Melanoma is the most aggressive cutaneous tumor. Neuropilin and tolloid-like 2 (NETO2) is closely related to tumorigenesis. However, the functional significance of NETO2 in melanoma progression remains unclear. Herein, we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients. Disrupting NETO2 expression markedly inhibited melanoma proliferation, malignant growth, migration, and invasion by downregulating the levels of calcium ions (Ca²⁺) and the expression of key genes involved in the calcium signaling pathway. By contrast, NETO2 overexpression had the opposite effects. Importantly, pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis. Overall, this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression, indicating that targeting NETO2 may effectively improve melanoma treatment.
Humans ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Melanoma/genetics* ; Membrane Proteins/genetics* ; Phosphorylation ; Signal Transduction

Humans ; Calcium/metabolism* ; Calmodulin ; Arrhythmias, Cardiac ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Myocytes, Cardiac/metabolism* ; Phosphorylation

CaMKII is essential for long-term potentiation (LTP), a process in which synaptic strength is increased following the acquisition of information. Among the four CaMKII isoforms, γCaMKII is the one that mediates the LTP of excitatory synapses onto inhibitory interneurons (LTP_E→I). However, the molecular mechanism underlying how γCaMKII mediates LTP_E→I remains unclear. Here, we show that γCaMKII is highly enriched in cultured hippocampal inhibitory interneurons and opts to be activated by higher stimulating frequencies in the 10-30 Hz range. Following stimulation, γCaMKII is translocated to the synapse and becomes co-localized with the postsynaptic protein PSD-95. Knocking down γCaMKII prevents the chemical LTP-induced phosphorylation and trafficking of AMPA receptors (AMPARs) in putative inhibitory interneurons, which are restored by overexpression of γCaMKII but not its kinase-dead form. Taken together, these data suggest that γCaMKII decodes NMDAR-mediated signaling and in turn regulates AMPARs for expressing LTP in inhibitory interneurons.
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Hippocampus/metabolism* ; Interneurons/physiology* ; Long-Term Potentiation/physiology* ; N-Methylaspartate/metabolism* ; Receptors, AMPA/physiology* ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Synapses/physiology*

Objective To study the stemness characteristics of uterine corpus endometrial carcinoma(UCEC)and its potential regulatory mechanism.Methods Transcriptome sequencing data of UCEC was obtained from The Cancer Genome Atlas.Gene expression profile was normalized by edgeR package in R3.5.1.A one-class logistic regression machine learning algorithm was employed to calculated the mRNA stemness index(mRNAsi)of each UCEC sample.Then,the prognostic significance of mRNAsi and candidate genes was evaluated by survminer and survival packages.The high-frequency sub-pathways mining approach(HiFreSP)was used to identify the prognosis-related sub-pathways enriched with differentially expressed genes(DEGs).Subsequently,a gene co-expression network was constructed using WGCNA package,and the key gene modules were analyzed.The clusterProfiler package was adopted to the function annotation of the modules highly correlated with mRNAsi.Finally,the Human Protein Atlas(HPA)was retrieved for immunohistochemical validation.Results The mRNAsi of UCEC samples was significantly higher than that of normal tissues(
Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Endometrial Neoplasms/genetics* ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mad2 Proteins ; Multigene Family ; Neoplastic Stem Cells ; Prognosis ; Securin

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; MEF2 Transcription Factors/metabolism* ; Muscle Fibers, Skeletal/metabolism* ; Muscle, Skeletal/metabolism* ; Myogenic Regulatory Factors/metabolism* ; Physical Conditioning, Animal/methods* ; Rats ; Signal Transduction

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Cyclic AMP Response Element-Binding Protein ; genetics ; metabolism ; Male ; MicroRNAs ; radiation effects ; Microwaves ; adverse effects ; Neuronal Plasticity ; radiation effects ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate ; genetics ; metabolism

Liquiritigenin (LQ) is a flavonoid that can be isolated from Glycyrrhiza radix. It is frequently used as a tranditional oriental medicine herbal treatment for swelling and injury and for detoxification. However, the effects of LQ on cognitive function have not been fully explored. In this study, we evaluated the memory-enhancing effects of LQ and the underlying mechanisms with a focus on the N-methyl-D-aspartic acid receptor (NMDAR) in mice. Learning and memory ability were evaluated with the Y-maze and passive avoidance tests following administration of LQ. In addition, the expression of NMDAR subunits 1, 2A, and 2B; postsynaptic density-95 (PSD-95); phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII); phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2); and phosphorylation of cAMP response element binding (CREB) proteins were examined by Western blot. In vivo, we found that treatment with LQ significantly improved memory performance in both behavioral tests. In vitro, LQ significantly increased NMDARs in the hippocampus. Furthermore, LQ significantly increased PSD-95 expression as well as CaMKII, ERK, and CREB phosphorylation in the hippocampus. Taken together, our results suggest that LQ has cognition enhancing activities and that these effects are mediated, in part, by activation of the NMDAR and CREB signaling pathways.
Animals ; Behavior Rating Scale ; Blotting, Western ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Cognition ; Glycyrrhiza ; Hippocampus ; In Vitro Techniques ; Learning ; Medicine, East Asian Traditional ; Memory ; Mice* ; N-Methylaspartate* ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Receptors, N-Methyl-D-Aspartate* ; Response Elements

Reducing [Mg2+]o to 0.1 mM can evoke repetitive [Ca2+]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg2+]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca2+ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg2+]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca2+ channel antagonist nimodipine, which blocked 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca2+]i spikes. The intracellular Ca2+ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca2+]i spikes. While Go6976, a specific inhibitor of PKCalpha had no effect on the tolerance, both the PKCepsilon translocation inhibitor and the PKCzeta pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca2+]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg2+]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca2+]i spike-induced activation of PKCepsilon and PKCxi, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.
Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Cell Death ; Cycloheximide ; N-Methylaspartate ; Neurons* ; Nimodipine ; Phosphatidylinositol 3-Kinases ; Phosphotransferases ; Rats* ; Receptors, AMPA ; Seizures

Kaempferol exerts cardioprotective actions through incompletely understood mechanisms. This study investigated the molecular mechanisms underlying the cardioprotective effects of kaempferol in sinus node dysfunction (SND) heart. Here, we demonstrate that angiotensin II (Ang II) infusion causes SND through oxidized calmodulin kinase II (CaMKII). In contrast to this, kaempferol protects sinus node against Ang II-induced SND. Ang II evoked apoptosis with caspase-3 activation in sinus nodal cells. However, kaempferol lowered the CaMKII oxidization and the sinus nodal cell death. To block the CaMKII oxidization, gene of p47phox, a cytosolic subunit of NADPH oxidase, was deleted using Cas9 KO plasmid. In the absence of p47phox, sinus nodal cells were highly resistance to Ang II-induced apoptosis, suggesting that oxidized-CaMKII contributed to sinus nodal cell death. In Langendorff heart from Ang II infused mice, kaempferol preserved normal impulse formation at right atrium. These data suggested that kaempferol protects sinus node via inhibition of CaMKII oxidization and may be useful for preventing SND in high risk patients.
Angiotensin II ; Animals ; Apoptosis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2* ; Calcium-Calmodulin-Dependent Protein Kinases ; Caspase 3 ; Cell Death ; Cytosol ; Heart ; Heart Atria ; Humans ; Mice ; NADPH Oxidase ; Plasmids ; Sick Sinus Syndrome* ; Sinoatrial Node*

This study was aimed to establish an experimental mouse model of combined transgenic inhibition of both multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and inward rectifier potassium current (Ik1), and to observe whether the specific inhibition of both CaMKII and Ik1 can bring about any effects on cardiac remodeling. Mice were divided into 4 groups: wild type (WT), CaMKII inhibited (AC3-I), Ik1 inhibited (Kir2.1-AAA) and combined inhibition of both CaMKII and Ik1 (AC3-I+Kir2.1-AAA). Mice in each group received electrocardiogram (ECG) and echocardiography examination. ECG in the condition of isoproterenol (ISO) injection was also checked. The whole cell patch clamp technique was used to measure Ik1 and the transient outward potassium current (Ito) from enzymatically isolated myocytes of left ventricle. In the condition of basal status, no significant changes of heart rate, PR interval and QRS interval were observed. No mouse showed ventricular arrhythmias in all of the 4 groups. After ISO injection, each group presented no significant ventricular arrhythmias either. The indexes measured by M-mode (motion-mode) and two-dimensional echocardiography had no significant differences among the four groups. Ik1 in AC3-I group was significantly higher than those in other three groups (P < 0.01) because of the results brought about by CaMKII inhibition. Among the latter three groups, both Kir2.1-AAA group and AC3-I+Kir2.1-AAA group had a significant reduced Ik1 compared with that of WT group, which was due to the Ik1 inhibition (P < 0.01). Ito in AC3-I group was higher than that of the other three groups (P < 0.01), but there were no significant differences in Ito among WT, Kir2.1-AAA and AC3-I+Kir2.1-AAA groups. Thus, combined transgenic myocardial CaMKII and Ik1 inhibition eliminated the up-regulation of Ik1 in CaMKII inhibited mice, and had no effects on cardiac remodeling including heart structure and function as well as arrhythmias at the basic and ISO conditions. The results of this study may provide a basis for the further investigation of combined inhibition of CaMKII and Ik1 in pathogenic cardiac remodeling.
Animals ; Arrhythmias, Cardiac ; Brugada Syndrome ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; physiology ; Cardiac Conduction System Disease ; Disease Models, Animal ; Electrocardiography ; Heart ; physiology ; Heart Conduction System ; abnormalities ; Heart Ventricles ; Isoproterenol ; Mice ; Mice, Transgenic ; Patch-Clamp Techniques ; Potassium Channels, Inwardly Rectifying ; physiology ; Up-Regulation ; Ventricular Remodeling