Davide Lovisolo

Intracellular calcium signals and control of cell proliferation: how many mechanisms?

The progression through the cell cycle in non‐transformed cells is under the strict control of extracellular signals called mitogens, that act by eliciting complex cascades of intracellular messengers. Among them, increases in cytosolic free calcium concentration have been long realized to play a crucial role; however, the mechanisms coupling membrane receptor activation to calcium signals are still only partially understood, as are the pathways of calcium entry in the cytosol. This article centers on the role of calcium influx from the extracellular medium in the control of proliferative processes, and reviews the current understanding of the pathways responsible for this influx and of the second messengers involved in their activation.

Blocking Ca2+ Entry: A Way to Control Cell Proliferation

Ca(2+) signalling is involved in virtually all cellular processes: among the others, it controls cell survival, proliferation and death regulating a plethora of intracellular enzymes located in the cytoplasm, nucleus and organelles. Changes in the cytosolic free Ca(2+) concentration may be due either to release from the intracellular Ca(2+) stores or to influx from the extracellular medium, through the opening of plasma membrane calcium-permeable channels. In particular, Ca(2+) entry from the extracellular space is a mechanism able to sustain long lasting intracellular Ca(2+) elevations: this signal, activated by many growth factors and mitogens in normal and tumoral tissues, is linked to DNA transcription and duplication, finally leading to cell proliferation. In the last years many informations have been provided about the transduction mechanisms related to Ca(2+) entry induced by mitogenic factors, mostly binding to tyrosine kinase receptors, but also to G-protein coupled ones. Nevertheless, some key points remain to be fully clarified: among them, the molecular structure of the Ca(2+) channels involved, their regulation by intracellular messengers, and the modes through which specificity is achieved. The increasing knowledge on Ca(2+) entry-dependent control of proliferation may provide a more satisfactory understanding of pathological alterations, including cancer progression and angiogenesis. A detailed description of the mechanisms that trigger Ca(2+) entry, and in particular the definition of calcium-permeable channels and their modulators at the molecular levels, will greatly improve our possibility to take advantage of Ca(2+) entry regulation as a therapeutic approach for the control of cell proliferation, designing antibodies or molecules with low side effects and specific channel blocker functions. The review will focus on this topic.

ARACHIDONIC ACID MEDIATES CALCIUM INFLUX INDUCED BY BASIC FIBROBLAST GROWTH FACTOR IN BALB-C 3T3 FIBROBLASTS

Basic fibroblast growth factor (bFGF), a peptide acting as a mitogen in different cell types, is able to induce a long lasting non capacitative calcium influx from the extracellular medium in Balb-c 3T3 mouse fibroblasts. This effect is mediated by the tyrosine kinase activity of bFGF receptors and the opening of voltage independent, agonist activated calcium channels. In this paper we investigate the signal transduction steps involved in this process using single cell calcium fluorimetry and electrophysiological techniques. One of the pathways initiated by the binding of growth factors to their tyrosine kinase receptors is the activation of cytosolic phospholipase A2 (cPLA2) and the release of arachidonic acid (AA) from the plasma membrane with the subsequent production of eicosanoids. We show here that, in our preparation, this pathway is involved in the opening of the bFGF-activated calcium permeable channels, through the activation of mitogen activated protein kinase (MAPK) and cPLA2. Evidence for direct involvement of AA is given by the finding that: (i) bFGF induces AA release from Balb-c 3T3 cells; (ii) blockers of AA metabolism are not effective; and (iii) the application of either arachidonic acid or its non metabolizable analogue 5,8,11,14-eicosatetraynoic acid (ETYA) reproduces the responses described for bFGF. Finally, single channel analysis indicates that bFGF, AA and ETYA can activate the same calcium permeable channel.

Control of Endothelial Cell Proliferation by Calcium Influx and Arachidonic Acid Metabolism: A Pharmacological Approach

In physiological conditions, endothelial cell proliferation is strictly controlled by several growth factors, among which bFGF and VEGF are the most effective. Both bind to specific tyrosine kinase receptors and trigger intracellular signal cascades. In particular, bFGF stimulates the release of arachidonic acid (AA) and its metabolites in many types of endothelial cells in culture. In bovine aortic endothelial cells, it has been suggested that AA is released by the recruitment of cytosolic phospholipase A2 (cPLA2). AA metabolites are involved in the control of both endothelial cell motility (mostly via the cyclooxygenase pathway) and proliferation (via the lipoxygenase (LOX) cascade). On the other hand, evidence has been provided for a proliferative role of AA‐induced calcium influx. By using a pharmacological approach, we have tried to elucidate the contribution to bovine aortic endothelial proliferation of the different pathways leading to production of AA and its metabolites. Two main informations were obtained by our experiments: first, AA release is not entirely due to cPLA2 involvement, but also to DAG lipase recruitment; second, cyclooxygenase derivatives play a role in the control of cell proliferation, and not only of motility. Moreover, by combining proliferation assays and single cell calcium measurements, we show that the blocking effect of carboxyamido‐triazole (CAI), an inhibitor of tumor growth and angiogenesis acting on calcium influx‐dependent pathways, including AA metabolism, is at least in part due to a direct effect on AA‐induced calcium influx. J. Cell. Physiol. 197: 370–378, 2003© 2003 Wiley‐Liss, Inc.

Expression and functional role of bTRPC1 channels in native endothelial cells.

We have analyzed the expression and localization of bovine transient receptor potential‐C1 (bTRPC1) in bovine aortic endothelial cells, and its possible involvement in the store‐independent calcium influx induced by basic fibroblast growth factor (bFGF). RT‐PCR experiments confirmed the existence of two btrpc1 mRNA isoforms; conversely, the btrpc3 gene was not transcribed. Anti‐TRPC1 antibody revealed the presence of the protein in the membrane‐rich compartment only. Application of anti‐TRPC1 during the response to bFGF caused a partial but significant reduction of calcium entry. This is the first evidence of TRP channel involvement in a non‐capacitative calcium influx induced by a biologically relevant agonist such as the angiogenic factor bFGF in native endothelial cells.

Interaction of Spherical Silica Nanoparticles with Neuronal Cells: Size‐Dependent Toxicity and Perturbation of Calcium Homeostasis

The effects of Stöber silica nanoparticles on neuronal survival, proliferation, and on the underlying perturbations in calcium homeostasis are investigated on the well-differentiated neuronal cell line GT1-7. The responses to nanoparticles 50 and 200 nm in diameter are compared. The 50-nm silica affects neuronal survival/proliferation in a dose-dependent way, by stimulating apoptotic processes. In contrast, the 200-nm silica does not show any toxic effect even at relatively high concentrations (292 μg mL−1). To identify the mechanisms underlying these effects, the changes in intracellular calcium concentration elicited by acute and chronic administration of the two silica nanoparticles are analyzed. The 50-nm silica at toxic concentrations generates huge and long-lasting increases in intracellular calcium, whereas the 200-nm silica only induces transient signals of much lower amplitude. These findings provide the first evidence that silica nanoparticles can induce toxic effects on neuronal cells in a size-dependent way, and that these effects are related to the degree of perturbation of calcium homeostasis.

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min) elevation of intracellular free calcium concentration ([Ca2+]i). Both the sustained [Ca2+]i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73-82], could be abolished either by chelation of extracellular calcium with EGTA or by SK&F 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SK&F 96365 was applied from the 4th to the 8th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G2/M phase. Both growth inhibition and G2/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.

A diamond-based biosensor for the recording of neuronal activity.

We have developed a device for recording the extracellular electrical activity of cultured neuronal networks based on a hydrogen terminated (H-terminated) conductive diamond. GT1-7 cells, a neuronal cell line showing spontaneous action potentials firing, could maintain their functional properties for days in culture when plated on the H-terminated diamond surface. The recorded extracellular electrical activity appeared in the form of well-resolved bursts of fast and slow biphasic signals with a mean duration of about 8ms for the fast and 60ms for the slow events. The time courses of these signals were in good agreement with those recorded by means of conventional microelectrode array (MEAs) and with the negative derivative of the action potentials intracellularly recorded with the patch clamp technique from single cells. Thus, although hydrophobic in nature, the conductive H-terminated diamond surface is able to reveal the spontaneous electrical activity of neurons mainly by capacitative coupling to the cell membrane. Having previously shown that the optical properties of H-terminated diamond allow to record cellular activity by means of fluorescent probes (Ariano, P., Baldelli, P., Carbone, E., Giardino, A., Lo Giudice, A., Lovisolo, D., Manfredotti, C., Novara, M., Sternschulte, H., Vittone, E., 2005. Diam. Relat. Mater. 14, 669-674), we now provide evidence for the feasibility of using diamond-based cellular biosensors for multiparametrical recordings of electrical activity from living cells.

Regulation of noncapacitative calcium entry by arachidonic acid and nitric oxide in endothelial cells

Several peptides, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), activate the release of arachidonic acid (AA) and nitric oxide (NO) in endothelial cells (ECs). Both messengers are involved in EC proliferation and vascular permeability and control calcium homeostasis in different ways. Interestingly, it has been recently suggested that NO acts as a downstream mediator of AA‐induced calcium entry in smooth muscle cells and isolated mouse parotid cells. In this paper, we have investigated the complex relationships that link intracellular calcium, AA, and NO in cultured endothelial cells. Using different experimental approaches, mainly simultaneous Ca2+ and NO fluorimetric confocal imaging, we provide evidence for a complex pathway leading to noncapacitative calcium entry (NCCE) in bovine aortic endothelial cells (BAECs). In particular, AA is able to induce NCCE through two different pathways: one dependent on eNOS recruitment and NO release, the other NO‐independent. Finally, we show that NO increase is involved in the control of BAEC proliferation.

Neuronal survival and calcium influx induced by basic fibroblast growth factor in chick ciliary ganglion neurons.

Basic fibroblast growth factor (bFGF/FGF2) exhibits widespread biological activities in the nervous system. However, little is known about the cascade of intracellular events that links the activation of its tyrosine kinase receptors to these effects. Here we report that, in ciliary ganglion neurons from chick embryo, this trophic factor significantly enhanced neuronal survival. The percentage of surviving neurons was reduced when intracellular calcium was chelated by adding a membrane‐permeable BAPTA ester to the culture medium, while antagonists of L‐ and N‐type voltage‐dependent calcium channels were ineffective. The ionic signals in response to bFGF stimulation have been studied using cytofluorimetric and patch‐clamp techniques. In single‐cell Fura‐2 measurements, bFGF elicited a long lasting rise of the cytosolic calcium concentration that was dependent on [Ca2+]o. In whole‐cell experiments, we observed a reversible depolarization of the membrane resting potential and an inward cationic current. Single channel experiments, performed in the cell‐attached configuration, provide evidence for the activation of two families of Ca2+‐permeable cationic channels. Moreover, inositol 1,4,5‐trisphosphate opens channels with similar properties, suggesting that this cytosolic messenger can be responsible for the calcium influx induced by bFGF.