The effects of NONRATT021972 lncRNA siRNA on PC12 neuronal injury mediated by P2X<Subscript>7</Subscript> receptor after exposure to oxygen-glucose deprivation

Adenosine triphosphate (ATP) participates in signal transmission by acting on P2X receptors, and the P2X₇ receptor is involved in the pathophysiological changes of ischemic injury. The PC12 cell line is a popular model system to study sympathetic neuronal function. Long noncoding RNAs (lncRNAs) are highly expressed in the nervous system and serve as regulatory RNAs. In this study, the effects of NONRATT021972 lncRNA siRNA on P2X₇-mediated PC12 neuronal injury after exposure to oxygen-glucose deprivation (OGD) were investigated. Our results showed that the viability of PC12 cells cultured with OGD or the P2X₇ agonist BzATP was significantly decreased. Treatment with NONRATT021972 siRNA reversed the decreased viability of PC12 cells under OGD conditions. The upregulated P2X₇ mRNA and protein levels in PC12 cells under OGD conditions or BzATP treatment were significantly decreased when pretreated with NONRATT021972 siRNA. Moreover, NONRATT021972 siRNA treatment effectively suppressed the increase in [Ca²⁺]_i induced by OGD or P2X₇ agonists (ATP or BzATP) in PC12 cells. Therefore, treatment with NONRATT021972 siRNA may decrease sympathetic neuronal injury induced by ischemia.     

LncRNA NONRATT021972 involved the pathophysiologic processes mediated by P2X<Subscript>7</Subscript> receptors in stellate ganglia after myocardial ischemic injury

Adenosine triphosphate (ATP) acts on P2X receptors to initiate signal transmission. P2X₇ receptors play a role in the pathophysiological process of myocardial ischemic injury. Long noncoding RNAs (lncRNAs) participate in numerous biological functions independent of protein translation. LncRNAs are implicated in nervous system diseases. This study investigated the effects of NONRATT021972 small interference RNA (siRNA) on the pathophysiologic processes mediated by P2X₇ receptors in stellate ganglia (SG) after myocardial ischemic injury. Our results demonstrated that the expression of NONRATT021972 in SG was significantly higher in the myocardial ischemic (MI) group than in the control group. Treatment of MI rats with NONRATT021972 siRNA, the P2X₇ antagonist brilliant blue G (BBG), or P2X₇ siRNA improved the histology of injured ischemic cardiac tissues and decreased the elevated concentrations of serum myocardial enzymes, creatine kinase (CK), CK isoform MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) compared to the MI rats. NONRATT021972 siRNA, BBG, or P2X₇ siRNA treatment in MI rats decreased the expression levels of P2X₇ immunoreactivity, P2X₇ messenger RNA (mRNA), and P2X₇ protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) in the SG compared to MI rats. NONRATT021972 siRNA treatment prevented the pathophysiologic processes mediated by P2X₇ receptors in the SG after myocardial ischemic injury.     

Requirement of reactive oxygen species-dependent activation of ASK1-p38 MAPK pathway for extracellular ATP-induced apoptosis in macrophage

Extracellular ATP, an autocrine or paracrine intercellular transmitter, is known to induce apoptosis in macrophages. However, the precise signaling mechanisms of ATP-induced apoptosis remain to be elucidated. Here we showed that activation of p38 mitogen-activated protein kinase (MAPK) plays a critical role in ATP-induced apoptosis. p38 activation and apoptosis in macrophages were induced by ATP. ATP-induced apoptosis was mediated in part by production of reactive oxygen species (ROS) derived from NOX2/gp91(phox), a component of the NADPH oxidase complex expressed in macrophages and neutrophils. Furthermore, ATP-induced ROS generation, p38 activation, and apoptosis were almost completely inhibited by selective P2X(7) receptor antagonists. We also found that ATP-induced apoptosis were diminished in ASK1-deficient macrophages accompanied by the lack of p38 activation. These results demonstrate that ROS-mediated activation of the ASK1-p38 MAPK pathway downstream of P2X(7) receptor is required for ATP-induced apoptosis in macrophages.     

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R^−/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [³H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R^−/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [³H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.     

Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages

Interaction of P2X7 receptor with P2X4 receptor has recently been suggested, but it remains unclear whether P2X4 receptor is involved in P2X7 receptor-mediated events, such as cell death of macrophages induced by high concentrations of extracellular ATP. Here, we present evidence that P2X4 receptor does play a role in P2X7 receptor-dependent cell death. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), and cell death via activation of P2X7 receptor. P2X4-knockdown cells, established by transfecting RAW264.7 cells with two short hairpin RNAs (shRNAs) targeting P2X4 receptor, showed a decrease of the initial peak of intracellular Ca(2+) after treatment with ATP, though pore formation and the P2X7-mediated activation of ERK1/2 and p38 MAPK were not affected. Intriguingly, P2X4 knockdown resulted in significant suppression of cell death induced by ATP or P2X7 agonist BzATP. In conclusion, our results suggest that P2X4 receptor is involved in P2X7 receptor-mediated cell death, but not pore formation or MAPK signaling.     

Differential crosstalk between P2X7 and arachidonic acid in activation of mitogen-activated protein kinases

Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca²⁺. The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors.The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.     

Differential crosstalk between P2X7 and arachidonic acid in activation of mitogen-activated protein kinases

Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca²⁺. The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors.The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.     

P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer's disease

Primary rat microglia stimulated with either ATP or 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O(2)(-)*). ATP and BzATP stimulate O(2)(-)* production through purinergic receptors, primarily the P2X(7) receptor. O(2)(-)* is produced through the activation of the NADPH oxidase. Although both p42/44 MAPK and p38 MAPK were activated rapidly in cells stimulated with BzATP, only pharmacological inhibition of p38 MAPK attenuated O(2)(-)* production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O(2)(-)* production to a greater extent than an inhibitor of p38 MAPK. Both ATP and BzATP stimulated microglia-induced cortical cell death indicating this pathway may contribute to neurodegeneration. Consistent with this hypothesis, P2X(7) receptor was specifically up-regulated around beta-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576).     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

周围神经损伤中P2Y12受体-p38MAPK通路的研究进展

周围神经损伤是临床中常见的神经损伤之一,神经胶质细胞和信号通路转导在周围神经损伤和再生修复中发挥重要作用。小胶质细胞的活化与周围神经损伤导致的神经损伤及疼痛密切相关,小胶质细胞是周围神经损伤与修复的关键场所。脊髓背角的小胶质细胞可被嘌呤信号通路的P2Y_(12)受体活化,进而导致p38MAPK磷酸化,造成相关神经损伤及感觉功能障碍。以脊髓背角的小胶质细胞为靶点,从P2Y_(12)受体-p38MAPK通路的角度可揭示周围神经损伤的部分可能机制。探究从嘌呤信号通路与小胶质细胞活化的新角度,将神经损伤后的P2Y_(12)受体与p38MAPK的磷酸化表达联系为P2Y_(12)受体-p38MAPK通路,可为临床治疗周围神经损伤提供新的思路。本文就周围神经损伤中P2Y_(12)受体-p38MAPK通路的研究进展作一综述。     

BMP4 Increases the Expression of TRPC and Basal [Ca2+]i via the p38MAPK and ERK1/2 Pathways Independent of BMPRII in PASMCs

Multiple abnormalities of bone morphogenetic protein (BMPs) signaling are implicated in the process of pulmonary arterial hypertension (PAH). BMP4 plays an important role during the process of pulmonary arterial remodeling and mutant of the principle BMP4 receptor, BMP receptors II (BMPRII), is found to associate with the development of PAH. However, the likely mechanism defining the contribution of BMPRII to BMP4 mediated signaling in pulmonary arterial smooth muscle cells (PASMCs) remains comprehensively unclear. We previously found that enhanced store operated calcium entry (SOCE) and basal intracellular calcium concentration [Ca²⁺]_i were induced by BMP4 via upregulation of TRPC1, 4 and 6 expression in PASMCs, and that BMP4 modulated TRPC channel expression through activating p38MAPK and ERK1/2 signaling pathways. In this study, BMPRII siRNA was used to knockdown BMPRII expression to investigate whether BMP4 upregulates the expression of TRPC and activating Smad1/5/8, ERK1/2 and p38MAPK pathway via BMPRII in distal PASMCs. Our results showed that knockdown of BMPRII: 1) attenuated BMP4 induced activation of P-Smad1/5/8, without altering BMP4 induced P-p38MAPK and P-ERK1/2 activation in PASMCs; 2) did not attenuate the BMP4-induced TRPC1, 4 and 6 expression; 3) did not affect BMP4-enhanced SOCE and basal [Ca²⁺]_i. Thus, we concluded that BMP4 activated Smad1/5/8 pathway is BMPRII-dependent, while the BMP4 – ERK/p-P38 – TRPC – SOCE signaling axis are likely mediated through other receptor rather than BMPRII.     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.     

Artepillin C Derived from Propolis Induces Neurite Outgrowth in PC12m3 Cells via ERK and p38 MAPK Pathways

We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 μM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.     

Nerve growth factor signaling involves interaction between the Trk A receptor and lysophosphatidate receptor 1 systems: nuclear translocation of the lysophosphatidate receptor 1 and Trk A receptors in pheochromocytoma 12 cells

We report here that the nerve growth factor (NGF) and lysophosphatidate (LPA) receptor signaling systems interact to regulate the p42/p44 MAPK pathway in PC12 cells. This is based upon several lines of evidence. First, the treatment of PC12 cells, which express LPA(1) receptors, with a sub-maximal concentration of LPA and NGF induced synergistic activation of p42/p44 MAPK. Second, the transfection of PC12 cells with LPA(1) receptor anti-sense construct, which reduced the expression of LPA(1), abrogated both LPA- and NGF-stimulated activation of p42/p44 MAPK. Third, the over-expression of recombinant LPA(1) receptor potentiated LPA- and NGF-dependent activation of p42/p44 MAPK. Fourth, the over-expression of C-terminal GRK2 peptide (which sequesters G-protein betagamma subunits) or beta-arrestin I clathrin binding domain (amino acids: 319-418) or pre-treatment of cells with pertussis toxin reduced the LPA- and NGF-dependent stimulation of p42/p44 MAPK. These findings support a model in which the Trk A receptor uses a G-protein-mediated mechanism to regulate the p42/p44 MAPK pathway. Such G-protein-mediated signaling is activated by the LPA(1) receptor as a means of cross-talk regulation with the Trk A receptor. Fifth, the treatment of cells with LPA induced the transactivation of the Trk A receptor. Sixth, LPA and/or NGF stimulated the translocation of tyrosine phosphorylated Trk A receptor and LPA(1) receptor to the nucleus. Taken together, these findings suggest that NGF and LPA exert cross-talk regulation both at the level of p42/p44 MAPK signaling and in the nuclear translocation of LPA(1) and Trk A receptors.     

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O(2)(-) production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O(2)(-) production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.