Cell Signaling and the Peroxiredoxin Based System Regulation of Transcription Factors Cell Cycle and Apoptosis

H2O2 is involved in diverse biological processes such as apoptosis, cell proliferation, differentiation, migration, immune response, and vascular remodeling. H2O2 is generated in cells not only as a byproduct of oxidative metabolism but also in response to various growth factors and cytokines, such as epidermal growth factor (EGF) [146], platelet-derived growth factor (PDGF) [147], transforming growth factor b1 (TGFP1) [148], interleukin 1 (IL-1) and TNF-a [149], insulin, angiotensin II [150, 151], via NADPH oxidase (Nox) activation and also via NADPH oxidase-independent mechanisms [152]. As H2O2 is involved in such a diverse array of cellular responses, its generation, concentration, localization and removal have to be tightly regulated (see also Chapters 2-4).

As modulators of the intracellular H2O2 concentration, peroxiredoxins play an important role in H2O2 signaling, especially because of their wide subcellular distribution and high affinity for H2O2. Unfortunately, the lack of a specific method for measuring intracellular H2O2 concentrations is a major hindrance in the study of H2O2 signaling [151]. Wood et al. [17] have proposed the "floodgate" model of Prdx signaling. According to this model, peroxiredoxins keep H2O2 levels low in the cell under resting conditions. An intracellular peroxide burst might lead to the overoxidation of peroxiredoxins (to cysteine sulfinic or sulfonic acid). As the overoxidized forms are enzymatically inactive, and the recycling ofthe overoxidized forms is slow, the transiently increased H2O2 level would trigger a signaling cascade of redox-regulated transcription factors. Diet et al. [153] have proposed that the activity of 2-Cys peroxiredoxins is not only influenced by H2O2 levels but also by the activity of sulfinyl reductase enzymes (i.e. Srx and sestrins). Several reviews have been published on the role of thioredoxins and peroxiredoxins in redox signaling [3, 15, 154-156].

Nuclear factor kB (NF-kB) and activator protein-1 (AP-1) are important redox-regulated transcription factors [157] (see also Chapters 11, 17 and 18). NF-kB is a multisubunit transcription factor, most often a heterodimer made up of a p50 and a p65 subunit, which is induced by a wide variety of agents such as H2O2,TNF-a, IL-1 and which, in turn, induces the expression of multiple genes involved in inflammatory, immune and acute phase responses [158-160]. In resting cells, NF-kB is present in an inactive form, bound to its inhibitor, IkB, in the cytoplasm. Upon activation, IkB dissociates from the complex, unmasking nuclear location sequences (NLS)inp50andp65. NF-kB then translocates into the nucleus (see [161] and references therein).

Box 6.1: Chaperone Proteins

Chaperones are proteins that facilitate the correct folding of denatured and nascent proteins (called chaperonins), or prevent proteins from aggregating and help maintain the unfolded state of proteins whilst they are transported (called molecular chaperones). Chaperones can be found in all cellular compartments. They inhibit protein aggregation via binding to hydrophobic residues on proteins. They often have a characteristic barrel-shape (chaperonin), or a semicircular three-dimensional structure, similar to a pair of boxing gloves (molecular chaperones). Families of chaperone proteins include the Hsp40 family (heat shock protein 40), Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsps and a-crystallin. Additional molecular chaperones are calnexin and calreticulin, protein disulfide isomerases (PDIs), peptidyl prolyl isomerases and Hsp70 co-chaperones.

As examples of chaperone function, posttranslational translocation of proteins is assisted by BiP, an Hsp70 family member in the endoplasmic reticulum, which prevents the proteins from returning to the cytosol by binding to them during transmembrane transport and releasing them in the lumen. Calnexin and calreticulin are calcium-binding proteins located in the endoplasmic reticulum. They bind to posttranslationally modified glycoproteins, prevent them from aggregating and ensure glycoproteins are correctly folded before leaving the ER. Hsp90 chaperones stabilize steroid hormone receptors.

The activity of NF-kB is regulated at multiple levels by the Prdx-based system via the modulation of H2O2 concentrations, depending on tissue and subcellular distribution. Prdx 4 induces NF-kB via induction of the phosphorylation and thus degradation of IkB-a [68]. Trx translocates from the cytoplasm to the nucleus in response to oxidative stress, such as TNF-a or UV exposure [162]. Nuclear translocation of Trx also increases following exposure to ionizing radiation [163] or phorbol ester treatment [100]. By forming a complex with the DNA-binding loop of p50, nuclear Trx 1 then enhances the DNA-binding activity of NF-kB [97,162]. In contrast, cytoplasmic Trx 1 inhibits NF-kB activity by inhibiting IkB degradation [162]. Similar to Trx 1, nuclear Prdx 1 also augments the activity of the NF-kB [32], while cytoplasmic Prdx 1 inhibits nuclear translocation of the transcription factor [32]. At the same time, Prdx 1 is itself regulated by NF-kB: a new kB site in the Prdx 1 promoter was recently identified by Wijayanti et al. [164]. Furthermore, by inhibiting the phorbol ester-dependent phosphorylation of Ser276 of the p65 subunit of NF-kB - via an IkB-independent pathway - lipopolysaccharide inhibited the phorbol ester-dependent activation of Prdx 1 [164].

Apart from NF-kB, the Prdx system also interferes with the transcription factor AP-1. In its commonest form, the transcription factor AP-1 is composed of a Fos and Jun subunit, which are activated at the transcriptional level upon induction of oxidative stress (see also Chapter 11, for reviews on AP-1 see [165-168]). DNA-binding of AP-1 is modulated by the redox status of a conserved cysteine residue in its DNA-recognition site (reviewed in [157]). The activity of AP-1, similar to NF-kB, is influenced by the Prdx-based system at multiple levels, and the individual proteins of the Prdx-based system are in turn regulated by AP-1.

Prdx 2 blocks the TNF-a-induced activation of AP-1 [169]. In contrast, Trx 1 enhances AP-1 activity: after translocation into the nucleus, Trx 1 acts as a hydrogen donor of redox factor 1 (Ref-1) - targeting redox active cysteine residues - which in turn increases the DNA-binding activity of AP-1 by reducing the conserved cysteine residue in the DNA-binding domain of AP-1 [100]. The transcription of Prdx 1 is regulated by AP-1. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, induces the transcriptional activity of Prdx 1 via two AP-1 sites (also called TPA-response elements) in the Prdx 1 promoter. This induction is mediated by a protein kinase C (PKC)/Ras/MEKK1/p38 MAPK signaling pathway [170].

An emerging new player in the transcriptional regulation of the Prdx-based system is the transcription factor Forkhead box O3 (FOXO3) [171,172]. FOXO3 is a member of the O subgroup of the forkhead transcription factors. It is regulated by the PI3K/ Akt signaling pathway and plays multiple roles in the initiation of apoptosis, cell-cycle arrest, stress-resistance and facilitation of DNA repair [173]. Prdx 3 contains two FOXO3-binding sites in its promoter region, and the baseline expression of Prdx 3 appears to depend on FOXO3 [171]. Chiribau et al. [171] recently demonstrated that FOXO3-dependent expression of Prdx 3 contributes to the oxidative stress resistance of human cardiac fibroblasts.

Thioredoxins are generally perceived as apoptosis inhibitors via signal cascade mechanisms, such as inhibition of the pro-apoptotic proteins phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and apoptosis signal-regulated kinase-1 (ASK1), as well as activation of the transcription factors NF-kB, AP-1 and SP-1 (reviewed in [174]). More recent studies, however, point toward a more complicated effect of Trx on apoptosis, such as in the case of differential NF-kB regulation depending on subcellular localization of Trx 1 [162].

Most studies support an antiapoptotic function of peroxiredoxins. Several studies have investigated the effect of Prdx depletion by RNA interference. Depletion of Prdx 3 resulted in increased sensitivity of HeLa cells to staurosporine or TNF-a-induced apoptosis [56]. Through its interaction with ASK1, Trx 1 is a negative regulator of apoptosis. Nadeau et al. [175] proposed a new model for ASK1 signaling: phosphorylation and oxidation (by H2O2 exposure) of ASK1, leads to interchain disulfide formation and thus the formation of an active and competent ASK1 signalosome. This signalosome then activates the downstream molecules MKK4/ 7 and JNK, and results in apoptosis. Trx 1 is a negative regulator of ASK1-induced apoptosis, as it covalently associates with the oxidized ASK1 and reduces it back to the inactive form [175]. Nuclear Trx 1 also protects MCF-7 breast cancer cells from cisplatin-induced cell death [176].

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a protein tyrosine phosphatase (PTP) tumor suppressor protein (see also Chapter 8). PTEN is negatively regulated by phosphorylation of tyrosine residues, but also by oxidative inactivation. PTPs contain an essential redox active cysteine residue which is in the thiolate state at neutral pH. The redox active Cys124 residue of PTEN is targeted by

Box 6.2: The Cell Cycle and Cell Cycle Arrest

The cell cycle, consisting of a growth and a chromosome cycle, is a process that leads to the duplication and division of cells. The two main phases of the eukaryotic cell cycle are the interphase and the M phase. The M phase consists of a mitosis and a cytokinesis step. During mitosis, chromosomes, which were replicated in the interphase, segregate. Cytokinesis means the division of the cell into two daughter cells. Cell growth and DNA replication is carried out during the interphase. The interphase can be further divided into G1, S and G2 phases. G1 is the first gap phase, and cells grow in mass during this phase. Cells may enter a quiescent, G0 phase, when they stop dividing. These cells may re-enter the G1 phase, upon adequate stimulation. DNA replicates during the S or synthetic phase. G2 is the second gap phase. Each of these phases is characterized by specific intracellular events, and progression from one phase to another is tightly controlled by cyclins and cyclin-dependent kinases (Cdks), of which there are various types. Cyclins and Cdks are themselves positively and negatively regulated by other enzymes.

Since control of the cell cycle determines if a cell divides or not, there are numerous attempts to influence this process, for instance as a means to stall tumor growth. Recent research has considered many different compounds as regulators of the cell cycle, for instance by targeting cyclin, or inhibition of Cdks. This area of research is vast and rapidly expanding. Here, we shall briefly mention the ability of the natural, sulfur-containing garlic compound diallyl-trisulfide to arrest the cell cycle of certain (cultured) prostate cancer cells at the G2/M transition.

The enzymes that control the cell cycle will be discussed in Box 16.3 in the context of DNA repair.

H2O2, resulting in the formation of a disulfide bond between Cys71 and Cys124 (reviewed in [155]). The disulfide form can be reduced back to the active, reduced state by Trx and GSH. PTEN is a key PTP in signaling pathways of cell growth and apoptosis. By removing a phosphate group, PTEN inactivates phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 is an activator of survival and cell growth via the PI-3-kinase/Akt Ser/Thr protein kinase pathway. Thus, via constitutive activation of Akt signaling, inactivation of PTEN is tumor-promoting (reviewed in [155]).

Trx 1 binds to PTEN, inhibiting its activity. Cysteine residues of protein tyrosine phosphatases (PTPs) and lipid phosphatase PTEN (a tumor suppressor) are also oxidized by peroxiredoxins [15]. In turn, loss of PTEN function causes downregula-tion of the expression of Prdx 1, 2, 5, 6, and Cu/Zn-SOD, and also leads to increased intracellular ROS levels in mouse embryonic fibroblasts [177].

Another signaling molecule targeted by the Prdx system is PDGF. The latter is a dimeric molecule connected by disulfide bonds. PDGF binds to the a- and b-PDGF protein tyrosine kinase receptors. Upon PDGF binding, PDGFR forms homo- or heterodimers. The subunits reciprocally phosphorylate tyrosine residues of one another, which leads to the activation of PDGFR and an increase of intracellular H2O2

levels. Activation of PDGFR results in increased cell proliferation, inhibition of apoptosis and reorganization of the cellular actin filaments inducing chemotaxis toward the PDGF gradient (reviewed in [178]). Physiologically, PDGF is an important player in wound-healing and development [178].

The PDGF signal is terminated by the recruitment of Prdx 2 to the cell membrane, subsequent removal of H2O2, reactivation of oxidatively inactivated tyrosine phosphatases and finally, dephosphorylation of PDGFR. Lack of Prdx 2 results in increased H2O2 production, prolonging the activation of PDGFR, which in turn enhances cellular response to PDGF, leading to increased cell proliferation and migration [49]. Albeit overexpression of Prdx 2 is known to inhibit apoptosis in malignant tumors, in this case, Prdx 2 acts as a tumor suppressor by inhibiting PDGF signaling and cell proliferation.

Molecules of the Prdx-based system also influence a number of other cell signaling pathways, most of which are less well understood. For instance, decrease of Prdx 1 expression was associated with decreased VEGF expression in Prdx 1 antisense transfected lung cancer cells [179]. Trx 1 activates hypoxia-inducible factor a (HIF-a, see Chapter 9), and this in turn leads to elevated VEGF expression [180]. C-terminal modification of TR 1 by an electrophile leads to the disruption of p53 conformation and triggers apoptosis [181].

In addition, some players of the Prdx-based system directly interact with cell cycle proteins: Prdx 1 binds to the Src homology 3 (SH3) domain of c-Abl, a cell cycle protein of cytostatic function, inhibiting its tyrosine kinase activity. Furthermore, Prdx 1 has been shown to be upregulated in cells entering the S phase [24, 26].

Peroxiredoxins and sulfiredoxins are often perceived as modulators of H2O2 signaling, but there is emerging evidence that the Prdx-based system also provides a link between H2O2 and "NO signaling [153]. In murine macrophages, "NO upregulates the expression of Prdx 1 and Prdx 6, both at the mRNA and the protein levels [153]. Importantly, "NO also promotes the expression of Srx. Thus, by increasing not only the amount of Prdx enzymes, but also the proportion of redox active molecules, "NO indirectly acts as an antioxidant [153].

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