Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.     

Hyperactivation of MAPK induces loss of ERalpha expression in breast cancer cells.

ERalpha-negative breast tumors tend to overexpress growth factor receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key intermediate in the signal transduction pathways of these receptors. High levels of constitutive Raf kinase (Deltaraf) activity imparts ERalpha- positive MCF-7 breast cancer cells with the ability to grow in the absence of estrogen. Deltaraf transfectants maintained in estrogen-depleted media showed greatly diminished responses to 17beta-estradiol or the pure antiestrogen ICI 182,780. Western blotting, ligand binding, and immunohistochemistry assays revealed a loss of ERalpha protein expression, and ribonuclease protection assays indicated that this correlated with loss of ERalpha message. In examining the basal expression of estrogen-induced genes in the stable transfectants or in transient cotransfection assays with an estrogen-response element- reporter construct and Deltaraf or constitutively active MAPK kinase (DeltaMEK), no ligand- independent activation of ERalpha was observed. Transient expression of Deltaraf and double-label immunostaining showed ERalpha was lost in those cells that transiently expressed Deltaraf. Abrogation of Raf signaling via treatment with the MEK inhibitors PD 098059 or U0126 resulted in reexpression of ERalpha. Similar studies performed with MCF-7 cells overexpressing epidermal growth factor receptor or c-erbB-2 confirmed that hyperactivation of MAPK resulted in down-regulation of ERalpha that was reversible by MEK inhibition or transfection with dominant negative ERK1 and ERK2 constructs. These data suggest that the hyperactivation of MAPK in epidermal growth factor receptor- or c-erbB-2-overexpressing breast cancer cells is directly responsible for generation of an ERalpha-negative phenotype and, more importantly, that this process may be abrogated by inhibiting these pathways, thus restoring ERalpha expression.     

Acid increases proliferation via ERK and p38 MAPK-mediated increases in cyclooxygenase-2 in Barrett's adenocarcinoma cells

Cyclooxygenase-2 (COX-2) has been linked to neoplastic progression in Barrett's esophagus. Acid exposure has been shown both to activate the MAPK pathways and to increase COX-2 protein expression in Barrett's metaplasia, but it is not known whether these effects are interrelated. We hypothesized that acid-induced activation of the MAPK pathways mediates an increase in COX-2 expression in Barrett's esophagus, and we tested this hypothesis in a Barrett's-associated adenocarcinoma cell line (SEG-1). We exposed SEG-1 cells to acidic or neutral media in the presence and absence of two MAPK inhibitors: U-0126 (an ERK inhibitor) or SB-203580 (a p38 inhibitor). We quantitated COX-2 protein levels using an enzyme immunometric assay and COX-2 mRNA levels using real-time PCR. We also determined how acid affects the activity of the COX-2 promoter and mRNA stability. Compared with SEG-1 cells exposed to neutral media, acid-exposed cells exhibited a 2.8-fold increase in COX-2 mRNA levels within 30 min. Both U-0126 and SB-203580 attenuated the acid-induced increase in COX-2 mRNA. Acid significantly increased COX-2 protein expression and promoter activity, and both of these effects were abolished by treatment with U-0126 and SB-203580. Acid exposure also stabilized COX-2 mRNA levels, an effect that was abolished by U-0126 but not by SB-203580. We conclude that acid increases COX-2 expression through activation of the MAPK pathways. Acid-induced activation of both ERK and p38 causes a significant increase in COX-2 promoter activity, and acid-activated ERK stabilizes COX-2 mRNA. These findings suggest potential mechanisms whereby acid reflux might promote carcinogenesis in Barrett's esophagus.     

ERK activation and mitogenesis in human airway smooth muscle cells

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE(2) and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.     

ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells

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.     

Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma ) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes

We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.     

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs

The roles of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH(2)-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.     

Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines

Dietary conjugated linoleic acid (CLA) reduces body fat in animals and some humans. Here we show that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, when added to cultures of stromal vascular cells containing newly differentiated human adipocytes, caused a time-dependent decrease in triglyceride content, insulin-stimulated glucose and fatty acid uptake, incorporation into lipid, and oxidation compared with controls. In parallel, gene expression of peroxisome proliferator-activated receptor-gamma and many of its downstream targets were diminished by trans-10, cis-12 CLA, whereas leptin gene expression was increased. Prior to changes in gene expression and metabolism, trans-10, cis-12 CLA caused a robust and sustained activation of mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling. Furthermore, the trans-10, cis-12 CLA-mediated activation of MEK/ERK could be attenuated by pretreatment with U0126 and pertussis toxin. In parallel, pretreatment with U0126 blocked the ability of trans-10, cis-12 CLA to alter gene expression and attenuate glucose and fatty acid uptake of the cultures. Intriguingly, the induction by CLA of MEK/ERK signaling was linked to hypersecretion of adipocytokines interleukin-6 and interleukin-8. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA decreases the triglyceride content of newly differentiated human adipocytes by inducing MEK/ERK signaling through the autocrine/paracrine actions of interleukins-6 and 8.     

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16^INK4A and p21^CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.     

Pharmacological inhibitors of extracellular signal-regulated protein kinases attenuate the apoptotic action of cisplatin in human myeloid leukemia cells via glutathione-independent reduction in intracellular drug accumulation

It has been reported that inhibition of extracellular signal-regulated protein kinases (ERKs) attenuates the toxicity cisplatin (cis-platinum (II)-diammine dichloride) in some cell types. This response was here investigated using human myeloid leukemia cells. Cisplatin stimulated ERK1/2 phosphorylation and caused apoptosis in U-937 promonocytic cells, an effect which was attenuated by the MEK/ERK inhibitors PD98059 and U0126. While ERK1/2 activation was a general phenomenon, irrespective of the used cell type or antitumour drug, the MEK/ERK inhibitors only reduced cisplatin toxicity in human myeloid cells (THP-1, HL-60 and NB-4), but not in RAW 264.7 mouse macrophages and NRK-52E rat renal tubular cells; and failed to reduce the toxicity etoposide, camptothecin, melphalan and arsenic trioxide, in U-937 cells. U0126 attenuated cisplatin-DNA binding and intracellular peroxide accumulation, which are important regulators of cisplatin toxicity. Although cisplatin decreased the intracellular glutathione (GSH) content, which was restored by U0126, treatments with GSH-ethyl ester and dl-buthionine-(S,R)-sulfoximine revealed that GSH does not regulate cisplatin toxicity in the present experimental conditions. In spite of it, PD98059 and U0126 reduced the intracellular accumulation of cisplatin. These results suggest that GSH-independent modulation of drug transport is a major mechanism explaining the anti-apoptotic action of MEK/ERK inhibitors in cisplatin-treated myeloid cells.     

IL-10 synergistically enhances GM-CSF-induced CCR1 expression in myelomonocytic cells

CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).     

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is up-regulated by flurbiprofen and other non-steroidal anti-inflammatory drugs in human colon cancer HT29 cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit prostaglandin synthetic enzyme, cyclooxygenases (COXs), as well as to exhibit anti-tumor activity although at much higher concentrations. 15-Hydroxyprostaglandin dehyrogenase (15-PGDH), a key prostaglandin catabolic enzyme, was recently shown to be a tumor suppressor. Effects of NSAIDs on 15-PGDH expression were therefore examined. Flurbiprofen and several other NSAIDs were found to induce 15-PGDH expression in human colon cancer HT29 cells. Flurbiprofen, the most active one, was also shown to induce 15-PGDH expression in other types of cancer cells. Induction of 15-PGDH expression appeared to occur at the stage of mRNA as levels of 15-PGDH mRNA were increased by flurbiprofen in HT29 cells. Levels of 15-PGDH were also found to be regulated at the stage of protein turnover. MEK inhibitors, PD98059 and U-0126, which inhibited ERK phosphorylation were shown to elevate 15-PGDH levels very significantly. These inhibitors did not appear to alter 15-PGDH mRNA levels but down-regulate matrix metalloproteinase-9 (MMP-9). This protease was shown to degrade and inactivate 15-PGDH suggesting that elevation of 15-PGDH levels could be due to inhibition of MMP-9 expression by these inhibitors. Similarly, flurbiprofen was also demonstrated to inhibit ERK activation and to down-regulate MMP-9 expression. Furthermore, flurbiprofen was shown to induce the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of MMP-9. The turnover of 15-PGDH was found to prolong in the presence of flurbiprofen as compared to that in the absence of this drug. Taken together, these results indicate that flurbiprofen up-regulates 15-PGDH by increasing the expression and decreasing the degradation of 15-PGDH in HT29 cells.     

Epidermal growth factor increases clathrin-dependent endocytosis and degradation of claudin-2 protein in MDCK II cells

Epidermal growth factor (EGF) decreases the mRNA and protein levels of claudin-2 (CLDN2) in Madin-Darby canine kidney (MDCK) II cells. Here we examined whether EGF affects the stability and intracellular distribution of CLDN2 protein. EGF decreased surface and total levels of CLDN2, which was inhibited by U0126, a MEK inhibitor. CLDN2 was co-localized at the tight junctions (TJs) with ZO-1, a scaffolding protein. The fluorescence signal for CLDN2 disappeared on treatment with EGF, which was inhibited by U0126. EGF accelerated the decrease in CLDN2 in the presence of cycloheximide, a translation inhibitor, indicating that EGF reduces the stability of the protein. Chloroquine, a lysosomal protease inhibitor, blocked the EGF-induced decrease in CLDN2 protein and caused the co-localization of CLDN2 with Lamp-1, a marker of lysosome. Monodancylcadaverine, an inhibitor of endocytosis, and clathrin siRNA blocked the EGF-induced decrease in CLDN2 and the translocation of CLDN2 from the TJs to the lysosome. EGF increased the association of CLDN2 with clathrin and adaptin α which was inhibited by U0126. These results suggest that EGF accelerates clathrin-dependent endocytosis and lysosomal degradation of CLDN2 protein mediated by the activation of a MEK/ERK pathway.