Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.

The calmodulin-dependent protein kinase II inhibitor KN-93 protects rat cerebral cortical neurons from N-methyl-D-aspartic acid-induced injury

In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyI-D-aspartic acid-induced injury. Results showed that, compared with N-methyi-D- aspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca2＋ concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 pM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin- dependent protein kinase II expression.

Tale of two kinases:Protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ in pre-diabetic cardiomyopathy

Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations,including insulin resistance,visceral fat accumulation,and dyslipidemias,which increase the risk for developing cardiovascular disease.Metabolic syndrome is associated with augmented sympathetic tone,which could account for the etiology of pre-diabetic cardiomyopathy.This review summarizes the current knowledge of the pathophysiological consequences of enhanced and sustainedβ-adrenergic response in pre-diabetes,focusing on cardiac dysfunction reported in diet-induced experimental models of pre-diabetic cardiomyopathy.The research reviewed indicates that both protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ play important roles in functional responses mediated byβ1-adrenoceptors;therefore,alterations in the expression or function of these kinases can be deleterious.This review also outlines recent information on the role of protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ in abnormal Ca^(2+)handling by cardiomyocytes from diet-induced models of pre-diabetic cardiomyopathy.

The cumulative analgesic effect of repeated electroacupuncture involves synaptic remodeling in the hippocampal CA3 region

In the present study, we examined the analgesic effect of repeated electroacupuncture at bilateral Zusanfi （ST36） and Yanglingquan （GB34） once a day for 14 consecutive days in a rat model of chronic sciatic nerve constriction injury-induced neuropathic pain. In addition, concomitant changes in calcium/calmodulin-dependent protein kinase II expression and synaptic ultrastructure of neurons in the hippocampal CA3 region were examined. The thermal pain threshold （paw withdrawal latency） was increased significantly in both groups at 2 weeks after electroacupuncture intervention compared with 2 days of electroacupuncture. In ovariectomized rats with chronic constriction injury, the analgesic effect was significantly reduced. Electroacupuncture for 2 weeks significantly diminished the injury-induced increase in synaptJc cleft width and thinning of the postsynaptJc density, and it significantly suppressed the down-regulation of intracellular calcium/ calmodulin-dependent protein kinase II expression in the hippocampal CA3 region. Repeated electroacupuncture intervention had a cumulative analgesic effect on injury-induced neuropathic pain reactions, and it led to synaptic remodeling of hippocampal neurons and upregulated calcium/calmodulin-dependent protein kJnase II expression in the hippocampal CA3 region.

GLYX-13 pretreatment ameliorates long-term isoflurane exposure-induced cognitive impairment in mice

Accumulating evidence indicates that inhalation anesthetics induce or increase the risk of cognitive impairment. GLYX-13（rapastinel） acts on the glycine site of N-methyl-D-aspartate receptors（NMDARs） and has been shown to enhance hippocampus-dependent learning and memory function. However, the mechanisms by which GLYX-13 affects learning and memory function are still unclear. In this study, we investigated these mechanisms in a mouse model of long-term anesthesia exposure. Mice were intravenously administered 1 mg/kg GLYX-13 at 2 hours before isoflurane exposure（1.5% for 6 hours）. Cognitive function was assessed using the contextual fear conditioning test and the novel object recognition test. The mRNA expression and phosphorylated protein levels of NMDAR pathway components, N-methyl-D-aspartate receptor subunit 2B（NR2B）-Ca2+/calmodulin dependent protein kinase II（CaMKII）-cyclic adenosine monophosphate response element binding protein（CREB）, in the hippocampus were evaluated by quantitative RT-PCR and western blot assay. Pretreatment with GLYX-13 ameliorated isoflurane exposure-induced cognitive impairment and restored NR2B, CaMKII and CREB mRNA and phosphorylated protein levels. Intracerebroventricular injection of KN93, a selective CaMKII inhibitor, significantly diminished the effect of GLYX-13 on cognitive function and NR2B, CaMKII and CREB levels in the hippocampus. Taken together, our findings suggest that GLYX-13 pretreatment alleviates isoflurane-induced cognitive dysfunction by protecting against perturbation of the NR2B/CaMKII/CREB signaling pathway in the hippocampus. Therefore, GLYX-13 may have therapeutic potential for the treatment of anesthesia-induced cognitive dysfunction. This study was approved by the Experimental Animal Ethics Committee of Drum Tower Hospital affiliated to the Medical College of Nanjing University, China（approval No. 20171102） on November 20, 2017.

Modulation of M4 muscarinic acetylcholine receptors by interacting proteins

Protein-protein interactions represent an important mechanism for posttranslational modifications of protein expression and function.In brain cells,surface-expressed and membrane-bound neurotransmitter receptors are common proteins that undergo dynamic protein-protein interactions between their intracellular domains and submembranous regulatory proteins.Recently,the Gφi/o -coupled muscarinic M4 receptor（M4R）has been revealed to be one of these receptors.Through direct interaction with the intracellular loops or C-terminal tails of M4Rs,M4R interacting proteins（M4RIPs）vigorously regulate the efficacy of M4R signaling.A synapse-enriched protein kinase,Ca2＋/calmodulin-dependent protein kinase II （CaMKII）,exemplifies a prototype model of M4RIPs,and is capable of binding to the second intracellular loop of M4Rs. Through an activity-and phosphorylation-dependent mechanism,CaMKII potentiates the M4R/Gφi/o-mediated inhibition of M4R efficacy in inhibiting adenylyl cyclase and cAMP production.In striatal neurons where M4Rs are most abundantly expressed,M4RIPs dynamically control M4R activity to maintain a proper cholinergic tone in these neurons.This is critical for maintaining the acetylcholine-dopamine balance in the basal ganglia,which determines the behavioral responsiveness to dopamine stimulation by psychostimulants.

Analgesic effect of gabapentin in a rat model for chronic constrictive injury

Background Gabapentin has been widely and successfully used in the clinic for many neuropathic pain syndromes since last decade, however its analgesic mechanisms are still elusive. Our study was to investigate whether Ca^2＋/calmodulin-dependent protein kinase Ⅱ （CaMKⅡ） contributes to the analgesic effect of gabapentin on a chronic constriction injury （CCI） model. Methods Gabapentin （2%, 100 mg/kg） or saline （0.5 ml/100 g） was injected intraperitoneally 15 minutes prior to surgery and then every 12 hours from postoperative day 0-4 to all rats in control, sham and CCI groups. The analgesic effect of gabapentin was assessed by measuring mechanical allodynia and thermal hyperalgesia of rats. Expression and activation of CaMKⅡ were quantified by reverse-transcriptional polymerase chain reaction and Western blotting. Results The analgesic effect of gabapentin on mechanical allodynia and thermal hyperalgesia was significant in the CCI model, with maximal reduction reached on postoperative day 8. Gabapentin decreased the expression of the total CaMKⅡ and phosphorylated CaMKⅡ in CCI rats. Conclusion The analgesic effect of gabapentin on CCI rats may be related to the decreased expression and phosphorylation of CaMKⅡ in the spinal cord.