Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Up‐regulation of TRPM6 transcriptional activity by AP‐1 in renal epithelial cells

Transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of magnesium in the kidney. We recently found that TRPM6 expression is up‐regulated by EGF, but the regulatory mechanism has not been clear. TRPM6 mRNA was endogenously expressed in HEK293 cells. TRPM6 mRNA expression was increased by EGF, which was inhibited by U0126, an MEK inhibitor. Promoter activity of human TRPM6 was observed in the TRPM6 5′‐flanking region from ?1,214 to ?718. This promoter activity was enhanced by EGF and inhibited by U0126. Three putative AP‐1 binding sites were identified within the region of ?1,214/?718. The mutation of the putative AP‐1 binding site (?741/?736) completely inhibited the EGF‐induced promoter activity. EGF increased p‐ERK1/2, c‐Fos, c‐Jun, and p‐c‐Jun levels, which were inhibited by U0126. The introduction of c‐Fos or c‐Jun siRNA inhibited the EGF‐induced promoter activity. A chromatin immunoprecipitation assay revealed that c‐Fos and c‐Jun bind to the AP‐1 binding site within the region of ?1,214/?718. These results suggest that EGF up‐regulates TRPM6 mRNA expression mediate via the activation of ERK/AP‐1‐dependent pathway. J. Cell. Physiol. 222: 481–487, 2010. © 2009 Wiley‐Liss, Inc.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Doppel-induced Purkinje cell death is stoichiometrically abrogated by prion protein

Activin A can induce erythroid differentiation, whereas basic fibroblast growth factor (bFGF) can maintain the undifferentiated status of erythroid progenitors. How these two factors together can affect the regulation of erythroid differentiation in hematopoietic cells has not been elucidated. This study demonstrates that bFGF antagonizes activin A-mediated growth inhibition and hemoglobin (Hb) synthesis in K562 cells. Analyses of mitogen-activated protein kinases revealed that activin A-induced p38 phosphorylation and inhibited ERK1/2 phosphorylation. In contrast, bFGF worked antagonistically to induce ERK1/2 phosphorylation and inhibited p38 phosphorylation in K562 cells. Furthermore, co-treatment of cells with activin A and bFGF decreased p38 phosphorylation and increased ERK1/2 phosphorylation. SB203580 inhibition of p38 activity eliminated activin A-mediated growth inhibition and Hb synthesis, whereas U0126 inhibition of ERK1/2 activity augmented the effects of activin A on K562 cells. These results suggest that bFGF can negatively modulate p38 and positively modulate ERK1/2 to antagonize activin A-mediated growth inhibition and Hb synthesis in K562 cells.     

P38 mitogen‐activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture

Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell‐based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes ‘dedifferentiation’ into fibroblastic cells. Mitogen‐activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up‐regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up‐regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow‐on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell‐based therapies.     

TNF‐α induces matrix metalloproteinase‐9 expression in A549 cells: Role of TNFR1/TRAF2/PKCα‐dependent signaling pathways

Matrix metalloproteinases (MMPs), in particular MMP‐9, have been shown to be induced by cytokines, including TNF‐α and contributes to airway inflammation. However, the mechanisms underlying TNF‐α‐induced MMP‐9 expression in human A549 cells remain unclear. Here, we report that TNF‐α‐induced MMP‐9 gene expression was mediated through the TNFR1/TRAF2/PKCα‐dependent signaling pathways in A549 cells, determined by zymographic, RT‐PCR, and Western blotting analyses. TNF‐α‐induced MMP‐9 expression was reduced by pretreatment with a TNFR Ab. Furthermore, TNF‐α‐induced TNFR1 and TRAF2 complex formation was revealed by immunoprecipitation using an anti‐TNFR1 Ab followed by Western blot analysis against an anti‐TRAF2 or anti‐TNFR1 Ab. In addition, TNF‐α‐induced MMP‐9 expression was also reduced by pretreatment with the inhibitor of PKCα (Gö6983), c‐Src (PP1), EGFR (AG1478), or PI3K (LY294002) or transfection with siRNAs of PKCα, Src, EGFR, Akt, p65, p300, and c‐Jun. On the other hand, TNF‐α stimulated the phosphorylation of c‐Src, EGFR, Akt, JNK1/2, and c‐Jun, which were inhibited by pretreatment with Gö6983. We also showed that TNF‐α induced Akt translocation and the formation of an Akt/p65/p300 complex. Pretreatment with the inhibitor of JNK1/2 (SP600125) but not the inhibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or PI3K (LY294002), markedly inhibited TNF‐α‐induced c‐Jun mRNA levels. Taken together, these data suggest that in A549 cells, TNF‐α induces MMP‐9 expression via the TNFR1/TRAF2/PKCα‐dependent JNK1/2/c‐Jun and c‐Src/EGFR/PI3K/Akt pathways. J. Cell. Physiol. 454–464, 2010. © 2010 Wiley‐Liss, Inc.     

Ouabain‐induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells

Cardiotonic steroid (CTS) ouabain is a well‐established inhibitor of Na,K‐ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain‐induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long‐term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain‐induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten‐micromolar ouabain leads to cell death, and we conclude that different effects of 1‐μM and 10‐μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.     

Role of mitogen‐activated protein kinase in Zn‐BC‐AM PDT‐induced apoptosis in nasopharyngeal carcinoma cells

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn‐BC‐AM was found to effectively induce apoptosis in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 cell line. Sustained activation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal kinase (JNK) as well as a transient increase in activation of extracellular signal‐regulated kinase (ERK) were observed immediately after Zn‐BC‐AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT‐induced apoptosis of HK‐1 cells. PD169316 also prevented the loss of Bcl‐2 and Bcl‐xL in PDT‐treated HK‐1 cells. However, inhibition of JNK with SP600125 had no effect on Zn‐BC‐AM PDT‐induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn‐BC‐AM PDT‐induced apoptosis. Further study showed that knockdown of the p38β isoform with siRNA also increased Zn‐BC‐AM PDT‐induced apoptosis, indicating that the anti‐apoptotic effect of PD169316 in PDT‐treated HK‐1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38β and ERK may enhance the therapeutic efficacy of Zn‐BC‐AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution. Copyright © 2010 John Wiley & Sons, Ltd.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N-terminal kinases (JNK) on the histamine-induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type-1 (H1) receptor-dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor-dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine-induced TH phosphorylation, we generated phospho-specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine-induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine-induced TH phosphorylation. Our data support the hypothesis that the up-regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.