Nuclear factor kappa B pathway
NFκB is a ubiquitous oxidative stress-sensitive transcription factor that plays a critical role in the regulation of a variety of genes important in cellular responses including inflammation, innate immunity, growth, and cell death. NFκB was first described in 1986 as a protein binding to the κ enhancer of lymphocytes . Subsequent studies revealed that NFκB is primarily a cytoplasmic factor that is expressed by virtually all cell types and that it constitutes a major inducible transcription factor whose modulation triggers a cascade of molecular events, some of which may be potential key targets for the treatment of inflammation. NFκB is a dimer, which classically consists of a p50 subunit and a trans-activating subunit p65 (or RelA); other NFkB subunits are p52 RelB or c-Rel. Unlike RelA, RelB, and c-Rel proteins, which are directly synthesized as mature proteins, p50 and p52 are generated by proteolytic processing from their larger precursors NFκB1 (p105) and NFκB2 (p100), respectively [98, 99]. Under basal conditions, NFκB appears in a latent form in the cytoplasm of nonstimulated cells, forming a complex with its inhibitors, the IkappaBs (IκB-α and IκΒ-β) .
When cells are stimulated by various inducers such as ROS, pro-inflammatory cytokines, bacterial products such as LPS or by action of NFκB protein (NFAP), NFκB is activated by means of the phosphorylation and degradation of the IκBs. The phosphorylation of IκB involves a specific IκB kinase (IKK) complex consisting of a core of three subunits, two catalytic subunits (IKK-α and IKK-β), and the regulatory subunit NFκB essential modifier (NEMO or IKKγ). IKK-α and IKK-β contain functional kinase domains that are capable of phosphorylating IκB at specific N-terminal serine residues to initiate its ubiquitination. The degradation of IκB-α results in rapid changes in the induction of NFκB, whereas the degradation of IκB-β is associated with a prolonged activation of NFκB [101, 102]. After its liberation, NFκB translocates to the nucleus and binds to specific elements within the promoters of responsive genes (κB-sites) to activate their transcription .
Various studies have shown that melatonin modulates the NFκB signaling pathway during inflammation and that modulation can occur during early (regulation of oxidant levels, IKK activation) as well as late (binding of NFκB to DNA) stages of the response [104, 105]. Modulation of NFκB modifies the expression of genes involved in the inflammatory process including iNOS, COX-2, and proinflammatory cytokines .
As mentioned earlier, among the wide range of genes involved in inflammation that contain functional κB-sites is iNOS. Once activated, NFκB stimulates the expression of iNOS, with an increase in NO formation . The basis for the protective effects of antioxidants such as melatonin is consistent with interference at the NFκB transcriptional pathway. For example, experimental models of ischemia reperfusion have shown that antioxidants such as glycine simultaneously block oxidative stress, the activation of NFκB and NO production [108, 109]. Moreover, research on models of inflammation in isolated hepatocytes also confirms that the inhibition of NFκB activation by immunosuppressive agents is related to the reduction in iNOS . Studies documenting the ability of melatonin to reduce NFκB activation and iNOS expression have been carried out using J774 and RAW 264.7 murine macrophages . Experiments using old SAMP8 mice have determined that melatonin reduces the expression of NFκB, induces mRNA downregulation of iNOS, and improves liver function . Melatonin administration lowers exercise-related inflammation and oxidative stress in both skeletal and cardiac muscle, by preventing NFκB activation and normalizing IkBα and IKKα protein expression [60, 62]. Other studies have also shown that reduction in NFκB activation by melatonin protects against liver thioacetamide damage or ischemia/reperfusion after kidney transplantation [49, 111].
LPS-induced NO production in rat endothelial cells is blocked by melatonin through reduction in iNOS expression by an NFκB-dependent mechanism . Similar results were obtained when the indolamine was administered in vivo (30 mg/kg); it prevented LPS-induced iNOS expression in rat aorta  and inhibited NFκB transcription factor activation in macrophages .
Equivalent changes in NFκB activation and COX expression were also reported following melatonin use in different models. In vitro studies confirmed that melatonin can suppress both iNOS and COX-2 expression by the inhibition of p52 NFκB-binding ability in LPS-stimulated macrophages . Experiments using C6 glioma cells exposed to LPS/interferon-γ showed that melatonin blocks NFκB activation, with a minor IκB-α degradation, and reduces iNOS and COX-2 expression . Interestingly, NFκB activation but not COX-2 overexpression is abolished by melatonin administration in RAW264.7 macrophage-like cells when previously stimulated with the fimbriae of the oral anaerobe Porphyromonas gingivalis .
It has been reported that melatonin blocks pro-inflammatory cytokine production in rats by reducing NFκB and AP-1 translocation into the nucleus . In line with this, reduction in mRNA and protein levels of TNF-α and IL-1β, via NFκB inhibition, has been reported in old SAMP8 melatonin-treated mice where the indoleamine exerts a protective role on the age-related degeneration of the liver, pancreas, and after injury to the heart [55, 106, 115]. Acute ethanol administration to mice not only causes liver tissue damage and pro-inflammatory cytokine overexpression, but also induces NFκB nuclear translocation and increased IκB degradation, effects that are significantly inhibited by melatonin . Melatonin modulation of inflammation in experimental diabetic neuropathy is also related to the inhibition of the NFκB cascade, induction of a marked reduction in TNF-α and IL-6 levels, and downregulation of iNOS and COX-2 . In Alzheimer’s disease, melatonin and its metabolites regulate both proinflammatory signals and oxidative stress mediators, such as COX2 and iNOS, by restricting NFκB full integration . Blockade by melatonin of cardiac iNOS, COX-2, TNF-α, IL-1, and IL-6 overexpression following acute exercise is also related to its ability to inactivate the NFκB pathway . Moreover, it has been reported that melatonin reduces Prevotella intermedia LPS-induced production of NO and IL-6 through the inhibition of nuclear translocation and DNA-binding activity of NFκB p50 subunit .
NFκB activation also appears to be a necessary step in the transcriptional induction of adhesion molecules, as confirmed by the fact that blockers of NFκB inhibit VCAM-1 expression in endothelial cells stimulated by the TNF-α . Melatonin diminishes not only ICAM-1 but also pro-inflammatory cytokine expression in a rat model of IBD, via NFκB inhibition .
CREB-binding protein (CBP) and its homologue p300 are large nuclear molecules that have been implicated in the coordination of a variety of transcriptional pathways with chromatin remodeling. They possess histone acetylase activity, which is necessary to open the chromatin structure through an acetylation-induced conformational change in histone protein. Via this action, CBP/p300 acts as an activator of NFκB and CRE-binding proteins [119, 120]. Activation of the noncanonical NFκB signaling pathway involved in the proteolytic processing of NFκB p100 to p52 is tightly regulated, and overproduction of p52 leads to lymphocyte hyperplasia and transformation. In vitro studies have demonstrated that melatonin suppresses p52 NFκB binding by the inhibition of CBP/p300-dependent acetylation, impairing its transactivation in LPS-stimulated macrophages .
The nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 catalyzes the formation of poly(ADP-ribose) polymers from its substrate NAD+, leading to a depletion of this dinucleotide and a reduction in the rate of glycolysis. Because NAD+ functions as a cofactor in the tricarboxylic acid cycle and in glycolysis, NAD+ depletion could lead to a rapid fall in intracellular ATP and, ultimately, cell injury . PARP-1 was initially known for its facilitating role in the repair of DNA damage induced by oxidative/nitrosative stress , but has subsequently been shown to be involved in the pathophysiology of acute and chronic inflammatory diseases. Currently, it is recognized that PARP-1 also participates in the regulation of NFκB-mediated production of pro-inflammatory cytokines, and inhibition of PARP-1 with synthetic inhibitors reportedly reduces the DNA-binding activity of NFκB and the transcription of NFκB-mediated genes . It has been shown, using zymosan and carrageenan-induced experimental models of inflammation, that melatonin reduces PARP-1 activation and attenuates the reduction in NAD+. The overall effect of melatonin is a significant protection of cellular viability . Moreover, some investigations have demonstrated an important role of peroxynitrite and subsequent PARP-1 activation in arthritis, which is blocked by melatonin administration . Furthermore, melatonin’s anti-inflammatory effects in experimental colitis are also associated with a marked reduction in PARP-1 immunoreactivity . Experimental studies carried out by subplantar injected superoxide into the hindpaw, which evokes a potent thermal hyperalgesia, found that PARP-1 was activated at the time of maximal hyperalgesia, and this was blocked by melatonin. This suggests that the antihyperalgesic effect of melatonin is derived partly by the inhibition of superoxide-driven PARP-1 activation .
Other transcriptional pathways
A number of other critical factors may participate in melatonin’s anti-inflammatory effects. These include inhibition of transcription factors and related proteins such as hypoxia-inducible factor (HIF), nuclear factor erythroid 2-related factor 2 (Nrf2), CREB, activating transcription factor 1 (ATF-1), CCAAT/enhancer binding protein (C/EBP), STATs, peroxisome proliferator-activated receptors (PPARs), AP-1, and actions on the phosphoinositol phosphate (PI3K)/Akt signaling pathway.
The best characterized response to hypoxia is the induction of HIF-1, which governs the oxygen-dependent induction of erythropoyetin, vascular endothelial cell growth factor (VEGF), and glycolytic enzymes. HIF-1 consists of two subunits (α- and β-subunit). Whereas HIF-1β is constitutively expressed, HIF-1α is strictly regulated by the cellular oxygen tension. Under normoxic conditions, HIF-1α protein is post-translationally hydroxylated on specific proline residues to enable binding to von-Hippel–Lindau protein, which targets HIF-1α for ubiquitinylation and proteasomal degradation . Important roles for HIF-1 in inflammation also include the activation of the immune response and stimulation of angiogenesis and have been proposed, and different groups have provided evidence that HIFs play an integrative role in conditions of hypoxia and inflammation by increasing the expression of iNOS or pro-inflammatory cytokines [126, 127]. Melatonin reportedly downregulates HIF-1α protein expression, under both normoxic and hypoxic conditions in DU145, PC-3, and LNCaP prostate cancer cells, without changes in HIF-1α mRNA levels. These effects may be related to a downregulation on HIF-1α protein translation through a p70S6K inhibitory mechanism .
More recently, it was found that melatonin destabilizes hypoxia-induced HIF-1α protein levels owing to its antioxidant activity in HCT116 human colon cancer cells. Moreover, in the same study under hypoxia, melatonin suppressed HIF-1α transcriptional activity, leading to reduction in different angiogenic processes such as VEGF expression or in vitro tube formation, invasion, and migration of human umbilical vein endothelial cells (HUVEC) by hypoxia-stimulated conditioned media of HCT116 cells . These findings suggest that the indolamine could be useful in tumor suppression via the inhibition of HIF-1α-mediated angiogenesis.
In Alzheimer’s disease, HIF-1 may upregulate BACE-1 protein, a transmembrane aspartic protease that is the major β-secretase facilitating the Aβ cytotoxic peptide generation. Melatonin administration reportedly prevents the Aβ peptide production by reducing both the BACE-1 protein and its mRNA . Finally, it has been proposed that melatonin may protect foetuses from LPS-induced placental cellular stress by a pleiotropic action involving a significant attenuation in both HIF-1α and VEGF expressions .
Another transcription factor involved in combating oxidative stress and inflammation is Nrf2. Nrf2 is a basic leucine zipper transcription factor that regulates the expression of a number of detoxifying, antioxidant genes and modulates some inflammatory processes [130, 131]. The Nrf2 pathway increases heme oxygenase-1 (HO-1) expression, which strengthens antioxidant defense. It has been suggested that melatonin modulates neuroinflammation by limiting the NFκB activation cascade and oxidative stress through elevated Nrf2 expression, which might be responsible, at least in part, for its neuroprotective effect in diabetic neuropathy . Moreover, data obtained in a model of acute liver injury using dimethylnitrosamine indicate that melatonin administration increases Nrf2 expression, normalizes antioxidant activities, reduces NFκB activation, and leads to the inhibition of iNOS and pro-inflammatory cytokine expression . In a rabbit model of hepatic failure of viral origin, it has been recently demonstrated that melatonin administration activates Nrf2 signaling, which causes the normalization of antioxidant enzymes expression, and supports the potential hepatoprotective role of this indole . Other studies, using cyclophosphamide, have documented that melatonin treatment reduces bladder damage and apoptosis, increases Nrf2, HO-1, and quinone oxidoreductase-1 expressions, and inhibits the NFκB pathway . Furthermore, melatonin significantly reduced iNOS expression in cerulein-induced pancreatitis in rat by a mechanism related to Nfr2 and NFκB modulation .
The CREB family of transcription factors consists of cAMP-responsive activators in mammalian systems. These include CREB and ATF-1, which are induced by a variety of growth factors and inflammatory signals, and subsequently mediate the transcription of genes containing a cAMP-responsive element (CRE) transcription factor. Several inflammation-related genes possess this CRE, including IL-2, IL-6, IL-10, and TNF-α. All CREB family members have a basic region leucine zipper dimerization domain located at the carboxy-terminal end, and they bind to DNA target sequences, such as the CRE, by dimerization through a leucine zipper . The activation of CRE-binding proteins is modulated owing to phosphorylation by several kinases and is mediated by coactivators such as CBP and p300 . Melatonin decreases phosphorylation of CREB and ATF-1 transcription factors not only in wild-type animals but also in mt1/2−/− after focal cerebral ischemia, indicating the presence of a MT1/MT2 receptor-independent mechanism on CREB and ATF-1 activation .
The C/EBP family belongs to the large group of basic leucine zipper (bZip) transcription factors. All members of the C/EBP family have a C-terminal leucine zipper domain for dimerization and a basic domain for DNA binding, respectively . Several C/EBP family members are involved in regulating various aspects of inflammation and immunity in the liver and in cells of the myelomonocytic lineage . Moreover, C/EBPβ has emerged as a key transcriptional transactivator for COX-2 and iNOS expression in murine and human cells induced by proinflammatory mediators [138, 139]. Some studies have indicated that melatonin inhibits the C/EBPβ transcriptional activity working as a negative regulator of adipogenesis in mice . Recently, it was reported that in LPS-stimulated CRL1999 human vascular smooth muscle cells melatonin inhibited phosphorylation of C/EBPβ, reduced its DNA binding, and caused an important reduction in both COX-2 and iNOS expression, which attenuates inflammatory damage .
STAT proteins participate in the regulation of cellular responses to cytokines and growth factors. With the aid of Janus kinases (JAKs), the pathway transduces the signal carried by these extracellular polypeptides to the cell nucleus, where activated STAT proteins modify gene expression . Inflammatory cytokines can bind to receptors and activate the STAT pathway . In addition, STAT may be negatively regulated by the suppressors of cytokine signaling (SOCS) family proteins . Melatonin protects neurons from brain-contusion-induced oxidative insults and reduces upregulation of SOCS3 and IL-6 mRNA expression, and STAT1 inactivation; these actions could contribute to the neuroprotective effects of melatonin . Studies in LPS-stimulated BV2 microglial cells have shown that melatonin induces anti-inflammatory effects through STAT activation and downregulation of chemokine expression by the inhibition of NFκB . Melatonin inhibition of P. intermedia LPS-mediated production of NO and IL-6 is related not only to an NFκB-related mechanisms but also suppression of STAT1 signaling .
PPARs belong to the nuclear hormone receptor superfamily of ligand-activated transcription factors. Three isoforms (α, β/δ, and γ), encoded by different genes, have been identified. Moreover, it has highlighted the role of PPARs in regulating inflammatory responses . Because of this, PPAR-γ functions as a central regulator of differentiation, apoptosis, and inflammatory responses, etc. . PPAR-related melatonin anti-inflammatory effects remain unclear, but it has been noted that this indolamine is able to suppress PPAR-γ expression when human mesenchymal stem cell (hMSC) differentiation was analyzed .
AP-1 is a heterodimeric protein, composed of members of the basic region leucine zipper protein superfamily, specifically, the Jun, Fos, and activating transcription factor proteins, which regulate gene expression in response to a variety of stimuli including cytokines, growth factors, stress, and bacterial and viral infections . Melatonin blocks pro-inflammatory cytokine production in rats by reducing not only NFκB but also AP-1 translocation into the nucleus .
PI3K/Akt is another important signaling pathway related to both acute and chronic inflammatory disorders . PI3K inhibition seems to play a role in the modulation of HUVEC by melatonin . Studies using C6 glioma cells have demonstrated a melatonin antioxidant effect associated with inhibition of PI3K/Akt and NFκB . Finally, melatonin was able to inhibit PI3K phosphorylation in LPS-stimulated BV2 microglial cells, documenting its important anti-inflammatory effect .
Mitogen-activated protein kinases
Effects of melatonin on the binding capacity of transcription factors such as NFκB or AP-1 may be regulated through the inhibition of protein kinases involved in signal transduction, such as MAPKs. MAPKs have been implicated in many physiologic processes including cell proliferation, differentiation, and death, and their activation seems to be a key component in signal transduction associated with cell migration. MAPKs are a family of serine/threonine kinases with three major types in mammalian cells: the extracellular signal-regulated protein kinases (ERK), the p38 MAPKs, and the c-Jun NH2-terminal kinases (JNK) . MAPKs are activated by specific MAPK kinases (MAPKK), such as MEK1/2 for ERK, MKK3/6 for p38, or MKK4/7 for JNK . Among the MAPK family members, the ERK pathway is frequently activated by mitogens and growth factors, while inflammation is a main trigger for JNK and p38 . In the last few years, melatonin has been shown to modulate MAPKs by acting on several steps of the activation cascade, and consequently on downstream effectors.
Antiproliferative melatonin effects on HUVEC and C6 glioma cells seem to be associated with inactivation of not only PI3K/Akt and NFκB but also ERK1/2 and protein kinase C (PKC) pathways [150, 151]. More recently, it has been demonstrated that melatonin attenuates apoptotic liver damage in a fulminant hepatic failure model in rabbits by a mechanism implicating a reduction in phosphorylated JNK and tumor necrosis receptor-1 (TNFR-1) expression . However, increases in JNK1/2/3 and p38 expression, mainly through a MT1 receptor pathway, have been reported in HepG2 hepatocarcinoma cells, which may be related to the ability of melatonin to induce apoptosis and cell cycle arrest in these and other cancer cells [17, 155]. These apparently contradictory effects may be related to MT1/MT2 as a mechanism determining the receptor-mediated biological actions of melatonin. As an example, ERK activity is increased by melatonin in mouse neuroblastoma cells that express only MT1 receptors , whereas in HUVEC expressing both MT1 and MT2 receptors, ERK is inhibited by melatonin . Furthermore, it has been reported that melatonin decreased p38 in both mt1/2−/− and wild-type mice after focal cerebral ischemia, but only reduced JNK1/2 in wild type .