Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease

Polycystic kidney (PCK) rats exhibit a multiorgan cyst pathology similar to human autosomal recessive polycystic kidney disease, and are proposed as an animal model of Caroli's disease with congenital hepatic fibrosis (CHF). This study investigated the expression and function of selected components of the mitogen activated protein kinase (MAPK) pathway in cultured intrahepatic biliary epithelial cells (BECs) of PCK rats. Compared to the proliferative activity of cultured BECs of control rats, those of the PCK rats were hyperresponsive to epidermal growth factor (EGF). The increase in BEC proliferation was accompanied by overexpression of MAPK/extracellular signal-regulated protein kinase (ERK) kinase 5 (MEK5), and subsequent phosphorylation of ERK5 in vitro. The increased proliferative activity was significantly inhibited by the transfection of short interfering RNA against MEK5 mRNA. An EGF receptor tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839), also significantly inhibited the abnormal growth of cultured BECs of PCK rats. By contrast, treatment with PD98059 and U0126, inhibitors for MEK1/2, was less effective. These results suggest that the activation of the MEK5-ERK5 cascade plays a pivotal role in the biliary dysgenesis of PCK rats, and also provide insights into the pathogenesis of Caroli's disease with CHF. As the MEK5-ERK5 interaction is highly specific, it may represent a potential target of therapy.     

Fumarylacetoacetate, the metabolite accumulating in hereditary tyrosinemia, activates the ERK pathway and induces mitotic abnormalities and genomic instability

Patients suffering from the metabolic disease hereditary tyrosinemia type I (HT1), caused by fumarylacetoacetate hydrolase deficiency, have a high risk of developing liver cancer. We report that a sub-apoptogenic dose of fumarylacetoacetate (FAA), the mutagenic metabolite accumulating in HT1, induces spindle disturbances and segregational defects in both rodent and human cells. Mitotic abnormalities, such as distorted spindles, lagging chromosomes, anaphase/telophase chromatin bridges, aberrant karyokinesis/cytokinesis and multinucleation were observed. Some mitotic asters displayed a large pericentriolar material cloud and/or altered distribution of the spindle pole-associated protein NuMA. FAA-treated cells developed micronuclei which were predominantly CREST-positive, suggesting chromosomal instability. The Golgi complex was rapidly disrupted by FAA, without evident microtubules/tubulin alterations, and a sustained activation of the extracellular signal-regulated protein kinase (ERK) was also observed. Primary skin fibroblasts derived from HT1 patients, not exogenously treated with FAA, showed similar mitotic-derived alterations and ERK activation. Biochemical data suggest that FAA causes ERK activation through a thiol-regulated and tyrosine kinase-dependent, but growth factor receptor- and protein kinase C-independent pathway. Pre-treatment with the MEK inhibitor PD98059 and the Ras farnesylation inhibitor B581 decreased the formation of CREST-positive micronuclei by approximately 75%, confirming the partial contribution of the Ras/ERK effector pathway to the induction of chromosomal instability by FAA. Replenishment of intracellular glutathione (GSH) with GSH monoethylester abolished ERK activation and reduced the chromosomal instability induced by FAA by 80%. Together these results confirm and extend the previously reported genetic instability occurring in cells from HT1 patients and allow us to speculate that this tumorigenic-related phenomenon may rely on the biochemical/cellular effects of FAA as a thiol-reacting and organelle/mitotic spindle-disturbing agent.     

Regulation of c-Fos and Fra-1 by the MEK5-ERK5 pathway

BACKGROUND: ERK5 is the newest subfamily member of the mitogen-activated protein kinase (MAPK) family, and is activated by various extracellular signals including growth factors. MEK5 is a specific activator of ERK5. c-Fos and Fra-1, well-known immediate early gene products, are members of the AP-1 family. We previously reported that activation of the MEK5-ERK5 pathway is able to induce expression of c-Fos. RESULTS: We have found that activation of the MEK5-ERK5 pathway causes the phosphorylation and stabilization of c-Fos and Fra-1. Phosphorylation of c-Fos appears to be mediated by ERK5 and a kinase(s) lying downstream of ERK5, and the MEK5-ERK5 pathway-dependent phosphorylation sites on c-Fos are different from the ERK1/2 pathway-dependent ones. Interestingly, activation of the MEK5-ERK5 pathway, but not that of the ERK1/2 pathway, is found to markedly increase the transactivation activity of c-Fos. Furthermore, our results show that the C-terminal half of ERK5 is necessary for the maximal activation of the transactivation activity of c-Fos and Fra-1. CONCLUSION: These results reveal a role of the MEK5-ERK5 pathway in modulating the function of the Fos family proteins which is different from the role of the ERK1/2 pathway.     

Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 S6, similar to that in Alzheimer's disease

In Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.     

Distribution of ERK1/2 and ERK3 during normal rat fetal lung development

The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.     

Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4-independent activation of the ERK1/2 signal transduction pathway in macrophages

Synthetic lipopeptides based on bacterial lipoprotein are efficient activators for monocytes/macrophages inducing the release of interleukin (IL)-1, IL-6, tumour necrosis factor-alpha (TNF-alpha), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor kappaB (NFkappaB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4). We show that P3CSK4 activates mitogen-activated protein (MAP)-kinases ERK1/2 and MAP kinase (MAPK)-kinases MEK1/2 in bone-marrow-derived macrophages (BMDM) and in the macrophage cell line RAW 264.7. Additionally, we could detect differences between the P3CSK4 and lipopolysaccharide (LPS)-induced phosphorylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose-response using RAW 264.7 cells or BMDM from BALB/c and LPS responder mice (C57BL/10ScSn) or LPS non-responder mice (C57BL/10ScCr). The lipopeptide activated the MAPK-signalling cascade in both LPS responder and non-responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFkappaB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti-CD14. These data correspond to the reduction of phosphorylation of ERK1/2 after stimulation with P3CSK4 in the presence of anti-CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide-induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream-located MAP kinases ERK1/2.     

神经再生素激活PC12细胞ERK1/2的实验研究

本实验初步探讨了神经再生素 ( NRF)作用于 PC12细胞的信号传导途径。采用 MTT法检测 NRF对无血清培养的PC12细胞存活率的影响 ;用磷酸化的 Elk单克隆抗体 ,通过 Western blot法观察 NRF的不同浓度 ( 10、10 0、10 0 0 ng/ m l)及不同作用时间 ( 0 min、15 min、3 0 min、1h、2 h)对无血清培养的 PC12细胞 ERK1/ 2活性的影响 ;还运用 MEK1/ 2阻断剂 U0 12 6,观察NRF激活 ERK1/ 2是否需要其上游信号蛋白 MEK1/ 2的激活。结果表明 ,NRF作用于无血清培养的 PC12细胞 ,可以激活ERK1/ 2 ,存在明显的剂量效应关系和时间依赖作用 ,在 10 0 0 ng/ ml浓度下最大 ,作用 2 h时达峰值 ;U0 12 6作用后 ,可抑制 NRF作用于 PC12细胞引起的 ERK1/ 2的激活。由此可见 ,NRF对无血清培养的 PC12细胞具有保护作用 ,可能通过激活 ERK1/ 2磷酸化级联途径来介导这些作用。     

Anterograde and retrograde transport of active extracellular signal-related kinase 1 (ERK1) in the ligated rat sciatic nerve

Neurons are one of the most polarized cells and often the nerve terminals may be located long distances from the cell body, thus signal transduction in neurons unlike other cells may need to be conducted over large distances. The mitogen-activated protein/extracellular signal-regulated kinases (MAP kinases or ERKs) regulate a diverse array of functions and in neurons, the ERK signalling pathways appear to have an important role in activity-dependent regulation of neuronal function. Using the ligated rat sciatic nerve as an experimental model we previously showed that the ERK1/2, MAP/ERK kinase (MEK1/2) and the p110 catalytic subunit of PI3-kinase are transported in the rat sciatic nerve. We have extended these findings to determine if these proteins are transported in the active state using antibodies that specifically detect the active form of ERK1/2, MEK1/2 and AKT which is activated downstream of PI3-kinase. We show significant accumulation of active ERK1 on the proximal and distal sides of a nerve ligation after 16 h. Active ERK2 also appeared to be accumulating at the ligature, however this did not reach statistical significance. In contrast there was not any significant accumulation of active MEK1/2 or active AKT. A component of both active ERK1 and active ERK2 is present in between the two ligations suggesting they are also present in the surrounding Schwann cells and are activated in response to nerve injury.Taken together our results suggest that a component of the accumulation of active ERK1 on the distal and proximal side of the nerve ligations results from transport in the anterograde and retrograde direction in the rat sciatic nerve.     

JNK and ERK1/2 pathways have a dual opposite effect on the expression of BACE1

The activity of beta-secretase (BACE1), the endo-protease essential for the production of amyloid beta (Abeta) peptides, is increased in brain of late-onset sporadic Alzheimer's disease (AD), and oxidative stress is the potential cause of this event. Oxidative stress up-regulates the expression and the activity of BACE1 in cellular and animal models, through a mechanism that involves the increase of gamma-secretase cleavage on APP and the activation of c-jun N-terminal kinase/activator protein 1 (JNK/AP1) pathway. We further characterized the cellular pathways that control BACE1 expression under oxidative stress. We investigated the involvement of extracellular signal regulated MAP kinase (ERK1/2) pathway in the regulation of BACE1 expression, since it has been recently shown that ERK1/2 is an endogenous regulator of the gamma-secretase activity. We found that ERK1/2 pathway negatively modulates BACE1 expression and activity. Moreover, we observed that conditions that abrogate the gamma-secretase activity favor the activation of signalling pathways that promote cell survival, such as ERK1/2 and the serine/threonine kinase Akt/protein kinase B (Akt). These data suggest that the positive or negative cellular responses to oxidative stress parallel the activities of the beta- and the gamma-secretase. ERK1/2 and JNK pathways are involved in this bipartite response, which can lead to neurodegeneration or neuroprotection depending on the cellular and environmental conditions or cooperation with other signalling pathways such as Akt cascade.     

The role of ERK5 in T-cell signalling

The mitogen-activated protein kinase (MAPK) ERK5 plays an important role in mammary epithelial proliferation, endothelial cell survival and normal embryonic development. In nonhaematopoietic cells, mitogenic and stress signals activate the ERK5 cascade. Here, we investigated the role of the ERK5 pathway in T-cell activation and show that primary and leukaemic T cells express ERK5, whose activating phosphorylation is induced by antibodies against CD3 but not by phorbol myristate acetate treatment. ERK5 localized in the cytosol and nucleus in quiescent and activated T cells. In the latter, ERK5 phosphorylation was mainly observed in the nucleus. Selective activation of the ERK5 cascade by transfecting constitutively active MEK5 and wildtype ERK5 induced a reporter gene driven by the IL-2 promoter while barely affecting CD69 expression. These results suggest a new role for the ERK5 cascade in intracellular signalling in T cells.     

KSR is a scaffold required for activation of the ERK/MAPK module

Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. Here, we show that KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, we found that KSR facilitates the phosphorylation of MEK by RAF. We further show that KSR associates independently with RAF and MEK, and that these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair.     

Protein kinase-A activity in PRKAR1A-mutant cells,and regulation of mitogen-activated protein kinases ERK1/2

Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-alpha (RIalpha) of the cAMP-dependent kinase (PKA) holoenzyme; how RIalpha insufficiency leads to tumorigenesis remains unclear. In many cells PKA inhibits the extracellular receptor kinase (ERK1/2) cascade of the mitogen-activated protein kinase (MAPK) pathway leading to inhibition of cell proliferation. We investigated whether the PKA-mediated inhibitory effect on ERK1/2 is affected in CNC cells that carry germline PRKAR1A mutations. PKA activity both at baseline and after stimulation with cAMP was augmented in cells carrying mutations. Quantitative message analysis showed that the main PKA subunits expressed were type I (RIalpha and RIbeta) but RIalpha was decreased in mutant cells. Immunoblot assays of ERK1/2 phosphorylation by the cell- and pathway-specific stimulant lysophosphatidic acid (LPA) showed activation of this pathway in a time- and concentration-dependent manner that was prevented by a specific inhibitor. There was a greater rate of growth in mutant cells; forskolin and isoproterenol inhibited LPA-induced ERK1/2 phosphorylation in normal but not in mutant cells. Forskolin inhibited LPA-induced cell proliferation and metabolism in normal cells, but stimulated these parameters in mutant cells. These data were also replicated in a pituitary tumor cell line carrying the most common PRKAR1A mutation (c.578del TG), and an in vitro construct of mutant PRKAR1A that was recently shown to lead to augmented PKA-mediated phosphorylation. We conclude that PKA activity in CNC cells is increased and that its stimulation by forskolin or isoproterenol increases LPA-induced ERK1/2 phosphorylation, cell metabolism and proliferation. Reversal of PKA-mediated inhibition of this MAPK pathway in CNC cells may contribute to tumorigenesis in this condition.     

Involvement of the ERK mitogen-activated protein kinase in cell resistance to complement-mediated lysis

Sublytic doses of complement desensitize cells and make them resistant to lytic complement doses. This process, named complement-induced protection, requires calcium ion influx, protein kinase C activation and protein synthesis. The involvement of the extracellular signal-regulated kinase, ERK, in cell desensitization by sublytic complement was examined in erythroleukaemia K562 cells and in COS-7 cells. As shown here, ERK is activated in K562 and COS-7 cells within 10 min of sublytic immune attack and then shows a decline and a second peak of activation at 20 min. C7- and C8-deficient human sera have a small effect on ERK activity. However, a significant increase in ERK activation is observed when C7 or C8, respectively, is added back to these sera. Complement-induced ERK activation was blocked in cells treated with GF109203X or Go6976, two selective PKC inhibitors, as well as by treatment with PD098059, an inhibitor of MEK1, the ERK kinase. PD098059 treatment also sensitized K562 cells to complement-mediated lysis and prevented complement-induced protection. COS-7 cells transfected with a dominant-negative MEK plasmid were incapable of undergoing the process of complement-induced protection. In conclusion, cell desensitization by sublytic doses of the complement membrane attack complex involves a signalling cascade that includes PKC-mediated ERK activation.     

The extracellular signal-regulated kinase (ERK) pathway is involved in human sperm function and modulated by the superoxide anion

Our aim was to ascertain the role of the extracellular signal-regulated protein kinase (ERK) pathway in human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and its regulation by the superoxide anion (O(2)(-)*). Immunoblotting indicated the presence of Shc, Grb2, Ras(p21), Raf and ERK1 and 2 (ERK1/2) in spermatozoa. Grb2, Ras(p21), Raf and MEK inhibitors dose-dependently prevented sperm capacitation and protein tyrosine phosphorylation, without modifying sperm O(2)(-)* production. Therefore, the whole ERK cascade plays a role in capacitation, downstream of O(2)(-)* but upstream of protein tyrosine phosphorylation. Upon incubation with FCSu, the early (5 min) increase in ERK1/2 activity (as shown by double phosphorylation of the Thr-Glu-Tyr motif) was followed by an important decrease over the next 2 h; superoxide dismutase did not change this pattern. The phosphorylation of the Thr-Glu-Tyr motif present in other sperm proteins (16-33 kDa) also increased (5 min incubation with FCSu) and then progressively decreased, and this effect was regulated by O(2)(-)*, MEK and cAMP. The phospho-Ser/Thr-Pro content (characteristic of ERK1/2 substrates) of Triton-insoluble proteins (75 and 80 kDa) increased during capacitation and also appeared to be regulated by O(2)(-)* and the ERK pathway. Inhibition of ERK1/2 activation reduced lysophosphatidylcholine-induced acrosome reaction and the associated protein tyrosine phosphorylation. These results support a role for the ERK pathway in human sperm function.     

Partial characterization of a novel mitogen-activated protein kinase/extracellular signal-regulated kinase activator in airway smooth-muscle cells

We demonstrated previously that in bovine tracheal myocytes, pretreatment with either forskolin or histamine significantly reduces both platelet-derived growth factor (PDGF)- and epidermal growth factor- induced Raf-1 activation but fails to inhibit extracellular signal-regulated kinase (ERK) activation substantially, evidence of a Raf-1-independent ERK activation pathway. To identify Raf-1-independent upstream signaling intermediates of mitogen-activated protein kinase/ERK kinase-1 (MEK1), the dual-function kinase required and sufficient for ERK activation in these cells, lysates from forskolin and PDGF-treated bovine tracheal myocytes were resolved using ion exchange chromatography. Kinase activity for MEK1 was assessed by in vitro phosphorylation assay. In all experiments, the major peak of MEK1 phosphorylation activity was detected in fractions 18 through 26 (80 to 160 mM NaCl), with the peak fraction eluting at a NaCl concentration of 140 mM. The ability of these fractions to activate MEK1 was confirmed by examining the phosphorylation of myelin basic protein, a known substrate for ERKs, in the presence of functional MEK1 and ERK1. Fractions containing kinase activity were also probed with antibodies against MEK kinase-1, Raf-1, A-Raf, B-Raf, Mos, and Tpl-2. None of these proteins was detected in fractions containing peak kinase activity, suggesting the presence of a novel PDGF-stimulated, forskolin-insensitive MEK1 kinase. Further separation of fractions holding peak MEK phosphorylation activity by gel filtration suggested an apparent molecular mass of 40 to 45 kD. We conclude that PDGF-induced activation of MEK1 in bovine tracheal myocytes is mediated at least in part by a novel kinase.