NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis.

Immunosuppressive therapies using calcineurin inhibitors, such as cyclosporine A, are associated with a higher incidence of squamous cell carcinoma formation in mice and humans. Calcineurin is believed to suppress tumorigenesis in part through Nfatc1, a transcription factor expressed primarily in hair follicle bulge stem cells in mice. However, mice overexpressing a constitutively active Nfatc1 isoform in the skin epithelium developed increased spontaneous skin squamous cell carcinomas. Because follicular stem cells can contribute to skin tumorigenesis, whether the endogenous expression of Nfatc1 inhibits or enhances skin tumorigenesis is unclear. Here we show that loss of the endogenous expression of Nfatc1 suppresses the rate of DMBA/TPA-induced skin tumorigenesis. Inducible deletion of Nfatc1 in follicular stem cells before tumor initiation significantly reduces the rate of tumorigenesis and the contribution of follicular stem cells to skin tumors. We find that skin tumors from mice lacking Nfatc1 display reduced Hras codon 61 mutations. Furthermore, Nfatc1 enhances the expression of genes involved in DMBA metabolism and increases DMBA-induced DNA damage in keratinocytes. Together these data implicate Nfatc1 in the regulation of skin stem cell-initiated tumorigenesis via the regulation of DMBA metabolism.

In silico target fishing for the potential bioactive components contained in Huanglian Jiedu Tang (HLJDD) and elucidating molecular mechanisms for the treatment of sepsis.

The present study was designed to target fish for potential bioactive components contained in a Huang Lian Jie Du decoction (HLJDD) and identify the underlying mechanisms of action for the treatment of sepsis at the molecular level. he bioactive components database of HLJDD was constructed and the sepsis-associated targets were comprehensively investigated. The 3D structures of the PAFR and TXA2R proteins were established using the homology modelling (HM) method, and the molecular effects for sepsis treatment were analysed by comparing the bioactive components database and the sepsis targets using computational biology methods. The results of the screening were validated with biological testing against the human oral epidermal carcinoma cell line KB in vitro. We found that multiple bioactive compounds contained in the HLJDD interacted with multiple targets. We also predicted the promising compound leads for sepsis treatment, and the first 28 compounds were characterized. Several compounds, such as berberine, berberrubine and epiberberine, dose-dependently inhibited PGE2 production in human KB cells, and the effects were similar in the presence or absence of TPA. This study demonstrates a novel approach to identifying natural chemical compounds as new leads for the treatment of sepsis.

The antiallergic mechanisms of Citrus sunki and bamboo salt (K-ALL) in an allergic rhinitis model.

The antiallergic effects of traditional medicines have long been studied. Traditional Korean medicine, Citrus sunki and bamboo salt, has been used for the treatment of allergic diseases in Korea. K-ALL, composed of Citrus sunki and bamboo salt, is a newly prepared prescription for allergic patients. To develop the new antiallergic agent, we examined the effects of K-ALL through in vivo and in vitro models. K-ALL and naringin (an active compound of K-ALL) significantly inhibited histamine release from rat peritoneal mast cells. This inhibitory effect of K-ALL on histamine release was higher than effects from other known histamine inhibitors such as bamboo salt, Citrus sunki or disodium cromoglycate. K-ALL significantly inhibited systemic anaphylactic shock induced by the compound 48/80 and passive cutaneous anaphylaxis induced by the IgE. K-ALL also inhibited production and mRNA expression of inflammatory cytokines induced by phorbol 12-myristate 13-acetate and the calcium ionophore A23187 on HMC-1 cells (a human mast cell line). In the ovalbumin-induced allergic rhinitis animal model, rub scores, histamine, IgE, inflammatory cytokines and inflammatory cell counts were all reduced by the oral or nasal administration of K-ALL (pre and posttreatment). These results indicate the great potential of K-ALL as an active immune modulator for the treatment of mast cell-mediated allergic diseases.

Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors.

The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

A phase I clinical trial of 12- O-tetradecanoylphorbol-13-acetate for patients with relapsed/refractory malignancies.

Phorbol esters activate protein kinase C and modulate a variety of downstream cell signaling pathways. 12-O-tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester that induces differentiation or apoptosis in a variety of cell lines at low concentrations. A phase I dose escalation trial of TPA was undertaken for patients with relapsed or refractory malignancies. The starting dose was 0.063 mg/m2 and most patients were treated with an intravenous infusion of TPA on days 1-5 and 8-12 followed by a 2-week rest period prior to retreatment. Thirty-five patients were treated. A biological assay was used to monitor levels of TPA-like activity in the blood after treatment. Serious adverse events included individual episodes of gross hematuria, a grand mal seizure, syncope, and hypotension. Many patients had transient fatigue, mild dyspnea, fever, rigors, and muscular aches shortly after the infusion. Dose-limiting toxicities included syncope and hypotension at a dose of 0.188 mg/m2. Only a single patient had evidence of tumor response. These studies establish 0.125 mg/m2 as the maximally tolerated dose when TPA is administered on this schedule.

Determinación de estallido respiratorio en sangre total mediante la técnica de citometría de flujo / Determination of respiratory burst in total blood by means of flow cytometry technique

La capacidad de defensa de las células fagocíticas depende del llamado estallido respiratorio, el cual se caracteriza por una serie de reacciones generadas por la NADPH oxidasa, que lleva a la liberación de especies reactivas de oxígeno, letales para el microorganismo. El propósito de este estudio fue desarrollar un método simple para la evaluación cuantitativa del estallido respiratorio mediante Citometría de flujo para poder utilizarlo como estrategia diagnóstica en individuos con alteración inmunológicas u otras patologías clínicas. La estandarización se realizó con estudios de dosis-respuesta y tiempo-respuesta, tanto del activador forbol 12-miristato-13acetato (PMA) así como del fluorocromo 123-dihidrorodamina (DHR). Se analizaron 30 muestras de individuos normales provenientes de un banco de sangre para obtener los valores de referencia, los cuales fueron definidos como los valores de estallido respiratorio neto expresado en porcentaje de fluorescencia y que fluctuaron entre 85,3 y 95,0 con una desviación estándar de 2,4; y los expresados en intensidad media de fluorescencia (MFI), que fluctuaron entre 13,3 y 50,4 con una desviación estándar de 9,3. De esta forma, los valores se caracterizaron como aquellos iguales al promedio ± 2 desviaciones estándar, cubriéndose el 96,6 por ciento de la muestra. En conclusión, esta nueva forma de determinar el estallido respiratorio, ofrece la gran ventaja de visualizar directamente las células, dando la oportunidad de medir en forma simultánea tanto porcentaje como la intensidad media de fluorescencia celular, permitiendo obtener así resultados más exactos y precisos.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) modulate several biochemical pathways in human leukemic myeloid cells.

The neuropeptides Vasoactive-intestinal peptide (VIP) and Pituitary adenylate-cyclase activating protein (PACAP) increased cAMP levels in three out of five human myeloid leukemic cell lines tested while an increased in calcium intracytoplasmic levels was seen only in one cell line (HEL). This increase was phospholipase C, Pertussis toxin dependent and associated with an increase in c-fos and c-jun protein expression together with the formation of functional AP-1 transcriptional factor complex. Cell exposure to VIP or PACAP resulted in a decrease in HEL cell proliferation associated with a down-regulation of the erythroid marker, Glycophorin A. Both peptides were found to increase intra-cytoplasmic calcium levels in blasts isolated from patients with myeloid leukemia. Thus VIP and PACAP are involved in the physiology and pathophysiology of human myeloid cells.

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide.

Growth of Acanthamoeba castellanii was inhibited by chitosanoligosaccharide (up to 20 mg ml-1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.

Dramatic switching of protein kinase C agonist/antagonist activity by modifying the 12-ester side chain of phorbol esters.

A dramatic switching of PKC agonist and antagonist activity was observed by modification of the hydrophilicity of the 12-ester side chain of phorbol. Thus, phorbol ester 4 that contains a glycol at the 12-ester chain demonstrated a pure and significant antagonist ability of PKC; however, 3 that contains an alkanol at the 12-ester chain demonstrated a potent PKC agonist activity. On the basis of the structural difference between 3 and 4 and results of the partition assay in the Hela cell/PBS buffer system, we propose that 4 acts as a translocation poison of the PKC-phorbol ester complex. The approach of controlling the agonist/antagonist activity of phorbol esters by the nature (i.e., hydrophilicity, charge, and rigidity, etc.) of the 12-ester chain may be very useful for developing selective PKC inhibitors and a potential pharmaceutical compound for anticancer therapies.

A sensitive bioassay for measuring blood levels of 12-O-tetradecanoylphorbol-13-acetate (TPA) in patients: preliminary pharmacokinetic studies.

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation in myelocytic leukemia cells, and it has been shown to have activity in patients with acute myelocytic leukemia. Because attempts to develop a suitable mass spectrometry assay for TPA were unsuccessful (because of the lack of sufficient sensitivity), we developed a novel and highly sensitive blood level bioassay for TPA that measures ethyl acetate-extractable differentiating activity in blood. Differentiating activity in ethyl acetate extracts of blood was measured in HL-60 cells by measuring the formation of adherent cells. The sensitivity of the assay was approximately 0.1 ng TPA/ml blood. The assay for TPA has a high degree of specificity and does not measure deesterifed potential metabolites (phorbol, phorbol-13-acetate, or phorbol-12-myristate), and the presence of GM-CSF, G-CSF, interferon-alpha, or interferon-gamma does not interfere with the assay. Blood levels of TPA as measured by the bioassay immediately after an IV infusion of TPA (0.125 mg/m2; approximately 0.25 mg per patient) and 1 and 3 h later were 1.75 +/- 0.55, 0.93 +/- 0.54, and 0.69 +/- 0.42 ng/ml, respectively (mean +/- SD from eight infusions in five patients). Terminal half-lives were determined in a few patients where TPA blood levels were measured at multiple time intervals after the TPA infusion. In these patients, the terminal half-life was 11.1 +/- 3.9 h (from five infusions in four patients). To the best of our knowledge, this is the first analytical method for the measurement of TPA.

Protein kinase C pathway is involved in regulating the secretion of prostatic acid phosphatase in human prostate cancer cells.

The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4Alpha-phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5alpha-dihydrotestosterone (DHT) at the same concentration of 50 nM. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be involved in DHT action on PAcP secretion.

Effect of nitrite on endothelial function in isolated lung.

Nitrated tyrosine, implicated in protein dysfunction, is increased in various tissues in association with diverse pathological processes. Angiotensin converting enzyme (ACE) is a luminal vascular endothelial enzyme whose dysfunction is an early sign of endothelial injury. ACE contains a tyrosine critical for its enzymatic activity. Others have shown that nitrite exacerbates the ACE dysfunction of cultured endothelial cells in contact with activated polymorphonuclear neutrophils (PMN). We hypothesized that exogenous nitrite would enhance endothelial ACE dysfunction associated with PMN activation in the isolated lung. Rats received lipopolysaccharide (LPS) 2 h prior to isolated lung perfusion with Ficoll containing buffer. Either formyl-Met-Leu-Phe (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 10(-7) M) was used to activate PMN in lungs treated or not treated with 300-microM nitrite. A first pass indicator dilution method and first order reaction kinetics were used to determine ACE activity, while lung Ficoll content served as an index of vascular permeability. Both fMLP and PMA decreased endothelial ACE activity and increased pulmonary artery pressure, edema and vascular permeability. Exogenous nitrate did not potentiate the decrease in ACE activity, the lung injury or nitrotyrosine immunoreactivity of lung homogenates. In contrast to observations in cultured endothelial cells, our findings in the whole lung are compatible with the speculation of others that the rat lung has an unidentified factor, which minimizes accumulation of nitrated proteins.

Ability of okadaic acid and other protein phosphatase inhibitors to mimic the stimulatory effects of 12-O-tetradecanoylphorbol-13-acetate on hydroperoxide production in mouse epidermis in vivo.

The non-12-O-tetadecanoylphorbol-13-acetate (TPA)-type tumor promoters, okadaic acid (OA) and calyculin-A (CAL-A), which neither interact with the phorbol ester receptor nor directly activate protein kinase C, mimic the stimulatory effects of and thapsigargin on hydroperoxide (HPx) production in mouse epidermis in vivo. The time course and dose dependency for the stimulation of HPx production by O and TPA are similar. HPx production is maximally stimulated 16 h after two applications of 2 nmol of OA at a 48-h interval. However CAL-A is a stimulator of HPx production about 4 times more potent than OA or TPA. Combinations of TPA and OA or CAL-A have subadditive effects on HPx production. The discrepancies between the abilities of various serine/threonine protein phosphatase (PP) inhibitors to stimulate HPx production suggest that PP inhibition alone is not sufficient for this response. Cycloheximide, Ca2+ antagonists, oxypurinol, diphenyliodonium, nordihydroguaiaretic acid, bromophenacyl bromide, antiinflammatory agents, and antihistamines block or decrease OA-stimulated HPx production. Although most of these inhibitors may have more than one action, their effects suggest that protein synthesis, Ca2+, xanthine oxidase and NADPH oxidase activities, the lipoxygenase pathway of arachidonic acid metabolism, and vascular permeability may be involved in the inflammatory and HPx responses that occur after tumor promoter treatment. The increased HPx-producing activity of the epidermis, therefore, may be a common event resulting from the inflammatory and tumor-promoting actions of diverse TPA- and non-TPA-type agents.

New teleocidin-related metabolites, (-)-7-geranylindolactam V and blastmycetin F, from Streptoverticillium blastmyceticum.

Two new teleocidin-related metabolites, (-)-7-geranylindolactam-V [2] and blastmycetin F [3], were isolated from fermentation broths of the actinomycete Streptoverticillium blastmyceticum NA34-17, and their structures were determined by spectroscopic methods. Compound 2 bound strongly to phorbol ester receptors in a mouse epidermal particulate fraction, suggesting that it is a potent in vivo tumor promoter comparable to teleocidins A-1 [4] and B-4 [5].

Toxicokinetics of tumour promoters of mouse skin. II. Metabolism of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin and biological activities of metabolites.

The metabolism of the phorbol diester [20-3H]12-O-tetradecanoylphorbol-13-acetate [( 3H]TPA) was studied in the back skin of NMRI mice after topical administration of a single tumour-promoting dose, dp. Up to 72 h after administration most of the radioactivity recovered from the skin surface, and from the epidermis and dermis of the treated skin area was unchanged TPA, as determined by silica gel HPLC of extracts obtained from these skin fractions. The major TPA metabolite was less polar than TPA and chromatographed in the range of long-chain TPA-20-acylates. At 72 h, it accounted for about 25, 5 and 30% of total radio-activity extracted from skin surface, epidermis and dermis respectively. Of metabolites more polar than TPA, phorbol-13-acetate (PA) by far predominated over 12-O-tetradecanoylphorbol (TP) in both the epidermis and dermis, and by 72 h its relative amount was 3.9 and 2.4% in these skin fractions. Both phorbol monoesters, PA and TP, were not detected in skin surface extracts. In addition to metabolites, various autoxidation products of TPA were present in small amounts in the extracts from each of the skin fractions. The TPA-20-acylate fraction of metabolites isolated from the extracts of skin fractions at 24 h was separated further into the individual metabolites by the combined use of argentation and reversed-phase HPLC. These individual metabolites were identified by co-chromatography with authentic reference compounds. They were TPA-20-acylates carrying saturated fatty acids (16-26 carbon atoms), cis-mono-unsaturated fatty acids (16-24 carbon atoms), linoleic acid and arachidonic acid. The conjugation of TPA with long-chain fatty acids is the first example of a new route of xenobiotic metabolism in skin. When tested for irritant activity on the mouse ear, TPA-20-acylates were about one to two orders of magnitude less active than TPA. Similarly, when TPA-20-tetradecanoate was tested for tumour-promoting activity on the back skin of NMRI mice over a dose range of dp = 50-200 nmol applied twice weekly according to the computer-assisted standard protocol 16, it was found to be of only intermediate potency as compared to the highly potent tumour promoter TPA. The results of the present investigation indicate that metabolic conjugation of TPA with long-chain fatty acids to yield TPA-20-acylates is another pathway of metabolic deactivation of TPA, thus supporting the hypothesis that TPA itself is the ultimate tumour promoter in mouse skin.

Permeability of a non-TPA-type tumor promoter, okadaic acid, through lipid bilayer membrane.

The interaction of okadaic acid (OA) with lipid bilayer membranes was studied to obtain information on its incorporation into the target cell. OA, which possesses a polyether structure with a carboxylic acid, was extracted with a chloroform or n-octanol solution from a buffer solution, indicating the hydrophobicity of OA. However, the distribution of OA to dipalmitoylphosphatidylcholine membrane was so low that OA did not strongly induce perturbation in the membrane structure. On the other hand, OA permeated freely through the lipid membrane in a liquid-crystalline state. It was therefore suggested that OA permeates through cell membrane and binds to the receptor, for example, protein phosphatase, which exists either in the cytosol or in the cell membrane.

Induced differentiation of K562 leukemia cells: a model for studies of gene expression in early megakaryoblasts.

The K562 leukemia cell line has been extensively used in studies of erythroid differentiation but it has been less well appreciated that treatment of K562 cells with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) leads to loss of their erythroid properties and to acquisition of several megakaryoblastoid characteristics. These include synthesis and surface expression of glycoprotein IIIa, an increase in platelet peroxidase positivity, enhancement of thromboxane A2 receptors, and increased cell volume and DNA ploidy. TPA-treated K562 cells also synthesize and secrete platelet derived growth factor (PDGF), transforming growth factor beta 1 (TGF beta 1), urokinase-plasminogen activator (u-PA) and its specific inhibitor, type 1 plasminogen activator inhibitor (PAI-1). Induction of all these proteins, which have also been found in platelet granules (u-PA on platelet surface receptors) occurs at the level of mRNA accumulation. Therefore, in addition to facilitating studies and cloning of genes specific for erythroid differentiation, the K562 cells offer a tool to approach early steps of megakaryoblast commitment and differentiation. Observations made with the K562 cell line and several other leukemia cell lines co-expressing erythroid and megakaryocyte markers suggest that the erythroid and megakaryocyte lineages diverge from a common bipotent precursor cell.

Transmembrane signaling: tumor promoter distribution.

Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.