Ca²⁺ signaling, genes and the cell cycle

Abstract

Changes in the concentration and spatial distribution of Ca²⁺ ions in the cytoplasm constitute a ubiquitous intracellular signaling module in cellular physiology. With the advent of Ca²⁺ dyes that allow direct visualization of Ca²⁺ transients, combined with powerful experimental tools such as electrophysiological recordings, intracellular Ca²⁺ transients have been implicated in practically every aspect of cellular physiology, including cellular proliferation. Ca²⁺ signals are associated with different phases of the cell cycle and interfering with Ca²⁺ signaling or downstream pathways often disrupts progression of the cell cycle. Although there exists a dependence between Ca²⁺ signals and the cell cycle the mechanisms involved are not well defined and given the cross-talk between Ca²⁺ and other signaling modules, it is difficult to assess the exact role of Ca²⁺ signals in cell cycle progression. Two exceptions however, include fertilization and T-cell activation, where well-defined roles for Ca²⁺ signals in mediating progression through specific stages of the cell cycle have been clearly established. In the case of T-cell activation Ca²⁺ regulates entry into the cell cycle through the induction of gene transcription.

Introduction

Ca²⁺ signals have been implicated in various aspects of the cell cycle using different approaches and experimental systems. This has lead to a consensus in the field for a requirement for Ca²⁺ in cellular proliferation. The cell cycle is essential for organismal development and its deregulation leads to disastrous results such as tumor growth. The cell cycle consists of four phases, two growth phases G1 and G2 interspersed by the DNA synthesis phase (S) and the cell division phase (M) [1]. To ensure proper cellular proliferation and survival the cell cycle is unidirectional and sequential. This guarantees that DNA synthesis precedes cell division to ensure diploidy in the two daughter cells [1].

The idea that Ca²⁺ signals may be involved in cell cycle progression is quite attractive given the versatile nature of Ca²⁺ signaling. Intracellular Ca²⁺ signals tend to be complex in terms of their spatial localization, temporal features and amplitude [2;3]. Ca²⁺ signals can be very localized, limited to small region surrounding the mouth of a channel for example [4], or can take the form of Ca²⁺ release waves that sweep through the entire cell or even across multiple cells. They can range in duration from μseconds during vesicular exocytosis for example, to hours after fertilization in mammals [5]. In fact specificity in Ca²⁺ signaling is dependent on this versatility. Ca²⁺ transients activate different Ca²⁺ binding proteins with varying enzymatic activities such as kinases or phosphatases, or signal transducers such as calmodulin that then branch out and modulate a multitude of signaling pathways to affect cell function. Based on the association and dissociation constants of the different Ca²⁺ binding proteins, Ca²⁺ signals with disparate dynamics induce distinct cellular responses.

Intracellular Ca²⁺ signaling is possible because cells maintain a low Ca²⁺ background in the cytoplasm with concentrations of ∼100 nM. Ca²⁺ signals are generated due to Ca²⁺ influx from the extracellular space with concentration around 1-2 mM, or Ca²⁺ release from intracellular Ca²⁺ stores, primarily the endoplasmic reticulum (ER) with concentrations of 250-600 μM [6]. Channels and pumps on the ER and plasma membranes coordinately regulate Ca²⁺ homeostasis in the ER and cytoplasm.

Ca²⁺ signals have been detected at various stages of the cell cycle and manipulation of Ca²⁺ signaling often affects cellular proliferation. This generated the consensus that Ca²⁺ signals are essential for cellular proliferation. However, given the ubiquitous and versatile nature of Ca²⁺ signaling and the integration of Ca²⁺ signals with other signaling modules regulating cellular physiology, it is quite difficult to identify the specific signaling pathway downstream of the Ca²⁺ signals. However, in at least two cases, following fertilization and during T-cell activation, Ca²⁺ signals have been shown to be essential for cell cycle progression, and the mechanisms through which Ca²⁺ signals mediate these cell cycle transitions are well defined.

I. Ca²⁺ signals are required at fertilization to induce completion of meiosis

As is often the case in biology the first studies to observe a correlation between the cell cycle and Ca²⁺ signals were performed to address a different problem, that of the role of Ca²⁺ ions in ‘protoplasm gelation’ following cellular stimulation [7]. Cellular stimulation was thought to be associated with an increased viscosity in the cytoplasm and Ca²⁺ was implicated in the process. One model of cell stimulation that was used to test the involvement of Ca²⁺ ions in colloid chemical changes in the cell was following fertilization of sea urchin eggs. Hence the earliest study to observe changes in Ca²⁺ dynamics associated with the cell cycle was performed in the mid 1930s in the context of fertilization, where a Ca²⁺ rise was observed following fertilization in of the sea urchin Arabica eggs [7]. Later studies confirmed this observation using ⁴⁵Ca²⁺ fluxes also in sea urchin [8-10]. Interestingly, initial hypotheses regarding the role of Ca²⁺ in inducing cell cycle progression at fertilization argued that Ca²⁺ has a physiochemical role in the formation of the mitotic spindle through increasing cytoplasmic viscosity [9;11]. The Ca²⁺ rise at fertilization was also linked to the observed increase in cellular respiration [9;12]. These studies sparked an interest in the potential role and regulation of Ca²⁺ signals in egg activation at fertilization and their role in cell cycle progression.

Other investigators became interested in the role of Ca²⁺ in cellular motility, wound healing and cellular cleavage. The common thread among these different cellular processes is that each involves contractions of the cell cortex. These interests were instigated by the known role at the time of Ca²⁺ in muscle contraction [13;14], it was therefore attractive to investigate the role of Ca²⁺ signals in other forms of cellular contractions in non-excitable cells. In this case the Xenopus laevis oocyte and embryo were attractive systems for these studies given their unusually large size (∼1.3mm for the fully grown stage VI oocyte). Indeed Ca²⁺ was shown to play important roles in wound healing in the oocyte and cell cleavage during early embryonic divisions [13;14]. These studies also stimulated research in the area of Ca²⁺ signaling during fertilization, egg activation and cell cycle progression.

Direct visualization of a Ca²⁺ rise at fertilization was first accomplished using aequorin in the medaka fish [15], and later in sea urchin [16]. These fertilization-induced Ca²⁺ increases took the form of a propagating wave that swept through the entire egg [17]. However the fact that Ca²⁺ transients, although quite dramatic, were observed at fertilization did not necessarily establish a functional role for Ca²⁺ signals in egg activation. The correlation between Ca²⁺ and fertilization was extended to a causal relationship when an ionophore-mediated Ca²⁺ rise was shown to be sufficient to parthogenetically activate sea urchin eggs [10;18]. Because ionophore treatment was also effective at activating eggs from other species independently of sperm, this argued that Ca²⁺ is the universal egg activator [19], a prediction that was validated by future studies in several other species [20].

Subsequent studies characterized the fertilization-dependent Ca²⁺ release in mammals and other vertebrates and showed specificity in the spatial and temporal pattern of these signals, where in mammals an oscillatory Ca²⁺ signal that lasts several hours is observed, whereas in the frog Xenopus laevis for example only a single Ca²⁺ transient is induced at fertilization and takes the form of a sweeping Ca²⁺ wave [20-22]. These differing Ca²⁺ dynamics among species are vital in mediating the complex events that need to occur at fertilization to induce the transition from gametogenesis to embryogenesis [20;22;23]. Central among these is the block to polyspermy and the release of the CSF arrest leading to the completion of meiosis and transitioning into the mitotic embryonic divisions.

Although not discussed in details here, eggs acquire the ability to produce the specialized Ca²⁺ transient at fertilization following a dramatic remodeling of the Ca²⁺ signaling machinery during oocyte maturation, which encompasses entry and progression through meiosis [22]. Hence not only are Ca²⁺ signals important for cell cycle progression, but also the Ca²⁺ signaling machinery remodels during the cell cycle to service the specific needs of the cell during its development [22;23].

Block to polyspermy

The polyspermy block is discussed here as an example of how Ca²⁺ signals during the cell cycle, in this case the egg arrested in meiosis II, are important to regulate physiological processes outside the central cell cycle machinery that are nonetheless essential for proper progression through the cell cycle. The block to polyspermy is mediated by the Ca²⁺ transient at fertilization and is critical to prevent polyploidy in the zygote, which will lead to its demise. Different species have devised distinct mechanisms to prevent polyploidy. The urodelas allow multiple sperms to penetrate the egg at fertilization, but only one sperm pronucleus fuses with the egg pronucleus and additional sperm nuclei disintegrate [24]. In contrast, in the more common monospermic fertilization only a single sperm is allowed to fuse with the egg [25]. This represents a challenge for species with large oocytes such Xenopus laevis given the large surface area of the egg and the number of sperm it is exposed to at fertilization. Hence such species have developed an electrical fast block to polyspermy to rapidly prevent additional sperm from fusing with the egg. This fast block is mediated by membrane depolarization due to the opening of Ca²⁺-activated Cl^- channels following the initial Ca²⁺ transient at fertilization [26]. The Ca²⁺ rise at fertilization gates open Ca²⁺-activated Cl^- channels leading to an inward current (Cl^- leaving the cell) and hence membrane depolarization. This change in membrane potential instantaneously blocks further sperm fusion because Xenopus sperm fusion in voltage-dependent [27]. Sea urchin eggs similarly exhibit a fast block to polyspermy [28]. A more sustained block to polyspermy, which is also observed in other species, occurs following the modification of the extracellular matrix surrounding the egg, making it impenetrable to sperm [29]. This is again a Ca²⁺-dependent event due to the fusion of cortical granules as a Ca²⁺ wave sweeps through the egg [30]. Cortical granules release proteolytic and glycolytic enzymes that modify the egg extracellular matrix [29].

Release of the CSF-dependent meiosis II block

Following oocyte maturation vertebrate eggs arrest at metaphase of meiosis II until fertilization. In a landmark study Masui and Market defined two activities from frog eggs at different stages of maturation that turn out to be essential drivers of M-phase. Microinjection experiments showed that cytoplasm from frog eggs arrested at metaphase of meiosis II when injected into blastomeres was capable of arresting their division due to a cell cycle arrest at metaphase. This activity was termed cytostatic factor (CSF) [31].

The second activity from those experiments was identified as an activity able to induce oocyte entry into and progression through meiosis (i.e. oocyte maturation), when cytoplasm from mature from eggs was injected into immature oocytes arrest at the G2-M transition. This activity was termed maturation promoting factor (MPF) [31]. Biochemical purification and characterization of MPF showed that it is composed of the catalytic Cdk1 Ser/Thr kinase subunit and the regulatory cyclin B subunit [32-34]. The central role of MPF as the universal inducer of the G2-M transition was cemented when its catalytic Cdk1 subunit was shown to be equivalent to p34^Cdc2 the product of the Cdc2 gene in fission yeast, which is required for cell cycle progression [35].

Entry into mitosis requires the activation of MPF leading to nuclear envelope breakdown, chromosome condensation, and formation of the mitotic spindle in metaphase. In a similar vein MPF inactivation, which occurs during anaphase and telophase, is essential for mitotic exit. MPF inactivation reverses the early mitotic events leading to chromosome de-condensation, cell division and reformation of the nuclear envelope [36]. Cyclin B is targeted for degradation due to poly-ubiquitination and degradation by the 26S proteasome. Ubiquitination of cyclin B requires the anaphase-promoting complex (APC), which is a large molecular complex composed of 11 subunits and functions as an E3 ubiquitin ligase [36]. CSF maintains metaphase II meiotic arrest by inhibiting the APC and hence MPF degradation [37]. This is accomplished through a complex series of phosphorylation regulatory loops involving multiple cell cycle kinases, including Mos and the MAPK cascade, which are required to establish CSF arrest [37]. Ultimately however CSF arrest is regulated by the APC inhibitor Emi2 [38;39]. Emi2 binds to and inhibits the APC during metaphase II an activity that is stabilized through Emi2 phosphorylation by the Mos-MAPK-Rsk cascade, and dephosphorylation by protein phosphatasae 2A. The fertilization-induced Ca²⁺ transient leads to the activation of CaMKII, which phosphorylates Emi2 [40-42]. CaMKII phosphorylated Emi2 becomes a target for further phosphorylation by polo-like kinase. The dually phosphorylated Emi2 is targeted for proteasomal degradation [38]. This activates APC and releases the CSF-mediated arrest. This complex signal transduction cascade provides an elegant example of the versatility of Ca²⁺ signaling and its essential role in releasing the CSF-mediated metaphase II arrest.

In addition to the role CaMKII in releasing the CSF-mediated meiotic arrest, calcineurin a Ca²⁺-dependent phosphatase is also important in this transition, arguing that the Ca²⁺-dependent regulation affects both kinases and phosphatase to coordinately regulates the release of CSF arrest [43;44]

From the brief description of the history of Ca²⁺ signaling in fertilization one can define a clear role for Ca²⁺ in mediating the meiotic metaphase-to-anaphase transition of the cell cycle. Importantly, in this case the downstream effectors linking Ca²⁺ signals to the cell cycle are well delineated. Given the diverse and promiscuous role of Ca²⁺ signals in various aspects of cellular function, it is often quite difficult to clearly define downstream targets. In the case of fertilization a conversion of multiple fields over a long period of time allowed this to happen. This required a good understanding of the biochemistry of the cell cycle with the complex kinase cascades involved, and tools to directly visualize the spatial-temporal features of the Ca²⁺ signal at fertilization. In addition, the biology of the egg is well adapted for such studies as it provides a synchronous population of cells arrested at specific stages of meiosis.

II. Ca²⁺ signals during various stages of the cell cycle

The large size of oocytes allows easier experimental access to the cytoplasm in terms of the ability to insert ion sensitive electrodes or microinject aequorin to measure rapid transient Ca²⁺ changes. Such experiments in oocytes discussed in the previous section, coupled to the realization that the mitotic spindle is associated with intracellular membranes, lead to proposals for a role of Ca²⁺ signals in regulating the dynamics of mitosis. Initial attempts to test this idea were performed using the large endosperm cells of Haemanthus with various probes, and showed a correlation between anaphase onset and Ca²⁺ signals [45;46]. Similar studies aimed at identifying Ca²⁺ transients during the mitotic cell cycle of animal cells became possible with the development of membrane permeant Ca²⁺-sensitive fluorescent probes such as Fura-2 [47;48]. Ca²⁺ transients associated with nuclear envelope breakdown, metaphase to anaphase transition and cytokinesis were observed [47-49]. Initial descriptive studies were followed by mechanistic experiments aimed at defining a causal relationship between the observed Ca²⁺ transients during mitosis and subsequent cellular events. It was shown using chelators and Ca²⁺ injection that a Ca²⁺ transient is necessary and sufficient to induce nuclear envelope breakdown (NEBD) and chromatin condensation in sea urchin embryos, and IP₃-dependent Ca²⁺ release was implicated [50;51]. Interestingly, the Ca²⁺ dependency of these events required protein translation [51]. These observations were extended to mammalian cells, where it was shown in Swiss 3T3 fibroblasts using Ca²⁺ uncaging and mild chelation conditions that NEBD is dependent on Ca²⁺ transients, whereas the metaphase to anaphase transition was not as sensitive to alterations in Ca²⁺ signaling [52]. The fact that an inhibitory peptide to CaMKII was able to interfere with NEBD in sea urchin embryos argued that the Ca²⁺-dependency of NEBD was mediated by CaMKII [53].

Surprisingly significant differences were observed in terms of the Ca²⁺ dependence of nuclear envelop breakdown during meiosis as compared to mitosis. In both mouse and Xenopus oocytes, germinal vesicle breakdown occurred independently of Ca²⁺ transients [54;55]. The differential Ca²⁺-dependency of nuclear envelop breakdown in mitosis and meiosis is intriguing especially that in both cases MPF is required [56]. It was argued that GVBD may also be Ca²⁺-dependent through early events that occur before the initiation of oocyte maturation [54;57], this seems unlikely though since long term Ca²⁺-deprivation of Xenopus oocytes (up to 48hrs) does not affect their ability to undergo GVBD [55]. Therefore, the differential Ca²⁺-dependency of GVBD and NEBD may reflect distinct mechanisms controlling both processes.

Oscillations in the levels of IP₃ were also observed to coincide with the mitotic cell cycle in sea urchin embryos, and blocking the IP₃ receptor using heparin inhibited mitosis arguing for a potential functional role for these IP₃ spikes in mitosis progression [58]. In contrast inhibiting Ca²⁺ signals in starfish, mouse and Xenopus oocytes did not affect their ability to enter meiosis and complete GVBD [54;55;59].

Downstream of Ca²⁺ both calmodulin (CaM) and Ca²⁺-CaM-dependent protein kinase II (CaMKII) have been shown to play a role in cell cycle progression. Expression levels of CaM have been linked to cell cycle progression both experimentally and physiologically. CaM levels change during the cell cycle with a pronounced increase during the G1-S transition [60;61], and inducible expression of CaM in mouse cells affect progress through both the G1 and M-phases of the cell cycle [62]. Similarly, CaM in the fungus Aspergillus nidulans is required for the G2-M transition of the cell cycle [63;64], and genetic analyses in the yeast S. cerevisiae show that CaM is required for progression through mitosis [65-68]. Consistent with the idea that CaM effects on the cell cycle may be mediated by CaMKII, knockout of the CaMKII gene in Aspergillus result in a G2-M arrest, showing that CaMKII is required for entry into M-phase [69].Pharmacological inhibition of CaMKII in mammalian cells shows similar results to the knockout in Aspergillus, with an additional apparent requirement for CaMKII during the G1-phase of the cell cycle [70]. Furthermore, expression of a constitutively active CaMKII arrests the cell cycle in G2 [71].

In addition, increased CaM expression levels have been associated with cellular transformation [70]. A decrease in functional CaM levels in DT40 cells results in slower growth [72], and CaM inhibition in mammalian cells interferes with DNA replication and slows cellular proliferation [73;74]. Consistently, serum-dependent induction of DNA synthesis was shown to be dependent on extracellular Ca²⁺ and CaM in normal cells but not in transformed cells [75]. Phosphorylation of the retinoblastoma protein (Rb) also exhibited the same dependence on extracellular Ca²⁺ and CaM [75]. Rb phosphorylation by cyclin D/cdk4 and cyclin E/cdk2 relieves its inhibition of E2F transcription factors leading to the expression of genes required for entry into S-phase [76].

In addition to its well defined role in T-cell activation, calcineurin is also apparently involved in other aspects of cell cycle. During the metaphase to anaphase transition at fertilization in Xenopus eggs calcineurin has been shown to play a role although the details remain to be elucidated [43;44]. In pancreatic acinar cells cyclosporine (CsA) treatment inhibits CREB leading to a reduction in cyclin D expression and arrest in the G1-phase of the cell cycle [77]. However, CsA has also been reported to exhibit cell autonomous tumor promoting effects [78], arguing that inhibition of calcineurin activity depending on the cellular context can either lead to cell cycle arrest or promotion of cellular proliferation.

It is clear from these studies that Ca²⁺ signals play important roles during cell cycle progression. Interestingly however, it seems that the defects tend to be cell type specific arguing that Ca²⁺ signals may be used differentially in distinct cell types to modulate the cell cycle. In addition, the details of the mechanisms of action of Ca²⁺ signaling at different stages of the cell cycle are not we defined.

III. Role of Ca²⁺ signals in the induction of oocyte meiosis

In the addition to the CSF-mediated metaphase II arrest discussed above, vertebrate oocytes also arrest at an earlier stage during the G2-M transition during their growth and maturation in preparation for fertilization. As oocytes develop in the ovary they remain arrested at prophase of meiosis I in a G2-like state of the cell cycle with an intact germinal vesicle (nucleus) and active transcription [79-81]. In contrast, to the CSF-mediated arrest in meiosis II, this arrest is quite stable and can last for several decades in human oocytes as they grow and mature in a sequential fashion [82]. During this long arrest oocytes accumulate macromolecular components to allow them to undergo meiosis/oocyte maturation and then egg activation before transitioning to embryogenesis [79;83]. The signal inducing release from this meiotic arrest varies between species, but ultimately commits the oocyte to oocyte maturation in preparation for fertilization. In mammals meiotic arrest is relieved following release of the oocyte from the follicle at ovulation, whereas in Xenopus meiotic arrest is released in response to progesterone [84;85]. Independent of the signal however, oocyte maturation induces a complex kinase cascade that ultimately activates MPF and commits the oocyte to maturation. The details of this cascade are best defined in Xenopus oocytes with close parallels in mammals. Progesterone treatment results in a decrease in cAMP levels in Xenopus oocytes through the activation of a progesterone cell surface serpentine G-protein coupled receptor [86;87]. This induces poly-adenylation of oocyte mRNAs leading to accumulation of the oocyte specific MAP kinase kinase, Mos [88]. Induction of the MAPK cascade culminates in activation of p90Rsk (Rsk), which inhibits Myt1, a kinase that phosphorylates and inhibits MPF [88;89]. A parallel pathway to the MAPK cascade is also activated leading to the induction of Cdc25C, which dephosphorylates and activates MPF. Combined Cdc25C activation and inhibition of Myt1 lead to a dramatic rapid rise in MPF activity. MPF is composed of cyclin B and Cdk1 the catalytic submit which is maintained in an inactive state due to phosphorylation on Thr14 and Tyr15 mediated by Myt1 kinase [90;91]. Both residues localize to the ATP-binding loop of cdk, and are dephosphorylated by the dual-specificity phosphatase Cdc25 [92]. Dephosphorylation of these residues constitutes the rate limiting step in MPF activation [93].

Given the specific arrest stage of these oocytes at the G2-M transition, there has been a long standing interest in determining whether Ca²⁺ signals play a role in releasing their meiotic. The preponderance of the evidence argue that this is not the case and that release from meiotic arrest occurs independently from a Ca²⁺ signal. However there are reports arguing for a potential role of Ca²⁺ signals in the initiation of oocyte maturation. Incubation of oocytes in high Ca²⁺ and Mg²⁺ concentrations in the presence of ionophore was reported to induce oocyte maturation [94]. Similarly, Ca²⁺ electroporation was also effective at inducing oocyte maturation [95]. If indeed Ca²⁺ is important for the release from meiotic arrest, then one would expect Ca²⁺ transient to be associated with oocyte maturation. Such Ca²⁺ transients were reported following the induction of oocyte maturation in Xenopus by some groups [96-98], but could not be confirmed by others [99;100]. In addition, injection of IP₃ into the oocyte, which releases Ca²⁺ from intracellular stores, did not release meiotic arrest [101]. Furthermore, injection of Ca²⁺ chelators at high concentrations effectively blocked oocyte maturation [95;102]. However, this inhibition is apparently not due to Ca²⁺ chelation, but rather to chelation of transition metals, specifically Zn²⁺[103]. This is plausible since Ca²⁺ chelators such as BAPTA are also powerful chelators of transition metals [104;105]. Indeed it was shown that these chelators when injected into the oocyte inhibit the activation of Cdc25C, and hence MPF and commitment to meiosis [103]. Cdc25C directly binds Zn²⁺, and this is important for Cdc25C substrate recognition and dephosphorylation and activation of MPF [103]. Additional support against a role for Ca²⁺ signals in releasing meiotic arrest comes from a study where oocytes were completely deprived of Ca²⁺ signals by emptying Ca²⁺ stores and incubating the cells in nominally Ca²⁺-free media. Under these conditions oocytes entered meiosis in response to progesterone and underwent GVBD, with the associated activation of the kinase cascade that drives oocyte maturation [55]. However, in the absence of Ca²⁺ signals such oocytes were not able to complete meiosis I because formation of the bipolar spindle and hence polar body extrusion was defective [55]. Similar results were obtained in mouse oocytes [54]. Together these data argue that Ca²⁺ signals are not required for the release of meiotic arrest. This is especially the case when one evaluates the consistent effects of interfering with Ca²⁺ signaling on fertilization in different species as compared to release of oocyte meiotic arrest.

IV. Ca²⁺ signals induce cell cycle reentry of quiescent T-cells following antigen stimulation

Another well defined example for a role for Ca²⁺ signals in cell cycle resumption is during T-cell activation in response to antigen stimulation. Naïve antigen-specific T-cells undergo clonal expansion following antigen stimulation leading to the formation of effector T-cells that are crucial for the adaptive immune response [106]. Antigen stimulation results in cross-linking of the T-cell receptor (TCR)-CD3 complex, which leads to activation of several signaling cascades including PLCγ. PLCγ cleaves its substrate phosphatidylinositol 4,5-bisphosphate (PIP2), generating IP₃ and diacylglycerol (DAG). DAG activates both PKC and RasGRP, leading to activation of NF-κB and the MAPK cascade respectively [106;107]. IP₃ binds and gates the IP₃ receptor Ca²⁺ channel on the ER membrane hence releasing intracellular Ca²⁺ stores and leading to a transient Ca²⁺ rise. Store depletion activates the Ca²⁺-release activated Ca²⁺ (CRAC) channel on the cell membrane resulting in a sustained low amplitude Ca²⁺ influx that is essential for NFAT activation and IL-2 transcription [108]. Together these signaling pathways converge on maintaining NFAT, NF-κB and AP1 transcription factors activity leading to the transcription of genes required for cytokine production and T-cell activation [106;108]. Activation of T-cells results in their transition from the G0 quiescent phase to entry into the cell cycle leading to their proliferation and clonal expansion.

Ca²⁺ influx through SOCE activates calcineurin, a Ca²⁺-dependent phosphatase that dephosphorylates and activates NFAT. The role of calcineurin in T-cell activation was initially revealed in attempts to elucidate the mechanism of action of the immunosuppressive drugs cyclosporine A (CsA) and FK506. Each drug binds to a distinct immunophilin, cyclophilin and FKBP respectively. The immunophilin-immunosuppressant complexes directly bind to and competitively inhibit calcineurin [109;110]. The introduction of these immunosuppressants to the clinic had a dramatic impact on improving patient outcome following organ transplantation, arguing that the major target for these agents are cells of the immune system. Calcineurin is a Ser/Thr phosphatase composed of two tightly associated subunits A and B. Calcineurin A is the catalytic subunit and contains a CaM binding site and an auto-inhibitory domain that maintains the enzyme inactive in the absence of Ca²⁺[111]. Calcineurin B contains four EF-hands Ca²⁺ binding domains and has a similar dumbbell structure to CaM. At resting cytoplasmic Ca²⁺ concentrations (∼100nM) the high affinity Ca²⁺ binding site on calcineurin B are occupied but the phosphatase is inactive. Binding of Ca²⁺-CaM induces a dramatic increase in the V_max of the enzyme leading to its activation [112]. Ca²⁺ and CaM exhibit a high degree of cooperativity in terms of activating calcineurin with a Hill coefficient of ∼3 [112]. This allows the enzyme to respond to a threshold Ca²⁺ signal effectively and hence translate different Ca²⁺ dynamics into gene transcription [113].

NFAT at rest resides in the cytoplasm in a phosphorylated state that possesses a low DNA-affinity [114]. Calcineurin dephosphorylates NFAT, which unmasks its nuclear localization sequence leading to its translocation to the nucleus where it can induce gene transcription [115]. However, NFAT is rapidly phosphorylated by several kinases leading to its export back out of the nucleus. Hence, maintenance of NFAT dependent transcription requires a sustained Ca²⁺ signal to keep NFAT in the nucleus, explaining the dependence of T-cell activation on Ca²⁺ influx through SOCE. SOCE provides a low amplitude sustained Ca²⁺ influx following the initial Ca²⁺ release transient downstream of TCR engagement.

The activation of T-cells is critically dependent on Ca²⁺ influx through the store-operated Ca²⁺ entry pathway which provides the sustained Ca²⁺ influx signal. This is illustrated by the fact that patients with defective SOCE present with severe combined immunodeficiency (SCID) [116-118]. This examples highlights the critical role of Ca²⁺ signals, in this case Ca²⁺ influx from the extracellular space through the store-operated Ca²⁺ entry pathway (SOCE) in inducing re-entry into the cell cycle. Furthermore, it highlights the integration of Ca²⁺ signaling pathways with other signaling modules to induce gene transcription, cell cycle progression and cell activation. Defects in T-cell activation or inactivation can result in serious clinical defects such as autoimmunity or immunodeficiencies.

Store-operated Ca²⁺ entry (SOCE)

The critical role that SOCE plays in the activation of cells in the immune system generated significant interest in identifying it molecularly. Biophysical characterization of the SOCE current in immune cells was the first step in that endeavor. The SOCE current in immune cells was referred to as the Ca²⁺ release-activated current (I_CRAC) and is the best characterized SOCE current biophysically. It is highly Ca²⁺-selective and exhibits strong inward rectification [119;120]. The initial idea that Ca²⁺ store content regulates Ca²⁺ influx at the cell membrane was proposed in the capacitative model, which suggested that Ca²⁺ flows directly into the ER lumen [121]. Although we now know that Ca²⁺ influx through SOCE channels flows into the cytosol, this model generated significant interest in this ubiquitous Ca²⁺ entry mechanism. SOCE is not only critical in the context of cell cycle re-entry of T-cells but is also involved in various physiological processes ranging from secretion to muscle development [122;123]. Despite the intense interest in SOCE it took two decades to identify the molecular players involved through the use of large scale RNAi screens. These experiments identified stromal interaction molecule 1 (STIM1) as the ER Ca²⁺ sensor that senses Ca²⁺ store depletion and transmits the signal to the cell membrane. STIM1 is an ER membrane protein with luminal EF hands that sense Ca²⁺ store content [124;125]. The CRAC channel at the cell membrane, Orai1, was identified as a mutation in two siblings with severe combined immunodeficiency (SCID) coupled to the use of genome wide RNAi screens in Dropsophila [116;126;127]. Orai1 is an integral membrane protein with 4 transmembrane domains and N- and C-terminal cytoplasmic domains. Overexpression of STIM1 and Orai1 produces large I_CRAC-like currents that activated only after store depletion, are highly Ca²⁺ selectivity, and inward rectifying, which mirrors the properties of the endogenous I_CRAC[127-129].

STIM1 and Orai1 combine to couple Ca²⁺ store depletion to activation of Ca²⁺ influx at the cell membrane. Store depletion releases Ca²⁺ from the low affinity STIM1 EF-hand Ca²⁺ binding domain leading to clustering of STIM1 into large puncta that are stabilized in an ER domain that localizes 10-20nm below the cell membrane. These STIM1 puncta recruit Orai1 and gate it open thus stimulating Ca²⁺ entry at the cell membrane [130]. With the identification of STIM1 and Orai1, it became possible to directly test the requirement for SOCE in T-cell activation. Knockout studies of STIM1 and Orai1 coupled to identification of additional mutations in human patients with immunodeficiencies in both proteins nicely confirmed the essential role SOCE in T-cell activation, because the major phenotype in these patients and knockout animals was defects in the activation potential of cells of immune system leading to immunodeficiencies [131]. The absence of either STIM1 or Orai1 was also associated with congenital myopathy and ectodermal dysplasia consistent with the role of SOCE in myoblast differentiation [123;132].

Interestingly, in addition to the role of SOCE in mediating cell cycle progression in T-cells, the SOCE current itself is regulated during cell division. SOCE inactivates during both mitosis and meiosis arguing that this is a conserved mechanism of regulating Ca²⁺ influx during cellular division [133;134]. SOCE inactivation requires MPF activity [135]. Although speculative at this point, SOCE inactivation may prevent maverick Ca²⁺ signals during cellular division that may interfere with normal cell division especially given the potential role of Ca²⁺ signals during M-phase as discussed in details above [136]. SOCE inactivation was recently shown to be due to both internalization of the Orai1 channel and the inability of STIM1 to form clusters in response to store depletion in oocytes [137;138].

SOCE is not the only Ca²⁺ signaling pathway that is regulated during the cell cycle. In fact Ca²⁺ signaling are dramatically remodeled during oocyte meiosis. IP₃-dependent Ca²⁺ release is sensitized [139-142] and the plasma membrane Ca²⁺-ATPase (PMCA) is internalized [143;144]. This remodeling allow the egg to produce the fertilization-specific Ca²⁺ transient that is essential for egg activation [22].

Perspective

It is clear from this brief review that Ca²⁺ signals are associated with progression of the cell cycle. However, because Ca²⁺ signals are involved in so many aspects of cellular physiology it is tricky to define the relationship between Ca²⁺ signaling and the cell cycle machinery. That is interfering with Ca²⁺ signaling could eventually leads to cell cycle arrest through indirect mechanisms. However in at least two cases, at vertebrate fertilization and during T-cell activation, there is a well defined link between Ca²⁺ signals, with specialized dynamics, and cell cycle progression. In both cases cells are arrested at a specific stage of the cell cycle, for the egg it is in metaphase of meiosis II by CSF, and for T-cell it is in the quiescent G0-phase of the cell cycle. In fact Ca²⁺ is the universal signal for egg activation at fertilization in all species studied to date, despite the fact that eggs from different species arrest at different stages of meiosis [20]. Furthermore the source of the Ca²⁺ transients differs. At fertilization it is primarily due to Ca²⁺ release from intracellular stores, whereas for cells of the immune system it is through Ca²⁺ influx through the store-operated Ca²⁺ entry pathway. The combination of different Ca²⁺ signaling pathways in distinct cell types generates disparate Ca²⁺ dynamics: a sustained Ca²⁺ rise in T-cells, versus prolonged Ca²⁺ oscillations following fertilization in mammals. Moreover, interfering with Ca²⁺ signaling in different cell types results is varied effects on the cell cycle. Together these observations argue that Ca²⁺ signals have been adapted through evolution to fit specific needs in cell cycle progression in a cell type specific fashion based on the cell's developmental stage. Ca²⁺ is a fitting second messenger for such a function because resumption of the cell cycle requires activation of a multitude of signaling cascades in a specific temporal sequence. Calcium's versatility as a second messenger and ability to branch out and activate different signaling modules given the diversity of Ca²⁺-dependent enzymes and processes make it a well-adapted messenger for this function.