Francesca Luchetti

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

Multiple roles of matrix metalloproteinases during apoptosis

Structural, molecular and biochemical approaches have contributed to piecing together the puzzle of how matrix metalloproteinases (MMPs) work and contribute to various disease processes. However, MMPs have many unexpected substrates other than components of the extracellular matrix which profoundly influence cell behaviour, survival and death. With the current understanding of diverse/novel roles of matrix metalloproteinases—particularly their direct or indirect relevance for the early steps during programmed cell death—some seemingly contrasting results seem less surprising. To better target MMPs an appreciation of their many extracellular, intracellular and intranuclear functions, often acting in opposing directions with paradoxical roles in cell death, is carefully required.

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 prevents apoptosis induced by UV-B treatment in U937 cell line.

Abstract:  Melatonin influences circadian rhythms and acts as antioxidant and free radical scavenger. UV irradiation triggers multiple cellular events which lead to cell death, in particular to apoptosis; this process involves reactive oxygen species. Apoptotic machinery involves several pathways, in which mitochondria play crucial roles. In this work we have evaluated by means of cytometric, biochemical and ultrastructural approaches, if incubation of U937 promonocytic leukemia cells with melatonin may affect apoptotic behavior induced by UV‐B. The cell line was treated with 1 mm melatonin before and after UV‐B exposure. Melatonin pretreatment significantly reduced the number of apoptotic cells, as revealed by FITC Annexin‐V and propidium iodide assays (P < 0.005), as well as attenuated mitochondria alterations, as shown by ultrastructural morphology, Mito Tracker and JC‐1 staining, and cytochrome c (cyt c) release (P < 0.005). On the contrary, incubation with melatonin after UV‐B exposure significantly protect U937 cells from UV‐B induced alterations, showing a possible delay of the apoptotic machinery (as revealed by the presence of earlier stages of apoptosis and significant cyt c release). Our results suggest that, in our experimental model, melatonin may play a role as noncytotoxic anti‐apoptotic compound and, at least in part, may protect U937 cells from UV‐B induced mitochondria dysfunction/damage.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

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.

Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34+ (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte‐specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6–8 days, virtually all of the CD34‐derived cells expressed the early megakaryocytic CD61 antigen, while, after 15–16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15–16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing. Anat Rec 258:90–99, 2000.

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.

PHOSPHOLIPID REARRANGEMENT OF APOPTOTIC MEMBRANE DOES NOT DEPEND ON NUCLEAR ACTIVITY

Abstract The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Behavior of nucleolar proteins during the course of apoptosis in camptothecin‐treated HL60 cells

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin‐treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti‐nucleolar antibodies seen in various pathological disorders. J. Cell. Biochem. 78:264–277, 2000.