Marcella Arcangeletti

Melatonin signaling and cell protection function

Besides its well‐known regulatory role on circadian rhythm, the pineal gland hormone melatonin has other biological functions and a distinct metabolism in various cell types and peripheral tissues. In different tissues and organs, melatonin has been described to act as a paracrine and also as an intracrine and autocrine agent with overall homeostatic functions and pleiotropic effects that include cell protection and prosurvival factor. These latter effects, documented in a number of in vitro and in vivo studies, are sustained through both receptor‐dependent and ‐independent mechanisms that control detoxification and stress response genes, thus conferring protection against a number of xenobiotics and endobiotics produced by acute and chronic noxious stimuli. Redox‐sensitive components are included in the cell protection signaling of melatonin and in the resulting transcriptional response that involves the control of NF‐κB, AP‐1, and Nrf2. By these pathways, melatonin stimulates the expression of antioxidant and detoxification genes, acting in turn as a glutathione system enhancer. A further and converging mechanism of cell protection by this indoleamine described in different models seems to lie in the control of damage and signaling function of mitochondria that involves decreased production of reactive oxygen species and activation of the antiapoptotic and redox‐sensitive element Bcl2. Recent evidence suggests that upstream components in this mitochondrial route include the calmodulin pathway with its central role in melatonin signaling and the survival‐promoting component of MAPKs, ERK1/2. In this review article, we will discuss these and other molecular aspects of melatonin signaling relevant to cell protection and survival mechanisms.—Luchetti, F., Canonico, B., Betti, M., Arcangeletti, M., Pilolli, F., Piroddi, M., Canesi, L., Papa, S., Galli, F. Melatonin signaling and cell protection function. FASEB J. 24, 3603–3624 (2010). www.fasebj.org

ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells

The pineal gland hormone melatonin has been recently described to downregulate the intrinsic (or damage-induced) pathway of apoptosis in human leukocytes. These properties appear to depend on a specific mitochondrial signaling of melatonin which is associated with a lower generation of reactive oxygen species and a better control of redox-sensitive components such as the antiapoptotic protein Bcl-2. Other elements upstream in this signaling are expected to contribute regulatory roles that remain unexplored. The aim of this study was to investigate whether the extracellular signal-regulated kinase (ERK), which controls the balance between survival and death-promoting genes throughout the MAPK pathway, is involved in the antiapoptotic signaling of melatonin. Human monocytic U937 cells irradiated with UVB light were used as a model of stress-induced apoptosis. In this model we found that pharmacological concentrations of melatonin (1 mM) were able to decrease superoxide anion production, mitochondrial damage, and caspase-dependent apoptosis by improved Bcl-2 levels and decreased Cyt c release in the cytoplasm. Moreover, melatonin increased the phosphorylative activation of ERK 1/2 independently from the presence of UVB stress, and decreased the UVB-mediated activation of the stress kinases p38 MAPK and JNK. The ERK 1/2 inhibitor PD98059, but not the p38 MAPK inhibitor SB203580, abolished to different extents the effects that melatonin had on the UVB-induced ROS generation, mitochondrial dysfunction, and apoptosis. Using these inhibitors, a cross-talk effect between stress and survival-promoting kinases was tentatively identified, and confirmed the hierarchical role of ERK MAPK phosphorylation in the signaling of melatonin. In conclusion, melatonin sustains the activation of the survival-promoting pathway ERK MAPK which is required to antagonize UVB-induced apoptosis of U937 cells. This kinase mediates also the antioxidant and mitochondrial protection effects of this hormonal substance that may find therapeutic applications in inflammatory and immune diseases associated with leukocyte oxidative stress and accelerated apoptosis.

Melatonin regulates mesenchymal stem cell differentiation: a review

Among the numerous functions of melatonin, the control of survival and differentiation of mesenchymal stem cells (MSCs) has been recently proposed. MSCs are a heterogeneous population of multipotent elements resident in tissues such as bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, gaining thus increasing interest for tissue repair and restoration therapeutic protocols. Receptor‐dependent and receptor‐independent responses to melatonin are suggested to occur in these cells. These involve antioxidant or redox‐dependent functions of this indolamine as well as secondary effects resulting from autocrine and paracrine responses. Inflammatory cytokines and adipokines, proangiogenic/mitogenic stimuli, and other mediators that influence the differentiation processes may affect the survival and functional integrity of these mesenchymal precursor cells. In this scenario, melatonin seems to regulate signaling pathways that drive commitment and differentiation of MSC into osteogenic, chondrogenic, adipogenic, or myogenic lineages. Common pathways suggested to be involved as master regulators of these processes are the Wnt/β‐catenin pathway, the MAPKs and the, TGF‐β signaling. In this respect melatonin emerges a novel and potential modulator of MSC lineage commitment and adipogenic differentiation. These and other aspects of the physiological and pharmacological effects of melatonin as regulator of MSC are discussed in this review.

New Strategies to Prolong the In Vivo Life Span of Iron-Based Contrast Agents for MRI

Superparamagnetic iron oxide (SPIO) and ultra small superparamagnetic iron oxide (USPIO) nanoparticles have been developed as magnetic resonance imaging (MRI) contrast agents. Iron oxide nanoparticles, that become superparamagnetic if the core particle diameter is ~ 30nm or less, present R1 and R2 relaxivities which are much higher than those of conventional paramagnetic gadolinium chelates. Generally, these magnetic particles are coated with biocompatible polymers that prevent the agglomeration of the colloidal suspension and improve their blood distribution profile. In spite of their potential as MRI blood contrast agents, the biomedical application of iron oxide nanoparticles is still limited because of their intravascular half-life of only few hours; such nanoparticles are rapidly cleared from the bloodstream by macrophages of the reticulo-endothelial system (RES). To increase the life span of these MRI contrast agents in the bloodstream we proposed the encapsulation of SPIO nanoparticles in red blood cells (RBCs) through the transient opening of cell membrane pores. We have recently reported results obtained by applying our loading procedure to several SPIO nanoparticles with different chemical physical characteristics such as size and coating agent. In the current investigation we showed that the life span of iron-based contrast agents in the mice bloodstream was prolonged to 12 days after the intravenous injection of murine SPIO-loaded RBCs. Furthermore, we developed an animal model that implicates the pretreatment of animals with clodronate to induce a transient suppression of tissue macrophages, followed by the injection of human SPIO-loaded RBCs which make it possible to encapsulate nanoparticle concentrations (5.3-16.7mM Fe) higher than murine SPIO-loaded RBCs (1.4-3.55mM Fe). The data showed that, when human RBCs are used as more capable SPIO nanoparticle containers combined with a depletion of tissue macrophages, Fe concentration in animal blood is 2-3 times higher than iron concentration obtained by the use of murine SPIO-loaded RBCs.

Fas Signalling Promotes Intercellular Communication in T Cells

Cell-to-cell communication is a fundamental process for development and maintenance of multicellular organisms. Diverse mechanisms for the exchange of molecular information between cells have been documented, such as the exchange of membrane fragments (trogocytosis), formation of tunneling nanotubes (TNTs) and release of microvesicles (MVs). In this study we assign to Fas signalling a pivotal role for intercellular communication in CD4+ T cells. Binding of membrane-bound FasL to Fas expressing target cells triggers a well-characterized pro-apoptotic signalling cascade. However, our results, pairing up flow cytometric studies with confocal microscopy data, highlight a new social dimension for Fas/FasL interactions between CD4+ T cells. Indeed, FasL enhances the formation of cell conjugates (8 fold of increase) in an early time-frame of stimulation (30 min), and this phenomenon appears to be a crucial step to prime intercellular communication. Our findings show that this communication mainly proceeds along a cytosolic material exchange (ratio of exchange >10, calculated as ratio of stimulated cells signal divided by that recorded in control cells) via TNTs and MVs release. In particular, inhibition of TNTs genesis by pharmacological agents (Latruculin A and Nocodazole) markedly reduced this exchange (inhibition percentage: >40% and >50% respectively), suggesting a key role for TNTs in CD4+ T cells communication. Although MVs are present in supernatants from PHA-activated T cells, Fas treatment also leads to a significant increase in the amount of released MVs. In fact, the co-culture performed between MVs and untreated cells highlights a higher presence of MVs in the medium (1.4 fold of increase) and a significant MVs uptake (6 fold of increase) by untreated T lymphocytes. We conclude that Fas signalling induces intercellular communication in CD4+ T cells by different mechanisms that seem to start concomitantly with the main pathway (programmed cell death) promoted by FasL.

Pharmacological doses of melatonin induce alterations in mitochondrial mass and potential, bcl‐2 levels and K+ currents in UVB‐exposed U937 cells

Apoptosis is observed in ‘actively’ dying cells after the exposure to cell stressors such as ultraviolet light irradiation. Since melatonin has been proposed to act under stressful conditions as cell protection factor, in this study we examined the potential of this molecule when used at pharmacological concentrations to control mitochondrial damage and apoptotic signalling of UVB irradiated U937 human leukaemic cells. Moreover, the effect of melatonin treatment on electrophysiological properties and membrane K+ currents of irradiated U937 cells was investigated as functional aspects relevant to the anti‐apoptotic role of melatonin. The general effect is associated with the restoration of mass, number and membrane potential of mitochondria, with a lower caspase activation and bcl‐2 upregulation. In the presence of the caspase inhibitor ZVAD‐Fmk, melatonin seems to drive UVB stressed cells to follow the mitochondrial intrinsic pathway, interfering just at the mitochondrial level. Moreover, treatment with melatonin, as well as ZVAD‐Fmk, prevented the K+ current reduction observed late following the UVB insult application, by sparing cells from death; this result also indicates that the decrease of K+ leakage currents could represent a functional feature of apoptotic process in UV‐exposed U937 cells.

Flow cytometric analyses disclose intercellular communications in FasL-stimulated T cells: results and trouble shooting.

LIFE depends on the ability of cells to communicate with one another. Much of this crosstalk occurs at cell–cell contacts and is regulated by complex structural interface: neurological and immunological synapses that transmit cell–cell signals through the extracellular space, relying on mechanisms of ligand-receptor signalling across tight cell–cell junctions. In addition to these well-known examples, other cellular structures involved in cell–cell communication have been identified (1). A frequent result of cell–cell communication is collective population behavior that can potentially lead to complex phenotypes not observed in separate individual cells, for example, in development or tumor formation (2,3). Immune responses against pathogens or any foreign antigens require fine immune regulation, where cellular communications are mediated by either soluble or cell surface molecules. Generally, absorption, exosome production and uptake, internalization, and membrane nanotube formation are the probable mechanisms through which the membrane fragments and cytoplasmic content are transferred from one cell to another (4). The identification of new structures involved in cell-to-cell communication has led to the development of new analytical and imaging tools, which have allowed us to enhance our understanding of the ubiquitous phenomenon of cell-to-cell communication. Such tools include quantitative cell microscopy, now mainly represented by two distinct techniques: flow cytometry (FCM) and image cytometry (IC) (5). FCM allows analysis of cell suspensions, cell-by-cell quantification of optical signals, rapid analysis (several hundred cells per second), and cell sorting, whereas IC allows precise localization and quantification of optical signals emitted by each point of the observed field and image analysis (morphology and morphometry).

New role of oxysterols as regulators of adipogenic differentation in adipose-derived mesenchymal stem cells

Growing evidence indicates that adipose tissue (AT) represents a potential source of pluripotent mesenchymal stem cells. However, the mechanisms underlying the lineage-specific commitment of human adipose-derived stem cells (ASC) remain still not fully elucidated. Oxysterols are cholesterol oxide products resulting from non-enzymatic (ie, 7-Ketocolesterol) or enzymatic (ie, 5,6-Secosterol) oxidation of cholesterol, which are now emerging as reliable markers of adipose “oxidative stress” in vivo. Recent data suggest that, by regulating the adipogenic differentiation of ASC, lipid peroxidation products may play an important role in linking the adipose dysfunction with impairment of glucose homeostasis. In this study we combined a lipidomic approach with the subcutaneous (sc) microdialysis technique to characterize the adipose-derived profile of fatty acids (FA) and oxysterols in vivo. ASC were isolated from abdominal sc, mesemteric (MES) and omental (OM) fat specimens obtained from obese nondiabetic (OB) and type 2 diabetic (OBT2D) patients. Flow cytometry (FC) was used for the evaluation of cell viability, mitochondrial status and cell immunophenotyping. In AT interstitial fluid, abundant concentrations of oxysterols (7κC and 5,6-S) and fatty acids (lipokines) were found. Experimental challenging with 7κC and 5,6-S showed a different time-dose-response effect. Indeed, the MTT assay, we found that in ASC isolated from the sc depot 5,6-S (50 and 10μM) reduced cell viability after 24, 48 and 72 h, respectively. In contrast, in the same cell-type population, the effect of 7κC at 10μM was observed only after 72 h. FC analysis indicated a similar effect of both oxysterols even after short-time exposure either in ASC from the MES or the OM fat depot. Notably, cell challenge with 7κC and 5,6-S at 10, 5 and 1 μM, respectively, was accompanied by an impairment of mitochondrial status only in OM, but not in MES. Furthermore, both the oxysterols (10μM) downregulated the expression of stemness surface markers suggesting a different “susceptibility” of the ASC to lipid peroxidation cell damage. Accordingly, 7κC at 10 and 1μM impaired the adipogenic differentiation of sc and OM ASC isolated from either OB or T2DOB, and demodulated the mitochondrial activity of the differentiated adipocytes. Altogether, our results suggest human AT as a critical compartment for storage and secretion of lipokines and oxysterols, which, when in excess, appear to detrimentally modulate the mitochondrial activity and the adipogenic differentiation of adipose precursor cells. This work was partly funded from Fondazione Cassa di Risparmio di Perugia, project 2010.020.0098, “Ricerca Scientifica e Tecnologica”

Niemann-Pick B-lymphocytes show autophagic stress features

Niemann-Pick disease (NPD) type A and B are lysosomal storage disorders (LSD) due to the lack of acid sphingomyelinase (ASM) activity (Schuchman et al., 2001). The enzyme defect results in a pathological accumulation of sphingomyelin (SM) within lysosomes. In many LSD, an accumulation of undegraded substrates in lysosomes due to deficiency of specific lysosomal enzymes impairs the autophagic process (Settembre et al., 2008), but an imbalance of the of autophagic process in NPB cells has never been shown. The purpose of this study is to examine the autophagic response in NPB B lymphocytes by means of flow cytometry, confocal microscopy and western blot techniques. EBV-transformed B Lymphocytes from patients with Niemann-Pick type B were treated with nocodazole (NZ) and wortmannin (WM), two autophagy inhibitors, and rapamicyn (RM), an autophagic inductor. Furthermore we starved cells using a serum-free medium to activate the autophagic process. NPD lymphocytes treated by NZ and RM showed an opposite trend than the expected results for normal cells, in Acridine Orange, Lysotracker Green and CD63 staining, clearly suggesting an impairment of this cellular pathway. Instead, starved cells highlighted a normal behaviour for these markers, indicating a residual ability to enter the process. In conclusion such results suggest the involvement of autophagy and the impairment of lysosomal network before and during NPB cells response to the above-mentioned stimuli. These scenario characterize an imbalance between formation and degradation of autophagic vacuoles (autophagic stress).

Cell-to-cell communication triggered by Fas-FasL

Eukaryotic cells have developed a variety intercellular communication mechanisms for the exchange of molecular information between neighbouring cells (trogocytosis) including immunological synapse, tunnelling nanotubes formation and uptake of exosomes. These lead to the intercellular transfer of organelles, plasma membrane components and cytoplasmic molecules. It has been demonstrated that the information transfer between immune cells plays an important role in modulation of immune responses (IR). Apoptosis is a physiological process essential to the development and homeostatic maintenance of the immune system through the extinction of the IR and the deletion of autoreactive lymphoid cells. In this work we have evaluated cell to cell communication during FasL-mediated apoptosis in CD4 and Jurkat T cells. The omotypic exchange has been demonstrated by fluorescent probes PKH67, PKH26, CFSE, DiI. We analysed labelled cells after 30, 60 and 120 min of co-incubation and FasL treatment. Furthermore, pre-treatments with two trogocytosis-specific inhibitors were performed: latrunculin A (actin filament-disrupting agent) and PP2 (inhibitor of Src family kinases). Our results show that Fas stimulation induces membrane transfer (PKH67 and PKH26 labeling), a large exchange of CFSE fluorescence (cytosolic elements) and also transfer of DiI fluorescence (labeled endocytic vesicles). In addition, the pre-treatment with latrunculin A has shown a strong decrease of exchange mechanisms compared to PP2-treatment in both cell lines. Our data suggest that Fas also promotes intercellular communication among T cells highligthing its important role in the immune response regulation.