Rola Barhoumi

Dietary docosahexaenoic acid suppresses T cell protein kinase Cθ lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)θ, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCθ with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCθ effector pathway signaling, both AP-1 and NF-κB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCθ signaling axis.

A Ratiometric pH Reporter For Imaging Protein-dye Conjugates In Living Cells

A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emitted green fluorescence (ca. 520 nm) when excited at the donor (488 nm). Below pH 6.0 however, energy transfer is efficient; hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that was imported into endosomes or in the cytosol using the noncovalently bound carrier, Pep-1, at 37 and 4 degrees C, respectively. The more acidic environment of the endosomes was conspicuous from the red fluorescence of the probe.

n-3 Polyunsaturated Fatty Acids Suppress the Localization and Activation of Signaling Proteins at the Immunological Synapse in Murine CD4+ T Cells by Affecting Lipid Raft Formation

The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4+ T cells from wild-type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of protein kinase Cθ, phospholipase Cγ-1, and F-actin into the IS was suppressed. In addition, both the phosphorylation status of phospholipase Cγ-1 at the IS and cell proliferation as assessed by CFSE labeling and [3H]thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 μm monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.

Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function

There is experimental evidence that dietary fish oil, which contains the n−3 fatty acid family, i.e., EPA and DHA, protects against colon tumor development, in part by increasing apoptosis. Since mitochondria can act as central executioners of apoptosis, we hypothesized that EPA and DHA incorporation into colonocyte mitochondrial membranes, owing to their high degree of unsaturation, would enhance susceptibility to damage by reactive oxygen species (ROS) generated via oxidative phosphorylation. This, in turn, would compromise mitochondrial function, thereby initiating apoptosis. To test this hypothesis, colonic crypts were isolated from rats fed either fish oil, purified n−3 fatty acid ethyl esters, or corn oil (control). Dietary lipid source had no effect on colonic mitochondrial phospholipid class mole percentages, although incorporation of EPA and DHA was associated with a reduction in n−6 fatty acids known to enhance colon tumor development, i.e., linoleic acid (LNA) and its metabolic product, arachidonic acid (ARA). Select compositional changes in major phospholipid pools were correlated to alterations in mitochondrial function as assessed by confocal microscopy. The mol% sum of LNA plus ARA in cardiolipin was inversely correlated with ROS (P=0.024). Ethanolamine glycerophospholipid ARA (P=0.046) and choline glycerophospholipid INA (P=0.033) levels were positively correlated to mitochondrial membrane potential. In contrast, ethanolamine glycerophospholipid EPA (P=0.042) and DHA (P=0.024), levels were negatively correlated to mitochondrial membrane potential. Additionally, EPA and DHA levels in choline glycerophospholipids (P=0.026) were positively correlated with caspase 3 activity. These data provide evidence in vivo indicating that dietary FPA and DHA induce compositional changes in colonic mitochondrial membrane phospholipids that facilitate appotosis.

Arsenic trioxide downregulates specificity protein (Sp) transcription factors and inhibits bladder cancer cell and tumor growth

Arsenic trioxide exhibits antiproliferative, antiangiogenic and proapoptotic activity in cancer cells, and many genes associated with these responses are regulated by specificity protein (Sp) transcription factors. Treatment of cancer cells derived from urologic (bladder and prostate) and gastrointestinal (pancreas and colon) tumors with arsenic trioxide demonstrated that these cells exhibited differential responsiveness to the antiproliferative effects of this agent and this paralleled their differential repression of Sp1, Sp3 and Sp4 proteins in the same cell lines. Using arsenic trioxide-responsive KU7 and non-responsive 253JB-V bladder cancer cells as models, we show that in KU7 cells, < or =5 microM arsenic trioxide decreased Sp1, Sp3 and Sp4 and several Sp-dependent genes and responses including cyclin D1, epidermal growth factor receptor, bcl-2, survivin and vascular endothelial growth factor, whereas at concentrations up to 15 microM, minimal effects were observed in 253JB-V cells. Arsenic trioxide also inhibited tumor growth in athymic mice bearing KU7 cells as xenografts, and expression of Sp1, Sp3 and Sp4 was significantly decreased. Inhibitors of oxidative stress such as glutathione or dithiothreitol protected KU7 cells from arsenic trioxide-induced antiproliferative activity and Sp repression, whereas glutathione depletion sensitized 253JB-V cells to arsenic trioxide. Mechanistic studies suggested that arsenic trioxide-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide.

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds.

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.

Docosahexaenoic acid selectively inhibits plasma membrane targeting of lipidated proteins

Membrane localization of lipidated cytosolic signaling proteins is mediated by interactions between specific lipid anchors and membranes, but little is known about the regulatory role of membrane composition in lipidated protein membrane targeting. Here, using green fluorescent protein (GFP) chimeras and quantitative fluorescence microscopy in living mouse colonocytes, we show that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid (PUFA) with membrane lipid‐modifying properties, selectively inhibits plasma membrane (PM) targeting and increases the endomembrane localization of lipidated proteins that are cytoplasmic cargo in the exocytic pathway, without affecting the exocytic pathway itself. DHA selectivity seems to be dictated by the protein trafficking route, independent of the functional state of proteins and the location and composition of membrane anchors. DHA enrichment in cell membranes was required to elicit the inhibitory effect. These data reveal that membrane lipid composition influences cell signaling by modulating intracellular trafficking and localization of membrane proteins, providing a potential molecular mechanism for the documented health benefits of DHA.

Mares with Delayed Uterine Clearance Have an Intrinsic Defect in Myometrial Function

Abstract Persistent, postmating endometritis affects approximately 15% of mares and results in reduced fertility and sizable economic losses to the horse-breeding industry. Mares that are susceptible to postmating endometritis have delayed uterine clearance associated with reduced uterine contractility. Unfortunately, the mechanism for reduced uterine contractility remains an enigma. The present study examined the hypothesis that mares with delayed uterine clearance have an intrinsic contractile defect of the myometrium. Myometrial contractility was evaluated in vitro by measuring isometric tension generated by longitudinal and circular uterine muscle strips in response to KCl, oxytocin, and prostaglandin F2α (PGF2α) for young nulliparous mares, older reproductively normal mares, and older mares with delayed uterine clearance. In addition, intracellular Ca2+ regulation was evaluated using laser cytometry to measure oxytocin-stimulated intracellular Ca2+ transients of myometrial cells loaded with a Ca2+-sensitive fluorescent dye, fluo-4. For all contractile agonists, myometrium from mares with delayed uterine clearance failed to generate as much tension as myometrium from older normal mares. Oxytocin-stimulated intracellular Ca2+ transients were similar for myometrial cells from mares with delayed uterine clearance and from older normal mares, suggesting that the contractile defect did not result from altered regulation of intracellular Ca2+ concentration. Furthermore, no apparent age-dependent decline was observed in myometrial contractility; KCl-depolarized and oxytocin-stimulated longitudinal myometrium from young normal mares and older normal mares generated similar responses. However, circular myometrium from young normal mares failed to generate as much tension as myometrium from older normal mares when stimulated with oxytocin or PGF2α, suggesting possible age-related alterations in receptor-second messenger signaling mechanisms downstream of intracellular Ca2+ release. In summary, for mares with delayed uterine clearance, an intrinsic contractile defect of the myometrium may contribute to reduced uterine contractility following breeding.

n-3 Polyunsaturated Fatty Acids Suppress Mitochondrial Translocation to the Immunologic Synapse and Modulate Calcium Signaling in T Cells

Recent studies indicate that the process of Ag presentation induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the immunologic synapse (IS). This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2+ influx and Th cell activation. Recently, we demonstrated that n-3 polyunsaturated fatty acids (PUFAs) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFAs suppress CD4+ T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca2+-dependent Th cell activation. CD4+ cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were cocultured with anti-CD3–expressing hybridoma cells (145-2C11) for 15 min at 37°C, and mitochondrial translocation to the IS was assessed by confocal microscopy. Fat-1 mice exhibited a significantly (p < 0.05) reduced percentage of T cells with mitochondria which translocated to the IS; fat-1 (30%) versus wild type control (82%). Regarding the effect on the mitochondrial-to-cytosolic Ca2+ ratio, wild type cells showed significant increases at the IS (71%) and total cell (60%) within 30 min of IS formation. In contrast, fat-1 CD4+ T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2+ ratio was observed in wild type cells that were coincubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2+ (CRAC) channels, BTP2. These observations provide evidence that n-3 PUFAs modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2+entry.