The importance of 12R-lipoxygenase and transglutaminase activities in the hydration-dependent ex vivo maturation of corneocyte envelopes.

BACKGROUND: Terminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R-lipoxygenase (12R-LOX) and other enzymes before transglutaminase (TG) attaches ω-hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross-linked by TG that has been activated by cathepsin D (CathD). OBJECTIVES: The primary aim of this work was to demonstrate the impact of relative humidity (RH) during ex vivo CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R-LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on ex vivo CE maturation was tested at low, optimal and high RH. METHODS: The first and ninth tape strip of photo-exposed (PE) cheek and photo-protected (PP) post-auricular sites of healthy volunteers were selected. Ex vivo CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors. RESULTS: Irrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for ex vivo CE maturation. The inhibition of 12R-LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during ex vivo maturation in the presence of TG or 12R-LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN-27219. However, a protease inhibitor mix reversed the negative effect of overhydration. CONCLUSION: The study adds to the understanding of the roles of 12R-LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC.

CONTEXTE: Les kératinocytes à différenciation terminale acquièrent les enveloppes protéiniques des cornéocytes (ECP) complexées aux enveloppes lipidiques des cornéocytes (ELC). Ces deux composants structurels des enveloppes cornéocytaires (EC) subissent un processus de maturation en gagnant en hydrophobicité, en rigidité et en surface. Les linoléoyl-acyle-céramides sont traités par 12R-lipoxygénase (12R-LOX) et d'autres enzymes avant que la transglutaminase (TG) ne fixe les x-hydroxy-céramides à l'involucrine dans les ECP. Les protéines structurelles sont simultanément réticulées par la TG qui a été activée par la cathepsine D (CathD). OBJECTIFS: L'objectif principal de ces travaux visait à démontrer l'impact de l'humidité relative (HR) pendant la maturation de l'EC ex vivo. Des humidités relatives faible, optimale et élevée ont été retenues pour étudier l'effet des inhibiteurs de la protéase (IP) sur la maturation de l'EC et l'activité de la TG ; l'activité de CathD et 12R-LOX a également été mesurée à une HR optimale. Finalement, l'effet du glycérol sur la maturation de l'EC ex vivo a été testé à des humidités relatives faible, optimale et élevée. MÉTHODES: La première et neuvième bandes adhésives sur un site à l'arrière de l'oreille protégé de la lumière (photo-protégé, PP) et sur une joue exposée à la lumière (photo-exposée, PE) de volontaires sains ont été sélectionnées. La maturation de l'EC ex vivo a été évaluée par l'approche de la maturité relative d'EC (RCEM) reposant sur l'hydrophobicité et la rigidité de l'EC. Les deuxième et huitième bandes ont été exposées à l'humidité relative en présence d'inhibiteurs. RÉSULTATS: Indépendamment de la profondeur de bande adhésive, les EC des échantillons EP ont atteint la rigidité d'EC de la même manière que les EC matures du site PP, mais ces améliorations faisaient défaut en ce qui concerne l'hydrophobicité des EC. Une HR à 70 % était optimale pour la maturation de l'EC ex vivo. L'inhibition de l'activité du 12R-LOX a entraîné une rigidité accrue de l'EC, laquelle était réduite par l'inhibiteur de la TG. L'hydrophobicité des EC est restée inchangée pendant la maturation ex vivo en présence de l'inhibition de la TG ou du 12R-LOX. L'hydrophobicité des EC a été améliorée en présence de glycérol à une HR de 44 % et à une HR de 100 %, mais non à une HR de 70 %. L'activité de la TG a par ailleurs significativement diminué à une HR de 100 % par rapport à l'inhibiteur commercial LDN-27219. Cependant, un mélange inhibiteur de la protéase a inversé l'effet négatif de la surhydratation. CONCLUSION: L'étude renforce la compréhension des rôles de l'activité de la TG et du 12R-LOX dans la maturation de l'EC et donne de plus amples détails sur l'effet du glycérol sur la couche cornée (stratum corneum, SC).

12R-lipoxygenase activity is reduced in photodamaged facial stratum corneum. A novel activity assay indicates a key function in corneocyte maturation.

BACKGROUND: During the late stage of keratinocyte differentiation, corneocytes gain a strong protein-lipid structure: the corneocyte envelopes (CE), composed of the inner corneocyte protein envelope (CPE) and the outer corneocyte lipid envelope (CLE). The hydrophobicity of CEs depends on the covalent attachment of linoleoyl-acyl-ceramides by transglutaminases (TG). These ceramides are processed by a range of other enzymes, including 12R-lipoxygenase (12R-LOX), before the covalent attachment of the free ω-hydroxyceramides to the CPE surface to form the CLE. The mechanical strength of CE is obtained with the formation of isodipeptide bonds by TG. The increase in hydrophobicity and rigidity leads to CE maturation which supports the integrity and mechanical resistance of the stratum corneum (SC). OBJECTIVES: The aim of this work was to develop and validate a novel enzyme activity assay for 12R-LOX in tape strippings of photo-exposed (PE) cheek and photo-protected (PP) post-auricular SC of healthy Chinese volunteers (n = 12; age 25 ± 3 years). RESULTS: A fluorescence-based assay was developed with ethyl linoleic acid as the substrate and a polyclonal antibody against 12R-LOX as an inhibitor. The specificity was shown by the lack of effect by a LOX inhibitor (ML351) and an epidermal-type lipoxygenase 3 (eLOX3) antibody on the acquired 12R-LOX activity. Reduced 12R-LOX activity was observed in the outer compared to the inner SC layers. Moreover, dramatically lower activity was shown in the PE vs. PP samples. Furthermore, the enzyme activity has a positive correlation (r = 0.94 ± 0.03) with CE maturity, in particular hydrophobicity, and a negative correlation (r = -0.96 ± 0.01) with transepidermal water loss (TEWL). CONCLUSION: This novel enzyme assay revealed a lower 12R-LOX activity in tape strippings from PE cheek for the first time. This finding is in line with less mature CEs and higher TEWL compared to PP post-auricular samples. This study indicates a strong link between 12R-LOX activity and CE maturation and SC integrity.

CONTEXTE: Pendant le stade avancé de différenciation des kératinocytes, les cornéocytes acquièrent une solide structure protéines-lipides : l'enveloppe des cornéocytes (EC) composée de l'enveloppe protéinique des cornéocytes (EPC) interne et de l'enveloppe lipidique des cornéocytes (ELC) externe. L'hydrophobicité des EC dépend de la liaison covalente des linoléoyl-acyle-céramides par transglutaminases (TG). Ces céramides sont traités par un ensemble d'autres enzymes, y compris la 12R-lipoxygénase (12R-LOX), avant la liaison covalente des xhydroxycéramides à la surface de l'EPC pour former l'ELC. La force mécanique de l'EC est obtenue par la formation de liaisons isodipeptides par TG. L'augmentation de hydrophobicité et de la rigidité conduit à une maturation de l'EC qui soutient l'intégrité et la résistance mécanique de la couche cornée (stratum corneum, SC). OBJECTIFS: L'objectif de ce travail était de développer et de valider un test novateur de l'activité enzymatique pour le 12R-LOX par arrachage par bande sur une joue exposée à la lumière (photo-exposed, PE) et sur de la SC à l'arrière de l'oreille protégée de la lumière (photo-protected, PP) de volontaires sains chinois (n = 12; 25 ans ± 3 ans). RÉSULTATS: Un test de fluorescence a été développé avec de l'acide linoléique éthylique en tant que substrat et un anticorps polyclonal anti-12R-LOX en tant qu'inhibiteur. La spécificité a été démontrée par le manque d'effet d'un inhibiteur de LOX (ML351) et d'un anticorps anti-lipoxygénase 3 (eLOX3) de type épidermique sur l'activité du 12R-LOX acquise. Une réduction de l'activité du 12R-LOX a été observée dans les couches de SC externes par rapport aux couches internes. En outre, une activité considérablement plus faible a été démontrée dans les échantillons PE par rapport aux échantillons PP. De plus, l'activité enzymatique a une corrélation positive (r = 0,94 ± 0,03) avec la maturité de l'EC, en particulier l'hydrophobicité, et une corrélation négative (r = −0,96 ± 0,01) avec une perte d'eau transépidermique (transepidermal water los, TEWL). CONCLUSION: Ce dosage enzymatique novateur, à partir de tape stripping sur les joues exposée à la lumière, a révélé une activité 12RLOX plus faible pour la première fois. Cette découverte est cohérente avec des EC moins matures et une TEWL plus élevée par rapport aux échantillons PP de l'arrière de l'oreille. Cette étude indique un lien solide entre l'activité du 12R-LOX et la maturation de l'EC et l'intégrité de la SC.

A new approach to assess the effect of photodamage on corneocyte envelope maturity using combined hydrophobicity and mechanical fragility assays.

BACKGROUND: The maturity of the corneocyte envelope (CE) provides information about the barrier functionality of the stratum corneum (SC). Corneocytes are enclosed by the CE, a protein-lipid matrix, contributing to mechanical resistance and hydrophobicity of the SC. OBJECTIVES: The aim of the work was to develop a novel and robust approach to characterize CE maturity based on rigidity, hydrophobicity and surface area. This offers an alternative approach to the Nile red staining and antigenicity of involucrin to characterize the CE. The photoexposed (PE) cheek and photoprotected (PP) post-auricular sites were selected for investigation. METHODS: Nine tape strips were obtained from the cheek and post-auricular sites of healthy Caucasians. CEs on the first and last tape strip were subjected to sonication to assess rigidity, and Nile red staining to determine hydrophobicity per unit surface area. In addition, the presence of involucrin and lipids was assessed to determine CE maturity by examination of the red/green pixel ratio, percentage of involucrin expressing CEs and alternatively the ratio of fluorescence density. RESULTS: The CE rigidity was lower in the deeper SC layers of the cheek, whereas post-auricular CEs were mechanically more resistant. Post-auricular CEs from the superficial SC had a larger surface area with a stronger fluorescence signal than those from the cheek. Interestingly, those CEs from the deeper SC layers had similar surface areas in both anatomical sites but were significantly different in hydrophobicity. These three parameters can be summarized as a relative CE maturity index that expresses CE maturity more precisely with a higher sensitivity than the conventional involucrin and Nile red staining approach. CEs of the cheek surface are more mature than CEs in the deeper SC layer, whereas CEs obtained from the post-auricular surface are more mature than those from the cheek surface. CONCLUSION: The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

A fundamental investigation into aspects of the physiology and biochemistry of the stratum corneum in subjects with sensitive skin.

BACKGROUND: Sensitive skin is a poorly understood skin condition. Defects in stratum corneum (SC) barrier function and/or extrasensory neuronal networks in the epidermis are believed to be involved in the problem. OBJECTIVES: This study aimed to unravel the relationships between bleomycin hydrolase (BH) and calpain-1 (C-1), pyrrolidone carboxylic acid (PCA) levels, corneocyte maturation, transglutaminase (TG) and plasmin activities on the cheeks of subjects with sensitive skin. METHODS: Forty-eight female Caucasian subjects, Fitzpatrick skin phototypes II-III, with self-perceived sensitive facial skin, were assessed and underwent a capsaicin reactivity test. Expert grading of skin condition was conducted as well as the measurement of transepidermal water loss (TEWL), skin capacitance, SC cohesion and SC integrity. BH, C-1 and plasmin activities were measured as well as PCA levels, plasmin and TG activity. Differential Nile red and involucrin immunostaining was performed to assess corneocyte maturation and size. RESULTS: About 52% of the subjects reacted to capsaicin. There were no significant differences between the capsaicin-sensitive and non-capsaicin-sensitive subjects with reference to skin grading, TEWL, skin capacitance and SC cohesion. PCA levels and BH activity were lowest in the capsaicin-sensitive panel (P < 0.05) and were correlated in non-capsaicin-sensitive subjects (r = 0.72). The activity of TG was significantly lower (48%) in the capsaicin-sensitive subjects (P < 0.001) and their corneocytes were less mature and smaller (P ≤ 0.05). SC was estimated to be thinner (6.87 ± 0.28 vs. 8.68 ± 0.26 µm; P = 0.001) in the capsaicin-sensitive subjects with a corresponding shorter SC path length (83.2 ± 4.4 µm and 113.1 ± 4.5 µm; P = 0.001). CONCLUSIONS: Despite the physiological similarities between the two groups of sensitive skin subjects, differences in their biochemistry were clearly evident. Lower levels of PCA, BH and TG activities together with a greater number of smaller and immature corneocytes indicate inferior SC maturation in the capsaicin-sensitive subjects. The reduced maturation of corneocytes and thinner SC likely contributes to a greater penetration of capsaicin and the associated increased skin sensitivity.

Variation in the activities of late stage filaggrin processing enzymes, calpain-1 and bleomycin hydrolase, together with pyrrolidone carboxylic acid levels, corneocyte phenotypes and plasmin activities in non-sun-exposed and sun-exposed facial stratum corneum of different ethnicities.

BACKGROUND: Knowledge of the ethnic differences and effects of photodamage on the relative amounts of natural moisturizing factor (NMF) together with filaggrin processing enzymes in facial stratum corneum is limited. Our aim was to characterize the activities of calpain-1 (C-1), bleomycin hydrolase (BH) and the levels of pyrrolidone carboxylic acid (PCA) as a marker for total NMF levels and to relate them to plasmin activities and corneocyte maturation. METHODS: Enzyme activities, PCA levels and corneocyte maturation were determined from facial tape strippings of photoexposed cheek and photoprotected post-auricular areas (PA) of healthy Caucasian (C), Black African (BA) and albino African (AA) female subjects living in South Africa. RESULTS: PCA concentration levels were of the order AA > BA > C subjects, and the highest activities of BH were present in the AA subjects. BH activities were greater on the photoexposed sites for the BA and C subjects, but they were only numerically elevated in the AA subjects. Photoprotected sites had an increase in C-1 activity in pigmented groups (C and BA), whereas in the AA subjects, the opposite was measured. Plasmin activities were greater on the cheek compared with the PA site for the AA and C subjects, but the activity was low in the BA subjects. In both test sites, the AA, but not the BA and C subjects, had smaller, parakeratotic and less mature corneocytes. CONCLUSION: Variation in PCA levels has been found for different ethnic groups in this study (AA > BA > C subjects). The values in the AA subjects are surprising as one might expect that the lack of pigmentation, and thereby increased photodamage, might lead to lower levels. Increased BH, but not C-1 activity, was observed in the AA subjects indicating that BH is associated with PCA production to a greater extent. Surprisingly, corneocyte maturation is still impaired with elevated PCA levels in AA subjects. The higher levels of plasmin and BH activities on the cheeks, especially for AA and C subjects, suggest that they can be used as markers for epidermal photodamage.

Variation in stratum corneum protein content as a function of anatomical site and ethnic group.

OBJECTIVES: Quantification of stratum corneum (SC) protein levels from tape strippings is frequently used to investigate skin conditions, to correct for amounts of SC protein removed in SC biomarker studies and to determine distribution of topically applied ingredients. In recent years, a rapid and convenient method for SC protein quantification from tape strippings has become available using infrared densitometry (IRD). However, standard curves have only been generated for Caucasian forearm and shoulder SC and have been assumed to be correct not only for facial SC but also for SC samples of other ethnic groups. The aim of this study was to investigate whether the use of IRD for SC protein measurement is valid for other body sites such as the cheek and for measuring SC protein content of darkly pigmented skin types. METHODS: Ten Caucasian and ten Black African female subjects with self-assessed normal skin participated in the study. Tape strippings were collected from two different body sites (forearm and cheek). First from the tape strippings, the SC optical absorption was determined densitometrically. This obtained absorption (%) was compared with absolute SC protein extracted from the same tapes using a colorimetric microbicinchoninic acid (µBCA) assay. RESULTS: Higher amounts of SC protein were removed from the forearm compared with the cheek (P < 0.01). The absolute SC protein concentration quantified by µBCA assay and the absorption of SC proteins by IRD followed a similar profile. There was no significant difference found between the two ethnicities in SC protein (P > 0.05). The overall coefficient of determination (R(2) ) shows a good fit to the regression line between the two methods in both sites (forearm = 0.82, cheek = 0.77). Also, both ethnicities showed good correlation (R(2) ≥ 0.69, P = 0.01). CONCLUSIONS: Facial SC is morphologically distinct from the forearm, as demonstrated by the differences in amounts of SC removed. Although the data distribution in different subject groups varied, the regression was always quite similar between the two body sites and both ethnic groups. Also, the correlations were similar to previously published data on other body sites. The resultant calibration curves can be used as a rapid indirect protein assessment of tape strippings from the cheek.

Identification of superoxide dismutase as a potential urinary marker of carbon tetrachloride-induced hepatic toxicity.

The aim of this study was the identification of a novel protein marker of hepatotoxicity in rat urine. Rats were dosed by gavage with carbon tetrachloride (CCl(4)) to induce acute liver injury. Surface enhanced laser desorption/ionisation (SELDI) ProteinChip technology revealed the appearance of a 15.7 kDa protein in the CCl(4)-treated rat urine. One-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) identified an 18.4 kDa protein in the CCl(4)-treated rat urine. The appearance of either protein was coincident over a time course during which they first appeared at 12h post-dosing, peaked at 36h and had disappeared again within 3 days post-dosing. The protein was identified by in-gel digestion and nano-electrospray (nano-ES)-tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD activity was found to be increased by 61.4-fold in CCl(4)-treated rat urine. Western blots of tissue homogenates from the rats revealed a time-dependent loss of SOD-1 from the livers of CCl(4)-treated rats matching the time course of SOD-1 appearance in urine. SOD-1 is not specifically located in liver; however, its appearance in urine in response to acute CCl(4)-induced hepatotoxicity is a novel finding; this coupled with loss from the liver following injury suggests urinary SOD-1 may be a potential marker of hepatotoxicity.

Role of calcineurin in the regulation of human lung mast cell and basophil function by cyclosporine and FK506.

BACKGROUND AND PURPOSE: Cyclosporine and FK506 are thought to act by targeting the Ca2+-dependent protein phosphatase, calcineurin. The aim of the present study was to determine whether cyclosporine and FK506 stabilize mast cells and basophils by interacting with calcineurin. EXPERIMENTAL APPROACH: The effects of cyclosporine and FK506 on the IgE-mediated release of histamine from mast cells and basophils were evaluated. The presence of calcineurin in cells was determined by Western blotting. Ca2+-dependent protein phosphatase activities were assessed in cell extracts using a synthetic phosphorylated peptide that is known to serve as a substrate for calcineurin. KEY RESULTS: FK506 was about 100-fold more potent than cyclosporine as an inhibitor of IgE-dependent histamine release from mast cells and basophils. Immunoblotting of solubilized preparations of purified cells demonstrated the presence of calcineurin in mast cells and basophils. In enzyme assays, mast cells expressed approximately 7-fold higher Ca2+-dependent protein phosphatase activity than basophils. Whereas cyclosporine effectively inhibited Ca2+-dependent protein phosphatase activity in cell extracts, FK506 was considerably less effective. CONCLUSIONS AND IMPLICATIONS: FK506 and cyclosporine inhibit the stimulated release of histamine from mast cells and basophils. However, the ability of cyclosporine, but not FK506, to inhibit Ca2+-dependent protein phosphatase activity questions whether FK506 stabilizes mast cells and basophils by interacting with calcineurin.

Markers of experimental acute inflammation in the Wistar Han rat with particular reference to haptoglobin and C-reactive protein.

C-reactive protein (CRP), haptoglobin (Hp) and fibrinogen (Fbgn) are acute phase reactants (APRs), the blood levels of which increase during acute inflammation. However, although the levels of these APRs are used to monitor inflammation in man, their usefulness and sensitivity as markers of inflammation in rodents are less clear. We therefore wished to evaluate, in a comparative fashion, a prototype immunoassay for serum CRP, a commercial assay for serum Hp, and an automated assay for Fbgn, using a model of acute inflammation in the rat. Additionally, pro-inflammatory cytokines and serum protein fractions were also measured. The model of inflammation used was the intraperitoneal injection of Freund's complete adjuvant (FCA). In a concluding experiment, findings with Hp in the FCA rat model were validated in a toxicologically relevant study involving the induction of acute hepatic inflammation using the model hepatotoxicant carbon tetrachloride (CCl(4)). Female Wistar Han rats were treated with a single injection of FCA in a dose-response study (1.25-10.0 ml/kg, sampling at 36 h) and two time-course studies (over 40 h and 21 days). In a final experiment, rats were dosed with CCl(4) at 0.8 ml/kg and sampled over a 17-day period. In FCA and CCl(4) experiments, serum/plasma was prepared and tissues taken at autopsy for histological assessment (CCl(4) study only). In the dose-response study, serum CRP, Hp and plasma Fbgn were increased at all FCA dose levels at 36 h post-dosing. Serum alpha(2) and beta(1) globulin fractions were also increased, while albumin levels were decreased. In the 40-h time-course study, CRP levels peaked at 25-40 h post-dosing, to approximately 120% of control (as 100%). Hp levels increased to a maximum at 25 and 40 h post-dosing with values greater than 400% of control, and alpha(2) and beta(1) globulin fractions peaked at 30 and 40 h post-dosing to 221 and 187% of control, respectively. Increased serum interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) levels peaked at 20 h (11-fold) and 25 h (19-fold), respectively. In a 21-day time-course study, no increased CRP levels were measured despite elevated levels of Hp, which peaked at 36 h (approximately 7-fold above control), and remained elevated up to 21 days. IL-6 and IL-1beta levels peaked at 12 h (19-fold) and 24 h (28-fold), respectively. Liver histopathology of animals treated with CCl(4) showed centrilobular hepatocellular degeneration and necrosis (most significant at 36 h) with an inflammatory response (most significant at 48 h). Resolution of the lesion was complete by 4 days post-dosing. Serum alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase levels peaked at 36 h post-dosing. Hp levels increased maximally at 48 h (426% of control). We conclude that serum CRP is a poor marker of acute inflammation in the rat in comparison with serum Hp and plasma Fbgn. Between Hp and Fbgn, serum Hp is shown to be the most sensitive and useful marker of acute inflammation.

Regulation of mammalian acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) plays a critical role in the regulation of fatty acid metabolism and its two isoforms, ACCalpha and ACCbeta, appear to have distinct functions in the control of fatty acid synthesis and fatty acid oxidation, respectively. They are regulated by similar short-term mechanisms of allosteric activation by citrate, and reversible phosphorylation and inactivation, and there is clearly interaction between these mechanisms. AMP-activated protein kinase is the important physiological ACC kinase for both isoforms and yet there is a potential physiological role for cAMP-dependent protein kinase in the hormonally mediated inactivation of ACCalpha, and phosphorylation of ACCbeta in its unique N-terminus.

Role of protein phosphatases in the regulation of human mast cell and basophil function.

Many extracellular stimuli mediate physiological change in target cells by altering the phosphorylation state of proteins. These alterations result from the dynamic interplay of protein kinases, which mediate phosphorylations, and protein phosphatases, which catalyse dephosphorylations. The antigen-mediated aggregation of high-affinity receptors for IgE on mast cells and basophils triggers rapid changes in the phosphorylation of many proteins and culminates in the generation of inflammatory mediators involved in allergic inflammatory diseases such as asthma. Although protein kinases have an established role in this process, less is known about the involvement of protein phosphatases. This imbalance has been redressed in recent years by the availability of phosphatase inhibitors, such as okadaic acid, that facilitate investigations of the role of protein phosphatases in intact cells. Here we review a number of studies in which inhibitors of protein phosphatases have been used to shed light on the potential importance of these enzymes in the regulation of human mast cell and human basophil function.

The regulation of acetyl-CoA carboxylase--a potential target for the action of hypolipidemic agents.

ACC exists as two major isoforms ACC1 or ACC alpha, and ACC2 or ACC beta, and there is evidence that they play separate roles in the production of malonyl-CoA for fatty acid synthesis and the control of mitochondrial beta-oxidation, respectively. ACC alpha can be regulated at the level of gene expression, allosteric regulation of the enzyme, and reversible phosphorylation by AMP-PK. Emerging lines of research suggest that similar mechanisms of regulation exist for ACC beta. Its inactivation in heart and skeletal muscle through phosphorylation by AMP-PK is becoming well-established. ACC is an important target of certain hypolipidemic drugs such as the fibrates. This is not simply because ACC alpha inhibition decreases the synthesis of a lipid component of VLDL because fatty acids synthesized de novo in liver are not always major contributors to VLDL lipid (158); it is also because ACC beta inhibition leads to a decrease in malonyl-CoA levels and the disinhibition of fatty acid oxidation. Partitioning fatty acids towards oxidation and away from esterification is an important aspect of the lipid-lowering effects of fibrates. Fibrates could use any of the mechanisms of ACC regulation to decrease activity. They could repress ACC gene expression through the activation of PPAR alpha, and fibroyl-CoA esters could inhibit ACC allosterically just as TOFyl-CoA does. However, we have demonstrated a rapid inactivation of ACC in cultured rat hepatocytes by gemfibrozil that is mediated by activation of AMP-PK and the subsequent phosphorylation of ACC. The end result is the inhibition of hepatic fatty acid synthesis and a possible activation of beta-oxidation as evidenced by the increased production of ketone bodies. The mechanism through which fibrates activate the AMP-PK cascade, the role of PPAR alpha, the physiological responses of biosynthesis and oxidation and the use of these mechanisms by other hypolipidemic agents are areas of ongoing investigation.

Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils.

1. The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE-mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose-dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC50) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 300 nM (basophils) for okadaic acid. 2. Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC50; 0.02-0.1 nM) > or = microcystin (0.1 nM) > okadaic acid (70 nM). 3. The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71+/-3% and 49+/-13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I-2-sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1-selective inhibitor, inhibitor-1 (I-1). 4. Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A-restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates. 5. These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

The role of pyruvate dehydrogenase, phosphofructo-1-kinase and acetyl-CoA carboxylase in the regulation of fatty acid synthesis in the lactating rat mammary gland during the starved to re-fed transition.

Re-feeding 24-h-starved lactating rats resulted in a rapid (within 0.5 h) restoration of glucose uptake by the mammary gland and a slower (within 3 h) restoration of fatty acid synthesis. The rapid reactivation of glucose uptake (82% of fed value within 0.5 h of re-feeding) correlated with a rapid reactivation of 6-phosphofructo-1-kinase (6-PF-1-K) and glycolysis (as determined by a 97% decrease in the [fructose-6-phosphate]/[fructose-1,6-bisphosphate] ratio). This could not be fully explained by a fall (29%) in the tissue concentration of its allosteric inhibitor, citrate. The delayed reactivation of pyruvate dehydrogenase (PDH) correlated very closely with the delayed reactivation of fatty acid synthesis and explained the continued output of pyruvate and lactate within the first 0.5 h of re-feeding. PDH reactivation preceded the reactivation of acetyl-CoA carboxylase (ACC), which did not occur significantly until 1.5 h of re-feeding. ACC reactivation correlated with a decrease in the tissue concentration of citrate and a second late phase of 6-PF-1-K activation. It is clear that the important regulatory steps 6-PF-1-K, PDH and ACC, are reactivated asynchronously in the lactating mammary gland in response to re-feeding starved rats and that PDH is more important than ACC in the regulation of fatty acid synthesis.

Preliminary characterization of the role of protein serine/threonine phosphatases in the regulation of human lung mast cell function.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC50) the IgE-dependent release of histamine was 0.2 microM. Okadaic acid also inhibited the IgE-mediated generation of prostaglandin D2 (PGD2) and sulphopeptidoleukotrienes (sLT) with IC50 values of 0.2 microM and 0.6 microM respectively. 2. The IgE-mediated generation of histamine, PGD2 and sLT was inhibited by okadaic acid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid > okadaol > okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs. 3. Extracts of HLMC liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 microM), okadaic acid (73 +/- 4% inhibition, P < 0.0005), okadaol (26 +/- 7% inhibition, P < 0.05) and okadaone (8 +/- 7% inhibition, P = 0.52). The rank order of activity of okadaic acid > okadaol > okadaone parallels the activity of these compounds as inhibitors of isolated PPs. 4. Dephosphorylation of radiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15 +/- 3% (P < 0.001) by a low (2 nM) concentration of okadaic acid and by 88 +/- 4% (P < 0.0005) by a higher (5 microM) concentration of okadaic acid. Because 2 nM okadaic acid may act selectively to inhibit PP2A whereas 5 microM okadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC. 5. Inhibitor 2, a PP1-selective inhibitor, attenuated (71 +/- 3% inhibition, P < 0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC. 6. Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylated by extracts of purified HLMC and this activity was inhibited (81 +/- 8% inhibition, P < 0.005) by 2 nM okadaic acid suggesting that PP2A is resident in HLMC. 7. Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid-induced inhibition of mediator release from HLMC.

Regulation of human basophil function by phosphatase inhibitors.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM okadaic acid. 2. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 +/- 9% (P < 0.002), 48 +/- 14% (P < 0.05) and 8 +/- 7% (P = 0.31), respectively. 6. Extracts of purified human basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.