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The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1. Show less

Dynamic protein phosphorylation, a major cellular regulatory system, is tightly controlled by coordinating the reversible action of protein kinases and phosphatases. Recent evidence is consistent with sophisticated mechanisms that regulate both kinases and phosphatases in the cell. Dual specificity phosphatases, which act on phosphorylated serine, threonine, and tyrosine residues in proteins, are valid targets for drug discovery. Chemical complementation combines genetic manipulations with chemical biology and high-content multiparametric analyses and was developed as a screening approach to discover protein phosphatase inhibitors. Using a dual specificity mitogen-activated protein kinase phosphatase as an example, a detailed protocol, discussion of issues relating to data analysis, and high-throughput implementation of the chemical complementation approach to drug discovery is presented. Show less

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity. Show less

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation. Show less

We compared gene expression profiles between Runx2 null mutant mice and their wildtype littermates. Most Runx2-dependent genes in bones were different from those in teeth, implying that the target genes of Runx2 are tissue-dependent. In vitro experiments determined that Runx2 is a part of the FGF and BMP signaling pathways in tooth and bone development, respectively. Runx2 (Cbfa1) is expressed in the neural crest-derived mesenchyme of developing bone and tooth. Runx2 homozygous null mice lack bone through a failure in osteoblast differentiation and have arrested tooth development at the late bud stage. The aim of this study was to discover and compare the identities and the roles of Runx2 target genes in bone and tooth development. Wildtype and Runx2-/- tissue was collected from mouse embryos, and gene expression was compared by Affymetrix microarray analysis and radioactive in situ hybridization of embryonic tissue sections (E12-E14). Induction of target genes by growth factors in bone and tooth tissue was studied using in vitro experiments, including a novel method involving hanging-drop cultures and RT-PCR. Thirteen bone and four tooth genes were identified that are Runx2-dependent. The identities of these genes do not significantly overlap between bone and tooth, indicating tissue specificity of several genes regulated by Runx2. Genes downregulated in bone development in Runx2 null mutants were Bambi, Bmp4, Bono1, Dkk1, Fgf receptor1, Gli1, Lef1, Patched, Prostaglandin F receptor1, Tcf1, Tgfbeta1, Wnt10a, and Wnt10b. Several of these genes were induced by BMPs in bone tissue in a Runx2-independent manner. Genes downregulated in tooth development were Dkk1, Dusp6, Enpp1, and Igfbp3. These genes were all induced by fibroblast growth factors (FGFs) in dental tissue. FGF-induction of Dkk1 was completely dependent on Runx2 function. The contrasting identities and distinctive mechanisms that stimulate the expression of Runx2-dependent genes in bone and tooth development imply that the developmental roles of Runx2 in these separate tissues are different. In tooth development, Dkk1 may be a direct transcriptional target of Runx2. Bone genes were stimulated by BMP4 before the formation of the ossification center, suggesting that BMPs may mediate the early epithelial-mesenchymal interactions involved in bone formation. Show less

Antiestrogen resistance is a major clinical problem in the treatment of breast cancer. Altered growth factor signaling with estrogen receptor (ER)-alpha is associated with the development of resistance. Gene expression profiling was used to identify mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP3) whose expression was correlated with response to the antiestrogen tamoxifen in both patients and in vitro-derived cell line models. Overexpression of MKP3 rendered ER-alpha-positive breast cancer cells resistant to the growth-inhibitory effects of tamoxifen and enhanced tamoxifen agonist activity in endometrial cells. MKP3 overexpression was associated with lower levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in the presence of estrogen but that estrogen deprivation and tamoxifen treatment decreased MKP3 phosphatase activity, leading to an up-regulation of pERK1/2 MAPK, phosphorylated Ser(118)-ER-alpha, and cyclin D1. The MAPK/ERK kinase inhibitor PD98059 blocked tamoxifen-resistant growth. Accumulation of reactive oxygen species was observed with tamoxifen treatment of MKP3-overexpressing cells, and antioxidant treatment increased MKP3 phosphatase activity, thereby blocking resistance. Furthermore, PD98059 increased the levels of phosphorylated c-Jun NH(2)-terminal kinase (JNK) in tamoxifen-treated MKP3-overexpressing cells, suggesting an interaction between MKP3 levels, activation of ERK1/2 MAPK, and JNK signaling in human breast cancer cells. MKP3 represents a novel mechanism of resistance, which may be a potential biomarker for the use of ERK1/2 and/or JNK inhibitors in combination with tamoxifen treatment. Show less

Brain-derived neurotrophic factor (BDNF) has been reported to be critical for the development of cortical inhibitory neurons. However, the effect of BDNF on the expression of transcripts whose protein products are involved in gamma amino butric acid (GABA) neurotransmission has not been assessed. In this study, gene expression profiling using oligonucleotide microarrays was performed in prefrontal cortical tissue from mice with inducible deletions of BDNF. Both embryonic and adulthood ablation of BDNF gave rise to many shared transcriptome changes. BDNF appeared to be required to maintain gene expression in the SST-NPY-TAC1 subclass of GABA neurons, although the absence of BDNF did not alter their general phenotype as inhibitory neurons. Furthermore, we observed expression alterations in genes encoding early-immediate genes (ARC, EGR1, EGR2, FOS, DUSP1, DUSP6) and critical cellular signaling systems (CDKN1c, CCND2, CAMK1g, RGS4). These BDNF-dependent gene expression changes may illuminate the biological basis for transcriptome changes observed in certain human brain disorders. Show less

DUSP6/MKP-3, a specific inhibitor of MAPK1/ERK2, frequently loses its expression in primary pancreatic cancer tissues. This evidence suggests that constitutive activation of MAPK1 synergistically induced by frequent mutation of KRAS2 and the loss of function of DUSP6 plays key roles in pancreatic carcinogenesis and progression. By profiling of gene expressions associated with downregulation of MAPK1 induced by exogenous overexpression of DUSP6 in pancreatic cancer cells, we found that AURKA/STK15, the gene encoding Aurora-A kinase, which plays key roles in cellular mitosis, was among the downregulated genes along with its related genes, which included AURKB, TPX2 and CENPA. An association of expression and promoter activity of AURKA with MAPK activity was verified. Knockdown of ETS2 resulted in a reduction of AURKA expression. These results indicate that AURKA is a direct target of the MAPK pathway and that its overexpression in pancreatic cancer is induced by hyperactivation of the pathway, at least via ETS2. Show less

Protein tyrosine phosphatases have a central role in the maintenance of normal cellular functionality. For example, PTP1B has been implicated in insulin-resistance, obesity, and neoplasia. Mitogen-activated protein kinase phosphatase-1 (MKP-1 or DUSP1) dephosphorylates and inactivates mitogen-activated protein kinase (MAPK) substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The lack of readily available selective small molecule inhibitors of MKP family members has severely limited interrogation of their biological role. Inspired by a previously identified inhibitor (NSC 357756) of MKP-3, we synthesized seven NSC 357756 congeners, which were evaluated for in vitro inhibition against several protein phosphatases. Remarkably, none displayed potent inhibition against MKP-3, including the desamino NSC 357756 analog NU-154. Interestingly, NU-154 inhibited human PTP1B in vitro with an IC(50) value of 24 +/- 1 microM and showed little inhibition against Cdc25B, MKP-1, and VHR phosphatases. NU-126 [2-((E)-2-(5-cyanobenzofuran-2-yl)vinyl)-1H-indole-6-carbonitrile] inhibited MKP-1 and VHR in vitro but was less active against human MKP-3, Cdc25B, and PTP1B. The inhibition of MKP-1 by NU-126 was independent of redox processes. The benzofuran substructure represents a new potential scaffold for further analog development and provides encouragement that more selective and potent inhibitors of MKP family members may be achievable. Show less

Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in approximately 70% of cases. We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF. We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS(G12V) or BRAF(V600E). Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF(V600E) (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient in TPC1 and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being approximately 10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC50 0.1-0.25 and approximately 0.05 micromol/L for AAL-881 and LBT-613, respectively) than BRAF + lines (IC50 2.5-5 and 0.1-0.5 micromol/L, respectively). Growth inhibition was associated with G1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts. Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma. Show less

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by the extracellular deposition of transthyretin (TTR), especially in the PNS. Given the invasiveness of nerve biopsy, salivary glands (SG) from FAP patients were used previously in microarray analysis; mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) was down-regulated in FAP. Results were validated by RT-PCR and immunohistochemistry both in SG and in nerve biopsies of different stages of disease progression. MKP-3 was also down-regulated in FAP SG biopsies. Given the relationship between MKPs and MAPKs, the latter were investigated. Only extracellular signal-regulated kinases 1/2 (ERK1/2) displayed increased activation in FAP SG and nerves. ERK1/2 kinase (MEK1/2) activation was also up-regulated in FAP nerves. In addition, an FAP transgenic mouse model revealed increased ERK1/2 activation in peripheral nerve affected with TTR deposition when compared to control animals. Cultured rat Schwannoma cell line treatment with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by the receptor for advanced glycation end-products (RAGE). Moreover, caspase-3 activation triggered by TTR aggregates was abrogated by U0126, a MEK1/2 inhibitor, indicating that ERK1/2 activation is essential for TTR aggregates-induced cytotoxicity. Taken together, these data suggest that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration. Show less

We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention. Show less

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs. Show less

The epidermal growth factor receptor (EGFR) gene is commonly amplified and rearranged in glioblastoma multiforme leading to overexpression of wild-type and mutant EGFRs. Expression of wild-type EGFR ligands, such as transforming growth factor-alpha (TGF-alpha) or heparin-binding EGF (HB-EGF), is also often increased in gliomas resulting in an autocrine loop that contributes to the growth autonomy of glioma cells. Glioblastoma multiformes express a characteristic EGFR mutant (EGFRvIII, de 2-7) that does not bind ligand, signals constitutively, and is more tumorigenic than the wild-type receptor. However, the downstream signals that mediate this increased tumorigenicity are not well understood. We hypothesized that signals induced specifically by EGFRvIII and not the wild-type receptor are more likely to mediate its increased tumorigenic activity and examined the gene expression profiles resulting from inducible expression of comparable levels of either wild-type EGFR or EGFRvIII in a U251-MG glioma cell line. Expression of EGFRvIII resulted in specific up-regulation of a small group of genes. Remarkably, all these genes, which include TGFA, HB-EGF, EPHA2, IL8, MAP4K4, FOSL1, EMP1, and DUSP6, influence signaling pathways known to play a key role in oncogenesis and function in interconnected networks. Increased expression of EGFRvIII-induced genes was validated by real-time PCR. The mutant receptor does not bind ligand, and EGFRvIII-induced expression of TGF-alpha and HB-EGF suggests that EGFRvIII plays a role in generating an autocrine loop using the wild-type EGFR in glioma. It also raises the possibility that EGFRvIII may signal, at least in part, through the wild-type receptor. Indeed, we show that inhibiting the activity of HB-EGF, a potent mitogen, with neutralizing antibodies reduces cell proliferation induced by expression of EGFRvIII. This suggests that the EGFRvIII-HB-EGF-wild-type EGFR autocrine loop plays an important role in signal transduction by EGFRvIII in glioma cells. We also show by immunohistochemistry that HB-EGF expression correlates with the presence of EGFRvIII in glioblastoma multiforme. Thus, our study provides a new insight into oncogenic signaling by EGFRvIII and improves our understanding of how autocrine loops are generated in glioma. Show less

The dual-specificity phosphatase Pyst2-L was found to be over expressed in leukocytes derived from AML and ALL patients as well as in certain other solid tumors and lymphoblastoid cell lines. Pyst2-L, binds and dephosphorylates both pERKs and pJNKs proteins, and thus, plays a role in regulating the MAP kinase signaling pathway. In the present study, a comparative genomic application was used and sequence analysis of multi-organisms databases were searched in order to identify genes homologous to Pyst2-L. The Xenopus laevis MAP kinase phosphatase X17c gene and the Yeast nitrogen starvation-induced protein phosphatase Yvh1p gene were revealed to be highly homologous with Pyst2-L. Both X17c and Yvh1p genes play a role in cell cycle regulation. A down regulated expression of the Yvh1p gene occurred in Saccharomyces cerevisiae that were synchronized to the G2-phase of the cell cycle by alpha-factor. In conformity with this result, a reduction in Pyst2-L expression levels was observed in G2-phase-synchronized Human K562 cells. Finally, we were able to show that cells in highly crowded cultures express high levels of the Pyst2-L phosphatase. These observations may indicate that low levels of the Pyst2-L phosphatase are essential for the G2-phase of the cell cycle and that this phosphatase might play a role in signaling cascades induced by cellular crowding. Show less

Pancreatic ductal adenocarcinoma is one of the most fatal malignancies. Intensive investigation of molecular pathogenesis might lead to identifying useful molecules for diagnosis and treatment of the disease. Pancreatic ductal adenocarcinoma harbors complicated aberrations of alleles including losses of 1p, 6q, 9p, 12q, 17p, 18q, and 21q, and gains of 8q and 20q. Pancreatic cancer is usually initiated by mutation of KRAS and aberrant expression of SHH. Overexpression of AURKA mapping on 20q13.2 may significantly enhance overt tumorigenesity. Aberrations of tumor suppressor genes synergistically accelerate progression of the carcinogenic pathway through pancreatic intraepithelial neoplasia (PanIN) to invasive ductal adenocarcinoma. Abrogation of CDKN2A occurs in low-grade/early PanIN, whereas aberrations of TP53 and SMAD4 occur in high-grade/late PanIN. SMAD4 may play suppressive roles in tumorigenesis by inhibition of angiogenesis. Loss of 18q precedes SMAD4 inactivation, and restoration of chromosome 18 in pancreatic cancer cells results in tumor suppressive phenotypes regardless of SMAD4 status, indicating the possible existence of a tumor suppressor gene(s) other than SMAD4 on 18q. DUSP6 at 12q21-q22 is frequently abrogated by loss of expression in invasive ductal adenocarcinomas despite fairly preserved expression in PanIN, which suggests that DUSP6 works as a tumor suppressor in pancreatic carcinogenesis. Restoration of chromosome 12 also suppresses growths of pancreatic cancer cells despite the recovery of expression of DUSP6; the existence of yet another tumor suppressor gene on 12q is strongly suggested. Understanding the molecular mechanisms of pancreatic carcinogenesis will likely provide novel clues for preventing, detecting, and ultimately curing this life-threatening disease. Show less

Mutations of RAS, RAF, and PTEN, all important members of the RAS/MAPK and PI3K/AKT cascades, are reported in a variety of human tumors, including melanomas and endometrial cancer. In endometrial cancer, mutually exclusive mutations of PTEN and KRAS have been reported. On the other hand, mutation of BRAF is highly frequent, and mutually exclusive mutations of BRAF and NRAS have also been reported in melanomas. In this study, we elucidated the involvement of the up-regulation of RAS/MAPK and PI3K/AKT cascades in the pathogenesis of endometrial cancer and melanoma by analyzing the genes and molecules in these cascades. Twelve cell lines, six melanoma and six endometrial cancer, were analyzed; 4 (67%) of the 6 melanomas had gene mutations in the RAS/MAPK cascade, and a decrease or loss of PTEN expression was also observed. These results suggested that simultaneous up-regulations in these two cascades play important roles in carcinogenesis of melanocytes. However, no activation of AKT by phosphorylation was observed. On the other hand, 4 (67%) of the 6 endometrial cancer cell lines had mutually exclusive up-regulations in these cascades. However, two cell lines with up-regulation of the PI3K/AKT cascade also had up-regulation in the RAS/MAPK cascade induced by inactivation of DUSP6. These results suggest that simultaneous up-regulation of RAS/MAPK and PI3K/AKT cascades are crucial events in the pathogenesis of melanocytes, whereas up-regulation of either the RAS/MAPK or PI3K/AKT cascade is crucial for the majority of endometrial cancers. Show less

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors. Show less

Insulin is a key hormone that controls glucose homeostasis. In liver, insulin suppresses gluconeogenesis by inhibiting the transcriptions of phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. In insulin resistance and type II diabetes there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycemia. To search for novel genes that negatively regulate insulin signaling in controlling metabolic pathways, we screened a cDNA library derived from the white adipose tissue of ob/ob mice using a reporter system comprised of the PEPCK promoter placed upstream of the alkaline phosphatase gene. The mitogen-activated dual specificity protein kinase phosphatase 3 (MKP-3) was identified as a candidate gene that antagonized insulin suppression on PEPCK gene transcription from this screen. In this study, we showed that MKP-3 was expressed in insulin-responsive tissues and that its expression was markedly elevated in the livers of insulin-resistant obese mice. In addition, MKP-3 can activate PEPCK promoter in synergy with dexamethasone in hepatoma cells. Furthermore, ectopic expression of MKP-3 in hepatoma cells by adenoviral infection increased the expression of PEPCK and G6Pase genes and led to elevated glucose production. Taken together, our data strongly suggests that MKP-3 plays a role in regulating gluconeogenic gene expression and hepatic gluconeogenesis. Therefore, dysregulation of MKP-3 expression and/or function in liver may contribute to the pathogenesis of insulin resistance and type II diabetes. Show less

Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARbeta2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARbeta2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARbeta2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16. Show less

T lymphocytes play a central role in controlling adaptive immune responses. IL-2 critically regulates both T cell growth and death and is involved in maintaining peripheral tolerance, but the molecules involved in these and other IL-2 actions are only partially known. We now provide a comprehensive compendium of the genes expressed in T cells and of those regulated by IL-2 based on a combination of DNA microarrays and serial analysis of gene expression (SAGE). The newly identified IL-2 target genes include many genes previously linked to apoptosis in other cellular systems that may contribute to IL-2-dependent survival functions. We also studied the mRNA expression of known regulators of signaling pathways for their induction in response to IL-2 in order to identify potential novel positive and/or negative feedback regulators of IL-2 signaling. We show that IL-2 regulates only a limited number of these genes. These include suppressors of cytokine signaling (SOCS) 1, SOCS2, dual-specificity phosphatase (DUSP) 5, DUSP6 and non-receptor type phosphatase-7 (PTPN7). Additionally, we provide evidence that many genes expressed in T cells locate in chromosomal clusters, and that select IL-2-regulated genes are located in at least two clusters, one at 5q31, a known cytokine gene cluster, and the other at 6p21.3, a region that contains genes encoding the tumor necrosis factor (TNF) superfamily members TNF, LT-alpha and LT-beta. Show less

RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response. Show less

Macrophages exposed to hyperoxia in the lung continue to survive for prolonged periods. We previously reported (Nyunoya, T., Powers, L. S., Yarovinsky, T. O., Butler, N. S., Monick, M. M., and Hunninghake, G. W. (2003) J. Biol. Chem. 278, 36099-36106) that hyperoxia induces cell cycle arrest and sustained extracellular signal-related kinase (ERK) activity in macrophages. In this study, we determined the mechanisms of hyperoxia-induced ERK activation and how ERK activity plays a pro-survival role in hyperoxia-exposed cells. Inhibition of ERK activity decreased survival of hyperoxia-exposed macrophages. This was due, at least in part, to down-regulation of the pro-apoptotic Bcl-2 family member, BimEL. In determining the mechanism of ERK activation by hyperoxia, we found that ERK activation was not associated with hyperoxia-induced activation of the upstream ERK kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2. When we examined the ability of whole cell lysates from hyperoxia-exposed cells to dephosphorylate purified phosphorylated ERK, we found decreased ERK-directed phosphatase activity. Two particular ERK-directed phosphatases (protein phosphatase 2A and MAPK phosphatase-3) demonstrated decreased activity in hyperoxia-exposed cells. Moreover, whole cell lysates from normoxia-exposed cells depleted of PP2A or MAPK phosphatase-3 were also less able to dephosphorylate ERK. These data demonstrate that, in hyperoxia-exposed macrophages, sustained activation of ERK due to phosphatase down-regulation permits macrophage survival via effects on the balance between pro- and anti-apoptotic Bcl-2 family proteins. Show less

DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms. Show less

Our previous study indicated that DUSP6/MKP-3/PYST1 could act as a tumor suppressor in human pancreatic cancer. DUSP6 was frequently underexpressed in primary pancreatic cancer tissues by an unknown mechanism. In this study, we demonstrated that hypermethylation of the expressional control region of DUSP6 could account for its abrogation in cultured human pancreatic cancer cells and in primary pancreatic cancer tissues. First, we checked intrinsic transcriptional expression levels of DUSP6 by a quantitative real time PCR assay in 16 cultured pancreatic cancer cell lines and found that the cells could be classified into four groups: very-low-level expression, low-level expression, high-level expression, and very-high-level expression. We observed restored expression of DUSP6 after treatment with 5-azacytidine and trichostatin A, a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, respectively, in cells with intrinsically very-low-level and low-level expression of DUSP6. Using a sodium-bisulfite-modification assay, we found that CpG sequences in intron 1 of DUSP6 were heavily methylated in MIA PaCa-2 and PAN07JCK, both showing the very low level of intrinsic expression of the gene. On the other hand, no methylation in this region was detected in 14 other cell lines. We checked the methylation state of this region by a methylation-specific PCR method in 12 primary pancreatic cancer tissues and compared it with the expression state of DUSP6 investigated by immunohistochemistry. Methylation was detected in five of eight cases with abolished expressions of DUSP6, four of which were poorly differentiated adenocarcinoma. On the other hand, none of the four cases with preserved expression of DUSP6 showed methylation. The methylation state significantly correlated with both the abolishment of protein expression (p = 0.038) and the histological subtype of adenocarcinoma (p = 0.023) by chi-square test. These results indicate that hypermethylation of the CpG islands in intron 1 may account for the strong suppression of DUSP6 expression. Other mechanism(s) and/or other CpG sites outside of our investigation may have some influence upon expressional suppression. Our combined results suggest that hypermethylation with modification of histone deacetylation play an important role in transcriptional suppression of DUSP6 in human pancreatic cancer. Show less

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance. Show less

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening. Show less

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1. Show less