Down-regulation of Mcl-1 by inhibition of the PI3-K/Akt pathway is required for cell shrinkage-dependent cell death.

The anti-apoptotic effect of a chloride-bicarbonate exchange blocker has been previously examined in endothelial cells and cardiomyocytes. However, the anti-apoptotic effects of this blocker on epithelial cells and the mechanism of the anti-apoptotic effect remain unknown. We examined the anti-apoptotic effects of a chloride-bicarbonate exchange blocker in a renal epithelial cell line (MDCK cells). Changes in the expression of bcl-2 family proteins, which are known to have anti-apoptotic effects, were also examined. Staurosporine was used to induce apoptotic cell death in the MDCK cells. Staurosporine treatment was sufficient to induce apoptotic cell death, detected by propidium iodide and DNA ladder formation. A chloride-bicarbonate exchange blocker was added 24 h before the staurosporine treatment and during treatment. The chloride-bicarbonate exchange blocker inhibited the staurosporine-induced apoptosis in the MDCK cells in a dose-dependent manner. The expression of bcl-2 family gene products was detected by RT-PCR and Western blotting. No changes in the expression of Bax, Bid and Bik (pro-apoptotic proteins), or Bcl-2 (an anti-apoptotic protein) were detected. However, Mcl-1 expression was reduced by the staurosporine treatment, and this reduction was recovered when the chloride-bicarbonate exchange blocker was added. LY294002, a PI 3-kinase inhibitor, partially inhibited this anti-apoptotic effect. In conclusion, chloride-bicarbonate exchange blockers appear to offer cell-protective effects via Mcl-1 up-regulation.     

Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages

We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99–105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.     

Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway

Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.     

A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid

Background

Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable. In particular, Fourier transform infrared (FTIR) spectroscopy is a vibrational tool widely used for the study not only of isolated and purified biomolecules but also of complex biological systems, including intact cells and tissues. One of the main advantages of this non-invasive approach is that it allows obtaining a molecular fingerprint of the sample under investigation in a rapid and label-free way.

Methods

Here we present an FTIR characterization of DRM fractions purified from the human breast cancer cells MCF-7, before and after treatment with the omega 3 fatty acid docosahexaenoic acid (DHA), which was found to promote membrane microdomain reorganization.

Results and Conclusions

We will show that FTIR spectroscopy coupled with multivariate analysis enables to monitor changes in the composition of DRMs, induced in particular by the incorporation of DHA in cell membrane phospholipids.

General significance

This study paves the way for a new label-free characterization of specific membrane domains within intact cells, which could provide complementary information to the fluorescence approaches presently used.     

Inflammation and insulin resistance

Obesity-induced chronic inflammation is a key component in the pathogenesis of insulin resistance and the Metabolic syndrome. In this review, we focus on the interconnection between obesity, inflammation and insulin resistance. Pro-inflammatory cytokines can cause insulin resistance in adipose tissue, skeletal muscle and liver by inhibiting insulin signal transduction. The sources of cytokines in insulin resistant states are the insulin target tissue themselves, primarily fat and liver, but to a larger extent the activated tissue resident macrophages. While the initiating factors of this inflammatory response remain to be fully determined, chronic inflammation in these tissues could cause localized insulin resistance via autocrine/paracrine cytokine signaling and systemic insulin resistance via endocrine cytokine signaling all of which contribute to the abnormal metabolic state.     

Omega-3 fatty acids and metabolic syndrome: Effects and emerging mechanisms of action

Epidemiological, human, animal, and cell culture studies show that n−3 fatty acids, especially α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), reduce the risk factors of cardiovascular diseases. EPA and DHA, rather than ALA, have been the focus of research on the n−3 fatty acids, probably due to the relatively inefficient conversion of ALA to EPA and DHA in rodents and humans. This review will assess our current understanding of the effects and potential mechanisms of actions of individual n−3 fatty acids on multiple risk factors of metabolic syndrome. Evidence for pharmacological responses and the mechanism of action of each of the n−3 fatty acid trio will be discussed for the major risk factors of metabolic syndrome, especially adiposity, dyslipidemia, insulin resistance and diabetes, hypertension, oxidative stress, and inflammation. Metabolism of n−3 and n−6 fatty acids as well as the interactions of n−3 fatty acids with nutrients, gene expression, and disease states will be addressed to provide a rationale for the use of n−3 fatty acids to reduce the risk factors of metabolic syndrome.     

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling

Autophagy is essential for successful white adipocyte differentiation but the data regarding the timing and relevance of autophagy action during different phases of adipogenesis are limited.     

Involvement of nitric oxide in the promotion of cell survival by ceramide 1-phosphate

Macrophages play vital roles in inflammatory responses, and their number at sites of inflammation is strictly regulated by cell death and division. Here, we demonstrate that production of nitric oxide (NO) is a major mechanism whereby ceramide-1-phosphate (C1P) blocks apoptosis in macrophages. However, NO failed to stimulate macrophage proliferation. The prosurvival effect of C1P was blocked by inhibitors of inducible NO synthase. The antiapoptotic effect of C1P was also blocked by phosphatidylinositol 3-kinase or nuclear factor-kappa B inhibitors. Moreover, NO reversed the inhibitory effect of C1P on acid sphingomyelinase, but the prosurvival effect of C1P was independent of this action.     

Lipid changes in the aged brain: effect on synaptic function and neuronal survival

As the brain ages, cognitive and motor performance decline. This decline is thought to be largely due to the accumulation of damaging products from normal oxidative metabolism and to the perturbation of general body homeostasis and brain-circulation separation. Despite this abundance of insults, the aged brain contains few dead neurons, suggesting that aging must be paralleled by triggering or enhancing neuronal survival mechanisms. Recent evidence points to the contribution of changes in the lipid composition of membranes to both age-dependent cognitive decline and robust neuronal survival. In this review, we describe and discuss the current understanding of the roles of lipids in neuronal aging, with special attention to their influence on membrane fusion, neurotransmitter receptor dynamics and survival/death signaling pathways.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.     

A ménage à trois made in heaven: G-protein-coupled receptors, lipids and TRP channels

Recent technical and conceptual advances in lipid analysis have given us a glimpse into the true versatility of the lipidome and the complexity of lipid signaling species. Progress alike in protein chemistry and genetics has presented us with new signal pathways and molecular mechanisms for the lipid actions. G-protein-coupled receptors (GPCR) appear to play a central role in the regulation of many lipid signals and are also themselves targets for some of these. TRP channels have recently been acknowledged as one of the most important GPCR effectors; in many cases the signals from GPCRs to TRPs are mediated via lipid signals. This review aims at presenting a view into the complex lipid signaling networks, their possible regulation by GPCRs and the signals transmitted to the TRP channels. Critical views and possible shortcomings in the composition of the studies are also presented.     

Phospholipids: “Greasing the wheels” of humoral immunity

Phospholipids are major structural components of all cellular membranes. In addition, certain phospholipids execute regulatory activities that affect cell behavior, function and fate in critically important physiological settings. The influence of phospholipids is especially obvious in the adaptive immune system, where these macromolecules mediate both intrinsic and extrinsic effects on B and T lymphocytes. This review article highlights the action of lysophospholipid sphingosine-1-phosphate as a lymphocyte chemoattractant, the function of phosphatidylinositol phosphates as signaling conduits in lymphocytes and the role of phospholipids as raw materials for membrane assembly and organelle biogenesis in activated B lymphocytes. Special emphasis is placed on the means by which these three processes push humoral immune responses forward. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Old and new generation lipid mediators in acute inflammation and resolution

Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.