Nitric Oxide Signal Transduction and Its Role in Skin Sensitization.

Nitric oxide (NO) is a signaling molecule that plays a crucial role in numerous cellular physiological processes. In the skin, NO is produced by keratinocytes, fibroblasts, endothelial cells, and immune cells and is involved in skin functions such as vasodilation, pigmentation, hair growth, wound healing, and immune responses. NO modulates both innate and adaptive immune responses. As a signaling molecule and cytotoxic effector, NO influences the function of immune cells and production of cytokines. NO is a key mediator that protects against or contributes to skin inflammation. Moreover, NO has been implicated in skin sensitization, a process underlying contact dermatitis. It modulates the function of dendritic cells and T cells, thereby affecting the immune response to allergens. NO also plays a role in contact dermatitis by inducing inflammation and tissue damage. NO-related chemicals, such as nitrofatty acids and nitric oxide synthase (NOS) inhibitors, have potential therapeutic applications in skin conditions, including allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD). Further research is required to fully elucidate the therapeutic potential of NO-related chemicals and develop personalized treatment strategies for skin conditions.

Crosstalk between Autophagy and Inflammatory Processes in Cancer.

Inflammation is an adaptive response to tissue injury, which is a critical process in order to restore tissue functionality and homeostasis. The association between inflammation and cancer has been a topic of interest for many years, not only inflammatory cells themselves but also the chemokines and cytokines they produce, which affect cancer development. Autophagy is an intracellular self-degradative process providing elimination of damaged or dysfunctional organelles under stressful conditions such as nutrient deficiency, hypoxia, or chemotherapy. Interestingly, the signaling pathways that are involved in cancer-associated inflammation may regulate autophagy as well. These are (1) the toll-like receptor (TLR) signaling cascade, (2) the reactive oxygen species (ROS) signaling pathway, (3) the inflammatory cytokine signaling pathway, and (4) the IκB kinase (IKK)/Nuclear factor-κB (NF-κB) signaling axis. Moreover, the studies on the context-specific functions of autophagy during inflammatory responses in cancer will be discussed here. On that basis, we focus on autophagy inhibitors and activators regulating inflammatory process in cancer as useful candidates for enhancing anticancer effects. This review summarizes how the autophagic process regulates these key inflammatory processes and vice versa in various cancers.

The Role of Protein S-Nitrosylation in Protein Misfolding-Associated Diseases.

Abnormal and excessive nitrosative stress contributes to neurodegenerative disease associated with the production of pathological levels of misfolded proteins. The accumulated findings strongly suggest that excessive NO production can induce and deepen these pathological processes, particularly by the S-nitrosylation of target proteins. Therefore, the relationship between S-nitrosylated proteins and the accumulation of misfolded proteins was reviewed. We particularly focused on the S-nitrosylation of E3-ubiquitin-protein ligase, parkin, and endoplasmic reticulum chaperone, PDI, which contribute to the accumulation of misfolded proteins. In addition to the target proteins being S-nitrosylated, NOS, which produces NO, and GSNOR, which inhibits S-nitrosylation, were also suggested as potential therapeutic targets for protein misfolding-associated diseases.

Potential Therapeutic Approaches through Modulating the Autophagy Process for Skin Barrier Dysfunction.

Autophagy is an attractive process to researchers who are seeking novel potential treatments for various diseases. Autophagy plays a critical role in degrading damaged cellular organelles, supporting normal cell development, and maintaining cellular homeostasis. Because of the various effects of autophagy, recent human genome research has focused on evaluating the relationship between autophagy and a wide variety of diseases, such as autoimmune diseases, cancers, and inflammatory diseases. The skin is the largest organ in the body and provides the first line of defense against environmental hazards, including UV damage, chemical toxins, injuries, oxidative stress, and microorganisms. Autophagy takes part in endogenous defense mechanisms by controlling skin homeostasis. In this manner, regulating autophagy might contribute to the treatment of skin barrier dysfunctions. Various studies are ongoing to elucidate the association between autophagy and skin-related diseases in order to find potential therapeutic approaches. However, little evidence has been gathered about the relationship between autophagy and the skin. In this review, we highlight the previous findings of autophagy and skin barrier disorders and suggest potential therapeutic strategies. The recent research regarding autophagy in acne and skin aging is also discussed.

Role of Nitric Oxide and Protein S-Nitrosylation in Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a process in which damage is induced in hypoxic tissue when oxygen supply is resumed after ischemia. During IRI, restoration of reduced nitric oxide (NO) levels may alleviate reperfusion injury in ischemic organs. The protective mechanism of NO is due to anti-inflammatory effects, antioxidant effects, and the regulation of cell signaling pathways. On the other hand, it is generally known that S-nitrosylation (SNO) mediates the detrimental or protective effect of NO depending on the action of the nitrosylated target protein, and this is also applied in the IRI process. In this review, the effect of each change of NO and SNO during the IRI process was investigated.

A Molecular Perspective on the Potential Benefits of Metformin for the Treatment of Inflammatory Skin Disorders.

Due to its anti-hyperglycemic effect, metformin is the first-line medication for the treatment of type 2 diabetes, particularly in people who are obese. However, metformin is a drug with a very wide range of pharmacological properties and reports of its therapeutic effect on diseases including inflammation and cancer are increasing. Numerous research groups have reported that metformin has beneficial effects on a variety of inflammatory skin disorders including psoriasis, acanthosis nigricans, acne, hidradenitis suppurativa, and allergic contact dermatitis. According to these reports, in addition to the well-known action of metformin, that is, its anti-hyperglycemic effect, NF-kB inhibition and the resulting alteration to the cytokine network may be the potential targets of metformin. Its anti-hyperandrogenism effect has also been confirmed as the major action of metformin in some inflammatory skin diseases. Moreover, novel regulatory mechanisms, including autophagy and antioxidant processes, have been suggested as promising mechanisms of action for metformin in inflammatory skin disorders.

Adiponectin-Secretion-Promoting Phenylethylchromones from the Agarwood of Aquilaria malaccensis.

The therapeutic potential of adiponectin regulation has received interest because of its association with diverse human disease conditions, such as diabetes, obesity, atherosclerosis, and cancer. Phenylethylchromone derivatives from Aquilaria malaccensis-derived agarwood promoted adiponectin secretion during adipogenesis in human bone marrow mesenchymal stem cells, and 5,6-dihydroxy-2-(2-phenylethyl)chromone (1) was identified as a new chromone derivative. A target identification study with the most potent adiponectin-secretion-promoting phenylethylchromones, 6-methoxy-2-(2-phenylethyl)chromone (3) and 7-methoxy-2-(2-phenylethyl)chromone (4), showed that they are PPARγ partial agonists. Therefore, the diverse therapeutic effects of agarwood are associated with a PPARγ-mediated adiponectin-secretion-promoting mechanism.

Identification of fermented tea (Camellia sinensis) polyphenols and their inhibitory activities against amyloid-beta aggregation.

Thirty-three phenolic compounds were identified from the extract of fermented tea (Camellia sinensis L.), including three undescribed flavonoids, namely quamoreokchaside I-II and kamoreokchaside I, along with thirty known compounds. All isolates were tested to evaluate their inhibitory effects against amyloid-beta (Aß) aggregation through thioflavin-T (ThT) fluorescence-based assay and transmission electron microscopy (TEM). Among the isolates, three tea polyphenols, including (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), significantly decreased Aß aggregation at a concentration of 10 µg ml-1, compared to the positive control, Aß alone. The anti-Aß aggregation effects of CG, ECG, and EGCG were confirmed again via TEM, which were consistent with the ThT fluorescence-based assay. Moreover, CG and ECG provided stronger protection on SH-SY5Y cells against Aß-induced cytotoxicity than EGCG. Remarkably, CG showed more potent inhibitory activity than EGCG, the best-known anti-Aß aggregation agent from tea products.

Pathophysiological Role of S-Nitrosylation and Transnitrosylation Depending on S-Nitrosoglutathione Levels Regulated by S-Nitrosoglutathione Reductase.

Nitric oxide (NO) mediates various physiological and pathological processes, including cell proliferation, differentiation, and inflammation. Protein S-nitrosylation (SNO), a NO-mediated reversible protein modification, leads to changes in the activity and function of target proteins. Recent findings on protein-protein transnitrosylation reactions (transfer of an NO group from one protein to another) have unveiled the mechanism of NO modulation of specific signaling pathways. The intracellular level of S-nitrosoglutathione (GSNO), a major reactive NO species, is controlled by GSNO reductase (GSNOR), a major regulator of NO/SNO signaling. Increasing number of GSNOR-related studies have shown the important role that denitrosylation plays in cellular NO/SNO homeostasis and human pathophysiology. This review introduces recent evidence of GSNO-mediated NO/SNO signaling depending on GSNOR expression or activity. In addition, the applicability of GSNOR as a target for drug therapy will be discussed in this review.

Amber Light (590 nm) Induces the Breakdown of Lipid Droplets through Autophagy-Related Lysosomal Degradation in Differentiated Adipocytes.

Lipolysis in the adipocytes provides free fatty acids for other tissues in response to the energy demand. With the rapid increase in obesity-related diseases, finding novel stimuli or mechanisms that regulate lipid metabolism becomes important. We examined the effects of visible light (410, 457, 505, 530, 590, and 660 nm) irradiation on lipolysis regulation in adipocytes differentiated from human adipose-derived stem cells (ADSCs). Interestingly, 590 nm (amber) light irradiation significantly reduced the concentration of lipid droplets (LDs). We further investigated the lipolytic signaling pathways that are involved in 590 nm light irradiation-induced breakdown of LDs. Immunoblot analysis revealed that 590 nm light irradiation-induced phosphorylation of hormone-sensitive lipase (HSL) was insufficient to promote reduction of LDs. We observed that 590 nm light irradiation decreased the expression of perilipin 1. We found that 590 nm light irradiation, but not 505 nm, induced conversion of LC3 I to LC3 II, a representative autophagic marker. We further demonstrated that the lysosomal inhibitors leupeptin/NH4Cl inhibited 590 nm light irradiation-induced reduction of LDs in differentiated adipocytes. Our data suggest that 590 nm light irradiation-induced LD breakdown is partially mediated by autophagy-related lysosomal degradation, and can be applied in clinical settings to reduce obesity.

S-nitrosylation of fatty acid synthase regulates its activity through dimerization.

NO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys(1471)and Cys(2091), but not at Cys(1127), increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis.

Transnitrosylation from DJ-1 to PTEN attenuates neuronal cell death in parkinson's disease models.

Emerging evidence suggests that oxidative/nitrosative stress, as occurs during aging, contributes to the pathogenesis of Parkinson's disease (PD). In contrast, detoxification of reactive oxygen species and reactive nitrogen species can protect neurons. DJ-1 has been identified as one of several recessively inherited genes whose mutation can cause familial PD, and inactivation of DJ-1 renders neurons more susceptible to oxidative stress and cell death. DJ-1 is also known to regulate the activity of the phosphatase and tensin homolog (PTEN), which plays a critical role in neuronal cell death in response to various insults. However, mechanistic details delineating how DJ-1 regulates PTEN activity remain unknown. Here, we report that PTEN phosphatase activity is inhibited via a transnitrosylation reaction [i.e., transfer of a nitric oxide (NO) group from the cysteine residue of one protein to another]. Specifically, we show that DJ-1 is S-nitrosylated (forming SNO-DJ-1); subsequently, the NO group is transferred from DJ-1 to PTEN by transnitrosylation. Moreover, we detect SNO-PTEN in human brains with sporadic PD. Using x-ray crystallography and site-directed mutagenesis, we find that Cys106 is the site of S-nitrosylation on DJ-1 and that mutation of this site inhibits transnitrosylation to PTEN. Importantly, S-nitrosylation of PTEN decreases its phosphatase activity, thus promoting cell survival. These findings provide mechanistic insight into the neuroprotective role of SNO-DJ-1 by elucidating how DJ-1 detoxifies NO via transnitrosylation to PTEN. Dysfunctional DJ-1, which lacks this transnitrosylation activity due to mutation or prior oxidation (e.g., sulfonation) of the critical cysteine thiol, could thus contribute to neurodegenerative disorders like PD.

Resveratrol induces autophagy through death-associated protein kinase 1 (DAPK1) in human dermal fibroblasts under normal culture conditions.

Autophagy is an essential process degrading damaged components. Although resveratrol has various beneficial activities for health, little is known about the effects of resveratrol on autophagy in skin. We investigated whether resveratrol affects autophagy in human dermal fibroblasts grown in complete medium. We found that after the resveratrol treatment, LC3-II reached a maximum level at 8 h and then gradually decreased. By PCR array analysis, we identified death-associated protein kinase 1 (DAPK1) as a new target of resveratrol, and we confirmed that the expression level of DAPK1 was enhanced by resveratrol. We also demonstrated that DAPK1 knock-down by siRNA was sufficient to reduce resveratrol-induced autophagy but did not affect the phosphorylation level of AMP-activated kinase (AMPK), a well-known target of resveratrol. These data indicate that resveratrol-induced autophagy can be mediated by DAPK1, raising the possibility that some of the beneficial effects of resveratrol may be due to its regulation of DAPK1.

ATP induced microglial cell migration through non-transcriptional activation of matrix metalloproteinase-9.

In response to brain insults, microglia, the resident inflammatory cells in CNS, migrate into injured sites to initiate inflammatory responses in brain. ATP, released from apoptotic or necrotic cells induce chemoattractive responses but the mechanism is not clear yet. In this study, we investigated whether ATP modulates microglial migration by regulating the activity of matrix metalloproteinases (MMPs). ATP induced rapid microglial migration and increased the activity of MMP-9 in the culture supernatants (secreted compartments) in a concentration-dependent manner. The increased activity of secreted MMP-9 is due to the increased protein secretion, but not by the increased MMP-9 mRNA and protein expression. Inhibition of MMP-9 activity by treatment with specific inhibitors including GM6001 and SB-3CT prevented ATP-induced microglial migration. ATP-induced microglial migration was also inhibited by P2Y receptor antagonists including clopidogrel as well as PI3K inhibitor such as wortmanin. Taken together, ATP non-transcriptionally increased MMP-9 activity by activation of P2Y and PI3K. The results from the present investigation may provide further insights into the regulation of the activity of MMP-9 during microglial migration, which may play essential role in the regulation of inflammatory responses in pathological situations such as neurodegenerative disorders.

Urokinase-type plasminogen activator induces BV-2 microglial cell migration through activation of matrix metalloproteinase-9.

In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.

Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.

Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.

Uridine protects cortical neurons from glucose deprivation-induced death: possible role of uridine phosphorylase.

We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.

Activation of protease-activated receptor1 mediates induction of matrix metalloproteinase-9 by thrombin in rat primary astrocytes.

Thrombin plays an important role in diverse neurological processes such as proliferation, cell migration, differentiation and neuroinflammation. In this study, we investigated the effect of thrombin on matrix metalloprotease-9 (MMP-9) expression in rat primary astrocytes. Thrombin (1-10U/ml) induced a significant increase in MMP-9 activity as measured by gelatin zymography. Thrombin also increased MMP-9 mRNA expression. Among three isotypes of thrombin receptor, i.e. protease-activated receptor (PAR)-1, -3 and -4, PAR1 agonist (1-100muM) but not PAR3 and PAR4 agonist induced MMP-9 expression. Inhibition of thrombin-induced MMP-9 production by SCH 79797 (10-50nM), a selective PAR1 receptor antagonist, confirmed that PAR1 is a main receptor for thrombin-induced MMP-9 expression. In astrocytes, thrombin activated Erk1/2, and it was inhibited by PD98059. In this study, thrombin-induced MMP-9 expression was inhibited by PD98059. PAR1 agonist activated Erk1/2 and PD98059 inhibited PAR1 agonist-induced MMP-9 expression. MMP-9 promoter reporter assay confirmed the positive effect of ERK1/2 on MMP-9 expression. These results suggest that the activation of PAR1 mediates thrombin-induced MMP-9 expression through the regulation of Erk1/2.

Role of p38 MAPK on the down-regulation of matrix metalloproteinase-9 expression in rat astrocytes.

In spite of their pathophysiological importance in neuro-inflammatory diseases, little is known about the signal transduction pathways that lead to the induction of matrix metalloproteinases (MMPs) in the central nervous system. We reported previously that lipopolysaccharide (LPS) induced MMP-9 expression through ERK1/2 pathway in rat primary astrocytes (Glia 41:15-24, 2003). Here, we investigated the role of other MAPK pathways, including p38 and JNK/SAPK, on the regulation of MMP-9 expression in LPS-stimulated rat primary astrocytes. LPS activated both p38 and JNK in astrocytes. Treatment with a specific p38 MAPK inhibitor SB203580, but not JNK inhibitor SP600125, increased the LPS-stimulated MMP-9 expression in a concentration-dependent manner. Anti-inflammatory cytokines, including IFN-gamma and IL-4, activated p38 MAPK and decreased MMP-9 production in LPS-stimulated astrocytes. When p38 MAPK activation was blocked by SB203580, the inhibitory effects of these cytokines on MMP-9 induction were abolished. Finally, direct injection of SB203580 into the lateral ventricle of rat brain increased the LPS-induced MMP-9 activity in cerebral cortex. Altogether, these results suggest that p38 activation down-regulates the inflammatory stimulation-induced overexpression of MMP-9, both in primary astrocytes and in cerebral cortex. The elaborate interplay between ERK1/2 and p38 pathways provides a more sophisticated mechanism for regulating MMP-9 activity in neuroinflammatory diseases.