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.     

Reduced expression of flavocytochrome b558, a component of the NADPH oxidase complex,in neutrophils from patients with myelodysplasia

OBJECTIVE: Patients with myelodysplasia (MDS) show a disturbed production of ROS in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils. Because generation of ROS is mediated by the NADPH oxidase complex, a component of which is flavocytochrome b558, we investigated whether the expression of flavocytochrome b558 in neutrophils from MDS patients is affected. MATERIAL AND METHODS: Neutrophils were stimulated with fMLP and GM-CSF, and plasma membrane expression of flavocytochrome b558 and specific granule markers were assessed by fluorescence-activated cell sorting analysis. Protein levels of the flavocytochrome b558 subunits gp91phox and p22phox in whole neutrophil lysates were detected by Western blotting. RESULTS: Stimulation of neutrophils with GM-CSF and fMLP increased the flavocytochrome b558 plasma membrane expression. The fMLP-induced translocation of flavocytochrome b558 was reduced in neutrophils from MDS patients (140%+/-9% vs 180%+/-13%, p<0.05). Analysis of cell surface expression of markers of flavocytochrome b558 containing granules (CD35 and CD66b) indicated that exocytosis of these granules in response to fMLP stimulation was not affected in MDS patients. Western blot analysis demonstrated a decreased protein expression level of the flavocytochrome b558 subunits gp91phox and p22phox in neutrophils from MDS patients. CONCLUSION: Our results indicate both a lower basal protein level and a disturbed fMLP-induced increase in plasma membrane expression of flavocytochrome b558 in neutrophils from MDS patients, which together might play a role in decreased ROS production.     

Role of phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways in granulocyte macrophage-colony-stimulating factor failure to delay fas-induced neutrophil apoptosis in elderly humans

Fas-stimulated neutrophils from elderly individuals show impaired granulocyte macrophage-colony-stimulating factor (GM-CSF)-induced apoptosis cell rescue. Herein, this defect was found to be associated with a significant reduction in GM-CSF-mediated Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Using Akt and ERK1/2 inhibitors, we demonstrated that both kinases were critical for GM-CSF antiapoptotic effects. Whereas Akt inhibition also affected GM-CSF-dependent ERK1/2 phosphorylation, ERK1/2 inhibition did not affect GM-CSF-induced Akt phosphorylation, suggesting that phosphoinositide 3-kinase (PI3-K)/Akt and ERK1/2 are activated in series and that PI3-K is located upstream of ERK1/2 along the GM-CSF-dependent signaling pathway. No age-associated changes in GM-CSF receptor expression were observed. Interestingly, both suppressors of cytokine signaling (SOCS)1 and SOCS3 proteins were significantly higher in unstimulated neutrophils from elderly individuals and, unlike in young individuals, did not further increase following GM-CSF cell triggering. These results indicate that defective PI3-K/Akt/ERK1/2 activation, likely dependent on elevated SOCS1 and SOCS3 levels, may affect the GM-CSF capacity to delay neutrophil apoptosis in elderly persons.     

Hepatocyte growth factor stimulates nitric oxide production through endothelial nitric oxide synthase activation by the phosphoinositide 3-kinase/Akt pathway and possibly by mitogen-activated protein kinase kinase in vascular endothelial cells.

Hepatocyte growth factor (HGF) has recently been the focus of attention due to its angiogenic effects, which are similar to those of vascular endothelial growth factor (VEGF); because of these effects, HGF is considered to be a novel therapeutic agent against vascular disorders, including atherosclerotic angiopathies. Although nitric oxide (NO), which is derived from vascular endothelial cells (ECs), is also involved in angiogenesis, little is known regarding the interactions between HGF and NO. We therefore examined the effects of HGF on NO production as well as endothelial NO synthase (eNOS) phosphorylation, and investigated their mechanisms. In bovine aortic ECs, HGF induced a rapid (5 min) increase of NO production measured by diaminofluorescein-2 diacetate. Moreover, HGF rapidly (2.5 min) stimulated eNOS phosphorylation (Ser-1179) as determined by Western immunoblot analyses. Both of these effects were almost completely suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and were partially suppressed by the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126. HGF also stimulated Akt phosphorylation (Ser-473), which was completely suppressed by LY294002 and was partially suppressed by U0126. Moreover, HGF stimulated extracellular signal-regulated kinase 1/2 phosphorylation (Thr-202/Tyr-204), which was completely suppressed by U0126 and was partially suppressed by LY294002. Taken together, these results indicate that HGF not only phosphorylates eNOS through the PI3K/Akt pathway, but also partially through the MAPK pathway, and that these two pathways may interact. Compared with VEGF, HGF was more potent in both NO production and eNOS phosphorylation. Our study thus demonstrates a novel activity of HGF-the stimulation of NO production-which occurs via eNOS phosphorylation that may in turn be mediated by cross-talk between the PI3K/Akt and MAPK pathways.     

The involvement of phosphatidylinositol 3-kinase in gonadotropin-releasing hormone-induced gonadotropin alpha- and FSHbeta-subunit genes expression in clonal gonadotroph LbetaT2 cells

Akt/protein kinase B (Akt/PKB), which is activated by phosphatidylinositol-3 kinase (PI3-kinase), plays an important role in cell survival and cell proliferation. Using the well differentiated, clonal gonadotroph cell line, LbetaT2, we examined (1) whether Akt/PKB was activated by gonadotropin-releasing hormone (GnRH); (2) the contribution of PI3-kinase-Akt/PKB pathway in each of gonadotropin subunit gene expression; (3) crosstalk between extracellular signal-regulated kinase (ERK) and Akt/PKB pathways. Insulin-like growth factor-1 (IGF-1) was used as Akt/PKBs classic activator. Western blot analyses using antibodies specific for the phosphorylated forms of ERK and Akt/PKB demonstrated that both were rapidly phosphorylated following treatment with GnRH and IGF-1. Akt/PKB activation by GnRH and IGF-1 was completely eliminated in the presence of the PI3-kinase inhibitor, LY 294002, but not in the presence of an Akt/PKB inhibitor. Interestingly, the total amount of Akt/PKB protein was dramatically increased in the presence of LY 294002. Phosphorylation of ERK was significantly increased in the presence of LY 294002 alone, and was further increased when GnRH was used in combination with LY 294002. In experiments using a luciferase reporter construct containing the serum response element (SRE), a known target of the ERK pathway, LY 294002 but not the Akt/PKB inhibitor increased SRE-luciferase activity. GnRH-induced SRE-luciferase activity was significantly increased by LY 294002. GnRH stimulation resulted in gonadotropin LHbeta, FSHbeta, and alpha-subunit promoter activation, while IGF-1 failed to stimulate any of them. GnRH-induced gonadotropin promoter activities were not modulated in the presence of an Akt/PKB inhibitor, but treatment with LY 294002 or Wortmannin resulted in a significant increase in alpha- and FSHbeta-subunit promoter activation, both with and without GnRH. LY 294002, but not the Akt/PKB inhibitor, significantly inhibited cell proliferation. These results suggest that GnRH-induced gonadotropin gene expression is not regulated through the Akt/PKB pathway; however, PI3-kinase may be involved in the negative regulation of alpha- and FSHbeta-subunit gene expression as well as cell proliferation.     

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.     

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.     

Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway

OBJECTIVE: We tested whether ischemic postconditioning (IPostC) is protective in remodeled myocardium. METHODS: Post-myocardial infarct (MI)-remodeled hearts after permanent coronary artery ligation and one kidney one clip (1K1C) hypertensive hearts of male Wistar rats were exposed to 40 min of ischemia followed by 90 min of reperfusion. IPostC was induced by six cycles of 10 s reperfusion interspersed by 10 s of no-flow ischemia. Activation of reperfusion injury salvage kinases was measured using Western blotting and in vitro kinase activity assays. RESULTS: IPostC prevented myocardial damage in both MI-remodeled and 1K1C hearts, as measured by decreased infarct size and lactate dehydrogenase release, and improved function. The reduction in infarct size and the recovery of left ventricular contractility achieved by IPostC was less in 1K1C hearts, but was unchanged in MI-remodeled hearts when compared to healthy hearts. In contrast, the recovery of inotropy was unaffected in 1K1C hearts, but was less in MI-remodeled hearts. Inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway with LY294002 abolished the protective effects of IPostC on both disease models and healthy hearts. Western blot analysis in conjunction with in vitro kinase activity assays identified protein kinase B (PKB)/Akt but not p42/p44 extracellular-signal regulated kinase 1/2 (ERK1/2) as the predominant kinase in IPostC-mediated cardioprotection in remodeled hearts. IPostC increased phosphorylation of the PKB/Akt downstream targets eNOS, GSK3beta, and p70S6K in remodeled hearts. CONCLUSION: Our results offer evidence that IPostC mediates cardioprotection in the remodeled rat myocardium primarily via activation of the PI3K-PKB/Akt reperfusion injury salvage kinase pathway.     

Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients

OBJECTIVE: The phosphorylation state of the S6 ribosomal protein was measured in the peripheral blasts of 19 newly diagnosed patients with acute leukemia. METHODS: We employed a flow cytometry protocol that enabled correlated measurement of pS6, phosphorylation of extracellular signal-regulated kinase (pERK), and cluster differentiation surface markers. Baseline levels of pS6 in leukemic blasts were compared with those found when the samples were activated using stem cell factor, or exposed to rapamycin, LY294002, or the mitogen-activated protein kinase inhibitor U0126. RESULTS: Results showed a considerable degree of intra- and intertumoral heterogeneity in the constitutive levels of pS6. Rapamycin and LY294002 suppressed pS6 in 10 of 11 cases that showed increased basal levels, consistent with phosphatidylinositol 3 (PI3)-kinase/Akt/mTOR signaling being the predominant upstream signaling pathway. However, in 6 of 11 cases pS6 was also suppressed by U0126, indicating that the ERK pathway can significantly input to pS6. CONCLUSIONS: The constitutive activation of pS6 in acute leukemia patients likely reflects alterations in growth factor signaling that can be mediated by the ERK as well as the mTOR pathway, and could potentially have prognostic significance. As well as identifying aberrant signal transduction in leukemia patients, the flow cytometry methodology has potential for the pharmacodynamic monitoring of novel agents that inhibit ERK or PI3-kinase/Akt/mTOR signaling.     

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.     

Activation of Human Neutrophils by Granulocyte Colony-Stimulating Factor,Granulocyte-Macrophage Colony-Stimulating Factor,and Tumor Necrosis Factor α: Role of Phosphatidylinositol 3-Kinase

Stimulation of human neutrophils with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or tumor necrosis factor alpha (TNF) resulted in phosphorylation of Akt, the potency being GM-CSF > G-CSF = TNF, which was inhibited by wortmannin. The findings indicated that phosphatidylinositol 3-kinase (PI3K) is activated by these cytokines. The possible participation of PI3K in activation of neutrophil functions induced by these cytokines was explored with PI3K inhibitors (wortmannin and LY294002). Superoxide release and adherence induced by GM-CSF or TNF were inhibited by PI3K inhibitors. Actin reorganization and morphological changes induced by G-CSF or GM-CSF were also inhibited by wortmannin, whereas these responses induced by TNF were unaffected by wortmannin. These findings suggested that PI3K is differentially involved in cytokine-mediated activation of neutrophil functions depending on the cytokines used. The results also showed that activation of extracellular signal-regulated kinase, but not p38 mitogen-activated protein kinase, induced by these cytokines is partly mediated by PI3K activation.     

Platelet factor 4 inhibits FGF2-induced endothelial cell proliferation via the extracellular signal-regulated kinase pathway but not by the phosphatidylinositol 3-kinase pathway

Platelet factor 4 (PF-4) is a member of the chemokine family with powerful antiangiogenic properties. The mechanism by which PF-4 inhibits endothelial cell proliferation is unclear. We investigated the effects of PF-4 on the intracellular signal transduction induced by basic fibroblast growth factor (FGF2). We found that PF-4 (10 microg/mL) inhibited the FGF2-induced proliferation of adrenal cortex capillary endothelial (ACE) cells. The inhibition of MEK1/2 (mitogen-activated protein kinase kinase) by PD98059 or of PI3K (phosphatidylinositol 3-kinase) by Ly294002 abolished the proliferation induced by FGF2, suggesting that ACE cell proliferation required dual signaling through both the extracellular signal-regulated kinase (ERK) and PI3K pathways. Ly294002 had no significant effect on ERK phosphorylation, whereas PD98059 had a weak effect on the phosphorylation of Akt, suggesting that 2 separate cascades are required for ACE cell proliferation. The addition of PF-4 (10 microg/mL) significantly inhibited ERK phosphorylation (95%), showing that PF-4 acted directly on or upstream from this kinase. Surprisingly, PF-4 did not affect FGF2-induced Akt phosphorylation. This suggests that PF-4 disrupts FGF2 signaling via an intracellular mechanism of inhibition. To exclude the possibility that PF-4 inhibited the binding of FGF2 to only one FGF receptor, preferentially activating the ERK pathway, we investigated the effect of PF-4 on FGF2-induced ERK and Akt phosphorylation, using mutant heparan sulfate-deficient Chinese hamster ovary cells transfected with the FGF-R1 cDNA. The addition of PF-4 (1 microg/mL) significantly inhibited ERK phosphorylation (90%), with no effect on Akt phosphorylation, suggesting that PF-4 acts downstream from the FGF-R1 receptor. In conclusion, this is the first report showing that PF-4 inhibits FGF2 activity downstream from its receptor.     

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.     

Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and peroxisome proliferator-activated receptor gamma signaling

The molecular events of hyperglycemia-triggered increase in adipogenic induction of lipid accumulation remain unclear. We examined the effects of hyperglycemia on adipogenic induction of lipid accumulation and its involved signaling molecules, such as phosphoinositide 3-kinase (PI3K), ERKs, and peroxisome proliferator-activated receptor gamma (PPAR gamma). Bone marrow-derived mesenchymal stem cells (MSCs) isolated from FVB/N mice were capable of differentiating into adipocytes in adipogenic medium. The effects of high glucose (HG) (25.5 mm) were assessed in vitro by RT-PCR, ELISA, flow cytometry, immunostaining, and immunoblotting. The in vivo effect of hyperglycemia was further studied in streptozotocin (STZ)-induced diabetic FVB/N mice. Exposure of MSCs to HG enhanced adipogenic induction of lipid accumulation as compared with 5.5 mm glucose. HG increased PPAR gamma expression and PI3K activity and its downstream effector Akt phosphorylation during adipogenesis. Inhibition of PI3K/Akt activity with PI3K inhibitor LY294002 or by expressing the dominant negative p85 or Akt prevented the HG-enhanced PPAR gamma-dependent adipogenic induction of lipid accumulation. Moreover, HG increased the phosphorylation of ERK1/2 during adipogenesis. MAPK/ERK inhibitor PD98059 inhibited the PI3K activity, Akt phosphorylation, and lipid accumulation triggered by HG. PI3K inhibitor LY294002 did not affect the HG-increased ERK1/2 phosphorylation during adipogenesis. We next observed that adipogenic induction of lipid accumulation of MSCs isolated from STZ-induced diabetic mice is enhanced. Moreover, triglyceride, PPAR gamma expression, phosphorylated Akt and ERK1/2, and marrow fat in bones of STZ-diabetic mice were also increased. These results suggest that hyperglycemia enhances the adipogenic induction of lipid accumulation through an ERK1/2-activated PI3K/Akt-regulated PPAR gamma pathway.     

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

Stimulation of insulin-like growth factor (IGF) binding protein-3 synthesis by IGF-I and transforming growth factor-alpha is mediated by both phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways in mammary epithelial cells

IGF binding protein (IGFBP)-3 is an important regulator of mammary epithelial cell (MEC) growth and can enhance the ability of both IGF-I and epidermal growth factor ligands such as TGFalpha to stimulate MEC proliferation. Here we investigate the role of the phosphatidylinositol-3 kinase (PI3K) and MAPK pathways in the regulation of IGFBP-3 expression by IGF-I and TGFalpha in bovine MECs. Both growth factors stimulated DNA synthesis, although IGF-I was the stronger mitogen. IGF-I and TGFalpha also stimulated IGFBP-3 mRNA and protein levels. TGFalpha stimulated rapid, transient activation of Akt that was maximal at 5 min and diminished by 15 min. In contrast, IGF-I-induced Akt activation was maximal between 15 and 90 min and was sustained for 6 h. Although ERK 1/2 was maximally stimulated by TGFalpha between 5 and 15 min, IGF-I did not stimulate discernible activation of ERK 1/2. In addition, TGFalpha but not IGF-I induced rapid phosphorylation of Shc, whereas only IGF-I activated insulin receptor substrate-1. Pretreatment with the PI3K inhibitor LY294002 or knockdown of p85 with small interfering RNA inhibited IGF-I or TGFalpha-stimulated IGFBP-3 expression. Similarly, MAPK kinase-1 inhibitors PD98059 and U0126 each abolished TGFalpha-stimulated increases in IGFBP-3 mRNA levels. In contrast to TGFalpha, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MAPK kinase-1 inhibitors, indicating that IGF-I may activate alternative substrates of the PI3K pathway that are involved in IGFBP-3 regulation. In conclusion, stimulation of IGFBP-3 mRNA levels by mitogens is regulated through both the PI3K and MAPK pathways in bovine MECs.     

Simultaneous inhibition of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways augment the sensitivity to actinomycin D in Ewing sarcoma

Purpose  Ewing sarcoma cells, of which over 85% retain chimeric fusion gene EWS/Fli-1, are by and large more resistant to chemotherapeutics compared to nonneoplastic cells. The purpose of this study is to determine the role of EWS/Fli-1 fusion and its downstream targets regarding the cells’ resistance against actinomycin D (ActD), which is one of the most commonly used antitumor agents in combination chemotherapy of Ewing sarcomas. Methods  Cytotoxicity was measured by WST-8 assay. Caspase-dependent and -independent cell death was examined by fluorescence microscope. Protein expression was analyzed by western blotting. Caspase activity was determined by Caspase-Glo assay. Results  ActD-induced caspase-dependent apoptotic cell death to Ewing sarcoma TC-135 cells in a dose- and time- dependent manner. Knockdown of EWS/Fli-1 fusion by siRNA resulted in enhancement of ActD-induced apoptosis. ActD treatment activated both mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways although in a distinctive manner. Combined administration of U0126 (MEK inhibitor) and LY294002 (PI3K inhibitor) significantly enhanced ActD-induced apoptosis in vitro and suppressed xenograft tumor growth in vivo. Conclusions  The present study demonstrated for the first time that combination of U0126 and LY294002 can augment the cytotoxicity of ActD against Ewing sarcoma cells in vitro and in vivo. Our results indicate that further study on combination of conventional chemotherapies with MEK and PI3K inhibitors may be considered for innovative treatments of Ewing sarcoma patients.     

洛伐他汀经PI3K/Akt和ERK1/2信号通路抑制大鼠骨髓间充质干细胞凋亡

目的 体外以缺氧无血清条件模拟心肌梗死后的心脏缺血微环境,研究洛伐他汀能否抑制缺氧无血清引起的骨髓间充质干细胞(MSC)凋亡并探讨其机制.方法 以Hocchst33342染色荧光显微镜观察法及Annexin V/PI流式细胞术检测洛伐他汀的抗凋亡作用,并进一步采用Westernblot方法 检测洛伐他汀对线粒体凋亡途径的抑制作用以及对磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(Akt)途径和丝裂原活化的蛋白激酶(MAPK)的激酶(MEK)/细胞内信号调节蛋白激酶(ERK1/2)途径的激活作用.结果 0.01～1 μmol/L浓度范围的洛伐他汀能够有效地抑制缺氧无血清引起的MSC凋亡.洛伐他汀抑制线粒体凋亡途径,洛伐他汀抑制细胞色素C释放,降低天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)活化,从而保护线粒体功能.洛伐他汀的抗凋亡效应以及其抑制细胞色素C释放的作用均可被PI3K抑制剂LY294002和MEK抑制剂U0126阻断.洛伐他汀能够激活PI3K/Akt和MEK/ERK1/2两条细胞存活信号通路,分别导致Akt和GSK-3β及ERK1/2磷酸化.结论 洛伐他汀能够抑制线粒体凋亡途径,并激活PI3K/Akt和MEK/ERK1/2细胞存活通路,最终发挥抗缺氧无血清引起的MSC凋亡.该研究为提高移植干细胞的存活率提供了一种可能有效的干预措施.     

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.     

Involvement of Akt kinase in the action of STI571 on chronic myelogenous leukemia cells

To elucidate the role of mitogen-activated protein kinases (MAPKs) and Akt kinase in leukemogenesis caused by the breakpoint cluster region (BCR)-Abelson (ABL) tyrosine kinase oncoprotein, we examined the activities of MAPKs and Akt kinase and their roles in the action of STI571, a specific inhibitor of BCR-ABL tyrosine kinase, in chronic myelogenous leukemia (CML) cells. We found that extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase are constitutively active in the chronic phase of CML, blast crisis of CML, and the CML-derived K562 cell line. Both interferon-alpha and STI571 suppressed ERK1/2 activity in K562 cells. In contrast, Akt kinase activity was inhibited only by STI571. K562 cell proliferation was markedly suppressed by LY294002, a specific inhibitor of PI3K/Akt kinase, and STI571 but not by PD98059, a specific inhibitor of MEK1/2. In addition, caspase-3 was activated by treatment of cells with STI571 and LY294002 but not with PD98059. These data indicate that Akt kinase may play a role in the proliferation of CML leukemia cells and the action of STI571. Primary leukemia cells from patients with CML blast crisis did not show inhibition of ERK1/2 or Akt kinase activity and were resistant to caspase-3-associated apoptosis after treatment with STI571. These findings suggest that STI571 does not effectively block signaling molecules downstream of the BCR-ABL tyrosine kinase in some cases of CML blast crisis.