Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

Objective

To investigate whether lipoxin A₄ (LXA₄) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA₄-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA₄-induced HO-1 induction.

Methods

Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA₄ or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay.

Results

Pretreatment of the cells undergoing H/R lesion with LXA₄ significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA₄ on the cells undergoing H/R lesion. LXA₄ increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure.

Conclusion

The protection role of LXA₄ against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway.     

p38 MAPK Regulates Cavitation and Tight Junction Function in the Mouse Blastocyst

Blastocyst formation is essential for implantation and maintenance of pregnancy and is dependent on the expression and coordinated function of a series of proteins involved in establishing and maintaining the trans-trophectoderm ion gradient that enables blastocyst expansion. These consist of Na/K-ATPase, adherens junctions, tight junctions (TJ) and aquaporins (AQP). While their role in supporting blastocyst formation is established, the intracellular signaling pathways that coordinate their function is unclear. The p38 MAPK pathway plays a role in regulating these proteins in other cell types and is required for embryo development at the 8–16 cell stage, but its role has not been investigated in the blastocyst.

Hypothesis

p38 MAPK regulates blastocyst formation by regulating blastocyst formation gene expression and function.

Methods

Embryos were cultured from the early blastocyst stage for 12 h or 24 h in the presence of a potent and specific p38 MAPK inhibitor, SB 220025. Blastocyst expansion, hatching, gene family expression and localization, TJ function and apoptosis levels were analyzed.

Results

Inhibition of the p38 MAPK pathway reduced blastocyst expansion and hatching, increased tight junction permeability, affected TJP1 localization, reduced Aqp3 expression, and induced a significant increase in apoptosis.

Conclusion

The p38 MAPK pathway coordinates the overall events that regulate blastocyst formation.     

银屑病的研究进展及其与血红素氧合酶1的关系

银屑病是一种免疫调节紊乱的慢性炎性皮肤疾病,多为T细胞功能异常所致,具有发病率高、易复发、病情顽固等特征。该病还与遗传及感染、饮食、压力等环境因素相关。目前,银屑病的治疗方法多种,但仍无法根治。研究显示,血红素氧合酶1(heme oxygenase 1,HO-1)在斑块型银屑病患者皮损处明显升高。HO-1具有抗炎、抗凋亡和改变增殖的作用,它可能通过调节氧化应激而介导银屑病的发生、发展进程。本文综述近年来的研究结果,讨论银屑病的发病机理及HO-1在该病治疗中的可能作用,旨在为临床治疗银屑病提供一些新的理论基础。     

Expression of Heme Oxygenase Isozyme mRNAs in the Human Brain and Induction of Heme Oxygenase-1 by Nitric Oxide Donors

Abstract: Heme oxygenase is an essential enzyme in the heme catabolism that produces carbon monoxide (CO). This study was designed to examine the expression of two heme oxygenase isozyme mRNAs in the human brain and to explore the involvement of nitric oxide (NO) and various neuropeptides in the regulation of their expression. Northern blot analysis showed the expression of heme oxygenase-1 and heme oxygenase-2 mRNAs in every region of the brain examined, with the highest levels found in the frontal cortex, temporal cortex, occipital cortex, and hypothalamus. In a human glioblastoma cell line, T98G, treatment with any of three types of NO donors—sodium nitroprusside, 3-morpholinosydnonimine, and S -nitroso- l -glutathione—caused a significant increase in the levels of heme oxygenase-1 mRNA but not in the levels of heme oxygenase-2 and heat-shock protein 70 mRNAs. Sodium nitroprusside increased the levels of heme oxygenase-1 protein but not the levels of heat-shock protein 70 in T98G cells. The increase in content of heme oxygenase-1 mRNA caused by sodium nitroprusside was completely abolished by the treatment with actinomycin D. On the other hand, the levels of heme oxygenase isozyme mRNAs were not noticeably changed in T98G cells following the treatment with 8-bromo cyclic GMP, sodium nitrite, or various neuropeptides, such as calcitonin gene-related peptide, endothelin-1, and corticotropin-releasing hormone. The present study has shown the expression profiles of heme oxygenase-1 and -2 mRNAs in the human brain and the induction of heme oxygenase-1 mRNA caused by NO donors in T98G cells. These findings raise a possibility that the CO/heme oxygenase system may function in concert with the NO/NO synthase system in the brain.     

Calcineurin enhances MAPK phosphatase-1 expression and p38 MAPK inactivation in cardiac myocytes

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiac myocytes including mitogen-activated protein kinase (MAPK) and calcineurin-nuclear factor of activated T-cells. However, it is uncertain if individual regulatory pathways operate in isolation or if interconnectivity between unrelated pathways is required for the orchestration of the entire hypertrophic response. To this end, we investigated the interconnectivity between calcineurin-mediated cardiac myocyte hypertrophy and p38 MAPK signaling in vitro and in vivo. We show that calcineurin promotes down-regulation of p38 MAPK activity and enhances expression of the dual specificity phosphatase MAPK phosphatase-1 (MKP-1). Transgenic mice expressing activated calcineurin in the heart were characterized by inactivation of p38 and increased MKP-1 expression during early postnatal development, before the onset of cardiac hypertrophy. In vitro, cultured neonatal cardiomyocytes infected with a calcineurin-expressing adenovirus and stimulated with phenylephrine demonstrated reduced p38 phosphorylation and increased MKP-1 protein levels. Activation of endogenous calcineurin with the calcium ionophore decreased p38 phosphorylation and increased MKP-1 protein levels. Inhibition of endogenous calcineurin with cyclosporin A decreased MKP-1 protein levels and increased p38 activation in response to agonist stimulation. To further investigate potential cross-talk between calcineurin and p38 through alteration in MKP-1 expression, the MKP-1 promoter was characterized and determined to be calcineurin-responsive. These data suggest that calcineurin enhances MKP-1 expression in cardiac myocytes, which is associated with p38 inactivation.     

LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1)

LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.     

钴- 原卟啉对大鼠脑损伤区域血红素氧合酶-1 及损伤周边区脑含水量的影响

目的:研究探讨钴-原卟啉对大鼠脑损伤的保护作用。方法:用钴-原卟啉灌(50mg/kg)胃处理后,建立液压脑损伤模型,采用免疫组织化学SP三步法检测大鼠脑损伤区域血红素氧合酶-1(HO-1)的表达情况,用干湿重法测定损伤周边区脑含水量的变化。结果:正常组、假手术组大鼠HO-1均无表达;模型组脑损伤区域的HO-1阳性细胞数为(3.45±047);实验组12h、24h、3d和7dHO-1的阳性细胞数分别为:(10.9±1.35)、(10.62±1.88)、(12.4±1.57)和(10.99±2.42)。正常组、假手术组脑损伤周边区脑含水量分别为(78.7±0.4)%和(78.6±0.7)%;模型组脑含水量(89.3±0.3)%;实验组12h、24h、3d和7d脑含水量分别为:(83.1±0.3)%、(83.6±0.6)%、(83.9±0.4)%和(83.2±0.3)%。结论:钴-原卟啉(CoPP)能够诱导大鼠脑损伤区域HO-1的表达,减轻脑损伤周边区域的水肿程度,故钴-原卟啉对大鼠脑损伤具有保护作用。     

Deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the development of heart failure. There is a direct correlation between myocardial function and myocardial TNF levels in humans. TNF may induce local inflammation to exert tissue injury. On the other hand, suppressors of cytokine signaling (SOCS) proteins have been shown to inhibit proinflammatory signaling. However, it is unknown whether TNF mediates myocardial inflammation via STAT3/SOCS3 signaling in the heart and, if so, whether this effect is through the type 1 55-kDa TNF receptor (TNFR1). We hypothesized that TNFR1 deficiency protects myocardial function and decreases myocardial IL-6 production via the STAT3/SOCS3 pathway in response to TNF. Isolated male mouse hearts (n = 4/group) from wild-type (WT) and TNFR1 knockout (TNFR1KO) were subjected to direct TNF infusion (500 pg.ml(-1).min(-1) x 30 min) while left ventricular developed pressure and maximal positive and negative values of the first derivative of pressure were continuously recorded. Heart tissue was analyzed for active forms of STAT3, p38, SOCS3 and SOCS1 (Western blot analysis), as well as IL-1beta and IL-6 (ELISA). Coronary effluent was analyzed for lactate dehydrogenase (LDH) activity. As a result, TNFR1KO had significantly better myocardial function, less myocardial LDH release, and greater expression of SOCS3 (percentage of SOCS3/GAPDH: 45 +/- 4.5% vs. WT 22 +/- 6.5%) after TNF infusion. TNFR1 deficiency decreased STAT3 activation (percentage of phospho-STAT3/STAT3: 29 +/- 6.4% vs. WT 45 +/- 8.8%). IL-6 was decreased in TNFR1KO (150.2 +/- 3.65 pg/mg protein) versus WT (211.4 +/- 26.08) mice. TNFR1 deficiency did not change expression of p38 and IL-1beta following TNF infusion. These results suggest that deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta.     

Chronic Treatment with Paraquat Induces Brain Injury,Changes in Antioxidant Defenses System,and Modulates Behavioral Functions in Zebrafish

Paraquat (PQ) administration consists in a chemical model that mimics phenotypes observed in Parkinson’s disease (PD), due to its ability to induce changes in dopaminergic system and oxidative stress. The aim of this study was to evaluate the actions of PQ in behavioral functions of adult zebrafish and its influence on oxidative stress biomarkers in brain samples. PQ (20 mg/kg) was administered intraperitoneally with six injections for 16 days (one injection every 3 days). PQ-treated group showed a significant decrease in the time spent in the bottom section and a shorter latency to enter the top area in the novel tank test. Moreover, PQ-exposed fish showed a significant decrease in the number and duration of risk assessment episodes in the light–dark test, as well as an increase in the agonistic behavior in the mirror-induced aggression (MIA) test. PQ induced brain damage by decreasing mitochondrial viability. Concerning the antioxidant defense system, PQ increased catalase (CAT) and glutathione peroxidase (GPx) activities, as well as the non-protein sulfhydryl content (NPSH), but did not change ROS formation and decreased lipid peroxidation. We demonstrate, for the first time, that PQ induces an increase in aggressive behavior, alters non-motor patterns associated to defensive behaviors, and changes redox parameters in zebrafish brain. Overall, our findings may serve as useful tools to investigate the interaction between behavioral and neurochemical impairments triggered by PQ administration in zebrafish.     

Possible Role of Heme Oxygenase-1 and Prostaglandins in the Pathogenesis of Cerebral Malaria: Heme Oxygenase-1 Induction by Prostaglandin D2 and Metabolite by a Human Astrocyte Cell Line

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D₂ is abundantly produced in the brain and regulates the sleep response. Moreover, PGD₂ is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD₂ significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD₂ treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD₂ may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.     

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo^−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.     

巨噬细胞免疫调变信号：Raf—1,MAPKp44,MAPKp42和p38MAPK的研究

为了了解巨噬细胞免疫调变机理,我们应用ＬＰＳ和ＰＭＡ处理小鼠抑制性巨噬细胞,观察到Ｒａｓ下游信号分子ＡＦ－１,分裂原激活蛋白激酶ＭＡＰＫｐ４４,ＭＡＰＫｐ４２和ｐ３８ＭＡＰＫ均被活化,发现ｆｏｒｓｋｏｌｉｎ能增强ｐ３８ＭＡＰＫ的活性,进一步提示ＰＫＣ和ＰＡＫ途径增强了ｐ３８ＭＡＰＫ的磷酸化效应,为我们了解ＬＰＳ如何激活ｐ３８ＭＡＰＫ信号通路提供了一个新的机会／     

Rapid Changes in Cortical and Subcortical Brain Regions after Early Bilateral Enucleation in the Mouse

Functional sensory and motor areas in the developing mammalian neocortex are formed through a complex interaction of cortically intrinsic mechanisms, such as gene expression, and cortically extrinsic mechanisms such as those mediated by thalamic input from the senses. Both intrinsic and extrinsic mechanisms are believed to be involved in cortical patterning and the establishment of areal boundaries in early development; however, the nature of the interaction between intrinsic and extrinsic processes is not well understood. In a previous study, we used a perinatal bilateral enucleation mouse model to test some aspects of this interaction by reweighting sensory input to the developing cortex. Visual deprivation at birth resulted in a shift of intraneocortical connections (INCs) that aligned with ectopic ephrin A5 expression in the same location ten days later at postnatal day (P) 10. A prevailing question remained: Does visual deprivation first induce a change in gene expression, followed by a shift in INCs, or vice versa? In the present study, we address this question by investigating the neuroanatomy and patterns of gene expression in post-natal day (P) 1 and 4 mice following bilateral enucleation at birth. Our results demonstrate a rapid reduction in dorsal lateral geniculate nucleus (dLGN) size and ephrin A5 gene expression 24-hours post-enucleation, with more profound effects apparent at P4. The reduced nuclear size and diminished gene expression mirrors subtle changes in ephrin A5 expression evident in P1 and P4 enucleated neocortex, 11 and 8 days prior to natural eye opening, respectively. Somatosensory and visual INCs were indistinguishable between P1 and P4 mice bilaterally enucleated at birth, indicating that perinatal bilateral enucleation initiates a rapid change in gene expression (within one day) followed by an alteration of sensory INCs later on (second postnatal week). With these results, we gain a deeper understanding of how gene expression and sensory input together regulate cortical arealization and plasticity during early development.     

Dancing with p53: The role of p38MAPK in mitosis of p53-deficient tetraploid cells

Comment on: Vitale I, et al. Cell Cycle 2010; 9:2823-9.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.