Differential induction of TNF-alpha and NOS2 by mitogen-activated protein kinase signaling pathways during Mycobacterium bovis infection

The role of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of TNF-alpha and NOS2 production by human monocytes infected with Mycobacterium bovis BCG was examined. Inhibition studies showed that ERK1/2 and p38 MAPK activation were necessary for the monocyte response to M. bovis infection. Analysis of MAPK activation showed rapid phosphorylation of ERK1/2 and p38 in response to M. bovis BCG. Phosphorylation was not due to an autocrine effect of TNF-alpha secretion, since an anti-TNF-alpha antibody had no significant effect on the levels of p38 phosphorylation. The inhibitor PD98059 significantly reduced M. bovis BCG-induced TNF-alpha production and almost completely abrogated phosphorylation of ERK1/2; in addition the potent MEK inhibitor U0126 also abrogated phosphorylation. In contrast, studies using inhibitors selective for ERK1/2 and p38 showed that p38 plays an essential role in the induction of NOS2, whereas the role of ERK1/2 was minor. These results suggest that ERK1/2 and p38 kinases differentially regulate the M. bovis BCG-mediated induction of TNF-alpha and NOS2 in human monocytes.     

Requirement for ERK1/2 activation in the regulation of progesterone production in human granulosa-lutein cells is stimulus specific

This study was conducted to determine whether the ERK1/2 family of MAPKs can be modulated by physiological regulators of the human corpus luteum, and whether this activation is important for progesterone secretion in human granulosa-lutein (hGL) cells. Human LH (hLH), hCG, and agents that indirectly elevate cAMP [cholera toxin, forskolin, (Bu)(2)cAMP], time- and dose-dependently activated ERK1/2 in hGL cells. ERK1/2 activation was reduced by preincubation with PKA inhibitors, including myristoylated PKI, suggesting that cAMP mediates ERK1/2 activation. Two structurally distinct inhibitors of MAPK kinase (MEK), PD 98059 and U 0126, abrogated hLH/hCG-induced ERK1/2 activation, but had no effect on hLH-, hCG-, or 22R-hydroxycholesterol-stimulated progesterone secretion. In contrast, both inhibitors blocked cholera toxin-, forskolin-, and (Bu)(2)cAMP-induced ERK1/2 phosphorylation concomitant with a reduction in progesterone secretion. The known luteotropin, PGE(2), promoted MEK- and cAMP-dependent activation of ERK1/2, and inhibitors of either MEK or PKA decreased PGE(2)-induced progesterone synthesis. Our findings demonstrate that the requirement for ERK1/2 activation as a regulator of progesterone synthesis in hGL cells is stimulus dependent, and that the MEK inhibitor-sensitive step is distal to cAMP generation, but proximal to the conversion of cholesterol to pregnenolone.     

Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptides stimulate mitogen-activated protein kinase in the pituitary cell line GH4C1 by a 3',5'-cyclic adenosine monophosphate pathway

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP38) regulate anterior pituitary cell secretion and proliferation. In the somatolactotrope GH4C1 cell line, these effects are mediated through the type-II-like PACAP receptor (VPAC2) coupled to the cAMP pathway. In this study, the control of the extracellularly responsive kinases (ERKs) by VIP and PACAP38 was investigated in GH4C1 cells. VIP and PACAP38 increased ERK1 and ERK2 phosphorylation and were equipotent stimulators of both kinases. ERK activation was mimicked by cholera toxin, forskolin and 8bromo-cAMP. VIP and PACAP38 activation of ERK2 was blocked by the protein kinase A inhibitor H89, whereas the protein kinase C inhibitor GF109203X, or prior PMA-induced depletion of the protein kinases C, failed to inhibit VIP and PACAP38 activation of ERK2. In contrast, thyrotropin-releasing hormone (TRH) elicited ERK activation by a PKC-dependent process. ERK activation by VIP or PACAP38 and TRH were additive and both sensitive to the MEK inhibitors PD98059 and U0126. In parallel, U0126 reduced prolactin (PRL) mRNA levels induced by VIP. These results demonstrate for the first time that VIP and PACAP38 activate ERK in GH4C1 cells. Cyclic AMP increase is sufficient to elicit ERK activation in these cells and thus likely to represent the transduction pathway underlying VIP- and PACAP38-dependent ERK activation. This mechanism seems to be involved in VIP-induced PRL gene regulation.     

Cardioprotection by mild hypothermia during ischemia involves preservation of ERK activity

Cooling the ischemic heart by just a few degrees protects it from infarction without affecting its mechanical function, but the mechanism of this protection is unknown. We investigated whether signal transduction pathways might be involved in the anti-infarct effect of mild hypothermia (35°C). Isolated rabbit hearts underwent 30 min of coronary artery occlusion/2 h of reperfusion. They were either maintained at 38.5°C or cooled to 35°C just before and only during ischemia. Infarct size was measured. The effects of the protein kinase C inhibitor chelerythrine, the nitric oxide synthase inhibitor N (ω)-nitro-L: -arginine methyl ester (L: -NAME), the phosphatidylinositol 3-kinase antagonist wortmannin, or either of the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors PD98059 or U0126 on cooling's protection were examined. Myocardial ATP assays were performed and the level of phosphorylation of extracellular signal-regulated kinase (ERK) and MEK was examined by western blotting. To investigate an effect of cooling on protein phosphatase (PPase), a PPase inhibitor cantharidin was tested in the infarct model and the effect of mild hypothermia on PP2A activity in vitro was measured. Infarct size was 34.4 ± 2.2% of the ischemic zone in normothermic (38.5°C) hearts, but only 15.6 ± 8.7% in hearts cooled to 35°C during ischemia. Mechanical function was unaffected. Neither chelerythrine, L: -NAME, nor wortmannin had any effect, but both PD98059 and U0126 completely eliminated protection. Ischemia rather than reperfusion was the critical time when ERK had to be active to realize protection. Phosphorylation of ERK and MEK fell during normothermic ischemia, but during hypothermic ischemia phosphorylation of ERK remained high while that of MEK was increased. Cooling only slightly delayed the rate at which ATP fell during ischemia, and ERK inhibition did not affect that attenuation suggesting ATP preservation was unrelated to protection. Cantharidin, like cooling, also protected during ischemia but not at reperfusion, and its protection was dependent on ERK phosphorylation. However, mild hypothermia had a negligible effect on PP2A activity in an in vitro assay. Hence, mild hypothermia preserves ERK and MEK activity during ischemia which somehow protects the heart. While a PPase inhibitor mimicked cooling's protection, a direct effect of cooling on PP2A could not be demonstrated.     

Localizing extracellular signal–regulated kinase (ERK) in pharmacological preconditioning's trigger pathway

Acetylcholine (ACh) and opioid receptor agonists trigger the preconditioned phenotype through sequential activation of the epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3-K), Akt, and nitric oxide synthase (NOS), and opening of mitochondrial (mito) K(ATP) channels with the generation of reactive oxygen species (ROS). Although extracellular signal-regulated kinase (ERK) has recently been reported to be part of this pathway, its location has not been determined. To address this issue, we administered a 5-min pulse of ACh (550 microM) prior to 30 min of ischemia in isolated rabbit hearts. It reduced infarction from 30.4 +/- 2.2% of the risk zone in control hearts to 12.3 +/- 2.8% and co-administration of the MEK, and, therefore, downstream ERK inhibitor U0126 abolished protection (29.1 +/- 4.6% infarction) con.rming ERK's involvement. MitoK(ATP) opening was monitored in adult rabbit cardiomyocytes by measuring ROS production with MitoTracker Red. ROS production was increased by each of three G protein-coupled agonists: ACh (250 microM), bradykinin (BK) (500 nM), and the delta-opioid agonist DADLE (20 nM). Co-incubation with the MEK inhibitors U0126 (500 nM) or PD 98059 (10 microM) blocked the increased ROS production seen with all three agonists. Direct activation of its receptor by EGF increased ROS production and PD 98059 blocked that increase, thus placing ERK downstream of the EGF receptor. Desferoxamine (DFO) which opens mitoK(ATP) through direct activation of NOS also increased ROS. PD 98059 could not block DFO-induced ROS production, placing ERK upstream of NOS. In isolated hearts, ACh caused phosphorylation of both Akt and ERK. U0126 blocked phosphorylation of ERK but not of Akt. The PI3-K inhibitor wortmannin blocked both. Together these data indicate that ERK is located between Akt and NOS.     

Asbestos-induced activation of cell signaling pathways in human bronchial epithelial cells

Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blotting using phospho-specific antibodies demonstrated that the EGF receptor kinase inhibitor PD153035 decreased both the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and its upstream signal pathway, including mitogen-activate protein kinase/ERK kinase (MEK)1/2. Similarly, the MEK1/2 kinase inhibitor PD98059 also demonstrated the ability to decrease phosphorylation of ERK1/2. Crocidolite-induced phosphorylation of EGF receptor, ERK1/2, and MEK1/2 was reduced by transfection of BEAS-2B cells with a catalase vector, supporting a participation of oxidative stress in this pathway. These results show that crocidolite can activate the phosphorylation of EGF receptor and its downstream cell signal pathway in BEAS-2B cells and this is associated with the oxidative stress presented by the fibers.     

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.     

Netrin-1 induces angiogenesis via a DCC-dependent ERK1/2-eNOS feed-forward mechanism

Netrin-1 is critical for axonal pathfinding which shares similarities with formation of vascular network. Here we report that netrin-1 induction of angiogenesis is mediated by an increase in endothelial nitric oxide (NO*) production, which occurs via a DCC-dependent, ERK1/2-eNOS feed-forward mechanism. Exposure of mature aortic endothelial cells to netrin-1 resulted in a potent, dose-dependent increase in NO* production, detected by electron spin resonance. Scavenging NO* with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) abolished netrin-1 stimulated angiogenesis. Netrin-1-stimulated NO* production or angiogenesis was inhibited by DCC antibody, DCC small interfering RNA (siRNA), specific inhibitors (PD98059, U0126), or siRNAs for MEK1/2. PTIO attenuated ERK1/2 phosphorylation, indicating a feed-forward mechanism. Netrin-1 induced a time-dependent phosphorylation of eNOS(s1179, s116) and a rapid dephosphorylation of eNOS(t497). Only eNOS(s1179) was sensitive to U0126 or PTIO. These data characterized a mechanism whereby netrin-1 promotes angiogenesis, which may broadly relate to cardiovascular, neuronal and cancer physiology.     

MEK1/2 inhibitors potentiate UCN-01 lethality in human multiple myeloma cells through a Bim-dependent mechanism

The role of Bim in synergistic interactions between UCN-01 and MEK1/2 inhibitors in human multiple myeloma cells was investigated. Exposure of U266 or RPMI8226 cells to UCN-01 resulted in ERK1/2 activation-associated Bim(EL) phosphorylation/down-regulation, events abrogated by MEK1/2 inhibitors. Enforced activation of ERK1/2 by transfection with constitutively active MEK1 diminished the capacity of PD98059 but not PD184352 to block UCN-01-mediated Bim(EL) phosphorylation and to potentiate apoptosis. Cotreatment with MEK1/2 inhibitors increased the association of Bim(EL) with both Bcl-2 and Bcl-x(L) in UCN-01-treated cells, leading to Bax/Bak conformational change and Bax mitochondrial translocation. Down-regulation of Bim(EL) by shRNA substantially diminished UCN-01/MEK inhibitor-mediated Bax/Bak activation and apoptosis. Furthermore, transfection of cells with S65A Bim, a mutant resistant to UCN-01-mediated phosphorylation, significantly sensitized cells to UCN-01 lethality. Conversely, ectopic expression of either Bcl-2 or Bcl-x(L) did not alter UCN-01/MEK1/2 inhibitor-mediated modifications in Bim(EL) phosphorylation but largely prevented cell death. Finally, IL-6 or IGF-1 failed to prevent MEK1/2 inhibitors from blocking UCN-01-induced Bim(EL) phosphorylation/degradation or cell death. Collectively, these findings argue that UCN-01-mediated ERK1/2 activation leads to Bim(EL) phosphorylation/inactivation, resulting in cytoprotection, and that interference with these events by MEK1/2 inhibitors plays a critical role in synergistic induction of apoptosis by these agents.     

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.     

cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway

BACKGROUND/AIMS: To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion. METHODS: Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway. RESULTS: Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2. CONCLUSIONS: Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases.     

Akt和胞外信号调节激酶通路间的信号交流对内质网应激条件下肝癌细胞周期的调控

目的 研究内质网应激介导的磷脂酰肌醇3激酶(PI3K)/Akt和丝裂原活化蛋白激酶(MEK)/胞外信号调节激酶(ERK)途径间的信号交流及其对内质网应激条件下肝癌细胞周期的调控作用.方法 采用PI3K抑制剂LY294002、Akt激活型突变载体myr-Akt和MEK抑制剂U0126分别阻断或激活内质网应激介导的Akt和ERK活化,并利用Western blot和流式细胞技术分析内质网应激条件下PI3K/Akt和MEK/ERK途径间的信号交流及其对肝癌细胞株SMMC-7721、Hep3B和HepG2细胞周期的调控作用.数据处理采用Sperman等级相关分析,P＜0.05为差异有统计学意义.结果 阻断PI3K/Akt明显促进内质网应激介导的MEK/ERK活化,而过度激活PI3K/Akt则抑制内质网应激介导的MEK/ERK活化.阻断MEK/ERK对内质网应激介导的PI3K/Akt活化无影响.持续活化的Akt突变载体myr-Akt和MEK抑制剂U0126均明显抑制了内质网应激诱导的压力细胞G0/G1期阻滞.结论 PI3K/Akt和MEK/ERK信号途径在内质网应激肝癌细胞中存在信号交流,该信号交流对细胞周期起重要调控作用.     

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4β-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4α-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.     

Protein kinase C (PKC)-delta/-epsilon mediate the PKC/Akt-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 in MCF-7 cells stimulated by bradykinin

In this paper the signal transduction pathways evoked by bradykinin (BK) in MCF-7 breast cancer cells were investigated. BK activation of the B(2) receptor provoked: (a) the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2); (b) the translocation from the cytosol to the membrane of the conventional protein kinase C-alpha (PKC-alpha) and novel PKC-delta and PKC-epsilon; (c) the phosphorylation of protein kinase B (PKB/ Akt); (d) the proliferation of MCF-7 cells. The BK-induced ERK1/2 phosphorylation was completely blocked by PD98059 (an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK)) and by LY294002 (an inhibitor of phosphoinositide 3-kinase (PI3K)), and was reduced by GF109203X (an inhibitor of both novel and conventional PKCs); G?6976, a conventional PKCs inhibitor, did not have any effect. The BK-induced phosphorylation of PKB/Akt was blocked by LY294002 but not by PD98059. Furthermore, LY294002 inhibited the BK-provoked translocation of PKC-delta and PKC-epsilon suggesting that PI3K may be upstream to PKCs. Finally, the proliferative effects of BK were blocked by PD98059, GF109203X and LY294002. These observations demonstrate that BK acts as a proliferative agent in MCF-7 cells activating intracellular pathways involving novel PKC-delta/-epsilon, PKB/Akt and ERK1/2.     

Growth hormone reduces chloride secretion in human colonic epithelial cells via EGF receptor and extracellular regulated kinase

BACKGROUND AND AIMS: Growth hormone (GH) has been shown to alleviate symptoms in patients with Crohn's disease. Chloride secretion is important in driving intestinal fluid secretion. We examined whether GH inhibits chloride secretion induced by carbachol (CCh, a calcium-dependent pathway), and the downstream effectors responsible. METHODS: T(84) cells were pretreated with GH at various concentrations followed by CCh (100 micromol/L). Chloride secretion was assessed as changes in short circuit current ( triangle up I(sc)) in Ussing chambers. Tyrphostins AG1478 (an epidermal growth factor receptor [EGFr] inhibitor) and AG490 (a Janus kinase 2 [JAK2] inhibitor), SB203580 (a p38 inhibitor), and PD98059 (a MEK1 inhibitor) were used. RESULTS: GH inhibited CCh-induced chloride secretion at up to 10 nmol/L, but higher concentrations were less effective. GH caused tyrosine phosphorylation of JAK2 and EGFr. AG490 suppressed activation of JAK2 and EGFr in response to GH. AG1478 prevented GH activation of EGFr and reversed its inhibitory effect on chloride secretion. GH also induced activation of both p38 and ERK1/2. AG490 reversed GH-induced tyrosine phosphorylation of both ERK1/2 and p38, but AG1478 reversed that of ERK1/2 only. PD98059, but not SB203580, reversed the inhibitory effect of GH on chloride secretion. CONCLUSIONS: GH inhibits CCh-induced chloride secretion via a JAK2-dependent mechanism involving transactivation of EGFr and consequent recruitment of ERK1/2. Although activated, p38 does not contribute to the inhibitory effect of GH on secretion. These data elucidate mechanisms of GH inhibition of chloride secretion in intestinal epithelia, which may be relevant to therapeutic benefits of GH in Crohn's disease or other diarrheal diseases.     

Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.