Nitric Oxide and Electrophilic Cyclopentenone Prostaglandins in Redox signaling, Regulation of Cytoskeleton Dynamics and Intercellular Communication

Nitric oxide (NO) and electrophilic cyclopentenone prostaglandins (CyPG) are local mediators that modulate cellular response to oxidative stress in different pathophysiological processes. In particular, there is increasing evidence about their functional role during inflammation and immune responses. Although the mechanistic details about their relationship and functional interactions are still far from resolved, NO and CyPG share the ability to promote redox-based post-translational modification (PTM) of proteins that play key roles in cellular homeostasis, signal transduction and transcription. NO-induced S-nitrosylation and S-glutathionylation as well as cyclopentenone-mediated adduct formation, are a few of the main PTMs by which intra- and inter-cellular signaling are regulated. There is a growing body of evidence indicating that actin and actin-binding proteins are susceptible to covalent PTM by these agents. It is well known that the actin cytoskeleton is key for the establishment of interactions among leukocytes, endothelial and muscle cells, enabling cellular activation and migration. In this review we analyze the current knowledge about the actions exerted by NO and CyPG electrophilic lipids on the regulation of actin dynamics and cytoskeleton organization, and discuss some open questions regarding their functional relevance in the regulation of intercellular communication.

Modulation of mammary cancer cell migration by 15-deoxy-Δ12,14-prostaglandin J2: implications for anti-metastatic therapy

Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signalling. Electrophilic lipids affect redox signalling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood. In the present study, we investigate the effect of the electrophilic prostaglandin 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2) on metastatic properties of breast cancer cells. 15d-PGJ2 was shown to decrease migration, stimulate focal-adhesion disassembly and cause extensive F-actin (filamentous actin) reorganization at low concentrations (0.03–0.3 μM). Importantly, these effects seem to be independent of PPARγ (peroxisome-proliferator-activated receptor γ) and modification of actin or Keap1 (Kelch-like ECH-associated protein 1), which are known protein targets of 15d-PGJ2 at higher concentrations. Interestingly, the p38 inhibitor SB203580 was able to prevent both 15d-PGJ2-induced F-actin reorganization and focal-adhesion disassembly. Taken together, the results of the present study suggest that electrophiles such as 15d-PGJ2 are potential anti-metastatic agents which exhibit specificity for migration and adhesion pathways at low concentrations where there are no observed effects on Keap1 or cytotoxicity.

Accumulation of 15-deoxy-Δ12,14-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelch-like erythroid cell-derived protein with cap‘n’collar homology-associated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very low concentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97–99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.

Redox signalling: from nitric oxide to oxidized lipids

Cellular redox signalling is mediated by the post-translational modification of proteins in signal-transduction pathways by ROS/RNS (reactive oxygen species/reactive nitrogen species) or the products derived from their reactions. NO is perhaps the best understood in this regard with two important modifications of proteins known to induce conformational changes leading to modulation of function. The first is the addition of NO to haem groups as shown for soluble guanylate cyclase and the newly discovered NO/cytochrome c oxidase signalling pathway in mitochondria. The second mechanism is through the modification of thiols by NO to form an S-nitrosated species. Other ROS/RNS can also modify signalling proteins although the mechanisms are not as clearly defined. For example, electrophilic lipids, formed as the reaction products of oxidation reactions, orchestrate adaptive responses in the vasculature by reacting with nucleophilic cysteine residues. In modifying signalling proteins ROS/RNS appear to change the overall activity of signalling pathways in a process that we have termed 'redox tone'. In this review, we discuss these different mechanisms of redox cell signalling, and give specific examples of ROS/RNS participation in signal transduction.

Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products

The molecular mechanisms through which oxidized lipids and their electrophilic decomposition products mediate redox cell signalling is not well understood and may involve direct modification of signal-transduction proteins or the secondary production of reactive oxygen or nitrogen species in the cell. Critical in the adaptation of cells to oxidative stress, including exposure to subtoxic concentrations of oxidized lipids, is the transcriptional regulation of antioxidant enzymes, many of which are controlled by antioxidant-responsive elements (AREs), also known as electrophile-responsive elements. The central regulator of the ARE response is the transcription factor Nrf2 (NF-E2-related factor 2), which on stimulation dissociates from its cytoplasmic inhibitor Keap1, translocates to the nucleus and transactivates ARE-dependent genes. We hypothesized that electrophilic lipids are capable of activating ARE through thiol modification of Keap1 and we have tested this concept in an intact cell system using induction of glutathione synthesis by the cyclopentenone prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2. On exposure to 15-deoxy-Δ12,14-prostaglandin J2, the dissociation of Nrf2 from Keap1 occurred and this was dependent on the modification of thiols in Keap1. This mechanism appears to encompass other electrophilic lipids, since 15-A2t-isoprostane and the lipid aldehyde 4-hydroxynonenal were also shown to modify Keap1 and activate ARE. We propose that activation of ARE through this mechanism will have a major impact on inflammatory situations such as atherosclerosis, in which both enzymic as well as non-enzymic formation of electrophilic lipid oxidation products are increased.