[Retracted] Liraglutide protects renal mesangial cells against hyperglycemia­mediated mitochondrial apoptosis by activating the ERK­Yap signaling pathway and upregulating Sirt3 expression.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the TUNEL assay data shown in Fig. 1C on p. 2853 and Fig. 5H on p. 2857 were strikingly similar to data that had already been published in different form in different articles written by different authors at different research institutes, or were submitted for publication at around the same time (a number of of which have now been retracted). Owing to the fact that the contentious data in the above article had already been published, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 19: 2849­2860, 2019; DOI: 10.3892/mmr.2019.9946].

Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells.

Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.

Saikosaponin A Recovers Impaired Filaggrin Levels in Inflamed Skin by Downregulating the Expression of FRA1 and c-Jun.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter-reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter-reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the -343/+25 FLG promoter-reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

The Raf/LIN-45 C-terminal distal tail segment negatively regulates signaling in Caenorhabditis elegans.

Raf protein kinases act as Ras-GTP sensing components of the ERK signal transduction pathway in animal cells, influencing cell proliferation, differentiation, and survival. In humans, somatic and germline mutations in the genes BRAF and RAF1 are associated with malignancies and developmental disorders. Recent studies shed light on the structure of activated Raf, a heterotetramer consisting of Raf and 14-3-3 dimers, and raised the possibility that a Raf C-terminal distal tail segment (DTS) regulates activation. We investigated the role of the DTS using the Caenorhabditis elegans Raf ortholog lin-45. Truncations removing the DTS strongly enhanced lin-45(S312A), a weak gain-of-function allele equivalent to RAF1 mutations found in patients with Noonan Syndrome. We genetically defined three elements of the LIN-45 DTS, which we termed the active site binding sequence (ASBS), the KTP motif, and the aromatic cluster. In the context of lin-45(S312A), mutation of each of these elements enhanced activity. We used AlphaFold to predict DTS protein interactions for LIN-45, fly Raf, and human BRAF, within the activated heterotetramer complex. We propose distinct functions for the LIN-45 DTS elements: i) the ASBS binds the kinase active site as an inhibitor, ii) phosphorylation of the KTP motif modulates DTS-kinase domain interaction, and iii) the aromatic cluster anchors the DTS in an inhibitory conformation. Human RASopathy-associated variants in BRAF affect residues of the DTS, consistent with these predictions. This work establishes that the Raf/LIN-45 DTS negatively regulates signaling in C. elegans and provides a model for its function in other Raf proteins.

The Caenorhabditis elegans protein SOC-3 permits an alternative mode of signal transduction by the EGL-15 FGF receptor.

Fibroblast Growth Factors and their receptors (FGFRs) comprise a cell signaling module that can stimulate signaling by Ras and the kinases Raf, MEK, and ERK to regulate animal development and homeostatic functions. In Caenorhabditis elegans, the sole FGFR ortholog EGL-15 acts with the GRB2 ortholog SEM-5 to promote chemoattraction and migration by the sex myoblasts (SMs) and fluid homeostasis by the hypodermis (Hyp7). Cell-specific differences in EGL-15 signaling were suggested by the phenotypes caused by egl-15(n1457), an allele that removes a region of its C-terminal domain (CTD) known to bind SEM-5. To determine how mutations altered EGL-15 activity in the SMs and Hyp7, we used the kinase reporter ERK-KTR to measure activation of the ERK ortholog MPK-1. Consequences of egl-15(n1457) were cell-specific, resulting in loss of MPK-1 activity in the SMs and elevated activity in Hyp7. Previous studies of Hyp7 showed that loss of the CLR-1 phosphatase causes a fluid homeostasis defect termed "Clear" that is suppressed by reduction of EGL-15 signaling, a phenotype termed "Suppressor of Clear" (Soc). To identify mechanisms that permit EGL-15 signaling in Hyp7, we conducted a genetic screen for Soc mutants in the clr-1; egl-15(n1457) genotype. We report the identification of SOC-3, a protein with putative SEM-5-binding motifs and PH and PTB domains similar to DOK and IRS proteins. In combination with the egl-15(n1457) mutation, loss of either soc-3, the GAB1 ortholog soc-1, or the SHP2 ortholog ptp-2, reduced MPK-1 activation. We generated alleles of soc-3 to test the requirement for the SEM-5-binding motifs, finding that residue Tyr356 is required for function. We propose that EGL-15-mediated SM chemoattraction relies solely on the direct interaction between SEM-5 and the EGL-15 CTD. In Hyp7, EGL-15 signaling uses two mechanisms: the direct SEM-5 binding mechanism; and an alternative, CTD-independent mechanism involving SOC-3, SOC-1, and PTP-2. This work demonstrates that FGF signaling uses distinct, tissue-specific mechanisms in development, and identifies SOC-3 as a potential adaptor that facilitates Ras pathway activation by FGFR.

A rare cutaneous infection with Mycobacterium chelonae in a pediatric patient treated with trametinib for KRAS-mutated diffuse glioma.

Mitogen-activated extracellular signal-regulated kinase inhibitors (MEKi) represent a promising new therapy for pediatric patients with low-grade gliomas, which frequently have abnormal signaling within the mitogen-activated protein kinase (MAP kinase) pathway. However, understanding of long-term efficacy and toxicity is limited in pediatric glioma patients. This article describes a rare presentation of a widespread cutaneous infection with Mycobacterium chelonae in a pediatric patient with a low-grade glioma treated with trametinib.

[Corrigendum] Hispolon inhibits breast cancer cell migration by reversal of epithelial­to­mesenchymal transition via suppressing the ROS/ERK/Slug/E­cadherin pathway.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that, with the cell migration assay data shown in Fig. 7 on p. 901, the "TPA" and "TPA + U0126" panels were strikingly similar, such that data which were intended to show the results from differently performed experiments had apparently been derived from the same original source. In addition, it was noted that the "TPA + hispolon" and "TPA + NAC" data panels in Fig. 4B on p. 899 contained overlapping sections. Thirdly, a data panel was shared between Figs. 1 and 4, although this was intentional on the part of the authors as the same experiment was being portrayed in these figures.  The authors were able to re­examine their original data files, and realized that errors were made in asssembling Figs. 4B and 7. The revised versions of Figs. 4 and 7, now containing the correct data for the "TPA + NAC" experiment in Fig. 4B and the Control ("Ctrl") experiment in Fig. 7, are shown on the next two pages. The authors wish to emphasize that the corrections made to these figures do not affect the overall conclusions reported in the paper, and they are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this corrigendum. All the authors agree to the publication of this corrigendum, and also apologize to the readership for any inconvenience caused. [Oncology Reports 35: 896­904, 2016; DOI: 10.3892/or.2015.4445].

Streptolysin S induces proinflammatory cytokine expression in calcium ion-influx-dependent manner.

Anginosus group streptococci (AGS) are opportunistic pathogens that reside in the human oral cavity. The ß-hemolytic strains of Streptococcus anginosus subsp. anginosus (SAA) produce streptolysin S (SLS), a streptococcal peptide hemolysin. In recent clinical scenarios, AGS, including this species, have frequently been isolated from infections and disorders beyond those in the oral cavity. Consequently, investigating this situation will reveal the potential pathogenicity of AGS to ectopic infections in humans. However, the precise mechanism underlying the cellular response induced by secreted SLS and its relevance to the pathogenicity of AGS strains remain largely unknown. This study aims to elucidate the mechanism underlying the host cellular response of the human acute monocytic leukemia cell line THP-1 to secreted SLS. In THP-1 cells incubated with the culture supernatant of ß-hemolytic SAA containing SLS as the sole cytotoxic factor, increased Ca2+ influx and elevated expression of proinflammatory cytokines were observed. Significantly reduced expression of SLS-dependent upregulated cytokine genes under Ca2+-chelating conditions suggests that Ca2+ influx triggers SLS-dependent cellular responses. Furthermore, SLS-dependent enhanced expression of IL-8 was also implicated in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The findings presented in this study are crucial for a comprehensive understanding of the real pathogenicity of SLS-producing ß-hemolytic AGS in the latest clinical situations.

A molecular mechanism for angiotensin II receptor blocker-mediated slit membrane protection: Angiotensin II increases nephrin endocytosis via AT1-receptor-dependent ERK 1/2 activation.

Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.

Empagliflozin Prevent High-Glucose Stimulation Inducing Apoptosis and Mitochondria Fragmentation in H9C2 Cells through the Calcium-Dependent Activation Extracellular Signal-Regulated Kinase 1/2 Pathway.

A previous study showed that high-glucose (HG) conditions induce mitochondria fragmentation through the calcium-mediated activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) in H9C2 cells. This study tested whether empagliflozin could prevent HG-induced mitochondria fragmentation through this pathway. We found that exposing H9C2 cells to an HG concentration decreased cell viability and increased cell apoptosis and caspase-3. Empagliflozin could reverse the apoptosis effect of HG stimulation on H9C2 cells. In addition, the HG condition caused mitochondria fragmentation, which was reduced by empagliflozin. The expression of mitochondria fission protein was upregulated, and fusion proteins were downregulated under HG stimulation. The expression of fission proteins was decreased under empagliflozin treatment. Increased calcium accumulation was observed under the HG condition, which was decreased by empagliflozin. The increased expression of ERK 1/2 under HG stimulation was also reversed by empagliflozin. Our study shows that empagliflozin could reverse the HG condition, causing a calcium-dependent activation of the ERK 1/2 pathway, which caused mitochondria fragmentation in H9C2 cells.

Progress in the development of ERK1/2 inhibitors for treating cancer and other diseases.

The extracellular signal-regulated kinases-1 and 2 (ERK1/2) are ubiquitous regulators of many cellular functions, including proliferation, differentiation, migration, and cell death. ERK1/2 regulate cell functions by phosphorylating a diverse collection of protein substrates consisting of other kinases, transcription factors, structural proteins, and other regulatory proteins. ERK1/2 regulation of cell functions is tightly regulated through the balance between activating phosphorylation by upstream kinases and inactivating dephosphorylation by phosphatases. Disruption of homeostatic ERK1/2 regulation caused by elevated extracellular signals or mutations in upstream regulatory proteins leads to the constitutive activation of ERK1/2 signaling and uncontrolled cell proliferation observed in many types of cancer. Many inhibitors of upstream kinase regulators of ERK1/2 have been developed and are part of targeted therapeutic options to treat a variety of cancers. However, the efficacy of these drugs in providing sustained patient responses is limited by the development of acquired resistance often involving re-activation of ERK1/2. As such, recent drug discovery efforts have focused on the direct targeting of ERK1/2. Several ATP competitive ERK1/2 inhibitors have been identified and are being tested in cancer clinical trials. One drug, Ulixertinib (BVD-523), has received FDA approval for use in the Expanded Access Program for patients with no other therapeutic options. This review provides an update on ERK1/2 inhibitors in clinical trials, their successes and limitations, and new academic drug discovery efforts to modulate ERK1/2 signaling for treating cancer and other diseases.

Naturally Occurring Plant-Based Anticancerous Candidates as Potential ERK2 Inhibitors: In-Silico Database Mining and Molecular Dynamics Simulations.

The evolutionarily conserved extracellular signal-regulated kinase 2 (ERK2) is involved in regulating cellular signaling in both normal and pathological conditions. ERK2 expression is critical for human development, while hyperactivation is a major factor in tumor progression. Up to now, there have been no approved inhibitors that target ERK2, and as such, here we report on screening of a naturally occurring plant-based anticancerous compound-activity-target (NPACT) database for prospective ERK2 inhibitors. More than 1,500 phytochemicals were screened using in-silico molecular docking and molecular dynamics (MD) approaches. NPACT compounds with a docking score lower than a co-crystallized LHZ inhibitor (calc.-10.5 kcal/mol) were subjected to MD simulations. Binding energies (ΔGbinding) of inhibitor-ERK2 complexes over the MD course were estimated using an MM-GBSA approach. Based on MM-GBSA//100 ns MD simulations, the steroid zhankuic acid C (NPACT01034) demonstrated greater binding affinity against ERK2 protein than LHZ, with ΔGbinding values of -50.0 and -47.7 kcal/mol, respectively. Structural and energetical analyses throughout the MD course demonstrated stabilization of zhankuic acid C complexed with ERK2 protein. The anticipated ADMET properties of zhankuic acid C indicated minimal toxicity. Moreover, in-silico evaluation of fourteen ERK2 inhibitors in clinical trials demonstrated the higher binding affinity of zhankuic acid C towards ERK2 protein.

Exercise training-driven exosomal miRNA-323-5p activity suppresses adipogenic conversion of 3T3-L1 cells via the DUSP3/ERK pathway.

Adipose-derived stem cell (ASC)-released exosomes (ASCexos) have multiple biological activities. We examined the effect of ASCexos derived from the inguinal adipose tissue of exercise-trained rats (EX-ASCexos) on adipogenic conversion of 3T3-L1 cells and analyzed their microRNA (miRNA) expression profiles. Differentiation of 3T3-L1 cells into adipocytes was performed for 9 d with EX-ASCexos or ASCexos from sedentary control rats (SED-ASCexos), and the expression of proteins and miRNA involved in adipogenic differentiation were determined. EX-ASCexos but not SED-ASCexos attenuated 3T3-L1 adipocyte differentiation with increased phosph-Ser112PPARγ expression, the inactive form of PPARγ. These differentiated adipocytes were also accompanied by increased phosph-Thr202/Tyr204ERK and decreased dual-specificity phosphatase 3 (DUSP3) levels. The exosomal miRNAs miR-323-5p, miR-433-3p, and miR-874-3p were identified specifically in EX-ASCexos. Of these, miR-323-5p mimic replicated the EX-ASCexo-induced suppression of 3T3-L1 adipocyte differentiation and altered adipogenesis-related factor expression. In conclusion, exercise training-driven exosomal miR-323-5p suppressed 3T3-L1 adipogenesis by increasing phosph-Ser112PPARγ expression, while phosph-Thr202/Tyr204ERK accumulation inhibited DUSP3 expression.

Extracellular signal-regulated kinase is activated in podocytes from patients with diabetic nephropathy.

In the past few decades, the global prevalence of diabetes has provided us with a warning about future chronic complications. Diabetic nephropathy (DN) is the main cause of end-stage kidney disease. Podocytes in the glomerulus play a critical role in regulating glomerular permeability, and podocyte injury is one of the main causes of DN. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that plays critical roles in intracellular signal transduction. In human patients with DN, phosphorylated ERK (pERK), the active form of ERK, is increased in the glomeruli. However, information on the expression of pERK, specifically in podocytes in DN, is limited. Meanwhile, high glucose induces ERK activation in immortalized podocyte cell lines, suggesting the involvement of podocytic ERK in DN. We performed an immunohistochemical study using Wilms' tumor-1 (WT-1) as a podocyte-specific marker to investigate whether podocytic pERK levels are increased in patients with DN. In the glomeruli of the DN group, we observed remarkable co-staining for WT-1 and pERK. In contrast, the glomeruli of the control group contained only a few pERK-positive podocytes. Statistical analyses revealed that, relative to healthy controls, patients with DN showed significantly increased pERK expression levels in cells that were positive for WT-1 (DN: 51.3 ± 13.1% vs. control: 7.3 ± 1.6%, p = 0.0158, t-test, n = 4 for each group). This suggests that ERK activation in podocytes is involved in the pathogenesis of DN.

Bayesian mapping of protein kinases to vasopressin-regulated phosphorylation sites in renal collecting duct.

Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase ß (CDC42BPB).NEW & NOTEWORTHY Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2.

Neurotrophin-3 Rescues Striatal Synaptic Plasticity in Model of Neurodegeneration by PLC Signaling Activation.

BACKGROUND: Neurotrophins are essential factors for neural growth and function; they play a crucial role in neurodegenerative diseases where their expression levels are altered. Our previous research has demonstrated changes in synaptic plasticity and neurotrophin expression levels in a pharmacological model of Huntington's disease induced by 3-nitropropionic acid (3-NP). In the 3- NP-induced HD model, corticostriatal Long Term Depression (LTD) was impaired, but neurotrophin-3 (NT-3) restored striatal LTD. This study delves into the NT-3-induced signaling pathways involved in modulating and restoring striatal synaptic plasticity in cerebral slices from 3-NPinduced striatal degeneration in mice in vivo. METHODS: Phospholipase C (PLC), phosphatidylinositol-3-kinase (PI3K), and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways activated by NT-3 were analyzed by means of field electrophysiological recordings in brain slices from control and 3-NP treated in the presence of specific inhibitors of the signaling pathways. RESULTS: Using specific inhibitors, PLC, PI3K, and MEK/ERK signaling pathways contribute to NT3-mediated plasticity modulation in striatal tissue slices recorded from control animals. However, in the neurodegeneration model induced by 3-NP, the recovery of striatal LTD induced by NT-3 was prevented only by the PLC inhibitor. Moreover, the PLC signaling pathway appeared to trigger downstream activation of the endocannabinoid system, evidenced by AM 251, an inhibitor of the CB1 receptor, also hindered NT-3 plasticity recovery. CONCLUSION: Our finding highlights the specific involvement of the PLC pathway in the neuroprotective effects of NT-3 in mitigating synaptic dysfunction under neurodegenerative conditions.

Early short-term hypoxia promotes epidermal cell migration by activating the CCL2-ERK1/2 pathway and epithelial-mesenchymal transition during wound healing.

Background: Due to vasculature injury and increased oxygen consumption, the early wound microenvironment is typically in a hypoxic state. We observed enhanced cell migration ability under early short-term hypoxia. CCL2 belongs to the CC chemokine family and was found to be increased in early hypoxic wounds and enriched in the extracellular signal-regulated kinase (ERK)1/2 pathway in our previous study. However, the underlying mechanism through which the CCL2-ERK1/2 pathway regulates wound healing under early short-term hypoxia remains unclear. Activation of epithelial-mesenchymal transition (EMT) is a key process in cancer cell metastasis, during which epithelial cells acquire the characteristics of mesenchymal cells and enhance cell motility and migration ability. However, the relationship between epithelial cell migration and EMT under early short-term hypoxia has yet to be explored. Methods: HaCaT cells were cultured to verify the effect of early short-term hypoxia on migration through cell scratch assays. Lentiviruses with silenced or overexpressed CCL2 were used to explore the relationship between CCL2 and migration under short-term hypoxia. An acute full-thickness cutaneous wound rat model was established with the application of an ERK inhibitor to reveal the hidden role of the ERK1/2 pathway in the early stage of wound healing. The EMT process was verified in all the above experiments through western blotting. Results: In our study, we found that short-term hypoxia promoted cell migration. Mechanistically, hypoxia promoted cell migration through mediating CCL2. Overexpression of CCL2 via lentivirus promoted cell migration, while silencing CCL2 via lentivirus inhibited cell migration and the production of related downstream proteins. In addition, we found that CCL2 was enriched in the ERK1/2 pathway, and the application of an ERK inhibitor in vivo and in vitro verified the upstream and downstream relationships between the CCL2 pathway and ERK1/2. Western blot results both in vivo and in vitro demonstrated that early short-term hypoxia promotes epidermal cell migration by activating the CCL2-ERK1/2 pathway and EMT during wound healing. Conclusions: Our work demonstrated that hypoxia in the early stage serves as a stimulus for triggering wound healing through activating the CCL2-ERK1/2 pathway and EMT, which promote epidermal cell migration and accelerate wound closure. These findings provide additional detailed insights into the mechanism of wound healing and new targets for clinical treatment.

[Role and mechanism of cysteine and glycine-rich protein 2 in the malignant progression of neuroblastoma].

OBJECTIVE: To investigate the function and underlying mechanism of cysteine and glycine-rich protein 2 (CSRP2) in neuroblastoma (NB). METHODS: The correlation between the expression level of CSRP2 mRNA and the prognosis of NB children in NB clinical samples was analyzed in R2 Genomics Analysis and Visualization Platform. The small interfering RNA (siRNA) targeting CSRP2 or CSRP2 plasmid were transfected to NB cell lines SK-N-BE(2) and SH-SY5Y. Cell proliferation was observed by crystal violet staining and real-time cellular analysis. The ability of colony formation of NB cells was observed by colony-forming unit assay. Immunofluorescence assay was used to detect the expression of the proliferation marker Ki-67. Flow cytometry analysis for cell cycle proportion was used with cells stained by propidium iodide (PI). Annexin V/7AAD was used to stain cells and analyze the percentage of cell apoptosis. The ability of cell migration was determined by cell wound-healing assay. The level of protein and mRNA expression of CSRP2 in NB primary tumor and NB cell lines were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: By analyzing the NB clinical sample databases, it was found that the expression levels of CSRP2 in high-risk NB with 3/4 stages in international neuroblastoma staging system (INSS) were significantly higher than that in low-risk NB with 1/2 INSS stages. The NB patients with high expression levels of CSRP2 were shown lower overall survival rate than those with low expression levels of CSRP2. We detected the protein levels of CSRP2 in the NB samples by Western blot, and found that the protein level of CSRP2 in 3/4 INSS stages was significantly higher than that in 1/2 INSS stages. Knockdown of CSRP2 inhibited cell viability and proliferation of NB cells. Overexpression of CSRP2 increased the proliferation of NB cells. Flow cytometry showed that the proportion of sub-G1, G0/G1 and S phase cells and Annexin V positive cells were increased after CSRP2 deficiency. In the cell wound-healing assay, the healing rate of NB cells was significantly attenuated after knockdown of CSRP2. Further mechanism studies showed that the proportion of the proliferation marker Ki-67 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) were significantly decreased after CSRP2 knockdown. CONCLUSION: CSRP2 is highly expressed in high-risk NB with 3/4 INSS stages, and the expression levels of CSRP2 are negatively correlated with the overall survival of NB patients. CSRP2 significantly increased the proliferation and cell migration of NB cells and inhibited cell apoptosis via the activation of ERK1/2. All these results indicate that CSRP2 promotes the progression of NB by activating ERK1/2, and this study will provide a potential target for high-risk NB therapy.

METTL14 Promotes Proliferation, Migration, and Invasion in Endometriotic Stromal Cell Growth by Activating the ZEB1/MEK/ERK Pathway.

OBJECTIVE: The aim of this study was to explore the mechanism of methyltransferase-like 14 (METTL14) on human endometriotic stromal cell (ESC; HEM15A) proliferation, migration, and invasion to provide novel therapy for endometriosis (EMs). DESIGN: Normal human endometrial stromal cells (HESCs) and HEM15A cells were selected. Corresponding controlled experiments were performed to analyze whether overexpression of METTL14, N6-methyladenosine (m6A) methylated ZEB1 mRNA, upregulation of ZEB1, and activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) can affect the proliferation, migration, and invasion of HEM15A cells. Materials, Setting, and Methods: HEM15A and HESCs were cultured in vitro. HEM15A cells were treated with oe-METTL14 and oe-zinc finger E-box-binding protein 1 (ZEB1) plasmids, 3-deazaadenosine (3-DAA) and the MEK/ERK pathway inhibitor isoprenaline (ISO). After identifying HEM15A and HESCs, METTL14, ZEB1, p-ERK1/2/ERK1/2, and p-MEK/MEK levels, and cell proliferation, migration, and invasion were assessed. The modification sites of ZEB1 and m6A were predicted using SRAMP database, with an m6A modification level assessed by MeRIP. The binding of YT521-B homology domain 2 (YTHDF2) to ZEB1 messenger RNA (mRNA), and ZEB1 stability and mRNA level were tested. RESULTS: Compared with HESCs, METTL14 level in HEM15A was significantly reduced. METTL14 overexpression in HEM15A prominently increased its proliferation, migration, and invasion. METTL14 overexpression notably elevated m6A-methylated ZEB1 mRNA level and reduced the stability and expression of ZEB1 mRNA. Further m6A modification inhibition increased ZEB1 mRNA stability and mRNA and protein levels and decreased ZEB1 m6A modification level. ZEB1 upregulation partially reversed METTL14 overexpression-inhibited HEM15A proliferation, migration, and invasion. METTL14 inhibited the MEK/ERK signaling activation by regulating ZEB1, and the MEK/ERK signaling activation partly averted METTL14-suppressed proliferation, migration, and invasion. LIMITATIONS: The effects of METTL14 on other growth aspects of HEM15A cells and the relation between ZEB1 and m6A require further investigation. CONCLUSIONS: METTL14 lowered ZEB1 expression by regulating ZEB1 m6A modification levels, thereby inhibiting the activation of the MEK/ERK pathway and ESC proliferation, migration, and invasion.

MAPK pathway orchestrates gallid alphaherpesvirus 1 infection through the biphasic activation of MEK/ERK and p38 MAPK signaling.

Therapies targeting virus-host interactions are seen as promising strategies for treating gallid alphaherpesvirus 1 (ILTV) infection. Our study revealed a biphasic activation of two MAPK cascade pathways, MEK/ERK and p38 MAPK, as a notably activated host molecular event in response to ILTV infection. It exhibits antiviral functions at different stages of infection. Initially, the MEK/ERK pathway is activated upon viral invasion, leading to a broad suppression of metabolic pathways crucial for ILTV replication, thereby inhibiting viral replication from the early stage of ILTV infection. As the viral replication progresses, the p38 MAPK pathway activates its downstream transcription factor, STAT1, further hindering viral replication. Interestingly, ILTV overcomes this biphasic antiviral barrier by hijacking host p38-AKT axis, which protects infected cells from the apoptosis induced by infection and establishes an intracellular equilibrium conducive to extensive ILTV replication. These insights could provide potential therapeutic targets for ILTV infection.