Understanding the pathogenic significance of altered calcium-calmodulin signaling in T cells in autoimmune diseases.

Calcium/calmodulin-dependent protein kinase IV (CaMK4) serves as a pivotal mediator in the regulation of gene expression, influencing the activity of transcription factors within a variety of immune cells, including T cells. Altered CaMK4 signaling is implicated in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and psoriasis, which are characterized by dysregulated immune responses and clinical complexity. These conditions share common disturbances in immune cell functionality, cytokine production, and autoantibody generation, all of which are associated with disrupted calcium-calmodulin signaling. This review underscores the consequences of dysregulated CaMK4 signaling across these diseases, with an emphasis on its impact on Th17 differentiation and T cell metabolism-processes central to maintaining immune homeostasis. A comprehensive understanding of roles of CaMK4 in gene regulation across various autoimmune disorders holds promise for the development of targeted therapies, particularly for diseases driven by Th17 cell dysregulation.

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.

Calcium/calmodulin dependent protein kinase IV in trophoblast cells under insulin resistance: functional and metabolomic analyses.

BACKGROUND: Insulin resistance (IR) is an important determinant of glucose metabolic disturbance and placental dysplasia in gestational diabetes mellitus (GDM). Calcium/calmodulin dependent protein kinase IV (CAMK4) improves insulin IR induced by a high-fat diet (HFD). The current study sought to elucidate the role and potential mechanism of CAMK4 in GDM. METHODS: A GDM model was established in female C57BL/6J mice via HFD feeding for one week before mating and throughout gestation. The IR was elicited by 10-6 M insulin treatment for 48 h in HTR-8/SVneo cells and mouse primary trophoblast cells. The function of CAMK4 was investigated by transfection of overexpression plasmid in HTR-8/SVneo cells and infection of lentivirus loaded with CAMK4 encoding sequence in primary trophoblast cells. Real-time PCR, western blot, cell counting kit-8, transwell, wound healing, dual-luciferase reporter assay, and liquid chromatography/mass spectrometry-based untargeted metabolomics were performed to confirm the effects of CAMK4 on trophoblast cells. RESULTS: Decreased CAMK4 expression was found in the placenta of GDM mice. CAMK4 overexpression ameliorated IR-induced viability impairment, migratory and invasive capacity inhibition, autophagy blocking, insulin signaling inactivation and glucose uptake disorder in trophoblast cells. CAMK4 also transcriptionally activated orphan nuclear receptor NUR77, and the effects of CAMK4 were abrogated by silencing of NUR77. Metabolomics analysis revealed that CAMK4 overexpression caused alterations of amino acid, lipid and carbohydrate metabolism, which were important in GDM. CONCLUSION: Our results indicated that CAMK4/NUR77 axis may provide novel potential targets in GDM treatment.

Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.

Fasudil compensates podocyte injury via CaMK4/Rho GTPases signal and actin cytoskeleton-dependent activation of YAP in MRL/lpr mice.

Deposition of immune complexes in the glomerulus leads to irreversible renal damage in lupus nephritis (LN), of which podocyte malfunction arises earlier. Fasudil, the only Rho GTPases inhibitor approved in clinical settings, possesses well-established renoprotective actions; yet, no studies addressed the amelioration derived from fasudil in LN. To clarify, we investigated whether fasudil exerted renal remission in lupus-prone mice. In this study, fasudil (20 mg/kg) was intraperitoneally administered to female MRL/lpr mice for 10 weeks. We report that fasudil administration swept antibodies (anti-dsDNA) and attenuated systemic inflammatory response in MRL/lpr mice, accompanied by preserving podocyte ultrastructure and averting immune complex deposition. Mechanistically, it repressed the expression of CaMK4 in glomerulopathy by preserving nephrin and synaptopodin expression. And fasudil further blocked cytoskeletal breakage in the Rho GTPases-dependent action. Further analyses showed that beneficial actions of fasudil on the podocytes required intra-nuclear YAP activation underlying actin dynamics. In addition, in vitro assays revealed that fasudil normalized the motile imbalance by suppressing intracellular calcium enrichment, thereby contributing to the resistance of apoptosis in podocytes. Altogether, our findings suggest that the precise manners of crosstalks between cytoskeletal assembly and YAP activation underlying the upstream CaMK4/Rho GTPases signal in podocytes is a reliable target for podocytopathies treatment, and fasudil might serve as a promising therapeutic agent to compensate for the podocyte injury in LN.

Ca2+/calmodulin-dependent protein kinase IV attenuates inflammation and mitochondrial dysfunction under insulin resistance in C2C12 cells.

CaMKIV has been reported involved in the improvement of whole-body insulin sensitivity and mitochondrial biogenesis of skeletal muscle. Here, we first investigate the effects of CaMKIV on glucose metabolism, cell viability, inflammatory function, and mitochondrial function in palmitate-induced C2C12 cells of insulin resistance. Then we explored the potential mechanism of these effects. Differentiated C2C12 cells were treated with or without 100 ng/ml of CaMKIV under palmitate-induced insulin resistance. The results suggest palmitate induced insulin sensitivity, reduced glucose uptake, decreased cell viability, increased inflammatory factors, and caused mitochondrial dysfunction in C2C12 cells. Of note, CaMKIV reversed palmitate-induced insulin resistance, increased the reduction of glucose uptake, inhibited inflammatory response, and mitochondrial dysfunction, despite of no change in cells viabilities. However, these beneficial effects of CaMKIV were blocked by the downregulation of CREB1. Taken together, our data demonstrated CaMKIV prevents palmitate-induced insulin resistance, inflammatory response, and mitochondrial dysfunction through phosphorylated CREB1 in differentiated C2C12 cells.

Inhibition of calcium/calmodulin-dependent protein kinase IV in arthritis: dual effect on Th17 cell activation and osteoclastogenesis.

OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase IV (CaMK4) in the development of joint injury in a mouse model of arthritis and patients with RA. METHODS: Camk4-deficient, Camk4flox/floxLck-Cre, and mice treated with CaMK4 inhibitor KN-93 or KN-93 encapsulated in nanoparticles tagged with CD4 or CD8 antibodies were subjected to collagen-induced arthritis (CIA). Inflammatory cytokine levels, humoral immune response, synovitis, and T-cell activation were recorded. CAMK4 gene expression was measured in CD4+ T cells from healthy participants and patients with active RA. Micro-CT and histology were used to assess joint pathology. CD4+ and CD14+ cells in patients with RA were subjected to Th17 or osteoclast differentiation, respectively. RESULTS: CaMK4-deficient mice subjected to CIA displayed improved clinical scores and decreased numbers of Th17 cells. KN-93 treatment significantly reduced joint destruction by decreasing the production of inflammatory cytokines. Furthermore, Camk4flox/floxLck-Cre mice and mice treated with KN93-loaded CD4 antibody-tagged nanoparticles developed fewer Th17 cells and less severe arthritis. CaMK4 inhibition mitigated IL-17 production by CD4+ cells in patients with RA. The number of in vitro differentiated osteoclasts from CD14+ cells in patients with RA was significantly decreased with CaMK4 inhibitors. CONCLUSION: Using global and CD4-cell-targeted pharmacologic approaches and conditionally deficient mice, we demonstrate that CaMK4 is important in the development of arthritis. Using ex vivo cell cultures from patients with RA, CaMK4 is important for both Th17 generation and osteoclastogenesis. We propose that CaMK4 inhibition represents a new approach to control the development of arthritis.

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.

CaMK4 overexpression in polycystic kidney disease promotes mTOR-mediated cell proliferation.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of fluid-filled cysts, causing nephron loss and a decline in renal function. Mammalian target of rapamycin (mTOR) is overactive in cyst-lining cells and contributes to abnormal cell proliferation and cyst enlargement; however, the mechanism for mTOR stimulation remains unclear. We discovered that calcium/calmodulin (CaM) dependent kinase IV (CaMK4), a multifunctional kinase, is overexpressed in the kidneys of ADPKD patients and PKD mouse models. In human ADPKD cells, CaMK4 knockdown reduced mTOR abundance and the phosphorylation of ribosomal protein S6 kinase (S6K), a downstream target of mTOR. Pharmacologic inhibition of CaMK4 with KN-93 reduced phosphorylated S6K and S6 levels and inhibited cell proliferation and in vitro cyst formation of ADPKD cells. Moreover, inhibition of calcium/CaM-dependent protein kinase kinase-ß and CaM, two key upstream regulators of CaMK4, also decreased mTOR signaling. The effects of KN-93 were independent of the liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathway, and the combination of KN-93 and metformin, an AMPK activator, had additive inhibitory effects on mTOR signaling and in vitro cyst growth. Our data suggest that increased CaMK4 expression and activity contribute to mTOR signaling and the proliferation of cystic cells of ADPKD kidneys.

Calcium/calmodulin-dependent protein kinase IV promotes imiquimod-induced psoriatic inflammation via macrophages and keratinocytes in mice.

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-treated Camk4-/- mice compared to IMQ-treated WT mice. CaMK4 inhibits IL-10 production by macrophages, thus allowing excessive psoriatic inflammation. Deletion of Camk4 in 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 as S100A8 and CAMP. Taken together, these data indicate that CaMK4 regulates IMQ-induced psoriasis by sustaining inflammation and provides a potential target for psoriasis treatment.

CaMK4 Promotes Acute Lung Injury Through NLRP3 Inflammasome Activation in Type II Alveolar Epithelial Cell.

Background: Type 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. Methods: CaMK4 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-1ß and IL-18 were measured by ELISA. Results: Phosphorylation 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-1ß production. The inhibition of NLRP3 inflammasome and decreased IL-1ß/IL-18 production by KN-93 led to reduced inflammatory infiltration and ameliorated lung injury in LPS-induced ALI mice. Conclusion: CaMK4 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.

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.

Magnesium promotes osteogenesis via increasing OPN expression and activating CaM/CaMKIV/CREB1 pathway.

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 (Mg2+ ), one of the degradation products, could stimulate osteogenesis. Here, we investigated the osteogenesis property of magnesium both in vitro and in vivo, and to identify the cellular and molecular mechanisms that mediate these effects. Results showed that magnesium exerts a dose-dependent increase in the proliferation of MC3T3 and MG63 cells, and in the expression of osteopontin (OPN), a promising biomarker of osteogenesis. Subsequently, the protein-protein interaction (PPI) network analysis showed the interactions between calmodulin (CaM) and calmodulin-dependent protein kinase (CaMK) and CREB1. The ratio of p-CaMKIV/CaMKIV and p-CREB1/CREB were increased at protein level in MC3T3 and MG63 cells after treatment with Mg2+ . Dual-luciferase reporter gene assay showed that p-CREB1 could directly bind to OPN promoter and up-regulate the transcription of OPN after nuclear entry. Meanwhile, the expression of OPN and p-CREB1, which increased after Mg2+ treatment, was down-regulated by sh-CaMKIV or sh-CREB1. Moreover, the mineralized deposit and expression of OPN were reduced after treatment with an inhibitor of CaMKIV, KN93. In addition, massive cavities in the cortical bone around the Mg screw were showed in vivo after injection of KN93. These data indicated that the osteogenic effect of Mg is related to the activation OPN through CaM/CaMKIV/CREB1 signaling pathway.

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. (AU)

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.

Ginsenoside Re exhibits neuroprotective effects by inhibiting neuroinflammation via CAMK/MAPK/NF­κB signaling in microglia.

Ginsenoside Re (G­Re) is a panaxatriol saponin and one of the pharmacologically active natural constituents of ginseng (Panax ginseng C.A. Meyer). G­Re has antioxidant, anti­inflammatory and antidiabetic effects. The present study aimed to investigate the effects of G­Re 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 G­Re significantly inhibited the production of IL­6, TNF­α, nitric oxide (NO) and ROS in BV2 microglial cells, and that of NO in mouse primary microglia, without affecting cell viability. G­Re also inhibited the nuclear translocation of NF­κB, and phosphorylation and degradation of IκB­α. In addition, G­Re dose­dependently suppressed LPS­mediated phosphorylation of Ca2+/calmodulin­dependent protein kinase (CAMK)2, CAMK4, extracellular signal­regulated kinase (ERK) and c­Jun N­terminal kinases (JNK). Moreover, the conditioned medium from LPS­stimulated microglial cells induced HT22 hippocampal neuronal cell death, whereas that from microglial cells incubated with both LPS and G­Re ameliorated HT22 cell death in a dose­dependent manner. These results suggested that G­Re suppressed the production of pro­inflammatory mediators by blocking CAMK/ERK/JNK/NF­κB signaling in microglial cells and protected hippocampal cells by reducing these inflammatory and neurotoxic factors released from microglial cells. The present findings indicated that G­Re may be a potential treatment option for neuroinflammatory disorders and could have therapeutic potential for various neurodegenerative diseases.