Suppression of adriamycin-induced apoptosis by sustained activation of the phosphatidylinositol-3'-OH kinase-Akt pathway

The mechanisms by which growth factors trigger signal transduction pathways leading to protection against apoptosis are of great interest. In this study, we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on adriamycin (ADR)-induced apoptosis. Treatment of human epithelial MKN74 cells with ADR, a DNA topoisomerase IIalpha inhibitor, caused apoptosis. However, cells pretreated with HGF/SF, but not those pretreated with EGF, were resistant to this apoptosis. The protective effect of HGF/SF against the ADR-induced apoptosis was abolished in the presence of either LY294002, an inhibitor of phosphatidylinositol-3'-OH kinase (PI3-K) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, an inhibitor of Akt, thus implicating the activation of PI3-K-Akt signaling in the antiapoptotic action of HGF/SF. Immunoblotting analysis revealed that HGF/SF stimulated the sustained phosphorylation of Akt for several hours but that EGF stimulated the phosphorylation only transiently. Furthermore, ADR-induced activation of caspase-9, a downstream molecule of Akt, was inhibited for at least 24 h after HGF/SF stimulation, but it was not affected by EGF stimulation. Cell-surface biotin-labeling analysis showed that the HGF/SF receptor remained on the cell surface until at least 30 min after HGF/SF addition but that the EGF receptor level on the cell surface was attenuated at an earlier time after EGF addition. These results indicate that HGF/SF, but not EGF, transmitted protective signals against ADR-induced apoptosis by causing sustained activation of the PI3-K-Akt signaling pathway. Furthermore, the difference in antiapoptotic capacity between HGF/SF and EGF is explained, at least in part, by the delayed down-regulation of the HGF/SF receptor.     

Mechanisms for growth factor-induced pituitary tumor transforming gene-1 expression in pituitary folliculostellate TtT/GF cells

PTTG1, a securin protein, also behaves as a transforming gene and is overexpressed in pituitary tumors. Because pituitary folliculostellate (FS) cells regulate pituitary tumor growth factors by paracrine mechanisms, epidermal growth factor (EGF) receptor (EGFR)-mediated PTTG1 expression and cell proliferation was tested in pituitary FS TtT/GF cells. EGFR ligands caused up to 3-fold induction of Pttg1 mRNA expression, enhanced proliferating cell nuclear antigen, and increased entry of G0/1-arrested cells into S-phase. PTTG binding factor mRNA expression was not altered. EGF-induced Pttg1 expression and cell proliferation was abolished by preincubation of TtT/GF cells with EGFR inhibitors AG1478 and gefitinib. Phosphatidylinositol 3 kinase, protein kinase C, and MAPK, but not c-Jun N-terminal kinase and Janus activating kinase signaling regulated EGF-induced Pttg1, as well as proliferating cell nuclear antigen mRNA expression and entry into S-phase. EGF-induced EGFR and ERK1/2 phosphorylation was followed by rapid MAPK kinase/ERK kinase-dependent activation of Elk-1 and c-Fos. EGF-induced Pttg1 expression peaked at the S-G2 transition and declined thereafter. Pttg1 cell cycle dependency was confirmed by suppression of EGF-induced Pttg1 mRNA by blockade of cells in early S-phase. The results show that PTTG1 and its binding protein PTTG binding factor are expressed in pituitary FS TtT/GF cells. EGFR ligands induce PTTG1 and regulate S-phase, mediated by phosphatidylinositol 3 kinase, protein kinase C, and MAPK pathways. PTTG1 is therefore a target for EGFR-mediated paracrine regulation of pituitary cell growth.     

Mitogen activated protein kinase-dependent activation of c-Jun and c-Fos is required for neuronal differentiation but not for growth and stress response in PC12 cells

MAPK-dependent activation of AP-1 protein c-Jun is involved in PC12 cell differentiation and apoptosis. However, the role of other AP-1 proteins and their connection to MAPKs during growth, differentiation and apoptosis has remained elusive. Here we studied the activation of AP-1 proteins in response to ERK, JNK, and p38 signaling upon NGF, EGF and anisomycin exposures. All treatments caused different kinetics and strength of MAPK and AP-1 activities. NGF induced persistent ERK and AP-1 activities, whereas upon EGF and anisomycin exposures, their activities were only weakly and transiently induced. The sustained AP-1 activity was associated with concomitant c-Fos and c-Jun expression and phoshorylation, which were JNK and ERK dependent. While inhibition of the ERK, JNK, and p38 activities partially prevented AP-1 activity and suppressed differentiation, none of them was required for anisomycin-induced apoptosis. The importance of c-Fos and c-Jun as mediators of differentiation was demonstrated by the findings that the corresponding siRNAs suppressed NGF-induced neurite outgrowth. However, the capacity of c-Fos to promote differentiation required cooperation with Jun proteins. In contrast, Fra-2 expression was not required for the differentiation response. Together, the results show that sustained c-Jun and c-Fos activities mediate MAPK signaling and are essential for differentiation of PC12 cells.     

Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.     

Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling

Isoliquiritigenin (ISL, 4,2′,4′-trihydroxychalcone), which is found in licorice, shallot and bean sprouts, is a potent antioxidant with anti-inflammatory and anti-carcinogenic effects. The purpose of this study was to investigate the effects of ISL treatment on the migration, invasion and adhesion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0–20 μmol/L ISL with or without 10 μg/L epidermal growth factor (EGF). ISL inhibited basal and EGF-induced cell migration, invasion and adhesion dose dependently. ISL decreased EGF-induced secretion of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF), but increased TIMP-2 secretion in a concentration-dependent manner. In addition, ISL decreased the protein levels of integrin-α2, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), and mRNA levels of uPA, MMP-9, VEGF, ICAM and integrin-α2. Furthermore, basal and EGF-induced activator protein (AP)-1 binding activity and phosphorylation of Jun N-terminal kinase (JNK), c-Jun and Akt were decreased after ISL treatment. However, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase was not altered. The JNK inhibitor SP600125 inhibited basal and EGF-induced secretion of uPA, VEGF, MMP-9 and TIMP-1, as well as AP-1 DNA binding activity and cell migration. These results provide evidence for the role of ISL as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of prostate cancer cells. The inhibition of JNK/AP-1 signaling may be one of the mechanisms by which ISL inhibits cancer cell invasion and migration.     

Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.     

Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.     

Increased expression of Mcl-1 is responsible for the blockage of TRAIL-induced apoptosis mediated by EGF/ErbB1 signaling pathway

Epidermal growth factor (EGF) protects against death receptor induced apoptosis in epithelial cells. Herein, we demonstrate that EGF protection against tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis is mediated by increased expression of the Bcl-2 family member myeloid cell leukemia 1 (Mcl-1). EGF increased the mRNA and protein levels of Mcl-1. Furthermore, expression of ErbB1 alone or in combination with ErbB2 in NIH3T3 cells up-regulates Mcl-1 following EGF treatment. In addition, up-regulation of Mcl-1 by EGF is mediated through AKT and NFkappaB activation since kinase inactive AKT and DeltaIkappaB effectively blocks this up-regulation. NFkappaB was also critical for the ability of EGF to prevent TRAIL induced apoptosis as a dominant negative IkappaB (DeltaIkappaB) blocked NFkappaB activation, and relieved EGF protection against TRAIL mediated mitochondrial cytochrome-c release and apoptosis. Finally, anti-sense oligonucleotides directed against Mcl-1 effectively reduced the protein levels of Mcl-1 and blocked EGF protection against TRAIL induced mitochondrial cytochrome-c release and apoptosis. Taken together, EGF signaling leads to increased Mcl-1 expression that is required for blockage of TRAIL induced apoptosis.     

Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.