ERK5 inhibitor BIX02189 attenuates methamphetamine-induced hyperactivity by modulating microglial activation in the striatum.

Extracellular signal-regulated protein kinase 5 (ERK5) has various physiological functions. However, the physiological role of ERK5 in the treatment of mice with an illicit drug such as methamphetamine (METH) remains unknown. We revealed that mice treated with METH showed hyperactivity, and increased p-ERK5 and Iba1 (a microglia marker) levels in the striatum. Additionally, these changes were inhibited by pretreatment with the ERK5 inhibitor BIX02189. The results suggest that METH-induced hyperactivity is associated with the activation of microglia via p-ERK5 in the striatum. Thus, the ERK5 pathway components in the central nervous system are potential therapeutic targets for preventing METH addiction.

Reciprocal regulation of endothelial-mesenchymal transition by MAPK7 and EZH2 in intimal hyperplasia and coronary artery disease.

Endothelial-mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFß1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

The Melanocyte Lineage Factor miR-211 Promotes BRAFV600E Inhibitor Resistance.

Resistance to targeted therapy and immunotherapy remains a major obstacle in improving care for patients with advanced melanoma. MicroRNAs play important roles in regulating gene networks involved in disease progression and resistance to therapy in cancers such as melanoma. MicroRNA miR-211 contributes to melanocyte and melanoma biology and has been implicated in targeted therapy resistance. Lee et al. (2020) report a novel mechanism by which miR-211 promotes resistance to BRAFV600E inhibitor therapy via the upregulation of the extracellular signal-regulated kinase 5 signaling pathway.

Cigarette smoke upregulates SPRR3 by favoring c-Jun/Fra1 heterodimerization in human bronchial epithelial cells.

AIM: The airway epithelium of smokers exhibits upregulated SPRR3, an indicator of pathogenic keratinization. The mechanisms underlying this phenomenon require investigation. PATIENTS & METHODS: Human bronchial epithelial (HBE) SPRR3 expression was analyzed by smoking status. Primary HBE cells were exposed to cigarette smoke (CS). SPRR3 expression, SPRR3 promoter activity, AP-1 factor binding and AP-1 factors' effects were analyzed. RESULTS: Current smokers display SPRR3 upregulation relative to never smokers. CS upregulates SPRR3 transcription in an exposure-dependent manner. CS promotes c-Jun and Fra1 binding to the SPRR3-AP-1/TRE site. Wild-type c-Jun and Fra1 upregulate, whereas c-Jun and Fra1, dominant-negative mutants, suppress SPRR3 promoter activity. CONCLUSION: CS induces SPRR3 upregulation in HBE cells by promoting aberrant c-Jun/Fra1 dimerization.

ERK5 plays an essential role in gestational beta-cell proliferation.

OBJECTIVES: Restoring a functional beta-cell mass is a fundamental goal in treating diabetes. A complex signalling pathway network coordinates the regulation of beta-cell proliferation, although a role for ERK5 in this network has not been reported. This question was addressed in this study. MATERIALS AND METHODS: We studied the activation of extracellular-signal-regulated kinase 5 (ERK5) in pregnant mice, a well-known mouse model of increased beta-cell proliferation. A specific inhibitor of ERK5 activation, BIX02189, was intraperitoneally injected into the pregnant mice to suppress ERK5 signalling. Beta-cell proliferation was determined by quantification of Ki-67+ beta cells. Beta-cell apoptosis was determined by TUNEL assay. The extent of beta-cell proliferation was determined by beta-cell mass. The alteration of ERK5 activation and CyclinD1 levels in purified mouse islets was examined by Western blotting. RESULTS: Extracellular-signal-regulated kinase 5 phosphorylation, which represents ERK5 activation, was significantly upregulated in islets from pregnant mice. Suppression of ERK5 activation by BIX02189 in pregnant mice significantly reduced beta-cell proliferation, without affecting beta-cell apoptosis, resulting in increases in random blood glucose levels and impairment of glucose response of the mice. ERK5 seemed to activate CyclinD1 to promote gestational beta-cell proliferation. CONCLUSIONS: Extracellular-signal-regulated kinase 5 plays an essential role in the gestational augmentation of beta-cell proliferation. ERK5 may be a promising target for increasing beta-cell mass in diabetes patients.

Analysis of Nkx3.1:Cre-driven Erk5 deletion reveals a profound spinal deformity which is linked to increased osteoclast activity.

Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5 fl/fl (Erk5 fl/fl) mice by ~6-8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5 fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5 fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5 fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.

WNK lysine deficient protein kinase 1 regulates human endometrial stromal cell decidualization, proliferation, and migration in part through mitogen-activated protein kinase 7.

The differentiation of endometrial stromal cells into decidual cells, termed decidualization, is an integral step in the establishment of pregnancy. The mitogen-activated protein kinase homolog, WNK lysine deficient protein kinase 1 (WNK1), is activated downstream of epidermal growth factor receptor during decidualization. Primary human endometrial stromal cells (HESCs) were subjected to small interfering RNA knockdown of WNK1 followed by in vitro decidualization. This abrogated expression of the decidual marker genes, insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL), and prevented adoption of decidual cell morphology. Analysis of the WNK1-dependent transcriptome by RNA-Seq demonstrated that WNK1 regulates the expression of 1858 genes during decidualization. Gene ontology and upstream regulator pathway analysis showed that WNK1 regulates cell migration, differentiation, and proliferation. WNK1 was required for many of the gene expression changes that drive decidualization, including the induction of the inflammatory cytokines, C-C motif chemokine ligand 8 (CCL8), interleukin 1 beta (IL1B), and interleukin 15 (IL15), and the repression of transforming growth factor-beta (TGF-beta) pathway genes, including early growth response 2 (EGR2), SMAD family member 3 (SMAD3), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 4 (ITGA4), and integrin subunit beta 3 (ITGB3). In addition to abrogating decidualization, WNK1 knockdown decreased the migration and proliferation of HESCs. Furthermore, mitogen-activated protein kinase 7 (MAPK7), a known downstream target of WNK1, was activated during decidualization in a WNK1-dependent manner. Small interfering RNA knockdown of MAPK7 demonstrated that MAPK7 regulates a subset of WNK1-regulated genes and controls the migration and proliferation of HESCs. These results indicate that WNK1 and MAPK7 promote migration and proliferation during decidualization and regulate the expression of inflammatory cytokines and TGF-beta pathway genes in HESCs.

Research progress of the role and mechanism of extracellular signal-regulated protein kinase 5 (ERK5) pathway in pathological pain.

Extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1 (MAPK1), is an important member of ERK family, which is a subfamily of the large MAPK family. ERK5 is expressed in many tissues, including the dorsal root ganglion (DRG) neurons and the spinal cord. In this review, we focus on elaborating ERK5-associated pathway in pathological pain, in which the ERK5/CREB (cyclic adenosine monophosphate (cAMP)-response element-binding protein) pathway plays a crucial role in the transduction of pain signal and contributes to pain hypersensitivity. ERK5 activation in the spinal dorsal horn occurs mainly in microglia. The activation of ERK5 can be mediated by N-methyl-D-aspartate (NMDA) receptors. We also elaborate the relationship between ERK5 activation and nerve growth factor-tyrosine kinase A (NGF-TrkA), and the connection between ERK5 activation and brain-derived neurotrophic factor (BDNF) in pathological pain in detail.

Novel mechanisms of endothelial mechanotransduction.

Atherosclerosis is a focal disease that develops preferentially where nonlaminar, disturbed blood flow occurs, such as branches, bifurcations, and curvatures of large arteries. Endothelial cells sense and respond differently to disturbed flow compared with steady laminar flow. Disturbed flow that occurs in so-called atheroprone areas activates proinflammatory and apoptotic signaling, and this results in endothelial dysfunction and leads to subsequent development of atherosclerosis. In contrast, steady laminar flow as atheroprotective flow promotes expression of many anti-inflammatory genes, such as Kruppel-like factor 2 and endothelial nitric oxide synthase and inhibits endothelial inflammation and athrogenesis. Here we will discuss that disturbed flow and steady laminar flow induce pro- and antiatherogenic events via flow type-specific mechanotransduction pathways. We will focus on 5 mechanosensitive pathways: mitogen-activated protein kinases/extracellular signal-regulated kinase 5/Kruppel-like factor 2 signaling, extracellular signal-regulated kinase/peroxisome proliferator-activated receptor signaling, and mechanosignaling pathways involving SUMOylation, protein kinase C-ζ, and p90 ribosomal S6 kinase. We think that clarifying regulation mechanisms between these 2 flow types will provide new insights into therapeutic approaches for the prevention and treatment of atherosclerosis.

Interferon-γ induces leucine-rich repeat kinase LRRK2 via extracellular signal-regulated kinase ERK5 in macrophages.

The gene encoding leucine-rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon-γ (IFN-γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN-γ-mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal-regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN-γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP-1 cells and human peripheral blood monocytes stimulated the ERK5-LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5-dependent IFN-γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages. Leucine-rich repeat kinase 2 (LRRK2) is a major risk factor for the development of Parkinson's disease (PD). However, the role of LRRK2 in the affected neurons remains enigmatic. Recently, LRRK2 has been reported to be strongly expressed in the immune system. Here, we demonstrate that LRRK2 is induced by Interferon gamma via extracellular signal-regulated kinase 5 (ERK5) in macrophages, thus providing new insights in LRRK2 and ERK5 biology.

Role of ERK1, 2, and 5 in dopamine neuron survival during aging.

Extracellular signal-regulated kinases (ERKs) 1, 2, and 5 have been shown to play distinct roles in proliferation, differentiation, and neuronal viability. In this study, we examined ERK1, 2, and 5 expression and activation in the substantia nigra (SN), striatum (STR), and ventral tegmental area (VTA) during aging. An age-related decrease in phosphorylated ERK5 was observed in the SN and STR, whereas an increase in total ERK1 was observed in all 3 regions. In primary cultures of the SN and VTA, inhibition of ERK5 but not ERK1 and 2 decreased dopamine neuronal viability significantly. These data suggest that ERK5 is essential for the basal survival of SN and VTA dopaminergic neurons. This is the first study to examine ERK1, 2, and 5 expression and activation in the SN, STR, and VTA during aging, and the relative roles of ERK1, 2, and 5 in basal survival of SN and VTA dopaminergic neurons. These data raise the possibility that a decline in ERK5 signaling may play a role in age-related impairments in dopaminergic function.

An MAPK-dependent pathway induces epithelial-mesenchymal transition via Twist activation in human breast cancer cell lines.

BACKGROUND: Twist is an epithelial-mesenchymal transition (EMT) transcription factor that instigates cell invasion. Our research has shown that osteopontin (OPN) regulates the EMT factor Twist. The underlying signaling pathway is unknown. We hypothesized that OPN activates Twist to induce EMT in human breast cancer. METHODS: Potential kinases for Twist were identified using NetPhosK. Inhibitors of MEK1/2, JNK, p38 MAPK, and PI3K were applied to human breast cancer cells MDA-MB231 (OPN high). After 24 h, Twist was immunoprecipitated and incubated with phosphoserine. Expression of the Twist target protein, Bmi-1, was determined following 24-h osteopontin aptamer (APT) treatment; mutant aptamer (MuAPT) was used as the control. Scratch-wound assay was imaged 12, 24, and 48 h after APT and MuAPT treatment. RESULTS: MEK1/2 inhibition caused ≈ twofold decrease in Twist serine phosphorylation (P < .05). APT blockade of OPN in MB231 decreased Bmi1 protein twofold (P < .05). Aptamer-treated cells were significantly decreased in cell migration and wound closure in the scratch wound-assay (P < .001). CONCLUSION: We demonstrate that OPN extracellular binding to MB231 activates an autocrine MAPK intracellular signaling pathway resulting in Twist activation and promoting Bmi1 expression to further EMT initiation and cellular migration. Our results elucidate a previously undescribed role for OPN as a prime regulator of EMT in human breast cancer cells.

Statins protect human endothelial cells from TNF-induced inflammation via ERK5 activation.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) exert pleiotropic effects on the cardiovascular system, in part through a decrease in reactive oxygen species (ROS) formation and reduction of vascular inflammation. To elucidate the molecular mechanisms involved in these effects, we investigated the effect of statins on TNF-α-induced ROS production, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression in human aortic endothelial cells (HAECs). Exposure of HAECs to TNF-α caused production of ROS via Rac-1 membrane translocation and activation. The Rac-1 activation and ROS liberation mediated TNF-stimulated NF-κB activation and the subsequent VCAM-1 and ICAM-1 expression. Extracellular-signal-regulated kinase 5 (ERK5) plays a central role in inhibiting endothelial inflammation. Immune complex kinase assay of protein extracts from HAECs treated with atorvastatin revealed increased ERK5 activity in a time- and dose-dependent manner. In addition, pretreatment with atorvastatin inhibited TNF-α-induced ROS production and VCAM-1 and ICAM-1 expression. Chemical or genetic inhibition of ERK5 ablated the statins inhibition of Rac-1 activation, ROS formation, NF-κB, VCAM-1 and ICAM-1 expression induced by TNF-α. Taken together, statins, via ERK5 activation, suppress TNF-stimulated Rac-1 activation, ROS generation, NF-κB activation and VCAM-1 and ICAM-1 expression in human ECs, which provides a novel explanation for the pleiotropic effects of statins that benefit the cardiovascular system.

Extracellular signal-regulated kinase 5 (ERK5) mediates prolactin-stimulated adult neurogenesis in the subventricular zone and olfactory bulb.

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.

Role of big mitogen-activated protein kinase 1 (BMK1) / extracellular signal-regulated kinase 5 (ERK5) in the pathogenesis and progression of atherosclerosis.

Big mitogen-activated protein kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a newly identified member of the mitogen-activated protein (MAP) kinase family. BMK1 has been reported to be sensitive to various neuro-humoral factors and oxidative stress in various cells. In this review, we focused on the role of BMK1 in atherosclerosis in a cultured rat aortic smooth muscle cell model. Treatment with platelet-derived growth factor caused vascular smooth muscle cell (VSMC) migration in a BMK1 activation-dependent manner. H(2)O(2) caused BMK1 activation and VSMC death, including apoptosis of VSMCs. An inhibitory function for BMK1 against cell death from oxidative stress was discovered using siRNA techniques to downregulate the expression of BMK1. These findings suggest a role for BMK1 in the pathogenesis and/or progression of atherosclerosis.

ERK5 Regulates glucose-induced increased fibronectin production in the endothelial cells and in the retina in diabetes.

PURPOSE: Fibronectin (FN) production and deposition in the tissue is a characteristic feature of diabetic retinopathy. ERK5 is a recent member of the mitogen activated protein kinase (MAPK) family, which plays a critical role in cardiovascular development and maintaining endothelial cell integrity. The aim of this study was to investigate the role of ERK5 signaling in glucose-induced FN overproduction. METHODS: Dermal-derived human microvascular endothelial cells (HMVECs) and human retinal microvascular endothelial cells (HRMECs) were used in this study. FN mRNA levels and secreted FN protein levels were measured using real-time PCR and ELISA, respectively. Constitutively active MAPK/ERK kinase 5 (MEK5 [CAMEK5]) adenovirus was used to upregulate ERK5. Dominant negative MEK5 (DNMEK5) and ERK5 siRNA (siERK5) were used to downregulate ERK5. Parallel retinal tissues of diabetic rats were examined. RESULTS: A significant decrease of FN was observed at both protein and mRNA levels following CAMEK5 transduction in basal as well as in high glucose. DNMEK5 transduction led to further enhancement of glucose-induced increased FN expression. siERK5 treatment led to an increase of FN synthesis. Retinal tissues of diabetic rats showed FN upregulation and ERK5 downregulation. TGFß1 mRNA and phosphorylated Smad2 were markedly suppressed by CAMEK5 transduction with and without glucose treatment. On the other hand, siERK5 transfection enhanced TGFß1 mRNA expression. Exogenous nerve growth factor supplementation resulted in elevated phosphorylated and total ERK5 with and without glucose treatment. CONCLUSIONS: Our experiments demonstrated a novel mechanism of glucose-induced increased FN production in diabetic retinopathy, which is mediated through decreased ERK5 signaling.

ERK5 protein promotes, whereas MEK1 protein differentially regulates, the Toll-like receptor 2 protein-dependent activation of human endothelial cells and monocytes.

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.