Epidermal loss of Bcl6 exacerbates MC903-induced atopic dermatitis-like skin inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease where Th2-type immune responses are dominant. In the lesional skin of AD, keratinocytes show differentiation defects and secrete proinflammatory cytokines and chemokines, amplifying Th2-type responses in AD. We previously reported that inducible loss of B-cell lymphoma 6 (Bcl6), a transcription repressor and a master transcriptional regulator of follicular helper T cells and germinal center B cells, in the whole body results in upregulation of Th2-related cytokines in mouse skin. However, the role of Bcl6 in keratinocytes remains to be clarified. Here, we observed that BCL6 positively regulates the expression of keratinocyte differentiation markers and plasma membrane localization of adherence junctional proteins in keratinocyte cell culture. Although keratinocyte-specific loss of Bcl6 alone did not induce AD-like skin inflammation, it aggravates MC903-induced AD-like skin inflammation in mice. In addition, Bcl6 expression is decreased in the epidermis of lesional skin from MC903-induced AD-like skin inflammation in mice. These results strongly suggest that Bcl6 downregulation in keratinocytes contributes to the development and aggravation of AD-like skin inflammation in mice.

Foxp3 and Bcl6 deficiency synergistically induces spontaneous development of atopic dermatitis-like skin disease.

Atopic dermatitis (AD) is a common chronic skin disease caused by immune dysfunction, specifically the hyperactivation of Th2 immunity. AD is a complex disease with multiple factors contributing to its development; however, the interaction between these factors is not fully understood. In this study, we demonstrated that the conditional deletion of both the forkhead box p3 (Foxp3) and B-cell lymphoma 6 (Bcl6) genes induced the spontaneous development of AD-like skin inflammation with hyperactivation of type 2 immunity, skin barrier dysfunction, and pruritus, which were not induced by the single deletion of each gene. Furthermore, the development of AD-like skin inflammation was largely dependent on IL-4/13 signaling but not on immunoglobulin E (IgE). Interestingly, we found that the loss of Bcl6 alone increased the expression of thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33 in the skin, suggesting that Bcl6 controls Th2 responses by suppressing TSLP and IL-33 expression in epithelial cells. Our results suggest that Foxp3 and Bcl6 cooperatively suppress the pathogenesis of AD. Furthermore, these results revealed an unexpected role of Bcl6 in suppressing Th2 responses in the skin.

Phosphatidylinositol 4,5-bisphosphate is localized in the plasma membrane outer leaflet and regulates cell adhesion and motility.

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

Epidermal loss of phospholipase Cδ1 attenuates irritant contact dermatitis.

Irritant contact dermatitis (ICD) is one of the most common inflammatory skin diseases caused by exposure to chemical irritants. Since chemical irritants primarily damage keratinocytes, these cells play a pivotal role in ICD. One of the phosphoinositide-metabolizing enzymes, phospholipase C (PLC) δ1, is abundantly expressed in keratinocytes. However, the role of PLCδ1 in ICD remains to be clarified. Here, we found that croton oil (CrO)-induced ear swelling, a feature of ICD, was attenuated in keratinocyte-specific PLCδ1 knockout mice (PLCδ1 cKO mice). Dendritic epidermal T cells (DETCs), which have a protective role against ICD, were activated in the epidermis of the PLCδ1 cKO mice. In addition, the skin of CrO-treated PLCδ1 cKO mice showed increased infiltration of Gr1+CD11b+ myeloid cells. Of note, elimination of Gr1+CD11b+ myeloid cells restored CrO-induced ear swelling in PLCδ1 cKO mice to a similar level as that in control mice. Taken together, our results strongly suggest that epidermal loss of PLCδ1 protects mice from ICD through induction of Gr1+CD11b+ myeloid cells and activation of DETCs.

Phospholipase Cγ1 is required for normal irritant contact dermatitis responses and sebaceous gland homeostasis.

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte-specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.

Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes.

Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of the by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 1050 µM elevated intracellular Zn2+ levels by releasing intracellular Zn2+. HHQ at 10 µM increased cellular thiol content in a zinc-dependent manner. However, HHQ at 30-50 µM reduced cellular thiol content. Although the latter actions of HHQ (30-50 µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3-100 µM significantly protected cells against oxidative stress induced by H2O2. The process of cell death induced by H2O2 was delayed by HHQ, although both H2O2 and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10-30 µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10-30 µM exerted contrasting effects on cell death caused by oxidative stress and Ca2+ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects.

Obesity exacerbates imiquimod-induced psoriasis-like epidermal hyperplasia and interleukin-17 and interleukin-22 production in mice.

Psoriasis is a chronic inflammatory skin disorder that is accompanied by an imbalance between the proliferation and differentiation of keratinocytes. A number of studies have suggested an association between obesity and severe psoriasis; however, it remains to be clarified whether obesity exacerbates psoriasis. To address this unsolved question, we induced psoriasiform dermatitis in mouse models for obesity. We found that obesity exaggerated the severity of psoriasiform dermatitis induced by topical application of the Toll-like receptor (TLR) 7 agonist, imiquimod. Ear swelling and epidermal hyperplasia were more prominent in the obese mice than in the control mice. When compared to imiquimod-treated control mice, imiquimod-treated obese mice expressed higher levels of psoriasis mediators, interleukin-17A (IL-17A) and IL-22 in the skin. Food intake restriction partially abrogated enhanced ear swelling and cytokine overproduction in obese mice. Furthermore, the obesity environment and imiquimod treatment synergistically induced an IL-17A downstream molecule, regenerating islet-derived 3γ (Reg3γ), which is a critical molecule for psoriatic epidermal hyperplasia. Palmitic acid, one of the fatty acids released by subcutaneous adipocytes, increased the expression of REG3A (a human homologue of mouse Reg3γ) in both the HaCaT keratinocyte cell line and normal human keratinocytes. Taken together, these results strongly suggest that obesity exacerbates psoriasiform dermatitis in mice by upregulating IL-17A, IL-22 and Reg3γ.

Activation Mechanisms and Diverse Functions of Mammalian Phospholipase C.

Phospholipase C (PLC) plays pivotal roles in regulating various cellular functions by metabolizing phosphatidylinositol 4,5-bisphosphate in the plasma membrane. This process generates two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, which respectively regulate the intracellular Ca2+ levels and protein kinase C activation. In mammals, six classes of typical PLC have been identified and classified based on their structure and activation mechanisms. They all share X and Y domains, which are responsible for enzymatic activity, as well as subtype-specific domains. Furthermore, in addition to typical PLC, atypical PLC with unique structures solely harboring an X domain has been recently discovered. Collectively, seven classes and 16 isozymes of mammalian PLC are known to date. Dysregulation of PLC activity has been implicated in several pathophysiological conditions, including cancer, cardiovascular diseases, and neurological disorders. Therefore, identification of new drug targets that can selectively modulate PLC activity is important. The present review focuses on the structures, activation mechanisms, and physiological functions of mammalian PLC.

Characterization of uptake and metabolism of very long-chain fatty acids in peroxisome-deficient CHO cells.

Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex. Using this solubilizing technique, we examined the role of the peroxisome in the uptake and metabolism of VLCFAs in Chinese hamster ovary (CHO) cells. When wild-type CHO cells were incubated with saturated VLCFAs (S-VLCFAs), such as C23:0 FA, C24:0 FA, and C26:0 FA, extensive uptake was observed. Most of the incorporated S-VLCFAs were oxidatively degraded without acylation into cellular lipids. In contrast, in peroxisome-deficient CHO cells uptake of S-VLCFAs was marginal and oxidative metabolism was not observed. Extensive uptake and acylation of monounsaturated (MU)-VLCFAs, such as C24:1 FA and C22:1 FA, were observed in both types of CHO cells. However, oxidative metabolism was evident only in wild-type cells. Similar manners of uptake and metabolism of S-VLCFAs and MU-VLCFAs were observed in IFRS1, a Schwan cell-derived cell line. These results indicate that peroxisome-deficient cells limit intracellular S-VLCFAs at a low level by halting uptake, and as a result, peroxisome-deficient cells almost completely lose the clearance ability of S-VLCFAs accumulated outside of the cells.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics.

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

Nonspecific phospholipase C3 of radish has phospholipase D activity towards glycosylinositol phosphoceramide.

Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.

Phosphatidylinositol-specific phospholipase C enhances epidermal penetration by Staphylococcus aureus.

Staphylococcus aureus (S. aureus) commonly colonizes the human skin and nostrils. However, it is also associated with a wide variety of diseases. S. aureus is frequently isolated from the skin of patients with atopic dermatitis (AD), and is linked to increased disease severity. S. aureus impairs the skin barrier and triggers inflammation through the secretion of various virulence factors. S. aureus secretes phosphatidylinositol-specific phospholipase C (PI-PLC), which hydrolyses phosphatidylinositol and cleaves glycosylphosphatidylinositol-anchored proteins. However, the role of S. aureus PI-PLC in the pathogenesis of skin diseases, including AD, remains unclear. In this study, we sought to determine the role of S. aureus PI-PLC in the pathogenesis of skin diseases. PI-PLC was observed to enhance the invasion and persistence of S. aureus in keratinocytes. Besides, PI-PLC promoted the penetration of S. aureus through the epidermal barrier in a mouse model of AD and the human organotypic epidermal equivalent. Furthermore, the loss of PI-PLC attenuated epidermal hyperplasia and the infiltration of Gr-1+ cells and CD4+ cells induced by S. aureus infection in the mouse model of AD. Collectively, these results indicate that PI-PLC eases the entry of S. aureus into the dermis and aggravates acanthosis and immune cell infiltration in infected skin.

Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities.

Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.

Enrichment of hematopoietic stem/progenitor cells in the zebrafish kidney.

Hematopoietic stem cells (HSCs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. It has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. The double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a+ runx1+) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. Transcriptome analysis revealed that gata2a+ runx1+ cells showed typical molecular signatures of HSCs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. Transplantation assays demonstrated that long-term repopulating HSCs were highly enriched within the gata2a+ runx1+ fraction. In contrast, colony-forming assays showed that gata2a- runx1+ cells abundantly contain erythroid- and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.

Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables.

Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20 mg/100 g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2 mg/100 g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables.

Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation.

Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid.

N-(3-oxododecanoyl)-l-homoserine-lactone, a quorum sensing molecule, affects cellular content of nonprotein thiol content in rat lymphocytes: Its relation with intracellular Zn2.

Cellular actions of N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule of bacteria, were studied on rat thymocytes using a flow cytometer with appropriate fluorescent dyes to elucidate the effects of ODHL on host cells. A bell-shaped concentration-response relation was observed in the ODHL-induced changes in cellular glutathione content ([GSH]i). ODHL concentration-dependently increased intracellular Zn2+ levels ([Zn2+]i) and cellular O2- content ([O2-]i). The bell-shaped relation induced by ODHL can be explained as follows: a low concentration of ODHL is expected to induce moderate oxidative stress that intracellularly releases Zn2+ by converting thiols to disulfides. A slight elevation of [Zn2+]i may increase the [GSH]i. On the other hand, it is likely that a high concentration of ODHL causes severe oxidative stress that further causes both the decrease in [GSH]i and the increase in [Zn2+]i. Excessive increase in [Zn2+]i may augment oxidative stress that further decreases the [GSH]i. Other notable actions induced by ODHL included the elevation of [Zn2+]i by Zn2+ influx and the increase in [GSH]i under Zn2+-free conditions. Therefore, it is suggested that ODHL elicits diverse actions on host cells.

Cytometrical analysis of the adverse effects of indican, indoxyl, indigo, and indirubin on rat thymic lymphocytes.

Many businesses thrive by producing health supplements from agricultural products, as exemplified by the production of functional (or health) foods using plants traditionally cultivated in rural areas. Dyes, such as indican, indigo, indoxyl, and indirubin, present in dye plants, possess antibacterial, antifungal, and antiproliferative activities. However, these effects may also lead to cytotoxicity. Thus, studies on normal mammalian cells are necessary to identify cytotoxicity and prevent adverse effects of functional foods that contain these dyes. In this study, the effects of indican, indigo, indoxyl, and indirubin were evaluated by flow cytometry using appropriate fluorescent probes in rat thymic lymphocytes. Among the dyes analyzed, indirubin exerted distinct cellular activities. Treatment with indirubin (10-30 µM) increased the population of shrunken dead cells. The side scatter, but not forward scatter, increased in indirubin-treated living cells. It increased the population of annexin V-bound living and dead cells and that of dead cells without annexin V. Indirubin elevated intracellular Ca2+, but not Zn2+ levels. The cellular content of superoxide anions increased and that of glutathione decreased. Indirubin depolarized the cellular plasma and mitochondrial membranes. It did not potentiate or attenuate the cytotoxicity of A23187 (Ca2+ overload) and H2O2 (oxidative stress). The results suggested that indirubin induces both apoptotic and non-apoptotic cell death. It may be difficult to predict and prevent the adverse effects of indirubin due to its diverse activities on normal mammalian cells. Therefore, indirubin should be removed from products that contain dye plant extracts.

Diverse cellular actions of tert-butylhydroquinone, a food additive, on rat thymocytes.

Tertiary butylhydroquinone (TBHQ) is a food additive that possesses antioxidant activity. Its alternative applications have been explored in recent studies. However, there is controversy regarding safety. In this study using rat thymocytes, the cellular actions of TBHQ at sublethal concentrations were examined. TBHQ at concentrations of 3 µM or more elevated intracellular Zn2+ concentration ([Zn2+]i) in a dose-dependent manner, by increasing membrane Zn2+ permeability and releasing Zn2+ from cellular stores. TBHQ at 30 µM significantly increased side scatter (cell density) and the exposure of phosphatidylserine (PS) on cell membrane surfaces. It also decreased cellular glutathione (GSH) content without affecting cell lethality. Forward scatter was attenuated by 100 µM TBHQ. Thus, it is considered that TBHQ at sublethal concentrations (30 µM or less) exerts some adverse actions on cells. TBHQ at 10-30 µM attenuated the increase in cell lethality induced by hydrogen peroxide (H2O2), while potentiation of H2O2 cytotoxicity by 100 µM TBHQ was observed. The range of concentrations of TBHQ from benefit to toxicity under in vitro conditions may be 10-30 µM. Although TBHQ exhibits antioxidative actions at concentrations that are lower than those which elicit adverse cellular effects, sublethal levels of TBHQ cause some adverse actions that may be clinically concerned.