Eui Ju Choi

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.

The tumour suppressor RASSF1A regulates mitosis by inhibiting the APC–Cdc20 complex

The tumour suppressor gene RASSF1A is frequently silenced in lung cancer and other sporadic tumours as a result of hypermethylation of a CpG island in its promoter. However, the precise mechanism by which RASSF1A functions in cell cycle regulation and tumour suppression has remained unknown. Here we show that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression. RASSF1A localizes to microtubules during interphase and to centrosomes and the spindle during mitosis. The overexpression of RASSF1A induced stabilization of mitotic cyclins and mitotic arrest at prometaphase. RASSF1A interacts with Cdc20, an activator of the anaphase-promoting complex (APC), resulting in the inhibition of APC activity. Although RASSF1A does not contribute to either the Mad2-dependent spindle assembly checkpoint or the function of Emi1 (ref. 1), depletion of RASSF1A by RNA interference accelerated the mitotic cyclin degradation and mitotic progression as a result of premature APC activation. It also caused a cell division defect characterized by centrosome abnormalities and multipolar spindles. These findings implicate RASSF1A in the regulation of both APC–Cdc20 activity and mitotic progression.

Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase

Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43°C for 20 min) suppressed UV‐stimulated c‐Jun N‐terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co‐immunoprecipitation. Hsp72 also inhibited JNK‐dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV‐induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress‐activated signaling by directly inhibiting JNK.

Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP+- induced dopaminergic neuronal cell death: Role of caspases, ROS, and JNK

In this study, we examined the possibility that MPTP and 6‐hydroxydopamine (6‐OHDA) act on distinct cell death pathways in a murine dopaminergic neuronal cell line, MN9D. First, we found that cells treated with 6‐OHDA accompanied ultrastructural changes typical of apoptosis, whereas MPP+ treatment induced necrotic manifestations. Proteolytic cleavage of poly(ADP‐ribose)polymerase by caspase was induced by 6‐OHDA, whereas it remained uncleaved up to 32 h after MPP+ treatment and subsequently disappeared. Accordingly, 6‐OHDA‐ but not MPP+‐induced cell death was significantly attenuated in the presence of a broad‐spectrum caspase inhibitor, N‐benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluomethylketone (Z‐VAD‐fmk). As measured by fluorometric probes, the level of reactive oxygen species (ROS) significantly increased after 6‐OHDA treatment. In contrast, the level of dihydroethidium‐sensitive ROS following MPP+ treatment remained unchanged while a slight increase in dichlorofluorescin‐sentive ROS was temporarily observed. As demonstrated by immunoblot analysis, the level of superoxide dismutase was down‐regulated following 6‐OHDA treatment, whereas it remained unchanged after MPP+ treatment. Cotreatment of cells with antioxidants such as N‐acetylcysteine or Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride (MnTBAP, cell‐permeable superoxide dismutase mimetic) rescued 6‐OHDA‐ but not MPP+‐induced cell death, whereas inclusion of catalase or NG‐nitro‐l‐arginine had no effect in both cases. In addition, 6‐OHDA induced ROS‐mediated c‐Jun N‐terminal kinase (JNK) activation that was attenuated in the presence of N‐acetylcysteine or MnTBAP but not catalase or Z‐VAD‐fmk. In contrast, MPP+ has little effect on JNK activity, indicating that ROS and/or ROS‐induced cell death signaling pathway seems to play an essential role in 6‐OHDA–mediated apoptosis but not in MPP+‐induced necrosis in a mesencephalon‐derived, dopaminergic neuronal cell line. J. Neurosci. Res. 57:86–94, 1999.

Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1.

ABSTRACT Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43°C for 20 min) resulted in inhibition of H2O2-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H2O2-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H2O2-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.

Compromised MAPK signaling in human diseases: an update.

The mitogen-activated protein kinases (MAPKs) in mammals include c-Jun NH2-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). These enzymes are serine–threonine protein kinases that regulate various cellular activities including proliferation, differentiation, apoptosis or survival, inflammation, and innate immunity. The compromised MAPK signaling pathways contribute to the pathology of diverse human diseases including cancer and neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. The JNK and p38 MAPK signaling pathways are activated by various types of cellular stress such as oxidative, genotoxic, and osmotic stress as well as by proinflammatory cytokines such as tumor necrosis factor-α and interleukin 1β. The Ras–Raf–MEK–ERK signaling pathway plays a key role in cancer development through the stimulation of cell proliferation and metastasis. The p38 MAPK pathway contributes to neuroinflammation mediated by glial cells including microglia and astrocytes, and it has also been associated with anticancer drug resistance in colon and liver cancer. We here summarize recent research on the roles of MAPK signaling pathways in human diseases, with a focus on cancer and neurodegenerative conditions.

Activation of c-Jun N-terminal Kinase Antagonizes an Anti-apoptotic Action of Bcl-2

Bcl-2 is an intracellular membrane-associated protein that prevents cell death induced by a variety of apoptotic stimuli. A mechanism by which Bcl-2 exerts an anti-cell death effect is, however, not fully understood. In the present study, Bcl-2 suppressed cell death of N18TG neuroglioma cells caused by various apoptotic stresses, including etoposide, staurosporine, anisomycin, and ultraviolet irradiation. Concomitantly, Bcl-2 disrupted a signaling cascade to the c-Jun N-terminal kinase activation induced by the apoptotic stresses. Bcl-2 also prevented the etoposide-induced stimulation of MEKK1. Furthermore, overexpression of c-Jun N-terminal kinase antagonized the death-protective function of Bcl-2. These data suggest that suppression of the c-Jun N-terminal kinase signaling pathway may be critical for Bcl-2 action.

Selenite Inhibits the c-Jun N-terminal Kinase/Stress-activated Protein Kinase (JNK/SAPK) through a Thiol Redox Mechanism

Selenium, an essential biological trace element, has been shown to modulate functions of many regulatory proteins involved in signal transduction and to affect a variety of cellular activities including cell growth, survival, and death. The molecular mechanism by which selenium exerts its action on the cellular events, however, remains unclear. In our present study, we observed that selenite suppresses both the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase pathway in 293T cells. In contrast, selenite had little effect on the extracellular signal-regulated kinase pathway. Furthermore, selenite directly inhibited JNK/SAPK activity in vitro but not the p38 activity. The in vitro inhibition of JNK/SAPK by selenite was reversed by the addition of reducing agents such as dithiothreitol and β-mercaptoethanol. Replacement of cysteine 116 in JNK1 by serine abolished the inhibitory effect of selenite on JNK1 activity both in vitro and in vivo. Selenite also suppressed a c-Jun-dependent luciferase reporter activity stimulated through the JNK signaling pathway. Taken together, our findings strongly suggest that selenite differentially modulates the mammalian mitogen-activated protein kinase pathways and that it can repress the JNK/SAPK signaling pathway by inhibiting JNK/SAPK through a thiol redox mechanism.

Ca2+‐Mediated Activation of c‐Jun N‐Terminal Kinase and Nuclear Factor κB by NMDA in Cortical Cell Cultures

Abstract: We examined the possibility that c‐Jun N‐terminal kinase (JNK) and nuclear factor κB (NF‐κB) might be involved in intracellular signaling cascades that mediate NMDA‐initiated neuronal events. Exposure of cortical neurons to 100 µM NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca2+ ionophore, induced activation of JNK. The NMDA‐induced activation of JNK was abrogated in the absence of extracellular Ca2+, suggesting that Ca2+ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti‐NF‐κB antibody demonstrated nuclear translocation of NF‐κB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF‐κB in a Ca2+‐dependent manner. Treatment with 3 mM aspirin blocked the NMDA‐induced activation of JNK and NF‐κB. Neuronal death following a brief exposure to 100 µM NMDA was Ca2+ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca2+ influx is required for NMDA‐induced activation of JNK and NF‐κB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA‐induced activation of NF‐κB and JNK.

Inhibition of Apoptosis Signal-regulating Kinase 1 by Nitric Oxide through a Thiol Redox Mechanism

Nitric oxide is an endogenous thiol-reactive molecule that modulates the functions of many regulatory proteins by a thiol-redox mechanism. NO has now been shown to inhibit the activation of apoptosis signal-regulating kinase 1 (ASK1) in murine fibrosarcoma L929 cells through such a mechanism. Exposure of L929 cells to interferon-γ resulted in the endogenous production of NO and in inhibition of the activation of ASK1 by hydrogen peroxide. The interferon-γ-induced inhibition of ASK1 activity was blocked by NG-nitro-l-arginine, an inhibitor of NO synthase. Furthermore, the NO donor S-nitro-N-acetyl-dl-penicillamine (SNAP) inhibited ASK1 activity in vitro, and this inhibition was reversed by thiol-reducing agents such as dithiothreitol and β-mercaptoethanol. SNAP did not inhibit the kinase activities of MKK3, MKK6, or p38 in vitro. The inhibition of ASK1 by interferon-γ was not changed by 1H- (1,2,4)oxadiazolo[4,3-α]quinoxalin-1-one, an inhibitor of guanylyl cyclase nor was it mimicked by 8-bromo-cyclic GMP. Site-directed mutagenesis revealed that replacement of cysteine 869 of ASK1 by serine rendered this protein resistant to the inhibitory effects both of interferon-γ in intact cells and of SNAP in vitro. Co-immunoprecipitation data showed that NO production inhibited a binding of ASK1, but not ASK1(C869S), to MKK3 or MKK6. Moreover, interferon-γ induced the S-nitrosylation of endogenous ASK1 in L929 cells. Together, these results suggest that NO mediates the interferon-γ-induced inhibition of ASK1 in L929 cells through a thiolredox mechanism.