Death-associated protein kinase 1 is associated with cognitive dysfunction in major depressive disorder

We previously showed that death-associated protein kinase 1(DAPK1)expression is increased in hippocampal tissue in a mouse model of major depressive disorde and is related to cognitive dysfunction in Alzheimer's disease.In addition,depression is a risk factor for developing Alzheimer's disease,as well as an early clinical manifestation of Alzheimer's disease.Meanwhile,cognitive dysfunction is a distinctive feature of major depressive disorder.Therefore,DAPK1 may be related to cognitive dysfunction in major depressive disorder.In this study,we established a mouse model of major depressive disorder by housing mice individually and exposing them to chronic,mild,unpredictable stressors.We found that DAPK1 and tau protein levels were increased in the hippocampal CA3 area,and tau was hyperphosphorylated at Thr231,Ser262,and Ser396 in these mice.Furthermore,DAPK1 shifted from axonal expression to overexpression on the cell membrane.Exercise and treatment with the antidepressant drug citalopram decreased DAPK1 expression and tau protein phosphorylation in hippocampal tissue and improved both depressive symptoms and cognitive dysfunction.These results indicate that DAPK1 may be a potential reason and therapeutic target of cognitive dysfunction in major depressive disorder.

Thioridazine reverses trastuzumab resistance in gastric cancer by inhibiting S-phase kinase associated protein 2-mediated aerobic glycolysis

BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance.While S-phase kinase associated protein 2(Skp2)overexpression has been implicated in the malignant progression of GC,its role in regulating trastuzumab resistance in this context remains uncertain.Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products,there has been a lack of successful commercialization of drugs specifically targeting Skp2.AIM To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment.METHODS Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells.Q-PCR,western blot,and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression.A cell counting kit-8 assay,flow cytometry,a amplex red glucose/glucose oxidase assay kit,and a lactate assay kit were utilized to measure the proliferation,apoptosis,and glycolytic activity of GC cells in vitro.A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo.RESULTS The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab.Thioridazine demonstrated the ability to directly bind to Skp2,resulting in a reduction in Skp2 expression at both the transcriptional and translational levels.Moreover,thioridazine effectively inhibited cell proliferation,exhibited antiapoptotic properties,and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways.The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo,surpassing the efficacy of either monotherapy.CONCLUSION Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance,particularly when used in conjunction with lapatinib.This compound has potential benefits for patients with Skp2-proficient tumors.

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

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.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway

AIM To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on alpha-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway.

Inhibition of DNA-dependent Protein Kinase Catalytic Subunit by Small Molecule Inhibitor NU7026 Sensitizes Human Leukemic K562 Cells to Benzene Metabolite-induced Apoptosis

Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

Chronic stress causes protein kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the rat hippocampus and prefrontal cortex,but not in the myocardium

Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system.However,it is unknown whether the protein kinase C ε（PKCε）-aldehyde dehydrogenase 2（ALDH2） pathway is altered under chronic stress,and this study sought to address this question.A rat model of depression was established using a chronic unpredictable mild stress（CUMS） protocol.After experiencing CUMS for 4 weeks,the sucrose preference test and the forced swim test verified depressive-like behaviors.Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex,but was not altered in the myocardium.Western blot assays demonstrated reduced levels of ALDH2 and PKCε,but increased levels of 4-hydroxy-2-nonenal（4 HNE） adducts.Caspase-3 expression did not obviously alter,but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex.In the myocardium,expression of ALDH2,PKCε and 4 HNE adducts did not remarkably alter;while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated.Pearson＇s correlation test demonstrated that expression of 4 HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex,but not in the myocardium.In conclusion,chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex,but not in the myocardium.Moreover,4 HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.

Retinoic Aacid Diminished the Expression of MMP-2 in Hyperoxia-exposed Premature Rat Lung Fibroblasts through Regulating Mitogen-activated Protein Kinases

This study examined the effects of retinoic acid （RA）, PD98059, SP600125 and SB203580 on the hyperoxia-induced expression and regulation of matrix metalloproteinase-2 （MMP-2） and metalloproteinase-2 （TIMP-2） in premature rat lung fibroblasts （LFs）. LFs were exposed to hyperoxia or room air for 12 h in the presence of RA and the kinase inhibitors PD98059 （ERK1/2）, SP600125 （JNK1/2） and SB203580 （p38） respectively. The expression levels of MMP-2 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. MMP-2 activity was measured by zymography. The amount of p-ERK1/2, REK1/2, p-JNK1/2, JNK1/2, p-p38 and p38 was determined by Western blotting. The results showed that： （1） PD98059, SP600125 and SB203580 significantly inhibited p-ERK1/2, p-JNK1/2 and p-p38 respectively in LFs; （2） The expression of MMP-2 mRNA in LFs exposed to hyperoxia was decreased after treatment with RA, SP600125 and SB203580 respectively （P0.01 or 0.05）, but did not change after treatment with PD98059 （P0.05）. Meanwhile, RA, PD98059, SP600125 and SB203580 had no effect on the expression of TIMP-2 mRNA in LFs exposed to room air or hyperoxia （P0.05）; （3） The expression of pro- and active MMP-2 experienced no change after treatment with RA or SP600125 in LFs exposed to room air （P0.05）, but decreased remarkably after hyperoxia （P0.01 or 0.05）. SB203580 inhibited the expression of pro- and active MMP-2 either in room air or under hyperoxia （P0.01）. PD98059 exerted no effect on the expression of pro- and active MMP-2 （P0.05）. It was suggested that RA had a protective effect on hyperoxia-induced lung injury by down-regulating the expression of MMP-2 through decreasing the JNK and p38 activation in hyperoxia.

Increased endothelin receptor B and G protein coupled kinase-2 in the mesentery of portal hypertensive rats

AIM: To elucidate the mechanisms of mesenteric vasodilation in portal hypertension (PHT), with a focus on endothelin signaling. METHODS: PHT was induced in rats by common bile duct ligation (CBDL). Portal pressure (PP) was measured directly via catheters placed in the portal vein tract. The level of endothelin-1 (ET-1) in the mesenteric circulation was determined by radioimmunoassay, and the expression of the endothelin A receptor (ETAR) and endothelin B receptor (ETBR) was assessed by immunofluorescence and Western blot. Additionally, expression of G protein coupled kinase-2 (GRK2) and β-arrestin 2, which influence endothelin receptor sensitivity, were also studied by Western blot. RESULTS: PP of CBDL rats increased significantly (11.89 ± 1.38 mmHg vs 16.34 ± 1.63 mmHg). ET-1 expression decreased in the mesenteric circulation 2 and 4 wk after CBDL. ET-1 levels in the systemic circulation of CBDL rats were increased at 2 wk and decreased at 4 wk. There was no change in ETAR expression in response to CBDL; however, increased expression of ETBR in the endothelial cells of mesenteric arterioles and capillaries was observed. In sham-operated rats, ETBR was mainly expressed in the CD31+ endothelial cells of the arterioles. With development of PHT, in addition to the endothelial cells, ETBR expression was noticeably detectable in the SMA+ smooth muscle cells of arterioles and in the CD31+ capillaries. Following CBDL, increased expression of GRK2 was also found in mesenteric tissue, though there was no change in the level of β-arrestin 2. CONCLUSION: Decreased levels of ET-1 and increased ETBR expression in the mesenteric circulation following CBDL in rats may underlie mesenteric vasodilation in individuals with PHT. Mechanistically, increased GRK2 expression may lead to desensitization of ETAR, as well as other vasoconstrictors, promoting this vasodilatory effect.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-,mitogen-activated protein kinase-,and Nrf2-dependent pathway

BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Role of Protein Kinase C in the Activation of Store-operated Ca^(2+) Entry in Airway Smooth Muscle Cells

Store-operated Ca2＋ channels （SOCs） are plasma membrane Ca2＋ permeable channels activated by depletion of intracellular Ca2＋ store. Ca2＋ entry through SOCs is known as store-operated Ca2＋ entry （SOCE）, which plays an important role in the functional regulation of airway smooth muscle cells （ASMCs）. Protein kinase C （PKC） has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca2＋ fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca2＋-ATPase （SERCA） inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ε isoforms in rat ASMCs. PKCα-selective inhibitor G6976 and PKCε-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 （STIM1） and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.

Specific effects of c-Jun NH2-terminal kinaseinteracting protein 1 in neuronal axons

c-Jun NH2-terminal kinase（JNK）-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B（Trk B） anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.

Regulation of Ikaros function by casein kinase 2 and protein phosphatase 1

The Ikaros gene encodes a zinc finger,DNA-binding protein that regulates gene transcription and chromatin remodeling.Ikaros is a master regulator of hematopoiesis and an established tumor suppressor.Moderate alteration of Ikaros activity (e.g.haploinsufficiency) appears to be sufficient to promote malignant transformation in human hematopoietic cells.This raises questions about the mechanisms that normally regulate Ikaros function and the potential of these mechanisms to contribute to the development of leukemia.The focus of this review is the regulation of Ikaros function by phosphorylation/dephosphorylation.Site-specific phosphorylation of Ikaros by casein kinase 2 (CK2) controls Ikaros DNA-binding ability and subcellular localization.As a consequence,the ability of Ikaros to regulate cell cycle progression,chromatin remodeling,target gene expression,and thymocyte differentiation are controlled by CK2.In addition,hyperphosphorylation of Ikaros by CK2 leads to decreased Ikaros levels due to ubiquitinmediated degradation.Dephosphorylation of Ikaros by protein phosphatase 1 (PP1) acts in opposition to CK2 to increase Ikaros stability and restore Ikaros DNA binding ability and pericentromeric localization.Thus,the CK2 and PP1 pathways act in concert to regulate Ikaros activity in hematopoiesis and as a tumor suppressor.This highlights the importance of these signal transduction pathways as potential mediators of leukemogenesis via their role in regulating the activities of Ikaros.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

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.

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.

Synthesis of New 2-Phenylamino-4<i>H</i>-chromene-3-carbonitrile Derivatives and Their Effects on Tumor Cell Lines and against Protein Kinases

The synthesis of 2-phenylimino-4H-chromene-3-carbonitriles 6(a-d) in good overall yields using an efficient and practical methodology in 3 steps has been implemented in this present work. The first step was a heterocyclization between 2-hydroxybenzaldehyde 1 and propanedinitrile 2 which produced 2-iminocoumarin 3 which was submitted to nitrogen/nitrogen displacement in the presence of aromatic primary amine 4. In the third step, reduction of 5 led to the desired 2-phenylimino-4H-chromene-3-carbonitriles 6. Compounds 5(a-d) and 6(a-d) were evaluated for their potential in vitro cytotoxicity against six selected tumor cell lines (Huh7-D12, Caco2, MDA-MB231, HCT 116, PC3 and NCI-H727) and tested for their protein kinase inhibition on eight selected protein kinases. Among them, compounds 5c and 6b exhibited inhibition on HsCK1e (5c: 44% and 6b: 42% at 1 μM) and 5c for cytotoxicity on PC3 cell lines (63% at 25 μM).

Cyclic nucleotide phosphodiesterase 3B is connected to osteopontin and protein kinase CK2 in pancreatic <i>β</i>-cells

Islets from RIP-PDE3B mice, exhibiting β-cell specific overexpression of the cAMP/cGMP-degrading enzyme phosphodiesterase 3B (PDE3B) and dysregulated insulin secretion, were subjected to microarray analysis. We show that osteopontin (OPN) mRNA is increased in a dose-dependent manner in islets from RIP-PDE3B mice, as compared to wild-type islets. In addition, in silico analysis shows that PDE3B and OPN are interacting. Furthermore, OPN interacts with protein kinase CK2 ina distinct submodule of the protein-protein interaction network. We studied PDE3B and OPN proteins and, in some cases, also PDE1B and PDE4C, under conditions of relevance for insulin secretion. In the presence of forskolin, PDE inhibitors, insulin, or a protein kinase CK2 inhibitor, similar alterations in protein levels of PDE3B and OPN are shown. In summary, results from using a number of strategies demonstrate a connection between PDE3B and OPNas well as a role for protein kinase CK2 inpancreatic β-cells.