Extracellular Signal-regulated Kinase Activation Research Articles

Insights into cellular behavior and micromolecular communication in urothelial micrografts

Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the proliferative potential of different porcine urothelial micrograft sizes in vitro, and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation occurred predominately at the micrograft borders and that ERK inhibition led to decreased urothelial migration and proliferation. Finally, we successfully isolated extracellular vesicles from the micrograft culture medium and evaluated their contents and relevance within various enriched biological processes. Our findings substantiate the potential of applying urothelial micrografting in future tissue-engineering models for reconstructive urological surgery, and, furthermore, highlights certain mechanisms as potential targets for future wound healing treatments.

Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury

BackgroundCerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI.MethodsSH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett’s T3 test (for unequal homogeneity) were used for statistical tests.ResultsERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed.ConclusionInhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model.9RDMMKPHSZ2No4hHf87VtDVideo

Comparative study of the molecular mechanisms underlying the G protein and β-arrestin-dependent pathways that lead to ERKs activation upon stimulation by Dopamine D2 receptor.

Dopamine D2 receptor (D2 R) has been shown to activate extracellular signal-regulated kinases (ERKs) via distinct pathways dependent on either G-protein or β-arrestin. However, there has not been a systematic study of the regulatory process of D2 R-mediated ERKs activation by G protein- versus β-arrestin-dependent signaling since D2 R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2 R-mediated ERKs activation via these two pathways. Our results showed that G protein-dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, β-arrestin-dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein- and β-arrestin-dependent pathways mediated ERKs activation. PTX Gi/o inhibitor, GRK2-CT, AG1478 epidermal growth factor receptor (EGFR) inhibitor, and wortmannin PI3-K inhibitor all blocked G protein-dependent ERKs activation. In contrast, GRK2 and β-Arr2 played a main role in β-arrestin-dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho-ERKs, β-Arr2 and importinβ1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes.

Chemokine CCL7 mediates trigeminal neuropathic pain via CCR2/CCR3-ERK pathway in the trigeminal ganglion of mice.

Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

Protection of high-frequency low-intensity pulsed electric fields and brain-derived neurotrophic factor for SH-SY5Y cells against hydrogen peroxide-induced cell damage.

Neurodegenerative diseases (NDDs) pose a significant global health threat. In particular, Alzheimer disease, the most common type causing dementia, remains an incurable disease. Alzheimer disease is thought to be associated with an imbalance of reactive oxygen species (ROS) in neurons, and scientists considered ROS modulation as a promising strategy for novel remedies. In the study, human neural cell line SH-SY5Y was used in probing the effect of combining noninvasive high-frequency low-intensity pulsed electric field (H-LIPEF) and brain-derived neurotrophic factor (BDNF) in protection against hydrogen peroxide (H2O2)-induced neuron damage. Our result finds that the combination approach has intensified the neuroprotective effect significantly, perhaps due to H-LIPEF and BDNF synergistically increasing the expression level of the phosphorylated epidermal growth factor receptor (p-EGFR), which induces the survival-related mitogen-activated protein kinases (MAPK) proteins. The study confirmed the activation of extracellular signal-regulated kinase (ERK) and the downstream pro-survival and antioxidant proteins as the mechanism underlying neuron protection. These findings highlighted the potential of H-LIPEF combined with BDNF in the treatment of NDDs. Furthermore, BDNF-mimetic drugs combining with noninvasive H-LIPEF to patients is a promising approach worthy of further research. This points to strategies for selecting drugs to cooperate with electric fields in treating neurodegenerative disorders.

TAp73 Inhibits EMT and Cell Migration in Pancreatic Cancer Cells through Promoting SMAD4 Expression and SMAD4-Dependent Inhibition of ERK Activation.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-β. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-β/Smad signaling, while preventing non-canonical TGF-β signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-β1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73-but not the β isoform-interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-β-stimulated pro-invasive ERK activation as an underlying mechanism.

Ultrafast distant wound response is essential for whole-body regeneration

Injury induces systemic responses, but their functions remain elusive. Mechanisms that can rapidly synchronize wound responses through long distances are also mostly unknown. Using planarian flatworms capable of whole-body regeneration, we report that injury induces extracellular signal-regulated kinase (Erk) activity waves to travel at a speed 10-100 times faster than those in other multicellular tissues. This ultrafast propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. The morphological properties of muscles allow them to act as superhighways for propagating and disseminating wound signals. Inhibiting Erk propagation prevents tissues distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues shortly after the first injury. Our findings provide a mechanism for long-range signal propagation in large, complex tissues to coordinate responses across cell types and highlight the function of feedback between spatially separated tissues during whole-body regeneration.

Extracellular Heat Shock Protein 70 Increases the Glucocorticoid Receptor and Dual-Specificity Phosphatase 1 via Toll-like Receptor 4 and Attenuates Inflammation in Airway Epithelial Cells.

Heat shock protein 70 (HSP70) regulates the ligand binding of the glucocorticoid receptor (GR). In asthma patients, heat treatment increased both the GR expression and secretion of extracellular HSP70 (eHSP70) by bronchial epithelial cells (EC). The objective of this study was to assess the effects of eHSP70 on GR expression and the GR-dependent regulation of immune response in human bronchial ECs. Cells were treated with either eHSP70 or transfected with an expression vector for intracellular HSP70 (iHSP70). Ribonucleic acid (RNA) and protein levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Interleukin (IL-6 and IL-8) secretion was determined by enzyme linked immunosorbent assay (ELISA). The overexpression of iHSP70 decreased, while eHSP70 increased GR expression. In addition, eHSP70 increased the expression of the GR target dual-specificity phosphatase 1 (DUSP-1). In doing so, eHSP70 reduced the tumor growth factor (TGF)-β1-dependent activation of extracellular signal-regulated kinase (Erk)-1/2 and cyclic AMP response element binding protein (CREB) and the secretion of IL-6 and IL-8. Blocking the GR or Toll-like receptor 4 (TLR4) counteracted all eHSP70-induced effects. This study demonstrates a novel anti-inflammatory effect of eHSP70 by the signaling cascade of TLR4-GR-DUSP1, which inhibits TGF-β1-activated pro-inflammatory ERK1/2-CREB signaling and cytokine secretion. The findings suggest that eHSP70 might present a novel non-steroidal therapeutic strategy to control airway inflammation in asthma.

Targeting the adenosine monophosphate-activated protein kinase signalling pathway by bempedoic acid attenuates Angiotensin II-induced cardiac remodelling in renovascular hypertension in rats

The crosstalk between the renin-angiotensin system and Adenosine monophosphate-activated protein kinase (AMPK) gained significant interest due to their involvement in the pathogenesis of several cardiovascular diseases. Angiotensin II (Ang II) plays a crucial role in developing cardiac remodelling by inducing energy imbalance, inflammation, oxidative and endoplasmic reticulum stress, and transforming growth factor-β (TGF-β)-induced fibrosis. Ang II directly or through extracellular signal-regulated kinase (ERK) activation impairs AMPK signalling with well-known antioxidant, anti-inflammatory, and anti-fibrotic effects. AimThis study aimed to investigate the role of bempedoic acid, a novel antihyperlipidemic drug, in attenuating hypertension-induced cardiac remodelling in rats by modulating Ang II-induced damage and activating the AMPK signalling pathway. MethodSixty adult male Sprague Dawley rats were randomly allocated into the Sham control group, Hypertensive group, Captopril group (30 mg/kg), and Bempedoic acid group (30 mg/kg). Hypertension was induced by left renal artery ligation in all groups except the Sham control group. Treatment with captopril and bempedoic acid started 14 days post-surgy and lasted two weeks. Finally, Hemodynamic measurements and electrocardiographic examination were done followed by heart tissue samples collection for biochemical, histopathological, and immunohistochemical examinations. Key findingsBempedoic acid preserved the cardiac function and electrocardiogram patterns. It inhibited endoplasmic reticulum stress, exhibited antioxidant activity, and increased endothelial nitric oxide synthase activity. Bempedoic acid interfered with ERK signalling pathways, including nuclear factor-κB and TGF-β, exerting anti-inflammatory and anti-fibrotic effects. SignificanceThese findings indicate the cardioprotective and antihypertrophic activity of bempedoic acid, which are suggested to result from energy-independent AMPK downstream signalling activation.

Leukocyte Ig-like receptor A3 facilitates inflammation, migration and invasion of synovial tissue-derived fibroblasts via ERK/JNK activation.

Leukocyte Ig-like receptor A3 (LILRA3) is a soluble receptor belongs to the immunoglobulin superfamily. Our previous studies demonstrated that LILRA3 is a common genetic risk for multiple autoimmune diseases, including RA. Functional LILRA3 conferred increased risk of joint destruction in patients with early RA. We undertook this study to further investigate the pathological role of LILRA3 in joint inflammation of RA. Soluble LILRA3 was measured by ELISA. LILRA3 plasmids were transfected into human fibroblast-like synoviocytes (FLSs) using electroporation. Activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was determined by western blots. Cytokine transcripts were quantified by real-time PCR. Migratory and invasive capacities of FLSs were evaluated using transwell migration and Matrigel invasion assays. FLS apoptosis was analysed using flow cytometry. Colocalization of LILRA3, LILRB1 and HLA-G in RA-FLSs was visualized by immunofluorescence staining. Soluble LILRA3 was specifically expressed in synovial fluid and serum LILRA3 was significantly increased and positively correlated with disease activity/severity in RA patients. LILRA3 induced an increased expression of IL-6, IL-8 and MMP3 in RA-FLSs. In vitro LILRA3 stimulation or overexpression promoted RA-FLS migration and invasion, and enhanced phosphorylation of ERK/JNK. Inhibition of ERK/JNK resulted in suppression of IL-6/IL-8 expression in LILRA3-stimulated RA-FLSs. LILRA3 was co-localized with its homologue LILRB1 and shared ligand HLA-G in RA-FLSs. The present study provides the first evidence that soluble LILRA3 is a novel proinflammatory mediator involved in synovial inflammation by promoting RA-FLS activation, migration and invasion, probably through the ERK/JNK signalling pathways.

Minocycline and antipsychotics inhibit inflammatory responses in BV-2 microglia activated by LPS via regulating the MAPKs/ JAK-STAT signaling pathway

BackgroundAbnormal activation of microglia is involved in the pathogenesis of schizophrenia. Minocycline and antipsychotics have been reported to be effective in inhibiting the activation of microglia and thus alleviating the negative symptoms of patients with schizophrenia. However, the specific molecular mechanism by which minocycline and antipsychotics inhibit microglial activation is not clear. In this study, we aimed to explore the molecular mechanism of treatment effect of minocycline and antipsychotics on schizophrenia.MethodsMicroglia cells were activated by lipopolysaccharide (LPS) and further treated with minocycline, haloperidol, and risperidone. Then cell morphology, specific marker, cytokines, and nitric oxide production process, and the proteins in related molecular signaling pathways in LPS-activated microglia were compared among groups.ResultsThe study found that minocycline, risperidone, and haloperidol significantly inhibited morphological changes and reduced the expression of OX-42 protein induced by LPS. Minocycline significantly decreased the production of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β). Risperidone also showed significant decrease in the production of IL-6 and TNF-α, while haloperidol only showed significant decrease in the production of IL-6. Minocycline, risperidone, and haloperidol were found to significantly inhibit nitric oxide (NO) expression, but had no effect on inducible nitric oxide synthase (iNOS) expression. Both minocycline and risperidone were effective in decreasing the activity of c‑Jun N‑terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinases (MAPKs) signal pathway. Additionally, minocycline and risperidone were found to increase the activity of phosphorylated-p38. In contrast, haloperidol only suppressed the activity of ERK. Minocycline also suppressed the activation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), while risperidone and haloperidol only suppressed the activation of STAT3.ConclusionsThe results demonstrated that minocycline and risperidone exert stronger anti-inflammatory and neuroprotective effects stronger than haloperidol, through MAPKs and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathways in BV2 cells stimulated with LPS, revealing the underlying mechanisms of minocycline and atypical antipsychotics in the treatment of negative schizophrenia symptoms.

Exploring the 1,3-benzoxazine chemotype for cannabinoid receptor 2 as a promising anti-cancer therapeutic

The discovery of selective agonists of cannabinoid receptor 2 (CB2) is strongly pursued to successfully tuning endocannabinoid signaling for therapeutic purposes. However, the design of selective CB2 agonists is still challenging because of the high homology with the cannabinoid receptor 1 (CB1) and for the yet unclear molecular basis of the agonist/antagonist switch. Here, the 1,3-benzoxazine scaffold is presented as a versatile chemotype for the design of CB2 agonists from which 25 derivatives were synthesized. Among these, compound 7b5 (CB2 EC50 = 110 nM, CB1 EC50 > 10 μM) demonstrated to impair proliferation of triple negative breast cancer BT549 cells and to attenuate the release of pro-inflammatory cytokines in a CB2-dependent manner. Furthermore, 7b5 abrogated the activation of extracellular signal-regulated kinase (ERK) 1/2, a key pro-inflammatory and oncogenic enzyme. Finally, molecular dynamics studies suggested a new rationale for the in vitro measured selectivity and for the observed agonist behavior.

Comparison of Cytotoxicity and Genotoxicity in Three Types of Indirect Restorative Materials on Human Periodontal Stem Cells.

This study aimed to compare the cell toxicity and biological characteristics of Ketac GIC (glass-ionomer cement), Nexus RMGIC (resin-modified glass-ionomer cement), and RelyX RC (resin cement) in human periodontal stem cells (PDSCs). To compare the effects of Ketac GIC, Nexus RMGIC, and RelyX RC on PDSCs, the cements were diluted from 1:2 to 1:8. PDSCs were then treated with the serially diluted cements with or without N-acetyl-cysteine (NAC), and cell survival was measured using water-soluble tetrazolium salt (WST-1) assay. Intracellular reactive oxygen species (ROS) was measured using 2',7'-dichlorofluorescin diacetate (DCFDA), and western blot analysis was performed to observe phosphorylation and activation of extracellular signal-regulated kinase (ERK) by Nexus RMGIC or RelyX RC. Cell death and proliferation were dose-dependently reduced following Nexus RMGIC or RelyX RC treatment. In addition, Nexus RMGIC or RelyX RC showed an increase intracellular ROS generation compared to Ketac GIC. Pretreatment with NAC confirmed the suppression of cell toxicity and ROS generation induced by Nexus RMGIC or RelyX RC. Nexus RMGIC or RelyX RC activates ERK phosphorylation, not p38 phosphorylation, in PDSCs. This study showed that the treatment with Nexus RMGIC or RelyX generates intracellular ROS and cell death through the ERK signaling pathway in PDSCs. In contrast, these effects were not observed with Ketac GIC, indicating that resin-based materials may have cytotoxic and genotoxic effects on PDSCs.

CKBA suppresses mast cell activation via ERK signaling pathway in murine atopic dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin disorder. Mast cells play an important role in AD because they regulate allergic reactions and inflammatory responses. However, whether and how the modulation of mast cell activity affects AD has not been determined. In this study, we aimed to determine the effects and mechanisms of 3-O-cyclohexanecarbonyl-11-keto-β-boswellic acid (CKBA). This natural compound derivative alleviates skin inflammation by inhibiting mast cell activation and maintaining skin barrier homeostasis in AD. CKBA markedly reduced serum IgE levels and alleviated skin inflammation in calcipotriol (MC903)-induced AD mouse model. CKBA also restrained mast cell degranulation both in vitro and in vivo. RNA-seq analysis revealed that CKBA downregulated the extracellular signal-regulated kinase (ERK) signaling in BM-derived mast cells activated by anti-2,4-dinitrophenol/2,4-dinitrophenol-human serum albumin. We proved that CKBA suppressed mast cell activation via ERK signaling using the ERK activator (t-butyl hydroquinone) and inhibitor (selumetinib; AZD6244) in AD. Thus, CKBA suppressed mast cell activation in AD via the ERK signaling pathway and could be a therapeutic candidate drug for AD.

O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer's disease.

Aberrant activation of Extracellular Signal-Regulated Kinase (ERK) signaling is associated with Alzheimer's disease (AD) pathogenesis. For example, enhanced ERK signal activation mediated by Apolipoprotein E4 (APOE4), which is a critical genetic risk factor for AD, increases the transcription of amyloid precursor protein (APP). We hypothesize that O-linked N-acetylglucosamine (O-GlcNAc) regulates the phosphorylation and activation of ERK. O-GlcNAc is a single sugar post-translational modification that dynamically cycles on and off proteins in response to nutrient changes by the action of the enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, O-GlcNAc quickly returns to a baseline level after stimulus removal (called O-GlcNAc homeostasis). We did a serum reactivation time-course followed by western blot in SH-SY5Y neuroblastoma cells after long-term O-GlcNAcase (OGA) inhibition by Thiamet-G (TMG) treatment, O-GlcNAc transferase (OGT) knock-down (KD) and OGA KD. Brain tissues of C57BL6/J mice and 5XFAD Alzheimer's disease mice intra-peritoneally injected with TMG for 1 month and C57BL6/J mice intra-peritoneally injected with TMG for 6 months were also used for western blot. We found that ERK1/2 phosphorylation at Thr 202/Tyr204 and Thr183/Tyr185 (p-ERK) are amplified and hence ERK1/2 are activated after long-term OGA inhibition in SH-SY5Y cells. In addition to pharmacological treatment, genetic disruption of O-GlcNAc by OGT KD and OGA KD also increased p-ERK in SH-SY5Y cells suggesting O-GlcNAc homeostasis controls ERK signaling. To determine how O-GlcNAc regulates p-ERK, we probed the expression of phosphorylated mitogen-activated protein kinase-kinase (p-MEK) which phosphorylates and activates ERK and Dual specificity phosphatase-4 (DUSP4) which dephosphorylates and inactivates ERK in SH-SY5Y cells. p-MEK increases in TMG treated and OGT KD cells whereas total DUSP4 decreases in OGT KD and OGA KD cells with serum reactivation time course. Next, we probed the role of OGA inhibition in regulating ERK activation using mice brain-tissue samples. Interestingly, 6-month intra-peritoneal TMG injection in C57BL/6J mice showed an increase in amplitude of p-ERK and APP protein levels, indicating long-term OGA inhibition potentially contributes to AD progression. Furthermore, 1-month TMG injection was sufficient to increase the amplitude of p-ERK in 5XFAD AD mice brains suggesting AD phenotype contributes to the acceleration of ERK activation mediated by OGA inhibition. Together, these results indicate that disruptions to O-GlcNAc homeostasis amplify ERK signal activation in AD.

Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons

RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light–inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Combined Aurora Kinase A and CHK1 Inhibition Enhances Radiosensitivity of Triple-Negative Breast Cancer Through Induction of Apoptosis and Mitotic Catastrophe Associated With Excessive DNA Damage

There is an urgent need for biomarkers and new actionable targets to improve radiosensitivity of triple-negative breast cancer (TNBC) tumors. We characterized the radiosensitizing effects and underlying mechanisms of combined Aurora kinase A (AURKA) and CHK1 inhibition in TNBC. Different TNBC cell lines were treated with AURKA inhibitor (AURKAi, MLN8237) and CHK1 inhibitor (CHK1i, MK8776). Cell responses to irradiation (IR) were then evaluated. Cell apoptosis, DNA damage, cell cycle distribution, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and Phosphoinositide 3-Kinase (PI3K) pathways were evaluated in vitro. Transcriptomic analysis was performed to facilitate the identification of potential biomarkers. Xenograft and immunohistochemistry were carried out to investigate the radiosensitizing effects of dual inhibition in vivo. Finally, the prognostic effect of CHEK1/AURKA in TNBC samples in the The Cancer Genome Atlas (TCGA) database and our center were analyzed. AURKAi (MLN8237) induced overexpression of phospho-CHK1 in TNBC cells. The addition of MK8776 (CHK1i) to MLN8237 greatly reduced cell viability and increased radiosensitivity compared with either the control or MLN8237 alone in vitro. Mechanistically, dual inhibition resulted in inducing excessive DNA damage by prompting G2/M transition to cells with defective spindles, leading to mitotic catastrophe and induction of apoptosis after IR. We also observed that dual inhibition suppressed the phosphorylation of ERK, while activation of ERK with its agonist or overexpression of active ERK1/2 allele could attenuate the apoptosis induced by dual inhibition with IR. Additionally, dual inhibition of AURKA and CHK1 synergistically enhanced radiosensitivity in MDA-MB-231 xenografts. Moreover, we detected that both CHEK1 and AURKA were overexpressed in patients with TNBC and negatively correlated with patient survival. Our findings suggested that AURKAi in combination with CHK1i enhanced TNBC radiosensitivity in preclinical models, potentially providing a novel strategy of precision treatment for patients with TNBC.

Inverse agonism of lysophospholipids with cationic head groups at Gi-coupled receptor GPR82

GPR82 is an orphan G protein-coupled receptor (GPCR) that has been implicated in lipid storage in mouse adipocytes. However, the intracellular signaling as well as the specific ligands of GPR82 remain unknown. GPR82 is closely related to GPR34, a GPCR for the bioactive lipid molecule lysophosphatidylserine. In this study, we screened a lipid library using GPR82-transfected cells to search for ligands that act on GPR82. By measuring cyclic adenosine monophosphate levels, we found that GPR82 is an apparently constitutively active GPCR that leads to Gi protein activation. In addition, edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine), an artificial lysophospholipid with a cationic head group that exerts antitumor activity, inhibited the Gi protein activation by GPR82. Two endogenous lysophospholipids with cationic head groups, lysophosphatidylcholine (1-oleoyl-sn-glycero-3-phosphocholine) and lysophosphatidylethanolamine (1-oleoyl-sn-glycero-3-phosphoethanolamine), also exhibited GPR82 inhibitory activity, albeit weaker than edelfosine. Förster resonance energy transfer imaging analysis consistently demonstrated that Gi protein-coupled GPR82 has an apparent constitutive activity that is edelfosine-sensitive. Consistent data were obtained from GPR82-mediated binding analysis of guanosine-5'-O-(3-thiotriphosphate) to cell membranes. Furthermore, in GPR82-transfected cells, edelfosine inhibited insulin-induced extracellular signal-regulated kinase activation, like compounds that function as inverse agonists at other GPCRs. Therefore, edelfosine is likely to act as an inverse agonist of GPR82. Finally, GPR82 expression inhibited adipocyte lipolysis, which was abrogated by edelfosine. Our findings suggested that the cationic lysophospholipids edelfosine, lysophosphatidylcholine and lysophosphatidylethanolamine are novel inverse agonists for Gi-coupled GPR82, which is apparently constitutively active, and has the potential to exert lipolytic effects through GPR82.

Molecular insights from the crab Neohelice memory model.

Memory acquisition, formation and maintenance depend on synaptic post-translational machinery and regulation of gene expression triggered by several transduction pathways. In turns, these processes lead to stabilization of synaptic modifications in neurons in the activated circuits. In order to study the molecular mechanisms involved in acquisition and memory, we have taken advantage of the context-signal associative learning and, more recently, the place preference task, of the crab Neohelice granulata. In this model organism, we studied several molecular processes, including activation of extracellular signal-regulated kinase (ERK) and the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) transcription factor, involvement of synaptic proteins such as NMDA receptors and neuroepigenetic regulation of gene expression. All these studies allowed description of key plasticity mechanisms involved in memory, including consolidation, reconsolidation and extinction. This article is aimed at review the most salient findings obtained over decades of research in this memory model.