CaMKIV-Mediated Phosphorylation Inactivates Freud-1/CC2D1A Repression for Calcium-Dependent 5-HT1A Receptor Gene Induction.

Calcium calmodulin-dependent protein kinase (CaMK) mediates calcium-induced neural gene activation. CaMK also inhibits the non-syndromic intellectual disability gene, Freud-1/CC2D1A, a transcriptional repressor of human serotonin-1A (5-HT1A) and dopamine-D2 receptor genes. The altered expression of these Freud-1-regulated genes is implicated in mental illnesses such as major depression and schizophrenia. We hypothesized that Freud-1 is blocked by CaMK-induced phosphorylation. The incubation of purified Freud-1 with either CaMKIIα or CaMKIV increased Freud-1 phosphorylation that was partly prevented in Freud-1-Ser644Ala and Freud-1-Thr780Ala CaMK site mutants. In human SK-N-SH neuroblastoma cells, active CaMKIV induced the serine and threonine phosphorylation of Freud-1, and specifically increased Freud-1-Thr780 phosphorylation in transfected HEK-293 cells. The activation of purified CaMKIIα or CaMKIV reduced Freud-1 binding to its DNA element on the 5-HT1A and dopamine-D2 receptor genes. In SK-N-SH cells, active CaMKIV but not CaMKIIα blocked the Freud-1 repressor activity, while Freud-1 Ser644Ala, Thr780Ala or dual mutants were resistant to inhibition by activated CaMKIV or calcium mobilization. These results indicate that the Freud-1 repressor activity is blocked by CaMKIV-induced phosphorylation at Thr780, resulting in the up-regulation of the target genes, such as the 5-HT1A receptor gene. The CaMKIV-mediated inhibition of Freud-1 provides a novel de-repression mechanism to induce 5-HT1A receptor expression for the regulation of cognitive development, behavior and antidepressant response.

CaMK4: Structure, physiological functions, and therapeutic potential.

Calcium/calmodulin-dependent protein kinase IV (CaMK4) is a versatile serine/threonine kinase involved in various cellular functions. It regulates T-cell differentiation, podocyte function, tumor cell proliferation/apoptosis, ß cell mass, and insulin sensitivity. However, the underlying molecular mechanisms are complex and remain incompletely understood. The aims of this review are to highlight the latest advances in the regulatory mechanisms of CaMK4 underlying T-cell imbalance and parenchymal cell mass in multiple diseases. The structural motifs and activation of CaMK4, as well as the potential role of CaMK4 as a novel therapeutic target are also discussed.

Nuclear Binding Protein 2/Nesfatin-1 Affects Trophoblast Cell Fusion during Placental Development via the EGFR-PLCG1-CAMK4 Pathway.

Previous studies have shown that nuclear binding protein 2 (NUCB2) is expressed in the human placenta and increases with an increase in the syncytialization of trophoblast cells. This study aimed to investigate the role of NUCB2 in the differentiation and fusion of trophectoderm cells. In this study, the expression levels of NUCB2 and E-cadherin in the placentas of rats at different gestation stages were investigated. The results showed that there was an opposite trend between the expression of placental NUCB2 and E-cadherin in rat placentas in different trimesters. When primary human trophoblast (PHT) and BeWo cells were treated with high concentrations of Nesfatin-1, the trophoblast cell syncytialization was significantly inhibited. The effects of NUCB2 knockdown in BeWo cells and Forskolin-induced syncytialization were investigated. These cells showed a significantly decreased cell fusion rate. The mechanism underlying NUCB2-regulated trophoblast cell syncytialization was explored using RNA-Seq and the results indicated that the epidermal growth factor receptor (EGFR)-phospholipase C gamma 1 (PLCG1)-calmodulin-dependent protein kinase IV (CAMK4) pathway might be involved. The results suggested that the placental expression of NUCB2 plays an important role in the fusion of trophoblasts during differentiation via the EGFR-PLCG1-CAMK4 pathway.

CaMKIV/CREB/BDNF signaling pathway expression in prefrontal cortex, amygdala, hippocampus and hypothalamus in streptozotocin-induced diabetic mice with anxious-like behavior.

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.

CFNAs of RBCs affect the release of inflammatory factors through the expression of CaMKIV in macrophages.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Resveratrol ameliorates polycystic ovary syndrome via transzonal projections within oocyte-granulosa cell communication.

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 (CaMKIIß) 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 CaMKIIß. Conclusions: Our data indicated that upregulation of CaMKIIß 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 CaMKIIß activators provides new insight and a therapeutic target for PCOS, which is helpful for improving female reproduction.

Calcium/calmodulin-dependent protein kinase IV regulates vascular autophagy and insulin signaling through Akt/mTOR/CREB pathway in ob/ob mice.

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.

Atractylodin inhibits fructose-induced human podocyte hypermotility via anti-oxidant to down-regulate TRPC6/p-CaMK4 signaling.

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.

CaMK4 is a downstream effector of the α1G T-type calcium channel to determine the angiogenic potential of pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α1G-subtype of voltage-gated T-type Ca2+ channel. We have previously revealed that the α1G channel functions as a background Ca2+ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α1G-mediated Ca2+ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α1G-mediated Ca2+ entry to Akt phosphorylation remain incompletely understood. In this study, we demonstrate that Ca2+/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α1G-mediated Ca2+ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α1G/Ca2+/CaMK4 is independent of the α1G/Ca2+/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α1G-CaMK4 signaling complex that regulates the Ca2+-dominated angiogenic potential in PMVECs.

Atractylodis rhizoma water extract attenuates fructose-induced glomerular injury in rats through anti-oxidation to inhibit TRPC6/p-CaMK4 signaling.

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.

Sevoflurane induces inflammation of microglia in hippocampus of neonatal rats by inhibiting Wnt/ß-Catenin/CaMKIV pathway.

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/ß-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, ß-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, ß-Catenin and CaMKIV. These results suggested that sevoflurane inhibited Wnt/ß-Catenin/CaMKIV pathway. CONCLUSION: Sevoflurane induces inflammation of microglia in hippocampus of neonatal rats by inhibiting Wnt/ß-Catenin/CaMKIV pathway.

Aberrantly glycosylated IgG elicits pathogenic signaling in podocytes and signifies lupus nephritis.

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.

The Regulating Mechanism of Chrysophanol on Protein Level of CaM-CaMKIV to Protect PC12 Cells Against Aß25-35-Induced Damage.

OBJECTIVE: To investigate the neuroprotective effect of chrysophanol (CHR) on PC12 treated with Aß25-35, and the involved mechanism. METHODS: After the establishment of an AD cell model induced by Aß25-35, 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 Aß25-35, PC12 cells showed that increased cell viability and reduced apoptosis. The qRT-PCR results indicated that the deposition of Aß25-35 triggers a decrease in levels of CaM, CaMKK, CaMKIV, and tau in PC12 cells. In addition, Western blot results also suggested that Aß25-35 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.

Nicotine Rescues Depressive-like Behaviors via α7-type Nicotinic Acetylcholine Receptor Activation in CaMKIV Null Mice.

The nicotinic acetylcholine receptors (nAChRs) are essential for acetylcholine-mediated signaling. Two major functional subtypes of nAChR in the brain, α7-type and α4ß2-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-ß-erythroidine, an α4ß2-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.

CaMK4 promotes abortion-related Th17 cell imbalance by activating AKT/mTOR signaling pathway.

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.

CaMK4-dependent phosphorylation of Akt/mTOR underlies Th17 excessive activation in experimental autoimmune prostatitis.

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.

DJ-1 regulates tyrosine hydroxylase expression through CaMKKß/CaMKIV/CREB1 pathway in vitro and in vivo.

Lack of dopamine production and neurodegeneration of dopaminergic neurons in the substantia nigra are considered as the major characteristics of Parkinson's disease, a prevalent movement disorder worldwide. DJ-1 mutation leading to loss of its protein functions is a genetic factor of PD. In this study, our results illustrated that DJ-1 can directly interact with Ca2+ /calmodulin-dependent protein kinase kinase ß (CaMKKß) and modifies the cAMP-responsive element binding protein 1 (CREB1) activity, thus regulates tyrosine hydroxylase (TH) expression. In Dj-1 knockout mouse substantia nigra, the levels of TH and the phosphorylation of CREB1 Ser133 are significantly decreased. Moreover, Dj-1 deficiency suppresses the phosphorylation of CaMKIV (Thr196/200) and CREB1 (Ser133), subsequently inhibits TH expression in vitro. Furthermore, Knockdown of Creb1 abolishes the effects of DJ-1 on TH regulation. Our data reveal a novel pathway in which DJ-1 regulates CaMKKß/CaMKIV/CREB1 activities to facilitate TH expression.

MiR-129-5p inhibits liver cancer growth by targeting calcium calmodulin-dependent protein kinase IV (CAMK4).

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.

Hesperidin-CAMKIV interaction and its impact on cell proliferation and apoptosis in the human hepatic carcinoma and neuroblastoma cells.

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.

Aberrant Amygdala-Dependent Cued Fear Memory in Na+/Ca2+ Exchanger 1 Heterozygous Mice.

Na+/Ca2+ exchangers (NCXs) are mainly expressed in the plasma membrane and exchange one Ca2+ for three Na+, depending on the electrochemical gradients across the plasma membrane. NCXs have three isoforms, NCX1-3, encoded by distinct genes in mammals. Here, we report that heterozygous mice lacking NCX1 (NCX1+/-) exhibit impaired amygdala-dependent cued fear memory. NCX1+/- mice showed significant impairment in fear-related behaviors measured with the elevated-plus maze, light-dark, open-field, and marble-burying tasks. In addition, NCX1+/- mice showed abnormality in cued fear memory but not in contextual fear memory in a fear-conditioning task. In immunohistochemical analyses, NCX1+/- mice had significantly increased number of c-Fos-positive cells in the lateral amygdala (LA) but not in the central amygdala following fear-related tone stimuli. c-Fos expression peaked at 1 h. In concordance with the aberrant fear-related behaviors in NCX1+/- mice, enhanced long-term potentiation was also observed in the LA of these mice. Furthermore, enhancement of CaMKII or CaMKIV activity in the LA was observed in NCX1+/- mice by immunoblot analyses. In contrast, CaMKII+/- but not CaMKIV-/- mice insufficiently exhibited tone-induced cued fear memory and there was no increase in the number of c-Fos-positive cells in the LA. Altogether, the increased CaMKII activity and consequent c-Fos expression likely account for the dysregulation of amygdala-dependent cued fear memory in NCX1+/- mice.