Barbara Gajkowska

A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-γ at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in α-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular α-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

IGF-I, EGF, and sex steroids regulate autophagy in bovine mammary epithelial cells via the mTOR pathway

Mammary gland growth and involution are based on a dynamic equilibrium between proliferation and apoptosis of mammary epithelial cells (MEC). The main type of cell death responsible for bovine mammary gland involution is apoptosis, but MEC also exhibit morphological features of autophagy. The present study has been undertaken in order to examine factors, which are responsible for the regulation of autophagy in bovine MEC. We used a model of in vitro mammary gland involution known to be dependent on fetal bovine serum (FBS) deficiency in the culture of bovine BME-UV1 cells. We investigated the effects of insulin-like growth factor-1 (IGF-I) and epidermal growth factor (EGF) signaling, as well as sex steroids and rapamycin (a specific inhibitor of mammalian target of rapamycin, mTOR, kinase) on autophagy in the MEC line BME-UV1. Our main focus was on the role of mTOR in the regulation of autophagy by growth factors and hormones. Laser scanning cytometry, electron microscopy, Western-blot analysis, GFP-LC3 reporter-based expression analysis, and LysoTracker Green-related fluorescence were used to determine the activity of autophagy in BME-UV1 cells. We found that FBS deficiency induced both autophagy and apoptosis with the highest intensity of both processes after 48h of MEC exposure to the deficient medium (0.5% FBS). Addition of IGF-I or/and EGF to the FBS-deficient medium clearly diminished autophagy. We also show that IGF-I and EGF are involved in the activation of mTOR in bovine MEC, whereas inhibition of mTOR by rapamycin abrogated the suppressive effects of IGF-I and EGF on autophagy. This suggests that mTOR links IGF-I and EGF signaling in inhibiting the autophagy pathways. Contrary to IGF-I and EGF, 17beta-estradiol and progesterone exerted stimulatory effects on autophagy in bovine MEC. At the same time we observed a suppressive effect of both steroids on mTOR activation/phosphorylation. In conclusion, autophagy in bovine MEC undergoes complex regulation, where its activity is controlled by survival pathways dependent on IGF-I and EGF, which are involved in suppression of autophagy, and by pregnancy steroids, which act as inducers of the process.

Molecular basis of parthenolide-dependent proapoptotic activity in cancer cells

This review outlines the molecular events that accompany the anti-tumor action of parthenolide (PN). Parthenolide (PN) is naturally derived compound, isolated from plant Tanacetum parthenium. PN has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate that this sesquiterpene lactone might evoke the above-mentioned effects by indirect action on genes. PN was shown to inhibit NF-kappaB- and STATs-mediated antiapoptotic gene transcription. On one hand, the proapoptotic activity of PN includes stimulation of intrinsic apoptotic pathway with the higher level of intracellular ROS and modifications of Bcl-2 family proteins (conformational changes of Bak and Bax, Bid cleavage). On the other hand, PN amplifies the apoptotic signal through the sensitization of cancer cells to extrinsic apoptosis, induced by TNF-alpha. These effects are specific to tumor cells. Unique properties of PN make this agent a promising metabolic inhibitor to retard tumorigenesis and to suppress tumor growth.

Disruption of lipid rafts causes apoptotic cell death in HaCaT keratinocytes.

Abstract:  Lipid rafts are cholesterol‐enriched microdomains in plasma membranes. The functional activity of many membrane proteins, including death and growth factor receptors, depends on their insertion in lipid rafts. We have previously demonstrated the presence of lipid rafts in keratinocytes and shown that lipid rafts are involved in the control of keratinocyte proliferation and metabolic activity. In this work, we investigated the effect of lipid‐raft disruption on HaCaT keratinocyte survival. Lipid rafts could be disrupted or rearranged with cholesterol‐targeting detergents: methyl‐β‐cyclodextrin and filipin III. Moreover, cholesterol oxidation by a specific oxidase or blocking of cholesterol synthesis by mevastatin had a similar effect on lipid rafts. All cholesterol‐modifying substances caused cell death in a concentration‐dependent manner. More detailed studies on the effects of cyclodextrin revealed apoptotic cell death at concentrations ≥0.5% (w/v). The molecular mechanism of apoptosis precipitated by raft disruption remains unknown but does not seem to be dependent of either membrane permeabilization or cell‐cycle arrest imposed by cholesterol‐modifying compounds.

Role of nitric oxide in the brain during lipopolysaccharide-evoked systemic inflammation.

Although the inducible isoform of nitric oxide synthase (iNOS) is a well‐established source of nitric oxide (NO•) during inflammation of the central nervous system (CNS), little is known about the involvement of constitutive isoforms of NOS (cNOS) in the inflammatory process. The aim of this study was to compare the responses of the expression and activity of iNOS and the two cNOS isoforms, neuronal and endothelial (nNOS and eNOS, respectively), in the brain to systemic inflammation and their roles in the cascade of events leading to degeneration and apoptosis. A systemic inflammatory response in C57BL/6 mice was induced by intraperitoneal injection of lipopolysaccharide [LPS; 1 mg/kg body weight (b.w.)]. The relative roles of the NOS isoforms were evaluated after injection of NG‐nitro‐L‐arginine (NNLA; 30 mg/kg b.w.), which preferentially inhibits cNOS, or 1400W (5 mg/kg b.w.), an inhibitor of iNOS. Biochemical and morphological alterations were analyzed up to 48 hr after administration of LPS. Systemic LPS administration evoked significant ultrastructural alterations in brain capillary vessels, neuropils, and intracellular organelles of neurons, astrocytes, and microglia. Apoptotic/autophagic processes occurred in many neurons of the substantia nigra (SN), which coincided with exclusive enhancement of iNOS expression and activity in this brain region. Moreover, inhibitors of both iNOS and cNOS prevented LPS‐evoked release of apoptosis‐inducing factor (AIF) from SN mitochondria. Collectively, the results indicate that synthesis of NO• by both the inducible and constitutive NOS isoforms contribute to the activation of apoptotic pathways in the brain during systemic inflammation.

Tellurium-induced cognitive deficits in rats are related to neuropathological changes in the central nervous system

The effects of sodium tellurite 0.1 and 0.4 mg/kg were assessed in the Morris water maze. Two days after treatment rats were tested for acquisition (posttreatment days 3-6) and on seventh day on a spatial retention task. Tellurium treatment was found to cause significant impairment in retention of the spatial learning task. Locomotor disturbances were not the cause of the observed effects. Ultrastructural observations showed neuropathological changes in hippocampus subfields and prefrontal cortex with swelling of synapses, astrocytes and astrocytic processes around the vessels in the cerebral cortex neuropil. Severity of the observed changes in glial-neuronal unit was in correlation with the extent of learning impairment. A direct injury of Schwann cells with the secondary myoclasis was noted in the sciatic nerve. Our results indicate that acute treatment with sodium tellurite results in impairment of learning and spatial memory.

Subcellular redistribution of BAX during apoptosis induced by anticancer drugs.

BAX is the 192-amino acid, 21-kDa protein which is ubiquitously distributed in normal tissues and is regarded as a tumor suppressor sensitizing malignant cells to anticancer drugs. In spite of many studies, the molecular mechanism of BAX action is still obscure. In the present study subcellular BAX translocations in human colon adenocarcinoma COLO 205 cells exposed to various anticancer drugs [camptothecin (CPT), etoposide (ETO), staurosporine (STP), 2-chloro-2′-deoxyadenosine (2CdA) and nimesulide (NIM)] was examined. Cells were grown on coverslips under optimal conditions (10% FCS/DMEM) or were stimulated to apoptosis with the drugs examined. Laser scanning cytometry was applied for the quantitative analysis of BAX expression, and distribution in the cytoplasmic (BAX Cf) and nuclear (BAX Nf) area. BAX maximal pixel (BAX MP), the parameter corresponding to aggregation of BAX in the cell, was also measured. All examined drugs increased the number of cells with high BAX MP, reaching the peak at 60 min after drug administration. The most pronounced effect was in the case of 2CdA, CPT and STP. The increase in BAX MP was observed only when antibody recognizing the 43-61 amino acid sequence was used. When antibody binding the N-terminal epitope (11-30 amino acid sequence) was applied, the number of cells expressing high BAX MP significantly decreased. These results indicate that apoptotic stimuli delivered by anticancer drugs led to aggregation of BAX in cancer cells, which is dependent on BAX activation by its cleavage at the N-terminal epitope and exposure of the BH3 domain. It was shown that BAX Nf increased in cells treated with CPT, STP, ETO, 2CdA and NIM, whereas BAX Cf rose after STP and NIM. The increase in BAX Nf and, occurring in most treatments, the increase in the BAX Nf:Cf ratio indicates a BAX shift from the cytoplasm to the nucleus. Furthermore, staining with different antibodies showed that only the activated form of BAX was translocated to the nucleus. Immunoelectron microscopy revealed that CPT-induced apoptosis was associated with translocation of BAX from the cytosol to organellar membranes (mitochondrial, Golgi apparatus and endoplasmic reticulum) and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. The subcellular translocations of BAX preceded in time the appearance of morphological symptoms of apoptosis. In conclusion, (i) in spite of different molecular mechanisms of apoptosis induction by the anticancer drugs examined, BAX remains a common link in the chain of reactions leading to cell death, and (ii) BAX activation and subcellular translocations from the cytosol to organellar membranes and nucleus are key cellular responses to drugs bearing proapoptotic properties.

Systemic administration of lipopolysaccharide induces molecular and morphological alterations in the hippocampus.

A systemic inflammatory reaction may have detrimental effects on the organism, including the central nervous system. Previous studies have indicated that lipopolysaccharide (LPS)-evoked systemic inflammation induces pathological alterations in the mouse midbrain, especially in the substantia nigra. The aim of the present study was to investigate whether the hippocampus is also affected after an intraperitoneal (i.p.) injection of LPS. We focussed on the dynamics of proinflammatory gene expression and the processes leading to neuronal cell death. A systemic inflammatory response in C57BL/6 mice was induced by an i.p. injection of LPS (1mg/kg b.w.). The genetic, biochemical and morphological alterations were analysed up to 96h after LPS administration using quantitative PCR, immunochemical, immunocytochemical and electron microscopic methods. Real-time PCR analysis indicated an altered expression of several genes, mainly responsible for arachidonic acid release and metabolism, in the hippocampus 96h after the systemic administration of LPS. Three hours after LPS treatment, the level of mRNA for iNOS, COX-2 and TNFα was increased; then, after 6-24h, it rose for TLR4 and cPLA2. The expression of 5-LOX and 12-LOX was increased at 12-24 and 24-48h after LPS injection, respectively. Our data demonstrate for the first time the sequential activation of the expression of several pro-inflammatory genes responsible for the maintenance of the inflammatory response. Moreover, the electron microscopy studies presented the stimulation of apoptosis-inducing factor (AIF)-mediated death signalling and cathepsin B-related autophagy or necrosis. These biochemical and morphological alterations in the hippocampus, which were induced by systemic inflammation, may be responsible for the impairment of cognition function observed previously.

Mitofusin 2 (Mfn2): a key player in insulin-dependent myogenesis in vitro

We have previously shown that mitochondrial activity increases in response to insulin in differentiating muscle cells. Moreover, the protein kinase kinase/extracellular-signal-regulated kinase (MAPKK/ERK-MEK) inhibitor PD98059 accelerates insulin-mediated myogenesis, whereas the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or blockade of mitochondrial respiration abrogates insulin-mediated myogenesis. Our present study focuses on the mitochondrial transmembrane protein, hyperplasia suppressor gene/mitofusin2 (HSG/Mfn2), which regulates both mitochondrial fusion (as demonstrated by perinuclear mitochondria clustering) and insulin-dependent myogenesis in vitro. Increased mitochondrial length and interconnectivity are not observed after the inhibition of PI3-K activity with LY294002. Insulin induces Mfn2 and subunits I and IV of cytochrome-c oxidase (MTCOI and NCOIV) in L6 myoblasts. Inhibition of the MEK-dependent signalling pathway elevates the Mfn-2 protein level. The molecular mechanism of this phenomenon is unknown, although immunoprecipitation studies indicate that, during insulin-mediated myogenesis, Ras protein (an upstream activator of the MAPK/ERK1/2 cascade) interacts with HSG/Mfn2 in muscle cells. Interaction of Ras with Mfn2 continues unless insulin is present and is reduced after PD98059 co-treatment indicating that insulin-mediated myogenesis is increased by the inhibition of MEK, most probably by the lack of mitogenic signals opposing muscle differentiation. We conclude that insulin-mediated myogenesis depends on PI3-K activity, which stimulates mitochondrial activity and the extensive fusion of mitochondria. We further suggest that insulin stimulates the expression of Mfn2 protein, which in turn binds to Ras and inhibits the MEK-dependent signalling pathway. At the same time, the PI3-K-dependent signalling pathway is boosted, mitochondrial respiration increases and the rate of myogenesis is accelerated.

Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis

Abstract.  Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose‐dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl‐inositol‐3‐kinase (PI‐3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin‐stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI‐3K‐ and MEK‐dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI‐3K rather than MEK supports insulin‐mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473‐phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI‐3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome‐c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin‐dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.