Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes.

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

Intracellular Mg2+ protects mitochondria from oxidative stress in human keratinocytes.

Reactive oxygen species (ROS) are harmful for the human body, and exposure to ultraviolet irradiation triggers ROS generation. Previous studies have demonstrated that ROS decrease mitochondrial membrane potential (MMP) and that Mg2+ protects mitochondria from oxidative stress. Therefore, we visualized the spatio-temporal dynamics of Mg2+ in keratinocytes (a skin component) in response to H2O2 (a type of ROS) and found that it increased cytosolic Mg2+ levels. H2O2-induced responses in both Mg2+ and ATP were larger in keratinocytes derived from adults than in keratinocytes derived from newborns, and inhibition of mitochondrial ATP synthesis enhanced the H2O2-induced Mg2+ response, indicating that a major source of Mg2+ was dissociation from ATP. Simultaneous imaging of Mg2+ and MMP revealed that larger Mg2+ responses corresponded to lower decreases in MMP in response to H2O2. Moreover, Mg2+ supplementation attenuated H2O2-induced cell death. These suggest the potential of Mg2+ as an active ingredient to protect skin from oxidative stress.

Magnesium Supplementation Attenuates Ultraviolet-B-Induced Damage Mediated through Elevation of Polyamine Production in Human HaCaT Keratinocytes.

Magnesium ions (Mg2+) have favorable effects such as the improvement of barrier function and the reduction of inflammation reaction in inflammatory skin diseases. However, its mechanisms have not been fully understood. Microarray analysis has shown that the gene expressions of polyamine synthases are upregulated by MgCl2 supplementation in human HaCaT keratinocytes. Here, we investigated the mechanism and function of polyamine production. The mRNA and protein levels of polyamine synthases were dose-dependently increased by MgCl2 supplementation, which were inhibited by U0126, a MEK inhibitor; CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor; and Naphthol AS-E, a cyclic AMP-response-element-binding protein (CREB) inhibitor. Similarly, reporter activities of polyamine synthases were suppressed by these inhibitors, suggesting that MEK, GSK3, and CREB are involved in the transcriptional regulation of polyamine synthases. Cell viability was reduced by ultraviolet B (UVB) exposure, which was rescued by MgCl2 supplementation. The UVB-induced elevation of reactive oxygen species was attenuated by MgCl2 supplementation, which was inhibited by cysteamine, a polyamine synthase inhibitor. Our data indicate that the expression levels of polyamine synthases are upregulated by MgCl2 supplementation mediated through the activation of the MEK/GSK3/CREB pathway. MgCl2 supplementation may be useful in reducing the UVB-induced oxidative stress in the skin.

Real-time imaging of human epidermal calcium dynamics in response to point laser stimulation.

BACKGROUND: Changes of epidermal calcium ion concentration are involved in regulation of barrier homeostasis and keratinocyte differentiation. Moreover, intracellular calcium dynamics might play a role in skin sensation. But, although calcium dynamics of cultured keratinocytes in response to mechanical stresses has been well studied, calcium propagation in stimulated human epidermis is still poorly understood. OBJECTIVE: The aim of this study was to demonstrate a novel method for real-time measurement of calcium dynamics in response to point stimulation of human epidermis at the single-cell level. METHODS: We examined calcium propagation in cross-sectional samples of living human epidermis ex vivo, as well as in cultured human keratinocytes, by means of two-photon microscopy after stimulating cells in stratum granulosum with the emission laser of a two-photon microscope. RESULTS: Cells in different epidermal layers showed different responses, and those in stratum basale showed the greatest elevation of intracellular calcium. Calcium propagation in epidermis was inhibited in the presence of apyrase (which degrades adenosine triphosphate; ATP) or gap-junction blockers. In cultured keratinocytes, on the other hand, calcium propagated in a simple concentric wave-like manner from the stimulation site, and propagation was strongly suppressed by apyrase. CONCLUSION: Our results suggested that ATP and gap junctions play important roles in calcium propagation induced by point laser stimulation of the uppermost layer of epidermis. Our method should be broadly useful to study calcium dynamics, epidermal physiological mechanisms, and mechanisms of skin sensation at the single-cell level.

Concurrent Fowlpox and Candidiasis Diseases in Backyard Chickens with Unusual Pox Lesions in the Bursa of Fabricius.

Concurrent fowlpox and candidiasis diseases occurred in a backyard chicken flock. Four deceased chickens (one Nagoya breed and three white silkie chickens) were examined for diagnosis. At necropsy, white curd-like plaques were observed in the crop. Fungal elements that stained positive for Candida albicans with immunohistochemistry were distributed throughout the tongue, choanal mucosa, esophagus, and crop. Typical fowlpox lesions, composed of proliferating epithelial cells with ballooning degeneration and viral intracytoplasmic inclusions, were observed in the conjunctiva, nasal mucosa, and skin around the cloaca. Interestingly, hyperplastic interfollicular epithelium with rare virus inclusions was observed in the bursa of Fabricius (BF). Some bursal follicles were replaced by proliferating epithelial cells. These proliferating cells immunohistochemically stained positive for cytokeratin. PCR and subsequent genetic sequencing detected the C. albicans gene in the crop, and fowlpox virus genes in the BF. These results indicate that this outbreak was a rare presentation of fowlpox in spontaneously infected chickens, with unusual pox lesions in the BF.

Mathematical modeling of calcium waves induced by mechanical stimulation in keratinocytes.

Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

Frontiers in epidermal barrier homeostasis--an approach to mathematical modelling of epidermal calcium dynamics.

Intact epidermal barrier function is crucial for survival and is associated with the presence of gradients of both calcium ion concentration and electric potential. Although many molecules, including ion channels and pumps, are known to contribute to maintenance of these gradients, the mechanisms involved in epidermal calcium ion dynamics have not been clarified. We have established that a variety of neurotransmitters and their receptors, originally found in the brain, are expressed in keratinocytes and are also associated with barrier homeostasis. Moreover, keratinocytes and neurons show some similarities of electrochemical behaviour. As mathematical modelling and computer simulation have been employed to understand electrochemical phenomena in brain science, we considered that a similar approach might be applicable to describe the dynamics of epidermal electrochemical phenomena associated with barrier homeostasis. Such methodology would also be potentially useful to address a number of difficult problems in clinical dermatology, such as ageing and itching. Although this work is at a very early stage, in this essay, we discuss the background to our approach and we present some preliminary results of simulation of barrier recovery.

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5'-[γ-thio]triphosphate in vitro.

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT-PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5'-[γ-thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose-dependent manner. The ATPγS-induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.

Morphological and functional differences in coculture system of keratinocytes and dorsal-root-ganglion-derived cells depending on time of seeding.

Previous study indicated that in a coculture system of keratinocytes and dorsal-root-ganglion-derived (DRG) cells, mechanical stimulation of keratinocytes induced ATP-mediated calcium propagation and excitation of DRG cells. Here, we examined two different coculture systems of keratinocytes and DRG cells. In one, we seeded keratinocytes first and then seeded DRG cells on the keratinocytes. In this system, nerve fibres from DRG cells passed between keratinocytes. Mechanical stimulation of keratinocytes did not induce excitation of DRG cells. In the other, we seeded both cell types together. At first, each cell type grew separately, forming cell aggregates. Then, nerve fibres grew out from the DRG cell aggregates to keratinocyte aggregates and penetrated into them. In this system, mechanical stimulation of keratinocytes induced excitation of the nerve fibres, but the excitation was not completely blocked by apyrase, an ATP-degrading enzyme. These results suggest that coculture of keratinocytes and DRG can generate a variety of structures, depending on the seeding conditions.

Phosphodiesterase inhibitors block the acceleration of skin permeability barrier repair by red light.

We previously demonstrated that exposure to red light (550-670 nm) accelerates epidermal permeability barrier recovery after barrier disruption. Furthermore, we showed that photosensitive proteins, originally found in retina, are also expressed in epidermis. In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. In this study, we evaluate the role of phosphodiesterese 6 in the acceleration by red light of epidermal permeability barrier recovery. Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1-methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of the barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, whereas T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier.

Prevention of allergic asthma by vaccination with transgenic rice seed expressing mite allergen: induction of allergen-specific oral tolerance without bystander suppression.

This study tested the feasibility of oral immunotherapy for bronchial asthma using a newly developed subunit vaccine in which a fragment (p45-145) of mite allergen (Der p 1) containing immunodominant human and mouse T cell epitopes was encapsulated in endoplasmic reticulum-derived protein bodies of transgenic (Tg) rice seed. Allergen-specific serum immunoglobulin responses, T cell proliferation, Th1/Th2 cytokine production, airway inflammatory cell infiltration, bronchial hyper-responsiveness (BHR) and lung histology were investigated in allergen-immunized and -challenged mice. Prophylactic oral vaccination with the Tg rice seeds clearly reduced the serum levels of allergen-specific IgE and IgG. Allergen-induced CD4(+) T cell proliferation and production of Th2 cytokines in vitro, infiltration of eosinophils, neutrophils and mononuclear cells into the airways and BHR were also inhibited by oral vaccination. The effects of the vaccine were antigen-specific immune response because the levels of specific IgE and IgG in mice immunized with Der f 2 or ovalbumin were not significantly suppressed by oral vaccination with the Der p 1 expressing Tg rice. Thus, the vaccine does not induce nonspecific bystander suppression, which has been a problem with many oral tolerance regimens. These results suggest that our novel vaccine strategy is a promising approach for allergen-specific oral immunotherapy against allergic diseases including bronchial asthma.

Mechanical-stimulation-evoked calcium waves in proliferating and differentiated human keratinocytes.

Calcium dynamics in the epidermis play a crucial role in barrier homeostasis and keratinocyte differentiation. We have recently suggested that the electro-physiological responses of the keratinocyte represent the frontier of the skin sensory system for environmental stimuli. In the present study, we have evaluated the responses of proliferating and differentiated human keratinocytes to mechanical stress by measuring the intracellular calcium level. Before differentiation, mechanical stress induces a calcium wave over a limited area; this is completely blocked by apyrase, which degrades ATP. In the case of differentiated keratinocytes, the calcium wave propagates over a larger area. Application of apyrase does not completely inhibit this wave. Thus, in differentiated cells, the induction of calcium waves might involve not only ATP, but also another factor. Immunohistochemical studies indicate that connexins 26 and 43, both components of gap junctions, are expressed in the cell membrane of differentiated keratinocytes. Application of octanol or carbenxolone, which block gap junctions, significantly reduces calcium wave propagation in differentiated keratinocytes. Thus, signaling via gap junctions might be involved in the induction of calcium waves in response to mechanical stress at the upper layer of the epidermis.

Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases.

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Phosphorothioate analogues of alkyl lysophosphatidic acid as LPA3 receptor-selective agonists.

The metabolically stabilized LPA analogue 1-oleoyl-2-O-methyl-rac-glycerophosphorothioate (OMPT) was recently shown to be a potent subtype-selective agonist for LPA3, a G-protein-coupled receptor (GPCR) in the endothelial differentiation gene (EDG) family. Further stabilization was achieved by replacing the sn-1 O-acyl group with an O-alkyl ether. A new synthetic route for the enantiospecific synthesis of the resulting alkyl LPA phosphorothioate analogues is described. The pharmacological properties of the alkyl OMPT analogues were characterized for subtype-specific agonist activity using Ca2+-mobilization assays in RH7777 cells expressing the individual EDG family LPA receptors. Alkyl OMPT analogues induced cell migration in cancer cells mediated through LPA1. Alkyl OMPT analogues also activated Ca2+ release through LPA2 activation but with less potency than sn-1-oleoyl LPA. In contrast, alkyl OMPT analogues were potent LPA3 agonists. The alkyl OMPTs 1 and 3 induced cell proliferation at submicromolar concentrations in 10T 1/2 fibroblasts. Interestingly, the absolute configuration of the sn-2 methoxy group of the alkyl OMPT analogues was not recognized by any of the LPA receptors in the EDG family. By using a reporter gene assay for the LPA-activated nuclear transcription factor PPARgamma, we demonstrated that phosphorothioate diesters have agonist activity that is independent of their ligand properties at the LPA-activated GPCRs. The availability of new alkyl LPA analogues expands the scope of structure-activity studies and will further refine the molecular nature of ligand-receptor interactions for this class of GPCRs.

Structure-activity relationships of fluorinated lysophosphatidic acid analogues.

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions with seven-transmembrane G-protein-coupled receptors (GPCRs) and the nuclear hormone receptor PPARgamma. To identify receptor-selective LPA analogues, we describe a series of fluorinated LPA analogues in which either the sn-1 or sn-2 hydroxyl group was replaced by a fluoro or fluoromethyl substituent. We also describe stabilized phosphonate analogues in which the bridging oxygen of the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) or alpha-difluoromethylene (-CF(2)-) moiety. The sn-2- and sn-1-fluoro-LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. We first tested these LPA analogues on insect Sf9 cells induced to express human LPA(1), LPA(2), and LPA(3) receptors. While none of the analogues were found to be more potent than 1-oleoyl-LPA at LPA(1) and LPA(2), several LPA analogues were potent LPA(3)-selective agonists. In contrast, 1-oleoyl-LPA had similar activity at all three receptors. The alpha-fluoromethylene phosphonate analogue 15 activated calcium release in LPA(3)-transfected insect Sf9 cells at a concentration 100-fold lower than that of 1-oleoyl-LPA. This activation was enantioselective, with the (2S)-enantiomer showing 1000-fold more activity than the (2R)-enantiomer. Similar results were found for calcium release in HT-29 and OVCAR8 cells. Analogue 15 was also more effective than 1-oleoyl-LPA in activating MAPK and AKT in cells expressing high levels of LPA(3). The alpha-fluoromethylene phosphonate moiety greatly increased the half-life of 15 in cell culture. Thus, alpha-fluoromethylene LPA analogues are unique new phosphatase-resistant ligands that provide enantiospecific and receptor-specific biological readouts.

Prediction of response to risperidone treatment with respect to plasma concencentrations of risperidone, catecholamine metabolites, and polymorphism of cytochrome P450 2D6.

In the present study, we examined the relationships between plasma concentrations of risperidone and clinical responses, extrapyramidal symptoms, plasma levels of cotinine and caffeine, or cytochrome (cyp)2D6 genotypes. In addition, we also investigated the relationships between plasma levels of 3-methoxy-4-hydroxyphenylglycol (MHPG) or homovanillic (HVA) acid and clinical responses to risperidone. One hundred and 36 patients (male/female: 58/78, age 37+/-13 years) who met DSM-IV criteria for schizophrenia, schizoaffective disorder, delusional disorder and brief psychotic disorder, and who were being treated with risperidone alone, were evaluated regarding their clinical improvement and extrapyramidal symptoms using the Positive and Negative Syndrome Scale (PANSS) and Simpson and Angus (SAS), respectively, and plasma levels of cotinine, caffeine, MHPG and HVA were analysed by high-performance liquid chromatography. The cyp2D6*5 and *10 alleles were identified using the polymerase chain reaction. There was a positive correlation between plasma levels of risperidone plus 9-hydroxyrisperidone (active moiety) and SAS scores, but not the PANSS. Pretreatment HVA levels in responders were higher than those in nonresponders. In addition, there was a negative correlation between changes in HVA levels and improvement in PANSS scores. There was no association between plasma levels of risperidone and plasma levels of cotinine or caffeine. Furthermore, there were no differences in the risperidone/9-hydroxyrisperidone ratio, clinical improvements and extrapyramidal symptoms among cyp2D6 genotypes. These results indicate that pretreatment HVA levels and plasma concentrations of active moiety might play a part in predicting the clinical response and occurrence of extrapyramidal symptoms, respectively, when treating patients with risperidone.

Lysophosphatidic acid production and action: validated targets in cancer?

The completion of the human genome project, the evolution of transcriptional profiling and the emergence of proteomics have focused attention on these areas in the pathophysiology and therapy of cancer. The role of lysophospholipids as potential mediators in cancer pathophysiology, screening and management has taken a major leap forward with the recent cloning of several enzymes involved in the metabolism of lysophospholipids. Lysophospholipids, although small molecules, contain a high "informational" content. Differences include the nature of the phosphate head group, the regiochemistry of the fatty acyl chain on the glyceryl backbone, the presence of ether versus ester linkages to the backbone, and the length and saturation of the fatty acyl or alkyl chain. This informational content is sufficient to result in a marked structure function activity relationship at their cognate receptors. Thus the emerging discipline of "functional lipidomics" is likely to prove as important as genomics and proteomics in terms of early diagnosis, prognosis, and therapy. Lysophospholipid levels are elevated in vivo in a number of pathophysiological states including ascitic fluid from ovarian cancer patients indicating a role in the pathophysiology of this devastating disease. Although controversial, levels of specific lysophospholipids may be altered in the blood of cancer patients providing a potential mechanism for early diagnosis. Several of the enzymes involved in the metabolism of lysophospholipids are aberrant in ovarian and other cancers. Further, the enzymes are active in the interstitial space, rendering them readily accessible to the effects of inhibitors including antibodies, proteins, and small molecules. In support of a role for lysophospholipids in the pathophysiology of cancer, expression of receptors for lysophospholipids is also aberrant in cancer cells from multiple different lineages. All of the cell surface receptors for lysophospholipids belong to the G protein coupled receptor family. As over 40% of all drugs in current use target this family of receptors, lysophospholipid receptors are highly "druggable." Indeed, a number of highly specific agonists and antagonists of lysophospholipid receptors have been identified. A number are in preclinical evaluation as therapeutics. We look forward to the next several years when the role of lysophospholipids in physiology and the pathophysiology and management of cancer and other diseases are fully elucidated.

Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.

Autotaxin (ATX) is a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5'-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein-coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.