Hideaki Kamata

Redox regulation of cellular signalling.

Extracellular stimuli elicit a variety of responses, such as cell proliferation and differentiation, through the cellular signalling system. Binding of growth factors to the respective receptor leads to the activation of receptor tyrosine kinases, which in turn stimulate downstream signalling systems such as mitogen-activated protein (MAP) kinases, phospholipase Cgamma (PLCgamma) and phosphatidylinositol 3-kinase. These biochemical reactions finally reach the nucleus, resulting in gene expression mediated by the activation of several transcription factors. Recent studies have revealed that cellular signalling pathways are regulated by the intracellular redox state. Generation of reactive oxygen species (ROS), such as H2O2, leads to the activation of protein tyrosine kinases followed by the stimulation of downstream signalling systems including MAP kinase and PLCgamma. The activation of PLCgamma by oxidative radical stress elevates the cellular Ca2+ levels by flux from the intracellular Ca2+ pool and from the extracellular space. Such reactions in the upstream signalling cascade, in concert, result in the activation of several transcription factors. On the other hand, reductants generally suppress the upstream signalling cascade resulting in the suppression of transcription factors. However, it is well known that cysteine residues in a reduced state are essential for the activity of many transcription factors. In fact, in vitro, oxidation of NFkappaB results in its activation, whereas reductants promote its activity. Thus, cellular signalling pathways are generally subjected to dual redox regulation in which redox has opposite effects on upstream signalling systems and downstream transcription factors. Not only are the cellular signalling pathways subjected to redox regulation, but also the signalling systems regulate the cellular redox state. When cells are activated by extracellular stimuli, the cells produce ROS, which in turn stimulate other cellular signalling pathways, indicating that ROS act as second messengers. It is thus evident that there is cross talk between the cellular signalling system and the cellular redox state. Cell death and life also are subjected to such dual redox regulation and cross talk. Death signals induce apoptosis through the activation of caspases in the cells. Oxidative radical stress induces the activation of caspases, whereas the oxidation of caspases results in their inactivation. Furthermore, some cell-death signals induce the production of ROS in the cells, and the ROS produced in turn stimulate the cell-death machinery. All this evidence shows that the cells fate is determined by cross talk between the cellular signalling pathways and the cellular redox state through a complicated regulation mechanism.

IKK/NF-κB signaling: balancing life and death – a new approach to cancer therapy

IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling pathways play critical roles in a variety of physiological and pathological processes. One function of NF-kappaB is promotion of cell survival through induction of target genes, whose products inhibit components of the apoptotic machinery in normal and cancerous cells. NF-kappaB can also prevent programmed necrosis by inducing genes encoding antioxidant proteins. Regardless of mechanism, many cancer cells, of either epithelial or hematopoietic origin, use NF-kappaB to achieve resistance to anticancer drugs, radiation, and death cytokines. Hence, inhibition of IKK-driven NF-kappaB activation offers a strategy for treatment of different malignancies and can convert inflammation-induced tumor growth to inflammation-induced tumor regression.

Hydrogen peroxide activates IκB kinases through phosphorylation of serine residues in the activation loops

The cellular redox state regulates nuclear factor‐κB (NF‐κB) signaling systems. We investigated the effects of H2O2 on inhibitor of NF‐κB (IκB) kinases (IKKα and IKKβ), which phosphorylate IκB leading to its degradation and NF‐κB activation. Tumor necrosis factor (TNF) stimulation increased IKK activity within 10 min, and then IKK activity decreased gradually within 30 min in HeLa cells. Stimulation of the cells with H2O2 induced a slight activation of IKK within 30 min. Furthermore, co‐stimulation with TNF suppressed the downregulation of IKK and sustained the activation for more than 30 min. H2O2 also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H2O2 enhanced TNF‐induced NF‐κB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKα and Ser181 of IKKβ, in the activation loops was essential for the H2O2‐mediated activation of IKK. H2O2‐induced activation of IKKα and IKKβ was reduced by IKKβ and IKKα kinase‐negative mutants, respectively, indicating that IKKα and IKKβ were stimulated by H2O2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF‐κB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops.

AMP-activated Protein Kinase Activation Increases Phosphorylation of Glycogen Synthase Kinase 3β and Thereby Reduces cAMP-responsive Element Transcriptional Activity and Phosphoenolpyruvate Carboxykinase C Gene Expression in the Liver

AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3β (GSK3β) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3β within 15 min. Stimulation with AICAR or the GSK3β inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3β to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3β(S9G) but not wild-type GSK3β. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3β phosphorylation were very similar. Furthermore, AMPK-mediated GSK3β phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3β is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3β by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.

N‐Acetylcysteine suppresses TNF‐induced NF‐κB activation through inhibition of IκB kinases

Here, we used a reductant, N‐acetyl‐L‐cysteine (NAC), to investigate the redox‐sensitive step(s) in the signalling pathway from the tumor necrosis factor (TNF) receptor to nuclear factor κB (NF‐κB). We found that NAC suppressed NF‐κB activation triggered by TNF or by overexpression of either the TNF receptor‐associated death domain protein, TNF receptor‐associated factor 2, NF‐κB‐inducing kinase (NIK), or IκB kinases (IKKα and IKKβ). NAC also suppressed the TNF‐induced activation of IKKα and IKKβ, phosphorylation and degradation of IκB, and nuclear translocation of NF‐κB. Furthermore, NAC suppressed the activation of IKKα and IKKβ triggered by the overexpression of NIK. These results indicate that IKKα and IKKβ are subject to redox regulation in the cells, and that NAC inhibits NF‐κB activation through the suppression of these kinases.

On–off system for PI3-kinase–Akt signaling through S-nitrosylation of phosphatase with sequence homology to tensin (PTEN)

Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an “on–off” pattern of control of Akt signaling.

Suppression of Nerve Growth Factor-induced Neuronal Differentiation of PC12 Cells N-ACETYLCYSTEINE UNCOUPLES THE SIGNAL TRANSDUCTION FROM Ras TO THE MITOGEN-ACTIVATED PROTEIN KINASE CASCADE

The cellular redox state is thought to play an important role in a wide variety cellular signaling pathways. Here, we investigated the involvement of redox regulation in the nerve growth factor (NGF) signaling pathway and neuronal differentiation in PC12 cells. N-acetyl-L-cysteine (NAC), which acts as a reductant in cells both by its direct reducing activity and by increasing the synthesis of the cellular antioxidant glutathione, inhibited neuronal differentiation induced by NGF or by the expression of oncogenic ras in PC12 cells. NAC suppressed NGF-induced c-fos gene expression and AP-1 activation. These results suggest that neuronal differentiation and NGF signaling are subject to regulation by the cellular redox state. NAC also suppressed the NGF-induced activation of mitogen-activated protein kinases (MAPKs) and decreased the amount of tyrosine phosphorylation of MAPKs. The suppression of MAPK by NAC was independent of glutathione synthesis. In parallel with the suppression of MAPK, the activation of MAPK kinase kinase activity was also suppressed in the presence of NAC. In contrast, NGF-induced activation of Ras was not inhibited by NAC. The inhibitory effect of NAC on the MAPK cascade was independent of transcription and translation. Thus, NAC suppresses NGF-induced neuronal differentiation by uncoupling the signal transduction from Ras to the MAP kinase cascade in PC12 cells.

Nuclear IKKβ Is an Adaptor Protein for IκBα Ubiquitination and Degradation in UV-Induced NF-κB Activation

Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

Phospholipase C-delta1 contains a functional nuclear export signal sequence.

We have previously observed, using a green fluorescent protein (GFP) fusion system, that PLC-δ1 is localized mainly at the plasma membrane and in the cytosol, whereas little is present in the nucleus in Madin-Darby canine kidney cells (Fujii, M., Ohtsubo, M., Ogawa, T., Kamata, H., Hirata, H., and Yagisawa, H. (1999)Biochem. Biophys. Res. Commun. 254, 284–291). Herein, we demonstrate that PLC-δ1 has a functional nuclear export signal (NES) sequence in amino acid residues 164–177 of the EF-hand domain. The fluorescence of NES-disrupted GFP/PLC-δ1 expressed in Madin-Darby canine kidney cells was present not only at the plasma membrane and in the cytosol but also in the nucleus. Moreover, treatment with leptomycin B, a specific inhibitor of NES-dependent nuclear export, resulted in the accumulation of GFP/PLC-δ1 in the nucleus. A site-directed mutant containing a pleckstrin homology domain, which does not bind inositol 1,4,5-trisphosphate and cannot hydrolyze phosphatidylinositol 4,5-bisphosphate in vitro, accumulated in the nucleus to a much greater extent than wild-type GFP/PLC-δ1 after treatment with leptomycin B. These results suggest that PLC-δ1 is shuttled between the cytoplasm and the nucleus; its nuclear export is dependent on the leucine-rich NES sequence and its active nuclear import is regulated by an unidentified signal(s).

Nerve growth factor and forskolin prevent H2O2-induced apoptosis in PC12 cells by glutathione independent mechanism

PC12 cells died by apoptosis at relatively low concentrations of H2O2, of which cytotoxicity was effectively suppressed by nerve growth factor (NGF), forskolin, and dbt-cAMP. Treatment with NGF or forskolin for 24 h increased the level of cellular antioxidant glutathione (GSH) by 1.6-2.0-fold. However, both NGF and forskolin protected cells against H2O2-stress even when cellular GSH was depleted by treatment with L-buthionine-(S,R)-sulfoximine (BSO). The GSH-independent protection effects of NGF and forskolin did not require new protein or RNA synthesis. Exogenous expression of an oncogenic ras suppressed apoptosis caused by H2O2 indicating that Ras protein also plays a role in suppressing apoptosis caused by oxidative radical stress.