Biomedical subjects

Elena Rapizzi

Publications and source records attributed to Elena Rapizzi.

6 recordsLinked to original sources

PubMed2006

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis.

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.

Calcium↗

PubMed2006

Sphingosine 1-phosphate inhibits cell migration in C2C12 myoblasts.

This study shows that sphingosine 1-phosphate (S1P) exerts an anti-migratory action in C2C12 myoblasts by reducing directional cell motility and fully abrogating the chemotactic response to insulin-like growth factor-1. The anti-migratory response to S1P required ligation to S1P(2), being attenuated in myoblasts where the receptor was down-regulated by specific antisense oligodeoxyribonucleotides or small interfering RNA (siRNA) and conversely potentiated in S1P(2)-overexpressing myoblasts. The investigation of RhoA and Rac GTPases, critically implicated in cell motility regulation, demonstrated that RhoA was rapidly activated by S1P, while Rac1 was unaffected within the first 5 min but stimulated thereafter. RhoA, but not Rac activation, was identified as a S1P(2)-dependent pathway in experiments in which receptor expression was attenuated by siRNA treatment or up-regulated by S1P(2)-encoding plasmid transfection. Finally, by expression of the dominant negative mutant of RhoA, the GTPase was found implicated in the anti-migratory action of S1P, whereas modulation of Rac1 functionality unaffected the anti-chemotactic effect of S1P, ruling out a role for this protein in the biological response. Since S1P was previously shown to inhibit myoblast proliferation and stimulate myogenesis, the here identified novel biological activity is in favour of a complex physiological role of the sphingolipid in the process of muscle repair.

Animals↗

PubMed2005

Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction.

To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.

Adenosine Diphosphate↗

PubMed2004

Inhibition of poly(ADP-ribose) glycohydrolase by gallotannin selectively up-regulates expression of proinflammatory genes.

Poly(ADP-ribose)-polymerase-1 (PARP-1) and poly(ADP-ribose) (PAR) are emerging key regulators of chromatin superstructure and transcriptional activation. Accordingly, both genetic inactivation of PARP-1 and pharmacological inhibition of PAR formation impair the expression of several genes, including those of the inflammatory response. In this study, we asked whether poly(ADP-ribose) glycohydrolase (PARG), the sole depoly(ADP-ribosyl)ating enzyme identified so far, also regulates gene expression. We report the novel finding that inhibition of PARG by gallotannin triggered nuclear accumulation of PAR and concomitant PAR-dependent expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), but not of interleukin-1beta and tumor necrosis factor-alpha, in cultured RAW 264.7 macrophages. Remarkably, silencing of PARG by means of small interfering RNA selectively impaired gallotannin-induced expression of iNOS and COX-2. Consistent with a PAR-dependent transcriptional activation, increases of iNOS and COX-2 transcripts were not caused by activation of transcription factors such as nuclear factor-kappaB, activator protein-1, signal transducer and activator of transcription-1 or interferon regulatory factor-1, nor by mRNA stabilization. Overall, our data provide the first evidence that pharmacological inhibition of PARG leads to PAR-dependent alteration of gene expression profiles in macrophages.

Animals↗

PubMed2002

Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria.

Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.

Animals↗

PubMedDate not supplied

A role for calcium in Bcl-2 action?

Changes in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) translate a variety of extracellular signals into widely diverse intracellular effects, ranging from secretion to movement, proliferation and also cell death. As regards the last one, it has long been known that large [Ca(2+)](c) increases lead cells to death. More recently, experimental evidence has been obtained that the oncogene Bcl-2 reduces the state of filling of intracellular Ca(2+) stores and thus affects the Ca(2+) responses induced by physiological and pathological stimuli. In this contribution, we will discuss this effect and its significance for the mechanism of action of Bcl-2, an important checkpoint of the apoptotic process.

Animals↗