Ca2+ signals are involved in cell proliferation.

Intracellular Ca²⁺ is a versatile and universal second messenger involved in control of a large number of cellular and physiological functions in essentially all types of cells. Ca²⁺ signals regulate not only short-term cell functions such as exocytosis and muscle contraction but also play a pivotal role in control of long-term cell functions as cell proliferation. Ca²⁺ signals activate genes responsible for entry of resting cells into the cell cycle, promote DNA synthesis and stimulate different events at mitosis. A particular attention has been paid to the so-called store operated Ca²⁺ entry (SOCE), an ubiquitous Ca²⁺ entry pathway and the most important one in non-excitable cells. SOCE is activated after depletion of intracellular Ca²⁺ stores upon activation of G-protein coupled receptors and tyrosine kinase receptors. The mechanism linking depletion of Ca²⁺ stores to increased Ca²⁺ influx remains unknown. Controversy also exists on the molecular nature of the ion channels involved. It is believed that SOCE is mediated by members of the superfamily of TRP cation channels, particular the so-termed canonic members (TRPc1-7) and TRPV6. In addition, the downstream signaling pathways activated by SOCE and related to cell proliferation are not well known. The most studied cell model in this regard is the Jurkat T cell. In these cells, activation of the T cell receptor promotes sustained SOCE that activates of the nuclear factor of activated cells (NFAT) leading to expression of interleukin-2 and cell proliferation. Among others, NFAT is relevant in the transcriptional control of COX—2, an enzyme involved in tumor growth and angiogenesis. Thus, COX-2 and its tumor promoting activity can be primary regulated by Ca²⁺ signals although this possibility has not been addressed. In the last few years there is a wide interest in the role of COX-2 in tumor growth because multiple basic and clinical data indicate that several NSAIDs prevent tumor cell growth and protect against a number of cancers. The mechanism is unknown but some authors believe that the action of NSAIDs is due to its well established inhibition of COX-2. Recent data however strongly suggest that the chemopreventive action mechanism of NSAIDs is unrelated to their anti-COX-2 activity (Kashfi & Rigas, 2005). Regardless of the mechanism, what is true is that multiple evidence in T cells and other cell models indicate that SOCE is involved in cell proliferation control: i) Entry into the cell cycle is preceded by SOCE activation; ii) stimulation of cell proliferation induced by growth factors correlates with increased activity and/or gene expression of TRP channels related to SOCE; iii) SOCE inhibition by different means (extracellular Ca²⁺ removal, inorganic channel blockers and SOCE antagonists) prevent cell proliferation.

Ca²⁺ entry is regulated by mitochondria.

A relevant characteristic of SOCE and members of the TRP superfamily of cation channels is their strong Ca²⁺-dependent inactivation. Thus, the intracellular Ca²⁺ extruding systems are essential for sustaining SOCE. In the last few years, the study of Ca²⁺ signaling has evolved from the cellular to the subcellular level and has focused in the important role of Ca²⁺ microdomains and subcellular compartments in control of cellular and physiological functions. Several groups including our own have shown that mitochondria are the most important systems for Ca²⁺ extrusion For example, recently we have used low-affinity aequorin targeted to mitochondria to show that activation of voltage-gated Ca²⁺ channels generates high Ca²⁺ microdomains that are sensed by subpopulation of nearby mitochondria. This subpopulation takes up essentially all Ca²⁺ entering the cell undergoing huge increases in mitochondrial Ca²⁺ concentration to reach the mM level, a value several orders of magnitude larger than previously thought. This indicates the very important role of mitochondria in the clearing of cytosolic Ca²⁺ increases. Recently, it has been shown that mitochondrial Ca²⁺ uptake is essential to sustain SOCE in T cells. These authors have shown that mitochondrial uncouplers as FCCP that collapse the mitochondrial potential, the electromotive force for Ca²⁺ uptake by mitochondria, promote SOCE inactivation, the electromotive force for mitochondrial Ca²⁺ uptake, promote SOCE inactivation. Accordingly mitochondria has a two fold control on Icrac channels underlying SOCE in T cells. First, mitochondrial Ca²⁺ uptake facilitates the full emptying of intracellular Ca²⁺ stores thus promoting full Icrac activation. Second, the mitochondrial uptake of entering Ca²⁺ prevents the strong Ca²⁺-dependent inactivation of SOCE. Accordingly, mitochondria promotes SOCE activation and prevents its inactivation, thus leading to a sustained SOCE activity that is essential for T cell proliferation. Accordingly, mitochondria regulates SOCE and therefore it is expected to play a pivotal role in control of proliferation. Our group is aimed at establishing the role of mitochondria in cell proliferation.

Ca²⁺ entry into mitochondria and cell death.

Mitochondria are central to the life of eukaryotic cells. It is becoming increasingly clear that mitochondria also play a key role in cell death. This role of mitochondria is not due to a 'loss of function' leading to energetic deficit but to an 'active' process mediated by regulated effector mechanisms in a wide variety of conditions. Under certain conditions mitochondria release proapoptotic factors that lead irreversible to activation of a cascade of proteases that culminate in cell death. The release of proapoptotic factors such as cytochorme C is related to a huge increase in the permeability of the inner mitochondrial membrane. Several factors promote this permeability increase including oxydative stress, loss of mitochondrial potential and specially mitochondrial Ca²⁺ overload. Thus, mitochondria is a clear target for cell protective drugs intended to avoid the limiting step in cell death. We are interested in drugs that may modulate mitochondrial Ca²⁺ uptake acting as neuroprotective and cardioprotective agents.

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