Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.     

Role of the p38 mitogen-activated protein kinase pathway in the generation of the effects of imatinib mesylate (STI571) in BCR-ABL-expressing cells

Imatinib mesylate (STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, exhibits potent antileukemic effects in vitro and in vivo. Despite the well established role of STI571 in the treatment of chronic myelogenous leukemia, the precise mechanisms by which inhibition of BCR-ABL tyrosine kinase activity results in generation of antileukemic responses remain unknown. In the present study we provide evidence that treatment of CML-derived BCR-ABL-expressing leukemia cells with STI571 results in activation of the p38 mitogen-activated protein (MAP) kinase signaling pathway. Our data indicate that STI571 induces phosphorylation of the p38 and activation of its kinase domain, in KT-1 cells and other BCR-ABL-expressing cell lines. We also identify the kinases MAP kinase-activated protein kinase-2 and Msk1 as two downstream effectors of p38, activated during inhibition of BCR-ABL activity by STI571. Importantly, pharmacological inhibition of p38 reverses the growth inhibitory effects of STI571 on primary leukemic colony-forming unit granulocyte/macrophage progenitors from patients with CML. Altogether, our data establish that activation of the p38 MAP kinase signaling cascade plays an important role in the generation of the effects of STI571 on BCR-ABL-expressing cells. They also suggest that, in addition to activation of mitogenic pathways, BCR-ABL promotes leukemogenesis by suppressing the function of growth inhibitory signaling cascades.     

Activation of the mitogen- and stress-activated kinase 1 by arsenic trioxide

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces activation of the mitogen- and stress-activated kinase 1 (MSK1) and downstream phosphorylation of its substrate, histone H3, in leukemia cell lines. Such activation requires upstream engagement of p38 MAPK, as demonstrated by experiments using pharmacological inhibitors of p38 or p38alpha knock-out cells. Arsenic-induced apoptosis was enhanced in cells in which MSK1 expression was decreased using small interfering RNA and in Msk1 knock-out mouse embryonic fibroblasts, suggesting that this kinase is activated in a negative feedback regulatory manner to regulate As2O3 responses. Consistent with this, pharmacological inhibition of MSK1 enhanced the suppressive effects of As2O3 on the growth of primary leukemic progenitors from chronic myelogenous leukemia patients. Altogether, these findings indicate an important role for MSK1 downstream of p38 in the regulation of As2O3 responses.     

Arsenic trioxide arrests cells early in mitosis leading to apoptosis

Arsenic trioxide (As(2)O(3)) is highly effective in the treatment of acute promyelocytic leukemias that express the promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. However, evidence has accumulated that As(2)O(3) induces apoptosis regardless of PML-RARalpha status. Here we show that, at clinically relevant concentrations, As(2)O(3) causes S and G(2)M phase arrest of both PML-RARalpha-positive and -negative leukemia cell lines, thus inhibiting their growth. Apoptotic cells are generated predominately from the G(2)M fraction. Using several independent methods, we demonstrate that the cells accumulated in the G(2)M peak consist primarily of cells arrested in the early stages of mitosis, prophase, prometaphase and metaphase. In mitotic cells, there was significant activation of caspases, PARP cleavage, and morphological changes characteristic of apoptosis. Unlike microtubule-active drugs that arrest cells in metaphase, arsenic trioxide did not affect the architecture of microtubules. Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis.     

The p38 MAPK pathway mediates the growth inhibitory effects of interferon-alpha in BCR-ABL-expressing cells

The mechanisms by which interferon-alpha (IFN-alpha) mediates its anti-leukemic effects in chronic myelogenous leukemia (CML) cells are not known. We determined whether p38 MAPK is activated by IFN-alpha in BCR-ABL-expressing cells and whether its function is required for the generation of growth inhibitory responses. IFN-alpha treatment induced phosphorylation/activation of p38 in the IFN-alpha-sensitive KT-1 cell line, but not in IFN-alpha-resistant K562 cells. Consistent with this, IFN-alpha treatment of KT-1 (but not K562) cells induced activation of the small GTPase Rac1, which functions as an upstream regulator of p38. In addition, IFN-alpha-dependent phosphorylation/activation of p38 was induced by treatment of primary granulocytes isolated from the peripheral blood of patients with CML. To define the functional role of the Rac1/p38 MAPK pathway in IFN-alpha signaling, the effects of pharmacological inhibition of p38 on the induction of IFN-alpha responses were determined. Treatment of KT-1 cells with the p38-specific inhibitors SB203580 and SB202190 reversed the growth inhibitory effects of IFN-alpha. On the other hand, the MEK kinase inhibitor PD098059 had no effects, further demonstrating the specificity of these findings. To directly determine the significance of IFN-alpha-dependent activation of p38 in the induction of the anti-leukemic effects of IFN-alpha, we evaluated the effects of p38 inhibition on leukemic colony formation in bone marrow samples of patients with CML. IFN-alpha inhibited leukemic granulocyte/macrophage colony formation in a dose-dependent manner, whereas concomitant treatment with p38 inhibitors reversed such an inhibition. Thus, the Rac1/p38 MAPK pathway is activated by IFN-alpha in BCR-ABL-expressing cells and appears to play a key role in the generation of the growth inhibitory effects of IFN-alpha in CML cells.     

Activation of Raf1 and the ERK pathway in response to l-ascorbic acid in acute myeloid leukemia cells

L-ascorbic acid (LAA) shows cytotoxicity and induces apoptosis of malignant cells in vitro, but the mechanisms by which such effects occur have not been elucidated. In the present study, we provide evidence that the ERK MAP kinase pathway is activated in response to LAA (< 1 mM) in acute myeloid leukemia cell lines. LAA treatment of cells induces a dose-dependent phosphorylation of extracellular signal-regulated kinases (ERK) and results in activation of its catalytic domain. Our data also demonstrate that the small G protein Raf1 and MAPK-activated protein kinase 2 are activated by LAA as an upstream and a downstream regulator of ERK, respectively. Although the ERK pathway has been known to activate cell proliferation, pharmacologic inhibition of ERK reduces LAA-dependent apoptosis and growth inhibitory response of acute myeloid leukemia cell lines, suggesting that this signaling cascade positively regulates induction of apoptotic response by LAA.     

Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells

Bromocriptine, a dopamine D(2) receptor agonist, is a therapeutic agent for patients with prolactinoma and hyperprolactinemia. In this study we demonstrated that bromocriptine induced activation of p38 mitogen-activated protein (MAP) kinase, with concomitant induction of apoptosis in rat pituitary adenoma cell line GH3 cells. Treatment of GH3 cells for 48 h with bromocriptine increased the p38 MAP kinase activity up to 3- to 5-fold and simultaneously increased the number of apoptotic cells. Inclusion in the medium of SB212090 or SB203580, specific p38 MAP kinase inhibitors, completely abolished the bromocriptine-induced activation of p38 MAP kinase and significantly reduced the number of apoptotic cells. The bromocriptine-induced p38 MAP kinase activation was not prevented by S(-)-eticropride hydrochloride, a specific D(2) receptor antagonist. Treatment with either epidermal growth factor (EGF) or thyrotropin-releasing hormone (TRH), which stimulates p44/42 MAP kinase, rescued cells from the bromocriptine-induced apoptosis, with concomitant inhibition of the bromocriptine-induced p38 MAP kinase activation. These results suggest that bromocriptine induces apoptosis in association with p38 MAP kinase activation, and that the p44/42 MAP kinase signaling through EGF and TRH receptors has an opposing effect on p38 MAP kinase activation as well as on apoptosis induced with bromocriptine in GH3 cells.     

Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. Here we demonstrate that treatment of promonocytic U937 cells with arsenic trioxide leads to G2/M arrest which was associated with a dramatic increase in the levels of cyclin B and cyclin B-dependent kinase and apoptosis. We further show that apoptosis occurs after bcl-2 phosphorylation and caspase-3 activation followed by cleavage of PARP and PLC-gamma1 degradation and DNA fragmentation. The arsenic trioxide-induced apoptosis could be blocked by the protein synthesis inhibitor cycloheximide. In addition, pretreatment of U937 cells with the DNA polymerase inhibitor aphidicolin also blocked apoptosis, but did not cause the arrest of cells in the G2/M phase. The findings suggest that arsenic trioxide exerts its growth-inhibitory effects by modulating expression and/or activity of several key G2/M regulatory proteins. Furthermore, arsenic trioxide-mediated G2/M arrest correlates with the onset of apoptosis.     

TGF-beta1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production

Serum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-beta1 (TGF-beta1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-beta1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-beta1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-beta1. Neither SB203580 nor PD98059 has obvious effect on TGF-beta1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-beta1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-beta1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.     

Arsenic trioxide inhibits the growth of A498 renal cell carcinoma cells via cell cycle arrest or apoptosis

Previously, we showed that arsenic trioxide potently inhibited the growth of myeloma cells and head and neck cancer cells. Here, we demonstrate that arsenic trioxide inhibited the proliferation of all the renal cell carcinoma cell lines (ACHN, A498, Caki-2, Cos-7, and Renca) except only one cell line (Caki-1) with IC(50) of about 2.5-10 microM. Arsenic trioxide induced a G(1) or a G(2)-M phase arrest in these cells. When we examined the effects of this drug on A498 cells, arsenic trioxide (2.5 microM) decreased the levels of CDK2, CDK6, cyclin D1, cyclin E, and cyclin A proteins. Although p21 protein was not increased by arsenic trioxide, this drug markedly enhanced the binding of p21 with CDK2. In addition, the activities of CDK2- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Arsenic trioxide (10 microM) also induced apoptosis in A498 cells. Apoptotic process of A498 cells was associated with the changes of Bcl-(XL), caspase-9, caspase-3, and caspase-7 proteins as well as mitochondria transmembrane potential (Deltapsi(m)) loss. Taken together, these results demonstrate that arsenic trioxide inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis.     

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.     

Involvement of AMP-activated protein kinase and p38 mitogen-activated protein kinase in 8-Cl-cAMP-induced growth inhibition

8-Cl-cAMP (8-chloro-cyclic AMP), which induces differentiation, growth inhibition and apoptosis in various cancer cells, has been investigated as a putative anti-cancer drug. Although we reported that 8-Cl-cAMP induces growth inhibition via p38 mitogen-activated protein kinase (MAPK) and a metabolite of 8-Cl-cAMP, 8-Cl-adenosine mediates this process, the action mechanism of 8-Cl-cAMP is still uncertain. In this study, it was found that 8-Cl-cAMP-induced growth inhibition is mediated by AMP-activated protein kinase (AMPK). 8-Cl-cAMP was shown to activate AMPK, which was also dependent on the metabolic degradation of 8-Cl-cAMP. A potent agonist of AMPK, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) could also induce growth inhibition and apoptosis. To further delineate the role of AMPK in 8-Cl-cAMP-induced growth inhibition and apoptosis, we used two approaches: pharmacological inhibition of the enzyme with compound C and expression of a dominant negative mutant (a kinase-dead form of AMPKalpha2, KD-AMPK). AICAR was able to activate p38 MAPK and pre-treatment with AMPK inhibitor or expression of KD-AMPK blocked this p38 MAPK activation. Cell growth inhibition was also attenuated. Furthermore, p38 MAPK inhibitor attenuated 8-Cl-cAMP- or AICAR-induced growth inhibition but had no effect on AMPK activation. These results demonstrate that 8-Cl-cAMP induced growth inhibition through AMPK activation and p38 MAPK acts downstream of AMPK in this signaling pathway.     

Honokiol causes the p21WAF1-mediated G(1)-phase arrest of the cell cycle through inducing p38 mitogen activated protein kinase in vascular smooth muscle cells

Honokiol, an active component in extracts of Magnolia officinalis, has been proposed to play a role in anti-inflammatory, antioxidant activity, anti-angiogenic and anti-tumor activity. Although honokiol has a variety of pharmacological effects on certain cell types, its effects on vascular smooth muscle cells (VSMC) are unclear. This issue was investigated in the present study, honokiol was found to inhibit cell viability and DNA synthesis in cultured VSMC. These inhibitory effects were associated with G1 cell cycle arrest. Treatment with honokiol blocks the cell cycle in the G1 phase, down-regulates the expression of cyclins and CDKs and up-regulates the expression of p21WAF1, a CDK inhibitor. While honokiol did not up-regulate p27, it caused an increase in the promoter activity of the p21WAF1 gene. Immunoblot and deletion analysis of the p21WAF1 promoter showed that honokiol induced the expression of p21WAF1 and that this expression was independent of the p53 pathway. Furthermore, the honokiol-mediated signaling pathway involved in VSMC growth inhibition was examined. Among the relevant pathways, honokiol induced a marked activation of p38 MAP kinase and JNK. The expression of dominant negative p38 MAP kinase and SB203580, a p38 MAP kinase specific inhibitor, blocked the expression of honokiol-dependent p38 MAP kinase and p21WAF1. Consistently, blockade of p38 MAPK kinase function reversed honokiol-induced VSMC proliferation and cell cycle proteins. These data demonstrate that the p38 MAP kinase pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E/CDK2 complexes and honokiol-dependent VSMC growth inhibition. In conclusion, these findings concerning the molecular mechanisms of honokiol in VSMC provides a theoretical basis for clinical approaches to the use therapeutic agents in treating atherosclerosis.     

ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.     

Regulatory effects of mammalian target of rapamycin-mediated signals in the generation of arsenic trioxide responses

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/activated during treatment of leukemia cells with As(2)O(3), to regulate downstream engagement of mammalian target of rapamycin (mTOR) and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As(2)O(3) responses. Consistent with this, As(2)O(3)-dependent pro-apoptotic effects are enhanced in double knock-out cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As(2)O(3)-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with acute myeloid leukemia, we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, short interfering RNA-mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As(2)O(3). Altogether, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As(2)O(3), and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the anti-leukemic properties of As(2)O(3).     

Pramanicin induces apoptosis in Jurkat leukemia cells: A role for JNK,p38 and caspase activation

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.     

High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase

The mitogen-activated protein (MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM (control) or 25 mM (high) glucose or 5 mM glucose plus 20 mM mannitol (osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage (means +/- SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase (P < 0.001) and p42/44 MAP kinase (P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-beta1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P < 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-beta1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.     

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.