Sergio D. Catz

Transcriptional regulation of bcl-2 by nuclear factor kappa B and its significance in prostate cancer.

This work presents direct evidence that the bcl-2 gene is transcriptionally regulated by nuclear factor-κB (NF-κB) and directly links the TNF-α/NF-κB signaling pathway with Bcl-2 expression and its pro-survival response in human prostate carcinoma cells. DNase I footprinting, gel retardation and supershift analysis identified a NF-κB site in the bcl-2 p2 promoter. In the context of a minimal promoter, this bcl-2 p2 site 1 increased transcription 10-fold in the presence of the p50/p65 expression vectors, comparable to the increment observed with the consensus NF-κB site, while for the full p2 promoter region transcriptional activity was increased sixfold by over-expression of NF-κB, an effect eliminated by mutating the bcl-2 p2 site 1. The expression of Bcl-2 has been linked to the hormone-resistant phenotype of advanced prostate cancer. Here we show that an increase in the level of expression of Bcl-2 in the human prostate carcinoma cell line LNCaP observed in response to hormone withdrawal is further augmented by TNF-α treatment, and this effect is abated by inhibitors of NF-κB. Concomitantly, bcl-2 p2 promoter studies in LNCaP cells show a 40-fold increase in promoter activity after stimulation with TNF-α in the absence of hormone.

Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils

Nitric oxide (.NO) release, oxygen uptake and hydrogen peroxide (H2O2) production elicited by increasing phorbol 12‐myristate 13‐acetate (PMA) concentrations were measured in human neutrophils. Half‐maximal activities were sequentially elicited at about 0.0001–0.001 μg (.NO) and 0.001‐0.01 μg (H2O2). At saturated PMA concentrations, .NO production, oxygen uptake and H2O2 release were 0.56 ± 0.04, 3.32 ± 0.52 and 1.19±0.17 nmol · min−1 · 106 cells−1. .NO production accounts for about 30% of the total oxygen uptake. Luminol‐dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half‐maximally activated at about 0.001‐0.01 μg . Preincubation with N G‐monomethyl‐l‐arginine (l‐NMMA) decreased O2 uptake and .NO release but increased H2O2 production, while superoxide dismutase (SOD) increased .NO detection by 30%. Chemiluminescence was also reduced by preincubation with l‐NMMA and/or SOD. The results indicate that .NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of ″NO and O2 .− release favour the formation of other oxidants like peroxynitrite.

Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47PHOX occurs during the activation of the enzyme in intact cells. p47PHOX carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47PHOX is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47PHOX whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47PHOX by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47PHOX. These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase.

BCL-2 in prostate cancer: A minireview

Prostate cancer progression and the development of androgen-independent prostate cancer have been largely related to a number of genetic abnormality that affect not only the androgen receptor but also crucial molecules involved in the regulation of survival or apoptotic pathways. One of these molecules, the pro-survival protein BCL-2, has been associated with the development of androgen-independent prostate cancer due to its high levels of expression in androgen-independent tumors in advanced stages of the pathology. The upregulation of BCL-2 after androgen ablation in prostate carcinoma cell lines and in a castrated-male rat model further established a connection between BCL-2 expression and prostate cancer progression. This review focuses on the experimental evidence that associates BCL-2 expression with prostate carcinogenesis and cancer progression, and analyzes the evidence that links the phosphatidylinositol 3-kinase (PI 3-kinase)/nuclear factor kappa B (NF-κB) survival pathway with the upregulation of BCL-2. The way in which hormone ablation influences this survival pathway and the potential application of novel therapeutic strategies to overcome this anti-apoptotic mechanism is examined.

The Rab27a Effectors JFC1/Slp1 and Munc13‐4 Regulate Exocytosis of Neutrophil Granules

Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene‐targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13‐4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1‐downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13‐4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13‐4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13‐4‐deficient mouse model (Jinx), we demonstrate that Munc13‐4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13‐4‐deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.

Rab27a and Rab27b Regulate Neutrophil Azurophilic Granule Exocytosis and NADPH oxidase Activity by Independent Mechanisms

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27aash/ash), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27aash/ash neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a‐deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27aash/ash and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27‐deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27‐deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1-Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein.

The mechanism of cytoskeleton remodeling during exocytosis is not well defined. A combination of vesicular dynamics and functional studies shows that the Rab27a effector JFC1 and the RhoA-GTPase–activating protein Gem-interacting protein are necessary for RhoA regulation, actin depolymerization, and vesicular transport through the actin cortex during exocytosis.

Cross-talk between IRAK-4 and the NADPH oxidase

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GAP GMIP

The mechanism of cytoskeleton remodeling during exocytosis is not well defined. A combination of vesicular dynamics and functional studies shows that the Rab27a effector JFC1 and the RhoA-GTPase–activating protein Gem-interacting protein are necessary for RhoA regulation, actin depolymerization, and vesicular transport through the actin cortex during exocytosis.