Calcium/calmodulin-dependent protein kinase IV signaling pathway is upregulated in experimental necrotizing enterocolitis.

PURPOSE: 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. METHODS: 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 < 0.05 was considered significant. RESULTS: Intestinal injury was induced in the NEC mice and confirmed by histological scoring and inflammatory cytokine IL6. CaMKIV and its downstream target genes of CREM and IL17 were significantly elevated in NEC mice relative to control. Similarly, phosphorylated-CaMKIV (pCaMKIV), the active form of CaMKIV, was more notably expressed in the NEC ileal tissue relative to control ileal tissue. Elevated pCaMKIV protein expression was also confirmed by western blot. CONCLUSION: CaMKIV expression and activation are upregulated in experimental NEC suggesting a potential contributing factor in the pathogenesis of NEC.

Frankincense upregulates the hippocampal calcium/calmodulin kinase II-α during development of the rat brain and improves memory performance.

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.

Inhibition of CaMKIV relieves streptozotocin-induced diabetic neuropathic pain through regulation of HMGB1.

BACKGROUND: The pathogenesis of diabetic neuropathic pain is complicated and its underlying mechanisms remain unclear. Calmodulin-dependent protein kinases (CaMKs) IV (CaMKIV), one of CaMKs, regulates several transcription factors in pain mechanisms. High-mobility group box 1 (HMGB1) is a key mediator in diabetic neuropathic pain. This study aims to find the roles and mechanisms of CaMIV in diabetic neuropathic pain. METHODS: Diabetic animal models were constructed by injecting with streptozotocin (STZ) intraperitoneally. They were randomly divided into seven groups (n = 6 per group): Naive, Normal Saline, STZ, STZ + Sham, STZ + DMSO and STZ + KN93 (an inhibitor of CaMKIV) (50 µg), STZ + KN93 (100 µg), which received KN93 (50 or 100 µg) intrathecally after the administration of STZ. Phospho-CaMKIV (pCaMKIV) and HMGB1 expression in rat dorsal root ganglion (DRG) and RAW264.7 cell line were measured by western blot. Distribution of pCaMKIV immune reactivity in different subpopulations of DRG neurons was measured by double-immunofluorescence staining. RESULTS: The pCaMKIV and HMGB1 in DRG significantly increased after STZ administration, and pCaMKIV can regulate the expression of HMGB1 based on both cellular and animal models. Pretreatment with CaMKIV inhibitor attenuated STZ-induced mechanical allodynia and thermal hyperalgesia, as well as reduced HMGB1 expression in the DRG. CONCLUSIONS: This study demonstrates that CaMKIV can relieve STZ-induced diabetic neuropathic pain. The mechanism of this function depended on the process: pCaMKIV localized in the nuclei of DRG neurons and regulated HMGB1 which was an important mediator of neuropathic pain. These findings reported CaMKIV may be a potential target or important node in relieving diabetic neuropathic pain.

Inhibition of calcium(2+)/calmodulin-dependent protein kinase type IV ameliorates experimental nephrotic syndrome.

OBJECTIVE: Evidence has demonstrated that Ca(2+)/calmodulin-dependent protein kinase type IV (CaMKIV) contributes to altered cytokine production by promoting the production of inflammatory cytokines. This study aimed to explore the protective role and underlying mechanisms of CaMKIV inhibition in experimental nephrotic syndrome. METHODS: BALB/c mice received single intravenous injections of adriamycin (10 mg/kg) then were sacrificed at two, four and six weeks. In the second study, treatment with KN-93, a CaMKIV inhibitor, or vehicle administered via intraperitoneal injection was started five days after adriamycin injection. Functional and pathologic parameters, the presence of inflammatory infiltration and the expressions of pro-inflammatory cytokines were assessed. RESULTS: The CaMKIV protein expression levels were upregulated in the mice with adriamycin nephropathy, which was significantly inhibited by KN-93 (p<0.01). As compared with the vehicle-treated controls, KN-93 treatment resulted in marked suppression of proteinuria and serum creatinine at week 6 (p<0.01), but not at two weeks after induction of the disease. KN-93 inhibited glomerulosclerosis and the development of tubulointerstitial lesions. The renal alpha-smooth muscle actin (α-SMA) expression was also significantly suppressed by KN-93 treatment at week 6 (p<0.01). Moreover, KN-93 inhibited the renal monocyte chemoattractant protein-1 (MCP-1) expression, paralleled by a reduction in the interstitial infiltration of macrophages and T-cells (p<0.01). CONCLUSION: Our findings suggest that activation of CaMKIV signaling is involved in the progression of glomerular diseases with a proteinuric state. Our data therefore justify the development of small molecule CaMKIV inhibitors for the treatment of clinical nephrotic syndrome.

Involvement of CaMKIV in neurogenic effect with chronic fluoxetine treatment.

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that has been suggested to participate in fluoxetine (FLX)-induced phosphorylation of cyclic AMP-response element binding protein (CREB). CREB is a key transcription factor in adult neurogenesis. The present study aimed at evaluating whether CaMKIV is involved in adult hippocampal neurogenesis with FLX treatment. Effects of chronic FLX on hippocampal cell proliferation, survival and phenotypes were assessed using bromodeoxyuridine (BrdU) immunohistochemistry or BrdU/neuronal nuclei (NeuN)/S100ß immunofluorescence staining in wild-type (WT) and CaMKIV knockout (KO) mice. Expression and phosphorylation of CaMKIV and CREB were assessed using RT-PCR and Western blotting. The behavioural action with FLX was assessed in the novelty suppressed feeding test (NSF), which is considered neurogenesis-dependent. CaMKIV KO mice have reduced cell proliferation, but not survival in the dentate gyrus of hippocampus with chronic treatment of FLX when compared to wild littermates. Phenotype analysis showed that most newborn cells matured into neurons. Phosphorylation of CaMKIV was up-regulated in WT mice and phosphorylation of CREB was impaired in CaMKIV KO mice after FLX treatment. The behavioural effects of FLX in NSF were similar in both types. These data suggest that CaMKIV is involved in some aspects of FLX-promoting hippocampal neurogenesis.

Calcium/calmodulin-dependent protein kinase IV suppresses IL-2 production and regulatory T cell activity in lupus.

The activity of calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased in T cells from patients with systemic lupus erythematosus (SLE) and has been shown to reduce IL-2 production by promoting the effect of the transcriptional repressor cAMP responsive element modulator-α on the IL2 promoter. In this article, we demonstrate that T cells from MRL/lpr mice display increased levels of CaMK4 in the nucleus, and that genetic deletion of Camk4 results in improved survival. We demonstrate that absence of CaMK4 restores IL-2 production, curbs increased T cell activation, and augments the number and activity of regulatory T cells. Analogously, silencing of CaMK4 in T cells from patients with SLE increases the expression of FoxP3 on stimulation in the presence of TGF-ß. Our results demonstrate the importance of the serine/threonine kinase CaMK4 in the generation and function of regulatory T cells in patients with SLE and lupus-prone mice, and its potential to serve as a therapeutic target.

Curcuminoids enhance memory in an amyloid-infused rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease. There are a limited number of therapeutic options available for the treatment of AD. Curcuminoids (a mixture of bisdemethoxycurcumin, demethoxycurcumin and curcumin) is the main chemical constituent found in turmeric, a well known curry spice, having potential in the treatment of AD. The objective of this study was to investigate the effects of curcuminoid mixture and individual constituents on spatial learning and memory in an amyloid-beta (Abeta) peptide-infused rat model of AD and on the expression of PSD-95, synaptophysin and camkIV. Curcuminoid mixture showed a memory-enhancing effect in rats displaying AD-like neuronal loss only at 30 mg/kg, whereas individual components were effective at 3-30 mg/kg. A shorter duration treatment with test compounds showed that the curcuminoid mixture and bisdemethoxycurcumin increased PSD-95 expression in the hippocampus at 3-30 mg/kg, with maximum effect at a lower dose (3 mg/kg) with respective values of 470.5 and 587.9%. However, after a longer duration treatment, two other compounds (demethoxycurcumin and curcumin) also increased PSD-95 to 331.7 and 226.2% respectively at 30 mg/kg. When studied for their effect on synaptophysin in the hippocampus after the longer duration treatment, the curcuminoid mixture and all three individual constituents increased synaptophysin expression. Of these, demethoxycurcumin was the most effective showing a 350.1% increase (P<0.01) at 30 mg/kg compared to the neurotoxin group. When studied for their effect on camkIV expression after longer treatment in the hippocampus, only demethoxycurcumin at 30 mg/kg increased levels to 421.2%. These compounds salvaged PSD-95, synaptophysin and camkIV expression levels in the hippocampus in the rat AD model, which suggests multiple target sites with the potential of curcuminoids in spatial memory enhancing and disease modifying in AD.

Lanthanum chloride impairs memory, decreases pCaMK IV, pMAPK and pCREB expression of hippocampus in rats.

Surveys have reported that rare-earth elements (REEs) could impair cognitive functions of children. Experimental studies have shown the neurological adverse effects of REEs on animals. However, the mechanism underlying these impairments is unclear. Lanthanum is often selected to study the effects of REEs. The purpose of this study was to investigate the memory impairment induced by lanthanum chloride (LaCl3) exposure and the possible mechanism from the aspects of expression of CREB signal pathway and synaptic ultrastructure in the hippocampus. Lactational rats were exposed to 0%, 0.25%, 0.50%, and 1.0% LaCl3 in drinking water, respectively. Their offspring were exposed to LaCl3 by parental lactation for 3 weeks and then administrated with 0%, 0.25%, 0.50% and 1.0% LaCl3 in drinking water for 1 month. The results showed that 0.25%, 0.50%, and 1.0% LaCl3 exposure could significantly impair memory of young rats. Hippocampal pCaMK IV, pMAPK, pCREB, c-fos and egr1 expression were decreased significantly, and synaptic ultrastructure was negatively affected after LaCl3 exposure. These results indicate that LaCl3 exposure impairs memory of rats and this impairment may be attributed to the lower levels of pCaMK IV, pMAPK, pCREB, c-fos and egr1 expression and change of synaptic ultrastructure in hippocampus.

The heterogeneous nuclear ribonucleoprotein L is an essential component in the Ca2+/calmodulin-dependent protein kinase IV-regulated alternative splicing through cytidine-adenosine repeats.

The regulation of gene expression through alternative pre-mRNA splicing is common in metazoans and is often controlled by intracellular signaling pathways that are important in cell physiology. We have shown that the alternative splicing of a number of genes is controlled by membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) through CaMKIV-responsive RNA elements (CaRRE1 and CaRRE2); however, the trans-acting factors remain unknown. Here we show that the heterogeneous nuclear ribonucleoprotein (hnRNP) L is a CaRRE1 binding factor in nuclear extracts. An hnRNP L high affinity CA (cytidine-adenosine) repeat element is sufficient to mediate CaMKIV and hnRNP L repression of splicing in a location (3'-splice site proximity)-dependent way. Depletion of hnRNP L by RNA interference followed by rescue with coexpressed exogenous hnRNP L demonstrates that hnRNP L mediates the CaMKIV-regulated splicing through CA repeats in heterologous contexts. Depletion of hnRNP L also led to increased inclusion of the stress axis-regulated exon and a CA repeat-harboring exon under depolarization or with activated CaMKIV. Moreover, hnRNP L binding to CaRRE1 was increased by CaMKIV and, conversely, was reduced by pretreatments with protein phosphatases. Therefore, hnRNP L is an essential component of CaMKIV-regulated alternative splicing through CA repeats, with its phosphorylation likely playing a critical role.

Upregulation of calcium/calmodulin-dependent protein kinase IV improves memory formation and rescues memory loss with aging.

Previous studies have suggested that calcium/calmodulin-dependent protein kinase IV (CaMKIV) functions as a positive regulator for memory formation and that age-related memory deficits are the result of dysfunctional signaling pathways mediated by cAMP response element-binding protein (CREB), the downstream transcription factor of CaMKIV. Little is known, however, about the effects of increased CaMKIV levels on the ability to form memory in adult and aged stages. We generated a transgenic mouse overexpressing CaMKIV in the forebrain and showed that the upregulation of CaMKIV led to an increase in learning-induced CREB activity, increased learning-related hippocampal potentiation, and enhanced consolidation of contextual fear and social memories. Importantly, we also observed reduced hippocampal CaMKIV expression with aging and a correlation between CaMKIV expression level and memory performance in aged mice. Genetic overexpression of CaMKIV was able to rescue associated memory deficits in aged mice. Our findings suggest that the level of CaMKIV expression correlates positively with the ability to form long-term memory and implicate the decline of CaMKIV signaling mechanisms in age-related memory deficits.

Chronic ethanol intake-induced changes in open-field behavior and calcium/calmodulin-dependent protein kinase IV expression in nucleus accumbens of rats: naloxone reversal.

AIM: To investigate the effects of chronic ethanol intake on the locomotor activity and the levels of calcium/calmodulin-dependent protein kinase IV (CaM kinase IV) in the nucleus accumbens (NAc) of rats. Simultaneously, the effects of nonselective opioid antagonist (naloxone) on the CaM kinase IV expression in the NAc and ethanol consumption of rats were also observed. METHODS: Ethanol was administered in drinking water at the concentrations of 6% (v/v), for 28 d. The locomotor activity of rats was investigated in the open-field apparatus. CaM kinase IV levels in the NAc were analyzed using Western blotting. RESULTS: Rats consuming ethanol solution exhibited a significant decrease of ambulation activity, accompanied by a reduced frequency of explorative rearing in an open-field task on d 7 and d 14 of chronic ethanol ingestion, whereas presumed adaptation to the neurological effects of ethanol was observed on d 28. Chronic ethanol intake elicited a significant decrease of the CaM kinase IV expression in the nuclei, but not in the cytoplasm of the NAc on d 28. Naloxone treatment significantly attenuated ethanol intake of rats and antagonized the decrease of CaM kinase IV in the nuclei of NAc neurons. The cytosolic CaM kinase IV protein levels of the NAc also increased in rats exposed to ethanol plus naloxone. CONCLUSION: Chronic ethanol intake-induced changes in explorative behavior is mediated at least partly by changes in CaM kinase IV signaling in the nuclei of the NAc, and naloxone attenuates ethanol consumption through antagonizing the downregulation of CaM kinase IV in the NAc.

Genetic enhancement of trace fear memory and cingulate potentiation in mice overexpressing Ca2+/calmodulin-dependent protein kinase IV.

Long-term potentiation (LTP) is a key cellular model for studying mechanisms for learning and memory. Previous studies reported that the Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is critical for gene regulation, and behavioral learning and memory. Less is known about the roles of CaMKIV in cortical plasticity and trace fear memory. Here we have found that LTP was significantly enhanced in the anterior cingulate cortex (ACC) of the mice overexpressing CaMKIV. By contrast, neither alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated basal excitatory synaptic transmission nor N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents were affected. Furthermore, paired-pulse ratio in the transgenic mice is normal. In behavioral tests, we found that the CaMKIV transgenic mice exhibited significant enhancement in trace fear memory, while the acute sensory thresholds were not affected. Our results provide strong evidence that forebrain CaMKIV contributes to trace fear memory by enhancing synaptic potentiation in the ACC.

Adeno-associated virus (AAV)-mediated suppression of Ca2+/calmodulin kinase IV activity in the nucleus accumbens modulates emotional behaviour in mice.

BACKGROUND: Calcium/calmodulin-dependent protein kinase IV (CaMKIV) controls activity-dependent gene transcription by regulating the activity of the cyclic AMP response element binding protein (CREB). This signaling pathway is involved in gating emotional responses in the CNS but previous studies did not address the potential roles of CaMKIV in discrete brain regions. In the present study, we aimed at specifically dissecting the role of CaMKIV in the nucleus accumbens of adult mice. RESULTS: We used recombinant adeno-associated virus (rAAV)-mediated gene transfer of a dominant-negative CaMKIV variant (rAAV-dnCaMKIV) to inhibit endogenous CaMKIV in the nucleus accumbens. rAAV-dnCaMKIV treated animals were subjected to a battery of tests including, prepulse inhibition of the acoustic startle response, open field, social interaction and anxiety-related behaviour. We found that basal locomotor activity in the open field, and prepulse inhibition or startle performance were unaltered in mice infected with rAAV-dnCaMKIV in the nucleus accumbens. However, anxiogenic effects were revealed in social interaction testing and the light/dark emergence test. CONCLUSION: Our findings suggest a modulatory role of CaMKIV in the nucleus accumbens in anxiety-like behaviour but not sensorimotor gating.