP kinases are activated by phosphorylation of tyrosine and threonine, and dual specificity phosphatases (DUSPs) can inactivate MAP kinases by dephosphorylating both tyrosine and threonine. However, a role for DUSPs in thymocyte development has not been described. In this study, we have defined the subset of DUSP genes expressed in the murine thymus, and how their expression varies in different thymocyte subsets. Of the murine DUSP genes screened that could potentially dephosphorylate MAP kinases, we found 10 transcribed in the thymus. Seven of these 10 thymic DUSPs are true MAP kinase phosphatases based on the presence of a MAP kinase binding domain and demonstrated phosphatase activity against MAP kinases. Six of the seven thymic MAP kinase phosphatases have been shown to dephosphorylate extracellular regulated kinase (ERK). Quantitative PCR analysis of thymocyte populations isolated from different developmental stages revealed significant changes in DUSP expression as thymocytes progressed through development. Specifically, DUSPs 1, 4, and 5 significantly increase in expression as cells go from small, resting CD4/CD8 double positive cells to the CD4 single positive stage. Additionally, in vitro experiments showed that DUSPs could respond to TCR signaling, as anti-CD3 stimulation of thymocytes transiently increased transcription of six of the 10 thymic DUSP genes within 30 min. Notably, the ERK-specific phosphatase DUSP5 was upregulated 43-fold within 30 min, and returned to baseline within 24 h. Overall, we have identified a subset of DUSPs that could potentially regulate ERK activation in response to TCR signals in thymocytes.

The PAC-1 dual specificity phosphatase predicts poor outcome in serous ovarian carcinoma

Objective. Data regarding signal transduction pathways in human tumors are largely confined to cell line studies to date. We have recently reported on the activation and prognostic role of mitogen-activated protein kinases (MAPK) in ovarian carcinoma in effusions. The objective of the present study was to investigate the expression and clinical role of dual-specificity phosphatases (DUSP), inhibitors of MAPK signaling, in ovarian cancer cells at this site. Methods. Thirty-nine fresh frozen malignant effusions from patients diagnosed with serous ovarian carcinoma were studied for mRNA expression of the DUSP MKP-1, MKP-4, MKP-5, and PAC-1 using RT-PCR. DUSP expression was analyzed for possible correlation with patient age, disease stage, tumor grade, histological grade, chemotherapy status, and survival. Results. MKP-1 and PAC-1 mRNA were found in 36 and 37 effusions, respectively, with expression levels showing considerable variation. MKP-4 and MKP-5 were uniformly absent. MKP-1 showed no association with clinicopathologic parameters. However, PAC-1 expression was significantly higher in effusions obtained before the institution of treatment with both platinum compounds ( P = 0.029) and paclitaxel ( P = 0.036). In univariate survival analysis, high level of expression of PAC-1 mRNA predicted significantly worse overall survival compared to low expression (mean = 30 vs. 52 months, median = 25 vs. 46 months) ( P = 0.007). Conclusions. Despite the limited size of this cohort, our results present the first evidence supporting a clinical role for PAC-1 in ovarian carcinoma. In view of the improved outcome associated with activation of all three MAPK families, as well as their elevated expression and activation in post-chemotherapy specimens presented in our previous work, they also suggest that PAC-1 is a true negative regulator of MAPK in ovarian carcinoma cells in effusions.