Biomedical subjects

Sestina Falcone

Publications and source records attributed to Sestina Falcone.

8 recordsLinked to original sources

PubMed2006

Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events.

Macropinocytosis, a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes, has been studied mostly in relation to antigen presentation. Early membrane traffic events occurring in this process are, however, largely unknown. Using human dendritic cells we show that a marked increase in the rate of macropinocytosis occurs a few minutes after application of two markers (small latex beads or dextran), depends on a slow intracellular Ca2+ concentration ([Ca2+](i)) rise that precedes the PI3K-dependent step, and is preceded and accompanied by exocytosis of enlargeosomes compensating in part for the macropinocytic plasma membrane internalization. Unexpectedly, macropinosomes themselves, which share markers with endosomes, undergo Ca2+ -dependent exocytosis so that, after approximately 20 minutes of continuous bead or dextran uptake, an equilibrium is reached preventing cells from overloading themselves with the organelles. Large [Ca2+](i) increases induced by ionomycin trigger rapid (<1 minute) exocytic regurgitation of all macropinosomes, whereas endosomes remain apparently unaffected. We conclude that, in dendritic cells, the rate of macropinocytosis is not constant but increases in a regulated fashion, as previously shown in other cell types. Moreover, macropinosomes are not simple containers that funnel cargo to an endocytic pathway, but unique organelles, distinct from endosomes by their competence for regulated exocytosis and other membrane properties.

Calcium↗

PubMed2005

Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS.

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

Adipose Tissue↗

PubMed2005

Nitric oxide, ceramide and sphingomyelinase-coupled receptors: a tale of enzymes and messengers coordinating cell death, survival and differentiation.

The gaseous messenger nitric oxide plays a role in a variety of biological functions. Evidence accumulated over the last 7 years indicates that functions of nitric oxide in apoptosis growth and differentiation may originate in part from an interplay with signalling members of the sphingolipid family. Interactions between nitric oxide and sphingolipids have been shown to be multiple, to involve regulation of activity and expression of the enzymes responsible for the synthesis of nitric oxide and of those involved in the sphingolipid metabolic pathways. Recent evidence indicates that one of these interactions, namely the cross-talk of sphingomyelinases and their product ceramide with nitric oxide and its generating enzyme endothelial nitric oxide synthase, plays prominent roles during key patho-physiological processes such as inflammation, proliferation, death and differentiation.

Apoptosis↗

PubMed2004

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Adenosine Triphosphate↗

PubMed2004

Activation of acid sphingomyelinase and its inhibition by the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway: key events in Escherichia coli-elicited apoptosis of dendritic cells.

Depletion of dendritic cells (DCs) via apoptosis contributes to sepsis-induced immune suppression. The mechanisms leading to DC apoptosis during sepsis are not known. In this study we report that immature DCs undergo apoptosis when treated with high numbers of Escherichia coli. This effect was mimicked by high concentrations of LPS. Apoptosis was accompanied by generation of ceramide through activation of acid sphingomyelinase (A-SMase), was prevented by inhibitors of this enzyme, and was restored by exogenous ceramide. Compared with immature DCs, mature DCs expressed significantly reduced levels of A-SMase, did not generate ceramide in response to E. coli or LPS, and were insensitive to E. coli- and LPS-triggered apoptosis. However, sensitivity to apoptosis was restored by addition of exogenous A-SMase or ceramide. Furthermore, inhibition of A-SMase activation and ceramide generation was found to be the mechanism through which the immune-modulating messenger NO protects immature DCs from the apoptogenic effects of E. coli and LPS. NO acted through formation of cGMP and stimulation of the cGMP-dependent protein kinase. The relevance of A-SMase and its inhibition by NO/cGMP were confirmed in a mouse model of LPS-induced sepsis. DC apoptosis was significantly higher in inducible NO synthase-deficient mice than in wild-type animals and was significantly reduced by treatment ex vivo with NO, cGMP, or the A-SMase inhibitor imipramine. Thus, A-SMase plays a central role in E. coli/LPS-induced DC apoptosis and its inhibition by NO, and it might be a target of new therapeutic approaches to sepsis.

Animals↗

PubMed2004

Nitric oxide confers therapeutic activity to dendritic cells in a mouse model of melanoma.

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

Animals↗

PubMed2003

Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway.

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte-derived DCs to the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) during their maturation process induced by treatment with tumor necrosis factor alpha or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA-NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo-antigens, or cross-linking of the CD3-T cell receptor complex. This effect persists after removal of DETA-NO, depends on the generation of cyclic guanosine 5'-monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)-12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL-12 release was observed after DC exposure to DETA-NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.

CD40 Antigens↗

PubMed2002

Nitric oxide regulates oestrogen-activated signalling pathways at multiple levels through cyclic GMP-dependent recruitment of insulin receptor substrate 1.

The gaseous messenger nitric oxide (NO) contributes to biological effects of oestrogen in target tissues, including reproductive organs, bone, cardiovascular and central nervous systems. Vasodilation and anti-atherosclerotic properties of NO have been shown to play a role in these effects. The possibility that NO acts also through regulation of the signal transduction cascade triggered by oestrogen, instead, has never been investigated. To study this we have used the MCF-7 human breast cancer cell line, an established model for oestrogen signalling. Exposure of these cells to 17-beta-oestradiol (E(2)) in the presence of NO gave rise to activation of signalling events additional to those triggered by E(2) alone, namely tyrosine phosphorylation of specific proteins, including the insulin receptor substrate-1, with recruitment to this adapter of the phosphatidylinositol 3'-kinase and persistent activation of Akt (protein kinase B). Active Akt, in turn, prevented E(2) from activating p42/44 extracellular signal-regulated kinases (ERK 1/2). These effects of NO, which were mediated through generation of cyclic GMP and activation of the cGMP-dependent protein kinase I, initiated in the first minutes after administration of oestrogen. The consequences, however, were long lasting, as modulation of Akt and ERK 1/2 activities by NO was responsible for inhibition of E(2)-triggered cell growth and regulation of oestrogen responsive-element dependent gene transcription. Generation of NO is stimulated by both E(2) and growth factors known to contribute to the complex network of intracellular events regulating the biological actions of oestrogen. It is conceivable, therefore, that modulation by NO of E(2) early signalling, here described for the first time, has broad significance in regulating cellular responses to the hormone.

Blotting, Western↗