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.

Amelioration of mitochondrial dysfunction in heart failure through S-sulfhydration of Ca^2＋/calmodulin-dependent protein kinase Ⅱ

OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.

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 mitochondrial bioenergetics as a therapeutic strategy in Alzheimer＇s disease

Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.

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.

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations.

OsDMI3-mediated OsUXS3 phosphorylation improves oxidative stress tolerance by modulating OsCATB protein abundance in rice

Calcium(Ca^(2+))/calmodulin(CaM)-dependent protein kinase(CCaMK)is an important positive regulator of antioxidant defenses and tolerance against oxidative stress.However,the underlying molecular mechanisms are largely unknown.Here,we report that the rice(Oryza sativa)CCa MK(OsDMI3)physically interacts with and phosphorylates OsUXS3,a cytosol-localized UDP-xylose synthase.Genetic and biochemical evidence demonstrated that OsUXS3 acts downstream of OsDMI3 to enhance the oxidative stress tolerance conferred by higher catalase(CAT)activity.Indeed,OsUXS3 interacted with CAT isozyme B(OsCATB),and this interaction was required to increase OsCATB protein abundance under oxidative stress conditions.Furthermore,we showed that OsDMI3 phosphorylates OsUXS3 on residue Ser-245,thereby further promoting the interaction between OsUXS3 and OsCATB.Our results indicate that OsDMI3 promotes the association of OsUXS3 with OsCATB to enhance CAT activity under oxidative stress.These findings reveal OsUXS3 as a direct target of OsDMI3 and demonstrate its involvement in antioxidant defense.

Role of oxygen free radicals in the proliferation of myofibroblasts induced by AngII

Previous studies have demonstrated the important role of angiotension II(AngII)in promoting proliferation of myofibroblasts(myoFbs)and myocardial fibrosis.However,the underlying mechanisms and the role of oxygen free radicals in the proliferation of myofibroblasts induced by AngII are unclear.The present study was designed to shed light on this issue through exploration of AngII signaling pathways via in vitro experiments.Primary cultures of neonatal rat myoFbs were divided into five groups which were treated with AngII(10^(-8) to 10^(-6) M),AngII with the antioxidant N-acetyl-L-cysteine(NAC),or normal culture medium.We observed the proliferation of myoFbs as induced by AngII at different concentrations with MTT.Reactive oxygen species(ROS)levels in myoFbs were detected by monitoring the fluorescence of 2',7'-dichlorofluorescein.The contents and levels of oxygen free radicals(OH·)in the three groups were detected by spectrophotometer,immunocytochemical staining,and confocal fluorescence.Western blot and image analysis were used to measure membrane translocation and expression of phospho-protein kinase Ca.MyoFbs incubated with AngII(10^(-8) to 10^(-6) M)for 24 h increased their rate of proliferation,the content of OH·,and expression of ROS(P<0.01 vs.control group),whereas these parameters decreased in the presence of NAC.Immunocytochemistry,confocal fluorescence staining and image analysis showed that AngII could promote the translocation and expression of p-PKCα in membrane,and the antioxidant NAC blocked this increase(P<0.01).Western blot results also showed that NAC could inhibit the expression of p-PKCα.