Academic literature on the topic 'ERK5/BMK1'

Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

ERK5 (extracellular-signal-regulated kinase 5), also termed BMK1 [big MAPK1 (mitogen-activated protein kinase 1)], is the most recently discovered member of the MAPK family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling, regulating hypoxia, tumour angiogenesis and cell migration. This review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.

Serum and growth factors activate both the canonical extracellular signal-regulated kinase (ERK) 1/2 pathway and the ERK5/big mitogen-activated protein kinase 1 (BMK) 1 pathway. Pharmacological inhibition of the ERK1/2 pathway using PD98059 and U0126 prevents cyclin D1 expression and inhibits cell proliferation, arguing that the ERK1/2 pathway is rate limiting for cell-cycle re-entry. However, both PD98059 and U0126 also inhibit the ERK5/BMK1 pathway, raising the possibility that the anti-proliferative effect of such drugs may be due to inhibition of ERK5 or both pathways. Here we characterize the effect of the novel mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD184352, on the ERK1/2 and ERK5 pathways in the Chinese hamster fibroblast cell line CCl39. In quiescent cells, serum-stimulated ERK1 activity was completely inhibited by PD184352 with an IC50 below 1μM, whereas ERK5 activation was unaffected even at 20μM. Serum-stimulated DNA synthesis and cyclin D1 expression was inhibited by low doses of PD184352, which abolished ERK1 activity but had no effect on ERK5. Similarly, in cycling cells PD184352 caused a dose-dependent G1 arrest and inhibition of cyclin D1 expression at low doses, which inhibited ERK1 but were without effect on ERK5. These results indicate that the anti-proliferative effect of PD184352 is due to inhibition of the classical ERK1/2 pathway and does not require inhibition of the ERK5 pathway.

p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/Erk5 pathway as a critical mediator of endothelial Pak2 signaling.

We previously demonstrated that resistance to hypoxia is a common feature of Haematopoietic Stem Cells (HSC) and Leukemic Stem Cells (LSC), but also that a hypoxia-selectable LSC subset is likely to exists within any type of leukemia, including stabilized clonal cells lines. In Chronic Myeloid Leukemia (CML), either cell lines or CD34+ cells explanted from CML patients, hypoxia completely suppresses the expression of BCR/Ablprotein but not that of BCR/Abl transcript, so that hypoxia-resistant cells, while remaining genetically leukemic, are independent of BCR/Abl signaling for their maintenance in vitro and resistant to treatment with Imatinib-Mesylate (IM). Thus, hypoxia-selectable LSC are refractory to IM due to the lack of its molecular target. This is very well in keeping with the notions that: (i) IM, despite its impressive efficacy as first-line therapy for patients with chronic-phase CML, induces a state of Minimal Residual Disease (MRD), rather than cure; (ii) LSC most likely sustain MRD and are thereby responsible for the late relapses of CML; (iii) tissue hypoxia provides optimal conditions for the homing of normal and neoplastic stem cells. The Extracellular signal-Regulated Kinase 5 (ERK5) is a Mitogen-Activated Protein Kinase involved in the control of cell survival and proliferation, as well as in the pathogenesis of a number of cancers, including CML. ERK5 is activated by cytokines regulating stem cell compartments and participates to cell response to hypoxia. The main targets of the experimental work of this thesis were: 1) to explore the role of ERK5 in the maintenance of leukemia stem and progenitor cells; 2) to address the effects of different pharmacological MEK5/ERK5 inhibitors on the survival and proliferation of CML cells, either bulk or LSC populations; 3) to gather information useful to identify innovative (combination) treatment capable to eliminate the IM-insensitive LSC and thereby MDR. The selection of leukemic progenitor cells (LPC)/LSC, as well as the effects of pharmacological ERK5 pathway inhibition on hypoxia-selected LPC/LSC, were estimated by the Culture-Repopulating Ability (CRA) assay, a non-clonal assay which measures the culture-repopulating power of normal or leukemic hematopoietic cells. On the basis of entity and kinetics of repopulation of secondary cultures (LC2) where cell growth is unrestrained, it is possible to estimate the LSC or LPC content of cell populations subjected to a selective treatment in primary cultures (LC1). In the experiments reported here, the CRA assay was used to estimate the content of hypoxia-resistant CML cell populations with LSC/LPC and to test the effects on these cell subsets of genetic or pharmacologic inhibition of the ERK5 pathway. The inhibitors used were the ERK5-specific inhibitor XMD8-92 and the MEK5-specific inhibitorsBIX02188 and BIX02189. Genetic inhibition of ERK5 was obtained using lentiviral vectors expressing ERK5-specific short hairpin RNAs. The effects of the inhibiting treatments were tested on the KCL22 and K562 human stabilized CML cell lines, where ERK5 is constitutively activated. The results obtained indicated that all three inhibitors were inactive on the bulk of CML cell population with respect to cell number in culture. On the other hand, XMD8-92 determined an appreciable increase of the percentage of KCL22, but not K562, cells in the G0/G1 phase of cell cycle and a decrease of the percentage of cells in S phase. BIX02188 and BIX02189 did not exhibit any appreciable effect on cell cycle phase distribution in either K562 or KCL22 cell line. XMD8-92, but not BIX02188 or BIX02189, also determined an increase of p27kip expression in both cell lines and reduced the basal apoptosis occurring in untreated K562 cell cultures. In hypoxia, where constitutive activation of ERK5 was suppressed, BIX02188 or BIX02189 did not affect the cell number significantly in either cell line. XMD8-92 treatment, on the contrary, resulted in a significant, although not marked, increase of viable cell number. A block of progression of CML cells to the S phase and the increase of p27Kip operated by XMD8-92 were observed in both cell lines. Consistently, neither BIX02188 nor BIX02189, which did not induce a detectable G0/G1 accumulation, increased the expression of p27Kip. Taken together with the data obtained in normoxia, these results point to a cytostatic, rather than cytotoxic, effect of ERK5-specific inhibition. Moreover, the treatment with XMD8-92, but not BIX02188 orBIX02189, resulted in a significant reduction of hypoxia-induced apoptosis. These results, together with those obtained in normoxia, suggest that the protective effect of XMD8-92 against basal apoptosis occurring in control cultures in normoxia is enhanced in hypoxia. The treatment of KCL22 and K562 cells with BIX02188 or BIX02189 in normoxic LC1 reduced the ability to repopulate LC2, while that with XMD8-92 almost completely suppressed LC2 repopulation. On the other hand, when hypoxic LC1 were treated with BIX02188 or BIX02189, LC2 repopulation was almost immediate. On the contrary, the treatment with XMD8-92 completely suppressed LC2 repopulation. These results indicate that MEK5 inhibitors have a modest effect on CRA of BCR/Ablprotein-expressing CML, actually accelerating BCR/Ablprotein re-expression in cells rescued from hypoxia. On the contrary, both BCR/Ablprotein-expressing and BCR/Ablprotein-negative subsets were markedly sensitive to the treatment with XMD8-92. Thus, XMD8-92 was inactive on the bulk of CML cell population, but capable to suppress completely the BCR/Ablprotein-positive LPC and the hypoxia-selected, BCR/Ablprotein-negative LPC/LSC. To confirm the above results while overcoming problems of interpretation of data due to possible off-target effects of ERK5-inhibiting drugs, K562 cells were infected with lentiviral vectors expressing shRNA against ERK5 (shERK5). In hypoxia, genetic knockdown of ERK5, unlike its pharmacological inhibition, impaired CML cell survival. These differences are likely due to the well-known property of ERK5 to regulate a number of genes by direct interaction, independently of its kinase activity. On the other hand, ERK5 knockdown suppressed LC2 repopulation driven by hypoxia-selected cells as much as the inhibition of ERK5 enzymatic activity by XMD8-92. Since we found that XMD8-92 does not inhibit the overall survival of CML cells, but suppresses hypoxia-selected LSC-like cell subsets, we tested the effects of the XMD8-92/IM combination. IM markedly reduced the number of viable cells in normoxia as well as in hypoxia. The combination with XMD8-92 determined a marginal, if any, enhancement of the inhibitory effect of IM. LC2 repopulation was reduced, but not abolished, by IM treatment of normoxic or hypoxic LC1. Importantly, XMD8-92, alone or in combination with IM, suppressed completely LC2 repopulation. The effectiveness of XMD8-92 demonstrated on hypoxia-selected cell subsets of stabilized CML lines was also tested on primary cells explanted from CML patients. In keeping with what observed for cell lines, XMD8-92, alone or in combination with IM, impaired culture repopulation driven by hypoxia-selected LSC from CML patients. These results indicate that the combined treatment of XMD8-92 with IM may be an useful approach to try to eradicate CML together with the induction of remission, being XMD8-92 active on LPC/LSC but not cell bulk, and, viceversa, IM very active on the bulk but unable to suppress CML progenitors.

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Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function.
Biotechnology and Biological Sciences Research Council, the Medical Research Council and the Wellcome Trust