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Individuals with diabetes mellitus (DM) tend to manifest anxiety and depression, which could be related to changes in the expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV), transcription factor cyclic AMP-responsive element binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) in different brain regions. The objective of this study was to determine whether mice with type 1 diabetes (T1DM) induced with streptozotocin show a profile of anxious-type behaviors and alterations in the expression/activity of CaMKIV, CREB, pCREB and BDNF in different regions of the brain (prefrontal cortex, amygdala, hippocampus and hypothalamus) in comparison to non-diabetic mice (NDB). Mice with 3 months of chronic DM showed an anxious-like behavioral profile in two anxiety tests (Open Field and Elevated Plus Maze), when compared to NDB. There were significant differences in the expression of cell signaling proteins: diabetic mice had a lower expression of CaMKIV in the hippocampus, a greater expression of CREB in the amygdala and hypothalamus, as well as a lower pCREB/CREB in hypothalamus than NDB mice (P 0.05). This is the first study evaluating the expression of CaMKIV in the brain of animals with DM, who presented lower expression of this protein in the hippocampus. In addition, it is the first time that CREB was evaluated in amygdala and hypothalamus of animals with DM, who presented a higher expression. Further research is necessary to determine the possible link between expression of CaMKIV and CREB, and the behavioral profile of anxiety in diabetic animals.

Rationale: Polycystic ovary syndrome (PCOS) is closely linked to follicular dysplasia and impaired bidirectional oocyte-granulosa cell (GC) communication. Given that PCOS is a heterogeneous, multifactorial endocrine disorder, it is important to clarify the pathophysiology of this ovarian disease and identify a specific treatment. Methods: We generated PCOS rat models based on neonatal tributyltin (TBT) exposure and studied the therapeutic effect and mechanism of resveratrol (RSV), a natural plant polyphenol. Transcriptome analysis was conducted to screen the significantly changed pathways, and a series of experiments, such as quantitative real-time polymerase chain reaction (PCR), Western blot and phalloidin staining, were performed in rat ovaries. We also observed similar changes in human PCOS samples using Gene Expression Omnibus (GEO) database analysis and quantitative real-time PCR. Results: We first found that injury to transzonal projections (TZPs), which are specialized filopodia that mediate oocyte-GC communication in follicles, may play an important role in the etiology of PCOS. We successfully established PCOS rat models using TBT and found that overexpressed calcium-/calmodulin-dependent protein kinase II beta (CaMKIIβ) inhibited TZP assembly. In addition, TZP disruption and CAMK2B upregulation were also observed in samples from PCOS patients. Moreover, we demonstrated that RSV potently ameliorated ovarian failure and estrus cycle disorder through TZP recovery via increased cytoplasmic calcium levels and excessive phosphorylation of CaMKIIβ. Conclusions: Our data indicated that upregulation of CaMKIIβ may play a critical role in regulating TZP assembly and may be involved in the pathogenesis of PCOS associated with ovarian dysfunction. Investigation of TZPs and RSV as potent CaMKIIβ activators provides new insight and a therapeutic target for PCOS, which is helpful for improving female reproduction.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) has recently emerged as an important regulator of glucose metabolism and vascular function, but the underlying mechanism is not fully understood. Recently, we revealed that CaMKIV limits metabolic disorder and liver insulin resistance and regulates autophagy in high-fat diet-induced obese mice. In the present study, we demonstrated that CaMKIV was not only associated with improvement of glucose tolerance and insulin sensitivity in ob/ob mice but also involved in the regulation of vascular autophagy and mitochondrial biogenesis. Our in vitro data indicated that CaMKIV reversed autophagic imbalance and restored insulin sensitivity in palmitate-induced A7r5 cells with insulin resistance. However, the protective effects of CaMKIV were nullified by suppression of Akt, mTOR, or CREB, suggesting that CaMKIV inhibits autophagy and improves insulin signaling in insulin resistance cell models in an Akt/mTOR/CREB-dependent manner. CaMKIV reversed autophagic imbalance and insulin sensitivity in vascular tissues and vascular cells through Akt/mTOR/CREB signaling, which could be regarded as a novel opportunity for the treatment of insulin resistance.

Objective To investigate the effect of sevoflurane on inflammation of microglia in hippocampus of neonatal rats, and to investigate whether the related mechanism is related to Wnt/β-Catenin/CaMKIV pathway. Methods Neonatal rats were anesthetized with 2% or 3% sevoflurane for 4 h a day for 3 consecutive days. Water maze test was used to detect the effect of sevoflurane anesthesia on memory function of neonatal rats. H&E and Nissl staining were used to observe the pathological damage of hippocampal area of neonatal rats induced by sevoflurane anesthesia. The expression of microglial marker Iba-1 was detected by Immunofluorescence. Immunofluorescence and WB were used to detect the expression CD32b, CD86, TNF-α, IL-6, Wnt3a, β-Catenin and CaMKIV in hippocampus. To further explore the related mechanism, Wnt-3α inhibitor and activator was treated to study the effect of sevoflurane on microglial inflammation in hippocampus of neonatal rats. Results Sevoflurane anesthesia significantly increased escape latency time, reduced platform crossing times, and damaged the learning and memory ability of neonatal rats. H&E and Nissl staining results showed that sevoflurane anesthesia caused obvious damage to the hippocampus of neonatal rats. Sevoflurane anesthesia promoted the expression of Iba-1 and activated microglia. Sevoflurane anesthesia not only significantly increased the positive expression of CD32b, CD86, TNF-α and IL-6, but also decreased the expression of Wnt3a, β-Catenin and CaMKIV. These results suggested that sevoflurane inhibited Wnt/β-Catenin/CaMKIV pathway. Conclusion Sevoflurane induces inflammation of microglia in hippocampus of neonatal rats by inhibiting Wnt/β-Catenin/CaMKIV pathway.

BackgroundType II alveolar epithelial cell (AEC II), in addition to its roles in maintaining lung homeostasis, takes an active role in inflammatory response during acute lung injury (ALI). Ca2+/calmodulin-dependent protein kinase IV (CaMK4) activated by Ca2+/calmodulin signaling, has been implicated in immune responses. This study was to investigate the roles of CaMK4 in the development of ALI and the underlying mechanisms.MethodsCaMK4 inhibitor KN-93 was used to investigate the effects of CaMK4 on NLRP3 inflammasome activation. The effects of KN-93 on disease development of lipopolysaccharide (LPS)-induced ALI were also evaluated. The role of CaMK4 on NLRP3 inflammasome activation was explored in human AEC II cell line A549 using KN-93 or CaMK4 siRNA. NLRP3 inflammasome activation was measured by histology immunofluorescence and Western blot. IL-1β and IL-18 were measured by ELISA.ResultsPhosphorylation of CaMK4 and the expression of NLRP3 and Caspase-1 p20 were increased in the lungs of LPS-induced ALI mice, which was suppressed by KN-93 as measured by Western blot. Further, the activation of NLRP3 inflammasome was detected in AEC II from patients with acute respiratory distress syndrome (ARDS) and LPS-induced ALI mice. In vitro, inhibition or silencing CaMK4 in AEC II significantly inhibited NLRP3 inflammasome activation, resulting in reduced IL-1β production. The inhibition of NLRP3 inflammasome and decreased IL-1β/IL-18 production by KN-93 led to reduced inflammatory infiltration and ameliorated lung injury in LPS-induced ALI mice.ConclusionCaMK4 controls the activation of NLRP3 inflammasome in AEC II during LPS-induced ALI. CaMK4 inhibition could be a novel therapeutic approach for the treatment of ALI.

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is a complicated syndrome characterized by genitourinary pain in the absence of bacterial infection. Th17 cell-driven autoimmunity has been proposed as a cause of CP/CPPS. However, the factors that promote Th17-driven autoimmunity in experimental autoimmune prostatitis (EAP) and the molecular mechanisms are still largely unknown. Here, we showed that Th17 cells were excessively activated, and blockade of IL-17A could effectively ameliorate various symptoms in EAP. Furthermore, we revealed that calcium/calmodulin-dependent kinase Ⅳ (CaMK4), especially Thr196 p-CaMK4 was increased in the Th17 cells of the EAP group, which were activated by intracellular cytosolic Ca2+ . Pharmacologic and genetic inhibition of CaMK4 decreased the proportion of Th17 cells, and the protein and mRNA level of IL-17A, IL-22, and RORγt. The phosphorylation of CaMK4 was dependent on the increase in intracellular cytosolic Ca2+ concentration in Th17 cells. A mechanistic study demonstrated that inhibition of CaMK4 reduced IL-17A production by decreasing the phosphorylation of Akt-mTOR, which was well accepted to positively regulate Th17 differentiation. Collectively, our results demonstrated that Ca2+ -CaMK4-Akt/mTOR-IL-17A axis inhibition may serve as a promising therapeutic strategy for CP/CPPS.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is an upstream regulator of CaMKK-CaMKIV signaling cascade that activates various transcription factors, thereby regulating several cellular activities including, neuronal communication and immune response. Owing to the abnormal expression in cancer and neurodegenerative diseases, the CaMKIV has been considered a potential drug target. In the present study, we checked the binding affinity of plant-derived natural compounds viz., quercetin, ellagic acid (EA), simvastatin, capsaicin, ursolic acid, DL-α-tocopherol acetate, and limonin towards CaMKIV. Molecular docking and fluorescence binding studies showed that EA and quercetin bind to the CaMKIV with a considerable affinity in comparison to other compounds. Enzyme inhibition assay revealed that both EA and quercetin inhibit CaMKIV activity with their IC50 values in the micromolar range. To get atomistic insights into the mode of interactions, inhibition mechanism, and the stability of the CaMKIV-ligand complex, a 100 ns MD simulation analysis was performed. Both EA and quercetin bind to the catalytically important residues of active site pocket of CaMKIV forming enough stabilizing interactions presumably inhibiting enzyme activity. Moreover, no significant structural change in the CaMKIV was observed upon binding of EA and quercetin. In conclusion, this study illustrates the application of phytoconstituents in the development of therapeutic molecules targeting CaMKIV having implications in cancer and neurodegenerative diseases after in vivo validation.

Objective To investigate the neuroprotective effect of chrysophanol (CHR) on PC12 treated with Aβ25-35, and the involved mechanism. Methods After the establishment of an AD cell model induced by Aβ25-35, the cell survival rate was detected by MTT, cell apoptosis was assayed by Hoechst 33342 staining, mRNA expressions of calmodulin (CaM), calcium/calmodulin-dependent protein kinase kinase (CaMKK), calcium/calmodulin-dependent protein kinase IV (CaMKIV) and tau (MAPT; commonly known as tau) were determined by qRT-PCR, and protein levels of CaM, CaMKK, CaMKIV, phospho-CaMKIV (p-CaMKIV), tau and phospho-tau (p-tau) were detected by Western blot analysis. Results When pretreated with CHR before exposure to Aβ25-35, PC12 cells showed that increased cell viability and reduced apoptosis. The qRT-PCR results indicated that the deposition of Aβ25-35 triggers a decrease in levels of CaM, CaMKK, CaMKIV, and tau in PC12 cells. In addition, Western blot results also suggested that Aβ25-35 decreases the protein expression of CaM, CaMKK, CaMKIV, p-CaMKIV, and the ratio of p-tau to tau in PC12 cells. However, the above effects were significantly alleviated after the treatment of CHR. Conclusion CHR plays a neuroprotective role in AD though decreasing the protein level of CaM-CaMKK-CaMKIV and the expression of p-tau downstream.

Summary Aim of the study Valproic acid (VPA) is a widely used antiepileptic drug for epilepsy. However, approximately 30% of patients with epilepsy do not respond to this therapy even when it was appropriately used. In order to explore the potential genetic factors related to the VPA response, this pharmacogenetics study was conducted. Methods A total of one hundred and fifty-seven Chinese children with epilepsy who were administered with by VPA for at least one year were enrolled. Thirteen single-nucleotide polymorphisms (SNPs) located in eight genes involving targets and metabolic enzymes of VPA were genotyped. The frequencies of these polymorphisms and the effect of genotypes on the efficacy of VPA were analyzed. Results The frequencies of two SNPs, rs7668258 (uridine diphosphate glucuronosyltransferase-2B7, UGT2B7) and rs306104 (calmodulin-kinase 4, CaMK4) were associated with VPA responses. However, no association was found for the other SNPs. Furthermore, the polymorphism of UGT2B7 influenced the adjusted concentration (AC) in the responders rather than in the non-responders. Conclusion Two SNPs (UGT2B7 and CaMK4) were associated with VPA response, which may explain the pharmacological mechanism of VPA resistance to some extent.

Despite ongoing research efforts, mechanisms of brain aging are still enigmatic and need to be elucidated for a better understanding of age-associated cognitive decline. The aim of this study is to investigate aging in the prefrontal cortex region of human brain in a meta-analysis of transcriptome datasets. We analyzed 591 gene expression datasets pertaining to female and male human prefrontal cortex biopsies of distinct ages. We used hierarchical clustering and principal component analysis (PCA) to determine the influence of sex and age on global transcriptome levels. In sex-specific analysis we identified genes correlating with age and differentially expressed between groups of young, middle-aged and aged. Pathways and gene ontologies (GOs) over-represented in the resulting gene sets were calculated. Potential causal relationships between genes and between GOs were explored employing the Granger test of gene expression time series over the range of ages. The most outstanding results were the age-related decline of synaptic transmission and activated expression of glial fibrillary acidic protein (GFAP) in both sexes. We found an antagonistic relationship between calcium/calmodulin dependent protein kinase IV (CAMK4) and GFAP which may include regulatory mechanisms involving cAMP responsive element binding protein (CREB) and mitogen-activated protein kinase (MAPK, alias ERK). Common to both sexes was a decline in synaptic transmission, neurogenesis and an increased base-level of inflammatory and immune-related processes. Furthermore, we detected differences in dendritic spine morphogenesis, catecholamine signaling and cellular responses to external stimuli, particularly to metal (Zinc and cadmium) ions which were higher in female brains.

Activation of calcium/calmodulin-dependent protein kinase IV (CaMKIV) has been shown to increase intestinal injury and inhibit epithelial cell proliferation in dextran sulfate sodium (DSS)-induced colitis mice. However, the role of CaMKIV in necrotizing enterocolitis (NEC) is unknown. We aimed to study the expression and activation of CaMKIV in experimental NEC. Following ethical approval, NEC (n = 10) was induced in C57BL/6 mouse pups by hypoxia, gavage hyperosmolar formula feeding and lipopolysaccharide from postnatal days P5 to 9. Breastfed pups served as control (n = 10). Mouse pups were sacrificed on P9 and the terminal ileum was harvested. Gene NEC injury was scored blindly by three independent investigators. CaMKIV, CREM and IL17 gene expression, and CaMKIV and pCaMKIV protein expression were assessed. The data were compared using Mann–Whitney U test. P

Magnesium (Mg) based alloy has been used as a biodegradable implant for fracture repair with considerable efficacy, and it has been proved that magnesium ion (Mg

Background: MicroRNAs (miRNA) are known to play a key role in the etiology and treatment of epilepsy through controlling the expression of gene. However, miR-125a-5p in the epilepsy is little known. Epilepsy in rat models was induced by Pentylenetetrazol (PTZ) and miR- 125a-5p profiles in the hippocampus were investigated in our experiment. Also, the relationship between miR-125a-5p and calmodulin-dependent protein kinase IV (CAMK4) was identified and the related mechanism was also illustrated. Methods: The miR-125a-5p mRNA expression levels were evaluated by quantitative real time polymerase chain reaction (qRT-PCR). Western Blot (WB) was used to analyze the CAMK4 protein expression levels. Seizure score, latency and duration were determined based on a Racine scale. The enzyme-linked immunosorbent assay (ELISA) was used to analyze the inflammatory factor expression. The relationship between miR-125a-5p and CAMK4 was detected through dual luciferase assay. Results: Downregulation of miR-125a-5p was observed in the hippocampus of PTZ-induced epilepsy rats. The overexpression of miR-125a-5p attenuated seizure and decreased inflammatory factor level in the hippocampus of PTZ-induced rats. The miR-125a-5p alleviated epileptic seizure and inflammation in PTZ-induced rats by suppressing its target gene, CAMK4. Conclusion: miR-125a-5p may represent a novel therapeutic treatment for PTZ-induced epilepsy by preventing the activation of CAMK4.

Blood transfusions reportedly modulate the recipient's immune system. Transfusion-related immunomodulation has been suggested as a mechanism of some adverse clinical outcomes. Extracellular nucleic acids circulate in plasma and activate relevant immune responses, but little is known about their mechanism of action in transfusion-related immunomodulation (TRIM). The aim of this study was to investigate the effects of cell-free nucleic acids (CFNAs) produced by red blood cells (RBCs) on innate immunity, especially peripheral blood mononuclear cells (PBMCs) and macrophages, and to investigate the mechanism of action.Differentially expressed genes (DEGs) between PBMCs exposed to RBC-produced CFNA and normal PBMCs were analyzed by gene expression data combined with bioinformatics. KEGG and GO enrichment analyses were performed for the DEGs, and in vitro experiments were performed for the effects of key genes on the release of inflammatory factors from macrophages.Analysis of microarray data showed that exposure of monocytes to RBC-produced CFNAs increased the expression of genes involved in the innate immune response, including chemokines, chemokine receptors, and innate response receptors, and that calcium channel activity was highly regulated, with a key gene being CaMKIV. CaMKIV played a critical role in LPS-induced inflammatory factor release from macrophages, which was exacerbated by overexpression of the CaMKIV gene.RBCs regulate the release of inflammatory factors during blood transfusion by releasing CFNAs and affecting expression of the CaMKIV gene in PBMCs or macrophages, which is a potential regulatory mechanism of blood transfusion-related immune regulation and related adverse reactions.

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α

Ginsenoside Re (G-Re) is a panaxatriol saponin and one of the pharmacologically active natural constituents of ginseng (Panax ginseng C.A. Meyer). G-Re has antioxidant, anti-inflammatory and antidiabetic effects. The present study aimed to investigate the effects of G-Re on neuroinflammatory responses in lipopolysaccharide (LPS)-stimulated microglia and its protective effects on hippocampal neurons. Cytokine levels were measured using ELISA and reactive oxygen species (ROS) levels were assessed using flow cytometry and fluorescence microscopy. Protein levels of inflammatory molecules and kinase activity were assessed by western blotting. Cell viability was assessed by MTT assay; apoptosis was estimated by Annexin V apoptosis assay. The results revealed that G-Re significantly inhibited the production of IL-6, TNF-α, nitric oxide (NO) and ROS in BV2 microglial cells, and that of NO in mouse primary microglia, without affecting cell viability. G-Re also inhibited the nuclear translocation of NF-κB, and phosphorylation and degradation of IκB-α. In addition, G-Re dose-dependently suppressed LPS-mediated phosphorylation of Ca2+/calmodulin-dependent protein kinase (CAMK)2, CAMK4, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinases (JNK). Moreover, the conditioned medium from LPS-stimulated microglial cells induced HT22 hippocampal neuronal cell death, whereas that from microglial cells incubated with both LPS and G-Re ameliorated HT22 cell death in a dose-dependent manner. These results suggested that G-Re suppressed the production of pro-inflammatory mediators by blocking CAMK/ERK/JNK/NF-κB signaling in microglial cells and protected hippocampal cells by reducing these inflammatory and neurotoxic factors released from microglial cells. The present findings indicated that G-Re may be a potential treatment option for neuroinflammatory disorders and could have therapeutic potential for various neurodegenerative diseases.

High fructose has been reported to drive glomerular podocyte oxidative stress and then induce podocyte foot process effacement in vivo, which could be partly regarded as podocyte hypermotility in vitro. Atractylodin possesses anti-oxidative effect. The aim of this study was to explore whether atractylodin prevented against fructose-induced podocyte hypermotility via anti-oxidative property. In fructose-exposed conditionally immortalized human podocytes, we found that atractylodin inhibited podocyte hypermotility, and up-regulated slit diaphragm proteins podocin and nephrin, and cytoskeleton protein CD2-associated protein (CD2AP), α-Actinin-4 and synaptopodin expression, which were consistent with its anti-oxidative activity evidenced by up-regulation of catalase (CAT) and superoxide dismutase (SOD) 1 expression, and reduction of reactive oxygen species (ROS) production. Atractylodin also significantly suppressed expression of transient receptor potential channels 6 (TRPC6) and phosphorylated Ca2+/calmodulin-dependent protein kinase IV (CaMK4) in cultured podocytes with fructose exposure. Additionally, in fructose-exposed podocytes, CaMK4 siRNA up-regulated synaptopodin and reduced podocyte hypermotility, whereas, silencing of TRPC6 by siRNA decreased p-CaMK4 expression, inhibited podocyte hypermotility, showing TRPC6/p-CaMK4 signaling activation in podocyte hypermotility under fructose condition. Just like atractylodin, antioxidant N-acetyl-L-cysteine (NAC) could inhibit TRPC6/p-CaMK4 signaling activation to reduce fructose-induced podocytes hypermotility. These results first demonstrated that the anti-oxidative property of atractylodin may contribute to the suppression of podocyte hypermotility via inhibiting TRPC6/p-CaMK4 signaling and restoring synaptopodin expression abnormality.

Interstitial kidney inflammation is present in various nephritides in which serum interleukin 23 (IL-23) is elevated. Here we showed that murine and human renal tubular epithelial cells (TECs) expressing the IL-23 receptor (IL-23R) responded to IL-23 by inducing intracellular calcium flux, enhancing glycolysis, and upregulating calcium/calmodulin kinase IV (CaMK4), which resulted in suppression of the expression of the arginine-degrading enzyme arginase 1 (ARG1), thus increasing in situ levels of free L-arginine. Limited availability of arginine suppressed the ability of infiltrating T cells to proliferate and produce inflammatory cytokines. TECs from humans and mice with nephritis expressed increased levels of IL-23R and CaMK4 but reduced levels of ARG1. TEC-specific deletion of Il23r or Camk4 suppressed inflammation, whereas deletion of Arg1 exacerbated inflammation in different murine disease models. Finally, TEC-specific delivery of a CaMK4 inhibitor specifically curbed renal inflammation in lupus-prone mice without affecting systemic inflammation. Our data offer the first evidence to our knowledge of the immunosuppressive capacity of TECs through a mechanism that involves competitive uptake of arginine and signify the importance of modulation of an inflammatory cytokine in the function of nonlymphoid cells, which leads to the establishment of an inflammatory microenvironment. New approaches to treat kidney inflammation should consider restoring the immunosuppressive capacity of TECs.

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of “liquid kidney biopsy” approaches to diagnose LN.

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is associated with many diseases including cancer and neurodegenerative disorders and thus being considered as a potential drug target. Here, we have employed the knowledge of three-dimensional structure of CAMKIV to identify new inhibitors for possible therapeutic intervention. We have employed virtual high throughput screening of 12,500 natural compounds of Zinc database to screen the best possible inhibitors of CAMKIV. Subsequently, 40 compounds which showed significant docking scores (-11.6 to -10.0 kcal/mol) were selected and further filtered through Lipinski rule and drug likeness parameter to get best inhibitors of CAMKIV. Docking results are indicating that ligands are binding to the hydrophobic cavity of the kinase domain of CAMKIV and forming a significant number of non-covalent interactions. Four compounds, ZINC02098378, ZINC12866674, ZINC04293413, and ZINC13403020, showing excellent binding affinity and drug likeness were subjected to molecular dynamics simulation to evaluate their mechanism of interaction and stability of protein-ligand complex. Our observations clearly suggesting that these selected ligands may be further employed for therapeutic intervention to address CAMKIV associated diseases. Communicated by Ramaswamy H. Sarma.

CaMK4 has an important function in autoimmune diseases, and the contribution of CaMK4 in psoriasis remains obscure. Here, we show that CaMK4 expression is significantly increased in psoriatic lesional skin from psoriasis patients compared to healthy human skin as well as inflamed skin from an imiquimod (IMQ)-induced mouse model of psoriasis compared to healthy mouse skin.Camk4-deficient (Camk4−/−) mice treated with IMQ exhibit reduced severity of psoriasis compared to wild-type (WT) mice. There are more macrophages and fewer IL-17A+γδ TCR+cells in the skin of IMQ-treatedCamk4−/−mice compared to IMQ-treated WT mice. CaMK4 inhibits IL-10 production by macrophages, thus allowing excessive psoriatic inflammation. Deletion ofCamk4in macrophages alleviates IMQ-induced psoriatic inflammation in mice. In keratinocytes, CaMK4 inhibits apoptosis as well as promotes cell proliferation and the expression of pro-inflammatory genes such asS100A8andCAMP. Taken together, these data indicate that CaMK4 regulates IMQ-induced psoriasis by sustaining inflammation and provides a potential target for psoriasis treatment.

CaMKII and CaMKIV are calcium/calmodulin-dependent kinases playing a rudimentary role in many regulatory processes in the organism. These kinases attract increasing interest due to their involvement primarily in memory and plasticity and various cellular functions. Although CaMKII and CaMKIV are mostly recognized as the important cogs in a memory machine, little is known about their effect on mood and role in neuropsychiatric diseases etiology. Here, we aimed to review the structure and functions of CaMKII and CaMKIV, as well as how these kinases modulate the animals’ behavior to promote antidepressant-like, anxiolytic-like, and procognitive effects. The review will help in the understanding of the roles of the above kinases in the selected neurodegenerative and neuropsychiatric disorders, and this knowledge can be used in future drug design.

Effective treatment remains lacking for neuropathic pain (NP), a type of intractable pain. Low-intensity focused ultrasound (LIFU), a noninvasive, cutting-edge neuromodulation technique, can effectively enhance inhibition of the central nervous system (CNS) and reduce neuronal excitability. We investigated the effect of LIFU on NP and on the expression of potassium chloride cotransporter 2 (KCC2) in the spinal cords of rats with peripheral nerve injury (PNI) in the lumbar 4–lumbar 5 (L4–L5) section. In this study, rats received PNI surgery on their right lower legs followed by LIFU stimulation of the L4–L5 section of the spinal cord for 4 weeks, starting 3 days after surgery. We used the 50% paw withdraw threshold (PWT50) to evaluate mechanical allodynia. Western blotting (WB) and immunofluorescence (IF) were used to calculate the expression of phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), and KCC2 in the L4–L5 portion of the spinal cord after the last behavioral tests. We found that PWT50 decreased (P < 0.05) 3 days post-PNI surgery in the LIFU− and LIFU+ groups and increased (P < 0.05) after 4 weeks of LIFU stimulation. The expression of p-CREB and CaMKIV decreased (P < 0.05) and that of KCC2 increased (P < 0.05) after 4 weeks of LIFU stimulation, but that of p-ERK1/2 (P > 0.05) was unaffected. Our study showed that LIFU could effectively alleviate NP behavior in rats with PNI by increasing the expression of KCC2 on spinal dorsal corner neurons. A possible explanation is that LIFU could inhibit the activation of the CaMKIV–KCC2 pathway.

Background Atractylodis rhizoma, an aromatic herb for resolving dampness, is used to treat Kidney-related edema in traditional Chinese medicine for thousands years. This herb possesses antioxidant effect. However, it is not yet clear how Atractylodis rhizoma prevents glomerular injury through its anti-oxidation. Purpose Based the analysis of Atractylodis rhizoma water extract (ARE) components and network pharmacology, this study was to explore whether ARE prevented glomerular injury via its anti-oxidation to inhibit oxidative stress-driven transient receptor potential channel 6 (TRPC6) and its downstream molecule calcium/calmodulin-dependent protein kinase IV (CaMK4) signaling. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ARE components. Network pharmacology analysis was preliminarily performed. Male Sprague-Dawley rats were given 10% fructose drinking water (100 mL/d) for 16 weeks. ARE at 720 and 1090 mg/kg was orally administered to rats for the last 8 weeks. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity in rat kidney cortex were detected, respectively. In rat glomeruli, redox-related factors forkhead box O3 (FoxO3), SOD2 and catalase (CAT), podocyte slit diaphragm proteins podocin and nephrin, cytoskeleton proteins CD2-associated protein (CD2AP) and α-Actinin-4, as well as TRPC6, p-CaMK4 and synaptopodin protein levels were analyzed by Western Blotting. SOD2 and CAT mRNA levels were detected by qRT-PCR. Results 36 components were identified in ARE. Among them, network pharmacology analysis indicated that ARE might inhibit kidney oxidative stress. Accordingly, ARE up-regulated nuclear FoxO3 expression, and then increased SOD2 and CAT at mRNA and protein levels in glomeruli of fructose-fed rats. It reduced H2O2 and MDA levels, and increased SOD activity in renal cortex of fructose-fed rats. Subsequently, ARE down-regulated TRPC6 and p-CaMK4, and up-regulated synaptopodin in glomeruli of fructose-fed rats. Furthermore, ARE increased podocin and nephrin, as well as CD2AP and α-Actinin-4, being consistent with its reduction of urine albumin-to-creatinine ratio and improvement of glomerular structure injury in this animal model. Conclusions These results suggest that ARE may prevent glomerular injury in fructose-fed rats possibly by reducing oxidative stress to inhibit TRPC6/p-CaMK4 signaling and up-regulate synaptopodin expression. Therefore, ARE may be a promising drug for treating high fructose-induced glomerular injury in clinic.

Thyroid hormones influence brain development through the regulation of gene expression. Ca

Sepsis and shock states impose mitochondrial stress, and in response, adaptive mechanisms such as fission, fusion and mitophagy are induced to eliminate damaged portions of or entire dysfunctional mitochondria. The mechanisms underlying these events are being elucidated; yet a direct link between loss of mitochondrial membrane potential ΔΨm and the initiation of fission, fusion and mitophagy remains to be well characterized. The direct association between the magnitude of the ΔΨm and the capacity for mitochondria to buffer Ca(2+) renders Ca(2+) uniquely suited as the signal engaging these mechanisms in circumstances of mitochondrial stress that lower the ΔΨm. Herein, we show that the calcium/calmodulin-dependent protein kinase (CaMK) IV mediates an adaptive slowing in oxidative respiration that minimizes oxidative stress in the kidneys of mice subjected to either cecal ligation and puncture (CLP) sepsis or endotoxemia. CaMKIV shifts the balance towards mitochondrial fission and away from fusion by 1) directly phosphorylating an activating Serine616 on the fission protein DRP1 and 2) reducing the expression of the fusion proteins Mfn1/2 and OPA-1. CaMKIV, through its function as a direct PINK1 kinase and regulator of Parkin expression, also enables mitophagy. These data support that CaMKIV serves as a keystone linking mitochondrial stress with the adaptive mechanisms of mitochondrial fission, fusion and mitophagy that mitigate oxidative stress in the kidneys of mice responding to sepsis.

To elucidate the potential influences of miR-507 and CAMK4 on the progression of preeclampsia (PE).Placental tissues were collected from 24 PE pregnancies and 24 healthy pregnancies. The relative levels of miR-507 and CAMK4 in placental tissues were detected. In addition, expressions of apoptosis-associated genes in collected tissues were examined by both quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The influences of miR-507 and CAMK4 on proliferative and migratory abilities in HTR-8/SVneo cells were assessed by CCK-8 and transwell assay, respectively. The target relationship between miR-507 and CAMK4 was detected by Luciferase assay.MiR-507 was upregulated in placental tissues collected from PE pregnancies. Overexpression of miR-507 suppressed proliferative and migratory abilities, and stimulated apoptosis in HTR-8/SVneo cells. CAMK4 was the target gene of miR-507, which was downregulated in placental tissues collected from PE pregnancies and negatively correlated to miR-507 level. The knockdown of CAMK4 suppressed proliferative and migratory abilities, and stimulated apoptosis in HTR-8/SVneo cells, and these trends were abolished by silence of miR-507.Highly expressed miR-507 in PE pregnancies inhibits proliferative and migratory potentials, and induces apoptosis in trophoblasts by targeting CAMK4.

BackgroundCirculatory iron is a hazardous biometal. Therefore, iron is transported in a redox-safe state by a serum glycoprotein - transferrin (TF). Different organs acquire iron from the systemic circulation through a tightly regulated mechanism at the blood-tissue interface which involves receptor-mediated internalization of TF. Thus, abnormal TF trafficking may lead to iron dyshomeostasis associated with several diseases including neurodegeneration. Iron -induced toxicity can cause neuronal damage to iron-sensitive brain regions. Recently, it was discovered that CAMKK2, a calcium (Ca2+)/calmodulin-activated kinase, controls receptor-mediated TF trafficking in mouse tissues, specifically in the brain. The biological function of CAMKK2 is mediated through multiple downstream effectors. Both CAMKK2 and one of its downstream kinase, CAMK4, exhibit overlapping expression in mouse brain. The role of CAMK4 in vesicular transport has been reported and loss of CAMKK2 or CAMK4 leads to cognitive defects in mouse. Therefore, it was hypothesized that CAMKK2-CAMK4 signaling regulates receptor-mediated TF trafficking and iron homeostasis which may be responsible for the neuronal malfunction observed in CAMKK2- or CAMK4-deficient mice.MethodsCAMK4−/−mouse was used to study tissue-specific turnover of TF, TF-receptor (TFRC) and iron. CRISPR/Cas9-based CAMKK2 and/or CAMK4 deleted human embryonic kidney-derived HEK293 cell clones were used to study the molecular defects in receptor-mediated TF trafficking. Further, a “zero functional G protein” condition in HEK293 cell was exploited to study CAMKK2-CAMK4 signaling-mediated regulation of intracellular Ca2+homeostasis which was linked to calcium signaling during TF trafficking.ResultsLoss of CAMK4 leads to abnormal post-translational modifications (PTMs) and turnover of TF in mouse cerebellum and liver which was associated with iron dyshomeostasis in these tissues. The HEK293 cell-based study revealed that the absence of CAMKK2-CAMK4 signaling altered intracellular Ca2+homeostasis and lead to abnormal calcium signaling during TF trafficking. Also, CAMKK2-CAMK4 signaling deficiency affected the molecular interaction of TF and TF-receptor-associated protein complexes which indicated a potential failure in the recruitment of interacting proteins due to differential PTMs in TF.ConclusionOverall, this study established a novel mechanistic link between intracellular Ca2+ level, receptor-mediated TF trafficking, and iron homeostasis, all regulated by CAMKK2-CAMK4 signaling.Graphical abstract

The nicotinic acetylcholine receptors (nAChRs) are essential for acetylcholine-mediated signaling. Two major functional subtypes of nAChR in the brain, α7-type and α4β2-type, have a high affinity for nicotine. Here, we demonstrated that chronic exposure to nicotine at 0.03–0.3 mg/kg for 14 days rescued depressive-like behavior in calcium/calmodulin-dependent protein kinase IV (CaMKIV) null mice. Chronic exposure to nicotine together with methyllycaconitine, an α7-type nAChR antagonist, but not with dihydro-β-erythroidine, an α4β2-type nAChR antagonist, failed to rescue the depressive-like behavior and restore the reduced number of BrdU-positive cells in the dentate gyrus (DG) of CaMKIV null mice. Furthermore, chronic exposure to nicotine enhanced the PI3K/Akt and ERK/CREB pathways and increased BDNF expression in the DG of CaMKIV null mice. Similar to chronic exposure to nicotine, both PNU-282987 and GTS-21, α7-type nAChR agonists, significantly rescued depressive-like behavior, with a reduction in the number of BrdU-positive cells in the DG of CaMKIV null mice. Both PNU-282987 and GTS-21 also enhanced the PI3K/Akt and ERK/CREB pathways and increased brain-derived neurotrophic factor (BDNF) expression in the DG of CaMKIV null mice. Taken together, we demonstrated that chronic exposure to nicotine rescues depressive-like behavior via α7-type nAChR through the activation of both PI3K/Akt and ERK/CREB pathways in CaMKIV null mice.

Problem The balance of the immune microenvironment along the maternal-fetal interface is closely related to pregnancy outcomes, with excessive inflammatory reactions leading to the occurrence of pathological pregnancy outcomes such as abortion. CaMK4 has been reported to play a significant role in autoimmune diseases through the regulation of Th17 cells. However, whether CaMK4 is associated with spontaneous abortion or the immune microenvironment along the maternal-fetal interface remains unclear. Methods of study In this study, we constructed normal pregnancy and LPS-induced abortion models in mice, and a CaMK4 inhibitor called KN-93 was administered to investigate the changes in and mechanisms of the immune response. The expression of CaMK4 was evaluated in the uteroplacental complex and spleen. Furthermore, the infiltration and function of Th17 cells were estimated in peripheral tissues and the uteroplacental complex. Results The expression of CaMK4 in the uteroplacental complex and spleen was significantly higher in the LPS-treated group than in the normal pregnancy group. KN-93, the CaMK4 inhibitor, reversed fetal resorption and excessive inflammation. In detail, KN-93 led to reduced infiltration of Th17 cells into peripheral tissues and the uteroplacental complex, and the functions of Th17 cells were inhibited. In addition, CaMK4 promoted the AKT/mTOR signaling pathway, which is one of the mechanisms that regulate the immune microenvironment. Conclusion CaMK4 is a critical regulator that promotes the expansion of Th17 cells and enhances their functions through the AKT/mTOR signaling pathway. The inhibition of CaMK4 can reverse the immune imbalance along the maternal-fetal interface and improve pregnancy outcomes.

Diabetic retinopathy (DR) is considered a main cause for vision loss in diabetes. To our knowledge, there were no studies on the association of genetic variants with DR in Chinese Hui nationality. In our research, 34 single nucleotide polymorphisms (SNPs) that were reported to be associated with DR in other ethnics were genotyped in 123 subjects with DR and 12 subjects without DR among Chinese Hui population using MassARRAY system. Association analysis performed by PLINK showed three SNP loci rs2300782, rs2292239, and rs10491034 were correlated with DR incidence. Furthermore, the genotype frequency analysis and association analysis of SNP with DR stage revealed the GT and TT genotypes of rs2292239, CC genotype of rs2300782, and GG genotype of rs10491034 were risk genotypes and associated with the severity of DR, which may be helpful for the study of DR susceptibility in Chinese Hui population. Our study indicates the rs2300782 of gene CAMK4, rs2292239 of gene ERBB3, and rs10491034 of gene ARHGAP22 are associated with DR incidence and severity among Chinese Hui population.

High-fat diet (HFD) not only induces insulin resistance in liver, but also causes autophagic imbalance and metabolic disorders, increases chronic inflammatory response and induces mitochondrial dysfunction. Calcium/calmodulin-dependent protein kinase IV (CaMKIV) has recently emerged as an important regulator of glucose metabolism and skeletal muscle insulin action. Its activation has been involved in the improvement of hepatic and adipose insulin action. But the underlying mechanism is not fully understood. In the present study, we aimed to address the direct effects of CaMKIV in vivo and to evaluate the potential interaction of impaired insulin sensitivity and autophagic disorders in hepatic insulin resistance. Our results indicated obese mice receiving CaMKIV showed decreased blood glucose and serum insulin and improved insulin sensitivity as well as increased glucose tolerance compared with vehicle injection. Meanwhile, defective hepatic autophagy activity, impaired insulin signaling, increased inflammatory response and mitochondrial dysfunction in liver tissues which are induced by high-fat diet were also effectively alleviated by injection of CaMKIV. Consistent with these results, the addition of CaMKIV to the culture medium of BNL cl.2 hepatocytes markedly restored palmitate-induced hepatic insulin resistance and autophagic imbalance. These effects were nullified by blockade of cyclic AMP response element-binding protein (CREB), indicating the causative role of CREB in action of CaMKIV. Our findings suggested that CaMKIV restores hepatic autophagic imbalance and improves impaired insulin sensitivity via phosphorylated CREB signaling pathway, which may offer novel opportunities for treatment of obesity and diabetes.

Background Frankincense is an oleo gum resin derived from trees of genus Boswellia. It has favorable effects on memory formation. However, the probable underlying molecular mechanisms have not been assessed. Frankincense exerts some of its effects via activation of protein kinases. Calcium/calmodulin kinaseII (CaMKII) and CaMKIV are crucial mediators of learning and memory. We studied the effect of maternal injection of the aqueous extract of frankincense during gestation and lactation periods on spatial memory performance and the mRNA expression levels of the hippocampal CaMKIIand CaMKIV in the offspring rats. Methods Aqueous extract of Frankincense (50 and 100 mg/kg) or tap water was gavaged to distinct female rats during gestation and lactation periods. Memory performance was assessed in groups of male offspring using Morris water maze. In other groups of the offspring (with no memory test), the hippocampi of the juvenile rats were removed 30 days after labor. A real-time PCR method was used to measure the mRNA levels of CaMKII and CaMKIV. Results Frankincense improved spatial memory retrieval in the offspring rats in a dose-dependent manner. The mRNA expression of hippocampal CaMKIV was unchanged between groups. However, the mRNA expression of hippocampal CaMKII was dose-dependently upregulated in the rats, whose mothers had received frankincense. Conclusions Due to the crucial role of the CaMKII in memory formation, the results provide a molecular basis for the effect of administration of frankincense to mother rats on improvement of the memory in the offspring.

Background/Aims: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. Methods: C2C12 and murine myogenic precursor cells (MPCs) were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C2C12 cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. Results: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. Conclusions: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.

Acute and chronic kidney failure is common in hospitalized patients with COVID-19, yet the mechanism of injury and predisposing factors remain poorly understood. We investigated the role of complement activation by determining the levels of deposited complement components (C1q, C3, FH, C5b-9) and immunoglobulin along with the expression levels of the injury-associated molecules spleen tyrosine kinase (Syk), mucin-1 (MUC1) and calcium/calmodulin-dependent protein kinase IV (CaMK4) in the kidney tissues of people who succumbed to COVID-19. We report increased deposition of C1q, C3, C5b-9, total immunoglobulin, and high expression levels of Syk, MUC1 and CaMK4 in the kidneys of COVID-19 patients. Our study provides strong rationale for the expansion of trials involving the use of inhibitors of these molecules, in particular C1q, C3, Syk, MUC1 and CaMK4 to treat patients with COVID-19.

Background: Hypoxia-ischemia (HI) results in increased activation of Ca2+/calmodulin kinase IV (CaM kinase IV) mediated by Src kinase. Therapeutic hypothermia ameliorates neuronal injury in the newborn. Hypothesis: Inhibition of Src kinase concurrently with hypothermia further attenuates the hypoxia-induced increased activation of CaM kinase IV compared with hypothermia alone. Design/Methods: Ventilated piglets were exposed to HI, received saline or a selective Src kinase inhibitor (PP2), and were cooled to 33°C. Neuropathology, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and CaM kinase IV activity were determined. Results: The neuropathology mean score (mean ± SD) was 0.4 ± 0.43 in normoxia-normothermia (p < 0.05 vs. hypoxia-normothermia), 3.5 ± 0.89 in hypoxia-normothermia (p < 0.05 vs. normoxia-normothermia), 0.7 ± 0.73 in hypoxia-hypothermia (p < 0.05 vs. normoxia-normothermia), and 0.5 ± 0.70 in normoxia-hypothermia (p < 0.05 vs. hypoxia-normothermia). The CaM kinase IV activity in cerebral tissue (pmol Pi/mg protein/min; mean ± SD) was 2,002 ± 729 in normoxia-normothermia, 1,704 ± 18 in normoxia-hypothermia, 6,017 ± 2,510 in hypoxia-normothermia, 4,104 ± 542 in hypoxia-hypothermia (p < 0.05 vs. normoxia-hypothermia), and 2,165 ± 415 in hypoxia-hypothermia with PP2 (p < 0.05 vs. hypoxia-hypothermia). The hypoxic groups with and without hypothermia or Src kinase inhibitor were comparable in the levels of ATP and PCr, indicating that they were similar in their degree of energy failure prior to treatments. Hypothermia or Src kinase inhibitor (PP2) did not restore the ATP and PCr levels. Conclusions: Hypothermia and Src kinase inhibition attenuated apoptotic cell death and improved neuropathology after hypoxia. The combination of short-duration hypothermia with Src kinase inhibition following hypoxia further attenuates the increased activation of CaM kinase IV compared to hypothermia alone in the newborn swine brain.

A relationship between alcohol dependence (AD) and calcium/calmodulin-dependent protein kinase IV (CAMKIV) has been reported in a whole genome study of Korean AD patients. The purpose of the present study is to compare the frequency of CAMKIV genotypes and alleles between AD and control subjects in Korea.The present study includes 281 AD patients and 139 control subjects. Seven single nucleotide polymorphism of CAMKIV gene known to show significant separation ratio in Asians were searched in SNP database and previous studies related to CAMKIV gene. Polymerase chain reaction and restriction fragment length polymorphism techniques were used to analyze genotype of CAMKIV gene SNPs.Major TT genotype and T allele frequencies of rs 25917 in AD patients were significantly higher than those of control subjects (genotype frequency, p=0.002; allele frequency, p=0.001). Major CC genotype and C allele frequencies of rs 117590959 in AD patients were also significantly higher than those of control subjects (genotype frequency, p0.001; allele frequency, p=0.001). Major genotypes of rs25917 (p=0.002, odd ratio: 3.13, 95% CI: 1.54-6.38) and rs11790959 (p=0.002, odd ratio: 3.22, 95% CI: 1.52-6.81) showed significantly higher odds ratios associated with AD than minor genotypes in logistic regression.These results suggest that CAMKIV might be a candidate AD gene. Further research is needed to determine the precise relationship between CAMKIV and AD and the function of each SNP.

Recreational use of ketamine (KET) has been increasing worldwide. Previous studies have demonstrated that KET induced neurotoxicity; however, few studies have examined how alcohol (ALC) affects KET-induced neurotoxicity. In light of the fact that some KET abusers combine KET with ALC, the present study was aimed to investigate the effects of ALC on KET-induced neurotoxicity and the underlying mechanism in vitro. Our data revealed that co-treatment with ALC and KET was more detrimental to cell viability than KET single treatment in both PC12 cells and primary cultured rat cortical neurons. Furthermore, ALC exacerbated KET-induced apoptosis characterized by morphological changes and the sub-G1 phase increase, which were mitigated by the pretreatment of CNQX, a known alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainite (KA) receptor antagonist. In addition, ALC and KET co-treatment led to intracellular Ca2+ overload, down-regulation of p-Akt, p-CREB, PKA, CaMK-IV, Bcl-2 and BDNF expression and up-regulation of cleaved caspase-3 and Bax expression, which can be attenuated by CNQX pretreatment. These results indicate that the potentiation of ALC on KET-induced neurotoxicity was related to the down-regulation of CREB-related pathways. Our present study also indicates that ALC and KET co-abuse might cause serious neurotoxicity which should be conveyed to the public and drew enough attention.

Sigma-1 receptor (σ1r) activation could attenuate the learning and memory deficits in the AD model, ischemia model and others. In our previous study, the activation of σ1r increased the expression of brain-derived neurotrophic factor (BDNF), possibly through the NR2A-induced pathway, and σ1r agonists might function as neuroprotectant agents in vascular dementia. Here, we used σ1r knockout mice to confirm the role of σ1r. Furthermore, an antagonist of NR2A was first used to investigate whether the NR2A-induced pathway is the necessary link between σ1r and BDNF. The operation of brain ischemia/reperfusion was induced by bilateral common carotid artery occlusion for 20min in C57BL/6 and σ1r knockout mice as the ischemic group. A σ1r agonist, PRE084 (1mg/kg, i.p.), and NR2A antagonist, PEAQX (10mg/kg, i.p.), were administered once daily throughout the experiment. Behavioral tests were performed starting on day 8. On day 22 after brain ischemia/reperfusion, mice were sacrificed and brains were immediately collected and the injured and the hippocampus was isolated and stored at -80°C for western blot analysis. After ischemic operation, contrast with the σ1r knockout mice, PRE084 significantly ameliorated learning and memory impairments in the behavioral evaluation, and prevented the protein decline of BDNF, NR2A, CaMKIV and TORC1 expression in wild-type mice. However, the effects of PRE084 on CaMKIV-TORC1-CREB and BDNF, even for learning and memory impairment, were antagonized by the co-administration of PEAQX, an antagonist of NR2A. The activation of σ1r improves the impairment of learning and memory in the ischemia/reperfusion model, and the expression of BDNF, which may have been achieved through the NR2A-CaMKIV-TORC1 pathway.

Hepatitis B virus X protein (HBx) plays an important role in the progression of hepatocellular carcinoma. Here we reported that overexpression of HBx in hepatocellular carcinoma (HCC) cells could induce the secretion of high-mobility group box 1 (HMGB1) to promote invasion and metastasis of HCC in an autocrine/paracrine manner. HBx triggered an increase of cytoplasmic calcium and activated CAMKK/CAMKIV pathway, leading to subsequent translocation and release of HMGB1. HMGB1 neutralizing antibody, as well as calcium chelator or inhibitors of CAMKK/CAMKIV, could remarkably reduce invasion and metastasis of HCC cells in vitro and in a murine HCC metastasis model in vivo . Furthermore, the level of HMGB1 in patient serum and tumor tissues was positively correlated with HBV DNA load. We demonstrate an inverse relationship between HMGB1 in tumor cytoplasm and overall prognosis of HCC patients. Conclusion: HBx promotes the progression of HCC through translocation and secretion of HMGB1 from tumor cells via calcium dependent cascades. These data indicates that HMGB1 could serve as a novel prognostic biomarker and potential therapeutic target for HBV-related HCC.

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a multi-functional Ser/Thr kinase, associated with cerebral hypoxia, cancer and neurodegenerative diseases. Here, we report design, synthesis and biological evaluation of seven pyrimidine substituted novel inhibitors of CAMKIV. We successfully synthesized and extensively characterized (ESI-MS, 1H NMR and 13C NMR studies) seven compounds which are showing appreciable binding-affinity to the CAMKIV. Molecular docking and florescence binding studies revealed that compound 1 is showing very high binding free energy (ΔG =-11.52 kcal/mol) and binding-affinity (K= 9.2 x 1010 M−1) to the CAMKIV. We further performed MTT assay to check the cytotoxicity and anti-cancer activity of these compounds. An appreciable IC50 (39μM) value of compound 1 was observed on human hepatoma cell line and non-toxic till the 400 μM on human embryonic kidney cells. To ensure anticancer activity of all these compounds, we further performed propidiumiodide assay to evaluate cell viability and DNA content during the cell cycle. We found compound 1 is again showing a better anticancer activity on both human hepatoma and human embryonic kidney cell lines. This article is protected by copyright. All rights reserved.

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4(+) T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4(+) T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4(+) T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a key regulatory molecule of cell signaling, and thereby controls its growth and proliferation, including expression of certain genes. The overexpression of CAMKIV is directly associated with the development of different types of cancers. Hesperidin is abundantly found in citrus fruits and exhibits wide range of pharmacological activities including anti-inflammatory, antibacterial and anticancerous effects. We have investigated binding mechanism of hesperidin with the CAMKIV using molecular docking methods followed by fluorescence quenching and isothermal titration calorimetric assays. An appreciable binding affinity of hesperidin was observed with CAMKIV during fluorescence quenching and isothermal titration calorimetric studies. Efficacy of hesperidin to inhibit the growth of human hepatic carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cancer cell lines were investigated. Hesperidin has significantly reduced the proliferation of HepG2 and SH-SY5Y cells and induces apoptosis by activating the caspase-3-dependent intrinsic pathway through the upregulation of proapoptotic Bax protein. Hesperidin treatment reduces the mitochondrial membrane potential of HepG2 and SH-SY5Y cells. All these observations clearly anticipated hesperidin a potent inhibitor of CAMKIV which may be further exploited a newer therapeutic approach for the management of different cancer types.

This study was designed to investigate the mechanism by which miR-129-5p affects the biological function of liver cancer cells. The expression levels of miR-129–5p in liver cancer tissues and cells were, respectively, determined. Crystal violet staining and flow cytometry were used to detect cell proliferation and apoptosis. Wound healing assay and transwell assay were performed to test cell migration and invasion. The target gene of miR-129–5p was analyzed and verified by bioinformatics analysis and luciferase reporter assay. Tumorigenicity assays in nude mice were used to test the antitumor ability of calcium calmodulin-dependent protein kinase IV (CAMK4). miR-129–5p was found to be underexpressed in hepatocellular cancer tissues and cells and also to inhibit liver cells proliferation, migration, and invasion and promote apoptosis. CAMK4 was a direct target for miR-129–5p and was lowly expressed in liver cancer tissues and cells. CAMK4 was also found to inhibit liver cells proliferation, migration and invasion, and promote apoptosis. CAMK4 might exert an antitumor effect by inhibiting the activation of mitogen-activated protein kinase (MAPK). MiR-129–5p was a tumor suppressor with low expression in liver cancer tissues and cells. CAMK4, which is a direct target gene of miR-129–5p, could inhibit tumor by inhibiting the activation of MAPK signaling pathway.

In the present study, we examined the importance of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of cardiac function using genetically modified CaMKIV-null mice. RT-PCR analysis revealed decreased expression of voltage-dependent calcium channels in the cardiac myocytes of CaMKIV-null mice compared with wild-type mice. CaMKIV-null mice showed shortened QT time on electrocardiograms. Pharmacological analysis revealed decreased responsiveness to the β-adrenergic blocker propranolol in CaMKIV-null mice, whereas the plasma norepinephrine level was not affected. CaMKIV-null mice showed decreased baroreflex on electrocardiograms. Heart rate variability analysis showed unstable R-R intervals, a decreased low frequency power/high frequency power (LF/HF) ratio, and increased standard deviation of the normal to normal R-R intervals (SDNN) in CaMKIV-null mice, suggesting decreased responsiveness to β-adrenergic stimulation in CaMKIV-null mice. Atrial contraction analysis and cardiac action potential recording showed a decreased response to the β-adrenoceptor agonist isoproterenol in CaMKIV-null mice. Furthermore, fluorescence imaging in a CRE-hrGFP assay revealed a decreased response to isoproterenol in CaMKIV-null cardiac myocytes. Taken together, our data strongly suggest a significant effect of CaMKIV gene ablation on cardiac β-adrenergic signal transduction.

Mounting evidence suggests that epigenetic modifications play critical roles in the survival/death of stressed neurons. Chief among these modifications is the deacetylation of histones within the chromatin by histone deacetylases (HDACs). HDAC4 is highly expressed in neurons and is usually trapped in cytosol. However, tightly regulated signal-dependent shuttling of this molecule between cytosol and nucleus occurs. Here, we studied the intracellular trafficking of HDAC4 and regulatory mechanisms during stroke. HDAC4 translocated from the cytosol into the nucleus of neurons in response to stroke induced by middle cerebral artery occlusion (MCAO) in mice. Similar translocation was seen after oxygen-glucose deprivation (OGD) in cultured mouse neurons. Expression of nuclear-restricted HDAC4 increased neuronal death after OGD and worsened infarcts and functional deficits in mice following MCAO; however, expression of cytosolic-restricted HDAC4 did not affect outcome after ischemia. In contrast, HDAC4 knockdown with siRNA improved neuronal survival after OGD. Furthermore, expression of nuclear-restricted HDAC4 reduced the acetylation of histones 3 and 4 as well as the levels of pro-survival downstream molecules after OGD. Finally, genetic deletion of calcium/calmodulin-dependent protein kinase IV (CaMKIV) increased the nuclear accumulation of HDAC4 in MCAO model, while overexpression of CaMKIV reduced the levels of nuclear HDAC4 following OGD. When HDAC4 was inhibited, the neuroprotection provided by CaMKIV overexpression was absent during OGD. Our data demonstrate a detrimental role of the nuclear accumulation of HDAC4 following stroke and identify CaMKIV as a key regulator of neuronal intracellular HDAC4 trafficking during stroke.

The calcium/calmodulin-dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family, and is primarily involved in transcriptional regulation in lymphocytes, neurons and male germ cells. CAMKIV operates the signaling cascade and regulates activity of several transcription activators by phosphorylation, which in turn plays pivotal roles in immune response, inflammation and memory consolidation. In this review, we tried to focus on different aspects of CAMKIV to understand the significance of this protein in the biological system. This enzyme is associated with varieties of disorders such as cerebral hypoxia, azoospermia, endometrial and ovarian cancer, systemic lupus, etc., and hence it is considered as a potential therapeutic target. Structure of CAMKIV is comprised of five distinct domains in which kinase domain is responsible for enzyme activity. CAMKIV is involved in varieties of cellular functions such as regulation of gene expression, T-cell maturation, regulation of survival phase of dendritic cells, bone growth and metabolism, memory consolidation, sperm motility, regulation of microtubule dynamics, cell-cycle progression and apoptosis. In this review, we performed an extensive analysis on structure, function and regulation of CAMKIV and associated diseases.