Biol Res 35:195-202, 2002

IP₃ dependent Ca²⁺ signals in muscle cells are involved in
regulation of gene expression.

ENRIQUE JAIMOVICH AND MARIA ANGELICA CARRASCO

Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 6530499, casilla 70005, Santiago , Chile.

ABSTRACT

Potassium depolarization of cultured muscle cells was employed to study cellular responses linked to calcium signaling. Events occurring after depolarization include i) A transient increase of the IP₃ mass (2-10s); ii) A slow calcium transient (5 to 25s) that propagates as a low concentration wave along the myotube showing a distinct calcium transient at the level of cell nuclei. Due to the presence of IP₃ receptors both in the SR (A-band region) and in the nuclear envelope, these two events appear to be related; iii) Phosphorylation of mitogen activated kinases (ERK 1/2) and of the transcription factor CREB (30 s-10 min), as well as expression of the early genes c-fos, c-jun and egr-1 mRNA (5-15 min). Several independent pieces of evidence, including results obtained using inhibitors specific for individual steps, allowed us to connect these in a sequential manner. As the same type of signaling cascade can be triggered by oxidants, neurotransmitters and hormones, the ensemble of results allows us to propose a general model to describe signaling events that link membrane stimulation to regulation of gene transcription in skeletal muscle cells.

Key terms: Excitation-transcription; inositol trisphosphate; mitogen-activated kinases; CREB; early genes; dihydropyridine receptors.

INTRODUCTION

Increased use, decreased use and injury are known to induce different forms of muscle adaptation. However, the cellular mechanisms responsible for these changes remain unknown. Since the process of muscle adaptation is completely dependent on nerve activity and on nerve-muscle interaction, it constitutes a good model to study plasticity and regeneration of excitable cells. Cultured muscle cells allow the study of many functions characteristic of adult muscle, including growth, differentiation, adaptation and contraction.

Between 1981 and 1989, a number of papers were published demonstrating the presence in skeletal muscle membranes of the metabolic machinery needed to produce inositol (1,4,5) trisphosphate (IP₃) (Jaimovich, 1991). Indeed, IP₃ was capable of releasing calcium from internal stores in ruptured fibers (Rojas and Jaimovich, 1990), but no role for IP₃ in excitation-

contraction (E-C) coupling was apparent. To gain insight to this problem, studies in intact cells were necessary, and we choose cultured skeletal muscle (both primary culture and cell lines, some developed in our laboratories) as a model. A series of recent publications, as well as yet unpublished work, suggest a role for IP₃ in the regulation of nucleoplasmic calcium, leading to early gene expression in these cells. Most interestingly, dihydropyridine receptors (DHPR) appear again in a role as voltage sensors, now for excitation-transcription coupling.

In multiple types of mammalian cells the level of intracellular Ca²⁺ controls an enormous number of cell functions, including cell division, secretion, gene expression, cell motility, and muscle contraction (Berridge, 2000). Different qualitative and quantitative aspects of Ca²⁺ signals, including amplitude, frequency and localization, determine the specificity of cellular responses (see Petersen, 2002). The problem of Ca²⁺- dependent regulation of gene expression, and thus cellular differentiation, is especially intriguing to the developmental biologist. When one thinks of Ca²⁺ signals in skeletal muscle, the exquisite control of E-C coupling comes to mind. This involves DHPRs and activation of the ryanodine receptors (RyR), the Ca²⁺ release channels of the sarcoplasmic reticulum (SR). The complex regulation of RyR activity in skeletal and cardiac skeletal muscle, by Ca²⁺, Mg²⁺ and redox state, is discussed elsewhere (Hidalgo et al., 2002).

Assuming Ca²⁺ is employed as a second messenger to control functions other than those limited to contraction of skeletal muscle, it is likely that both the kinetics of the Ca²⁺ signals and the subcellular location, as well as the nature of the Ca²⁺ release channels involved, would be different from those of the channels linked to E-C coupling. In many cells, intracellular increases in Ca²⁺ are mediated by the IP₃ cascade (Berridge, 1993). The activation of various types of receptors in different cells induces the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate IP₃ and diacylglycerol, which are activators of intracellular Ca²⁺ channels and protein kinase C, respectively. Indeed, skeletal muscle fibers possess the basic molecular machinery that constitutes a functioning IP₃ messenger system, including phosphatidylinositol-4-kinase, phosphatidylinositol-4 phosphate-5-kinase (Hidalgo et al., 1986; Carrasco et al., 1988), phospholipase C (Carrasco et al., 1993), inositolphosphate phosphatases (Sanchez et al., 1991), IP₃ 3-kinase (Carrasco and Figueroa, 1995), and G-proteins (Scherer et al., 1987; Carrasco et al., 1994). Some of these enzyme activities, particularly that of phosphatidylinositol-4 phosphate-5-kinase, have been localized to the transverse tubule (T-tubule) system (Carrasco et al., 1988). In addition, IP₃-sensitive Ca²⁺ channels have been found in the SR (Volpe et al., 1986; Suarez-Isla et al., 1988). Thus, depolarization of the T-tubule system could lead to IP₃ production and release of internal Ca²⁺ in addition to Ca²⁺ release through the RyRs.

Slow calcium signals in skeletal muscle cells.

In skeletal muscle cells, Ca²⁺ release from internal stores may follow more than one set of kinetics and may have more than one function. Ca²⁺ waves, unrelated to Ca²⁺ spikes involved in E-C coupling, have been reported in both chicken and rodent myotubes (Flucher and Andrews, 1993; Powell et al., 1996). We have shown that the complex Ca²⁺ release response, induced by elevated potassium, involves two components with different kinetics (Jaimovich and Rojas, 1994; Jaimovich et al., 2000). One is a fast Ca²⁺ transient, known to be associated with the RyR and E-C coupling. The other a slower transient with as yet poorly defined causes and unknown functions. DHPRs appear to be the membrane voltage sensors for the slow calcium signal, since 10 µM nifedipine completely blocked the slow Ca²⁺ transient after K⁺ depolarization but only partially reduced the fast Ca²⁺ signal. In dysgenic myotubes, which do not express the alpha 1 subunit of the DHPR (GLT cell line), no calcium signal after depolarization was detectable (Araya et al., manuscript in preparation). After transfection of the alpha 1 subunit, K⁺ depolarization induced slow calcium transients that were similar to those present in normal C₂C₁₂ and NLT cell lines.

Also, depolarization of cultured muscle leads to an increase in IP₃ mass (Jaimovich et al., 2000). Recently, presence of the IP₃R has been shown both biochemically (Liberona et al., 1998) and immunocytologically (Jaimovich et al., 2000; Powell et al., 2001) in normal and diseased muscle, further suggesting a role for IP₃ signals. Biochemical localization of IP₃Rs to isolated nuclei of developing muscle further implicates the IP₃ cascade in nuclear signaling events (Liberona et al., 1998). To understand this possible role of IP₃Rs in the propagation of such Ca²⁺ transients and perhaps in the modulation of growth and development of skeletal muscle, we investigated the expression and subcellular localization of these receptors in cultured muscle. Furthermore, we explored the link between IP₃Rs and slow Ca²⁺ signals, and sought possible downstream targets for these signals in muscle cells.

Expression of mRNA and proteins of the IP₃R isotypes

At the mRNA and protein levels, three IP₃R isoforms have been previously demonstrated to be present in different proportions in a number of rodent cell lines (Newton et al., 1994; Wojcikiewicz, 1995; De Smedt et al., 1997). In particular, the study of mRNA expression performed by De Smedt et al., (1997) has shown that in three mouse skeletal cell lines, including C₂C₁₂ cells, IP₃R type 3 is clearly the most abundant transcript. The proportion of types 1 and 2 vary in these cell lines, but there was a tendency for type 2 to increase and for type 3 to decrease as differentiation proceeds. Our IP₃ binding results (Liberona et al., 1998; Jaimovich et al., 2000) show that at least two isoforms are present in the nuclear region. In addition, these binding studies indicate that a high proportion of the IP₃Rs are found in the nuclear fraction in both cell lines and primary cultures. Nuclear localization of specific IP₃R isoforms in skeletal muscle has not been described in the literature. To confirm and extend these findings we have looked at the expression (mRNA and protein) and the localization of the IP₃R (Powell et al., 2001a,b). Confocal microscopy of cultured mouse muscle, doubly labeled for the IP3R type 1 and proteins of known distribution, reveals that these receptors are localized to the I band of the sarcoplasmic reticulum and shows that the staining for IP₃R type 1 is continuous with staining of the nuclear envelope region.

Link between IP₃Rs and cytoplasmic Ca²⁺ transients

The slow calcium signal (monitored as the response to fluo-3) moves through the cytoplasm and seems to trigger even higher Ca²⁺ concentrations inside nuclei, perhaps by moving from the region of the nuclear envelope to the nucleoplasm. In fact, the slow calcium wave can be envisaged as having two components, a more rapid, diffuse component (slow-rapid) of low fluorescence intensity, that always precedes a localized high fluorescence wave (slow-slow component) that propagates from one nucleus to another.

The role of the IP₃Rs in the I-band SR region could be related to the diffuse, low fluorescence intensity, cytosolic component, we see always associated with localized nuclear Ca²⁺ increases. Since the cell does not contract during either part of the slow wave, this indicates that the overall cytosolic calcium concentration remains below the contraction threshold and that high fluorescence areas must be highly compartmentalized, most probably in the nuclear region (Estrada et al., 2000; Jaimovich et al., 2000). As expected, high concentrations of ryanodine eliminated the initial fast calcium increase associated with contraction, and interestingly it also eliminated the first or slow-rapid cytosolic propagation component as well. However the second slow calcium rise (slow-slow) phase was preserved. This suggests that a ryanodine sensitive calcium pool is involved in propagation of the slowrapid Ca²⁺ wave through the cytosol. The apparently different intracellular distribution of receptors, RyR in the SR membranes and IP₃Rs, at least in some developmental stages, concentrated in membranes associated with the nuclei, points to the presence of two separate calcium release systems (Humbert et al., 1996; Guihard et al., 1997; Liberona et al., 1998; Petersen et al., 1998). We propose that the role for the IP₃R is to maintain cytosolic calcium concentrations within the appropriate range in distinct sub-cellular regions and, more specifically, to control nuclear calcium release.

An important finding was that pre-incubation of cells with 2-APB (2-aminoethoxydiphenyl borate) inhibited slow Ca²⁺ signals (Estrada et al., 2001; Powell et al., 2001). This compound has been shown to be a fairly specific inhibitor of IP₃ mediated Ca²⁺ release. The complete absence of slow Ca²⁺ waves in the presence of 2-APB suggests that these signals are mediated by IP₃Rs. Similar results were obtained using other compounds interfering with the IP₃ system, such as the IP₃R blocker xestospongin-C, or the PLC inhibitor U-73122 (Estrada et al., 2001). As IP₃R channels require both IP₃ and Ca²⁺ for activation, the function of IP₃Rs located in the I-band SR region could be to propagate a local Ca²⁺ wave that would ensure Ca²⁺ availability in the nuclear region, required for local Ca²⁺ release and regulation of gene expression (see figure 1), without causing contraction.

Role of RyRs in slow calcium signals

In order to define the role of RyRs, in either component of the slow release process, we compared the calcium signals in muscle cells (1B5) from dyspedic mice, which do not express any of the RyR isoforms and lack E-C coupling (Buck et al., 1997; Moore et al., 1998; Fessenden et al., 2000; Kiselyov et al., 2000; Protasi et al., 2000; Ward et al., 2000) to C₂C₁₂ cells, which have wild type calcium signals (Estrada et al., 2001). Our data show that in the absence of RyR expression, cultured dyspedic skeletal myotubes retain the slow delayed intracellular calcium transient that is seen as a second phase of release after K⁺ depolarization in normal myotubes expressing RyR. This slow delayed release appears to require IP₃ receptors that are expressed in both cell types. Our experimental evidence to support this hypothesis is summarized as follows: First, fast and slow calcium signals are detected in primary culture myotubes, as well as in a mouse muscle cell line, like C₂C_12, following depolarization induced by high potassium. Second, 1B5 "dyspedic" mice muscle cells, which do not express any of the ryanodine receptor isoforms (confirmed by [³H]-ryanodine binding and immunocytochemistry), show particularly notable nuclear calcium increases in response to K⁺ depolarization. The kinetics of this transient is comparable to those of the slow signal observed in rat and mouse primary cultures (Jaimovich and Rojas 1994; Estrada et al., 2000; Jaimovich et al., 2000) or C₂C₁₂ cells. Both the fast calcium transient, responsible for E-C coupling, and the fast-slow wave, associated with signal propagation, are absent in 1B5 myotubes. The fast calcium signal is restored in these cells upon re-expression of RyR1, but not RyR3 (Moore et al., 1998; Fessenden et al., 2000; Protasi et al., 2000, Galaz et al., 2001). Third, [³H]-IP₃ binding and western blot analysis show the presence of IP₃Rs in both 1B5 and C₂C₁₂ muscle cells. The presence of IP₃R isoforms was confirmed by immunocytochemistry. Type 1 IP₃Rs were localized preferentially to the nuclear envelope and both type 1 and type 3 IP₃R immunoreactivity was higher in 1B5 cells compared to C₂C₁₂. Finally, in both 1B5 and C₂C₁₂ cells, K⁺ depolarization resulted in an increase in IP₃ mass (Estrada et al., 2001). The kinetics of the IP₃ mass transient is compatible with IP₃ release preceding the calcium transient (see Figure 1).

Fig. 1: The scheme represents our present knowledge of the pathways involved either as causes or consequences of slow calcium waves induced by membrane depolarization in skeletal muscle cells. DHPR appears as the voltage sensor in the T-tubule membrane, communicating a signal (via a G-protein?) to phospholipase C (PLC). IP3 is liberated and diffuses to receptors located both in the SR region and in the nuclear envelope. Subsequent calcium increase will affect intracellular pathways, such as ERK 1/2 phosphorylation, CREB phosphorylation and regulation of early gene expression.

Calcium regulation of signal transduction and transcription in skeletal muscle

Calcium involvement in signaling pathways that lead to changes in gene expression is a well-established phenomenon. We have reported that depolarization of myotubes induces calcium signals both in cytosol and nucleus via IP₃, as well as phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 and the cAMP-response element-binding protein (CREB) (Powell et al., 2001). We have also shown (Carrasco et al., manuscript in preparation) that K⁺-induced depolarization of rat myotubes elicits a transient increase in mRNA levels of the early genes c-fos, c-jun and egr-1. Both CREB phosphorylation and activation of early genes were inhibited by blocking ERK phosphorylation. All three events (CREB phosphorylation, early gene expression and ERK phosphorylation) were maintained in the absence of extracellular calcium, while caffeine induced an increase in intracellular calcium that mimicked the depolarization stimulus. Depolarization either in the presence of 2APB (inhibitor of IP₃ induced calcium release) or of cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the three early genes mentioned above. The intracellular pathways implicated so far from our studies are summarized in figure 1.

In neurons, a number of calcium-dependent effects on transcription factors, such as CREB, and on early genes, such as c-fos, have been described. Depolarization induces ERK activation (see Rosen et al., 1994; Robertson et al., 1999; Vanhoutte et al., 1999), CREB phosphorylation and c-fos activation (reviewed in Ghosh and Greenberg, 1995; Bito et al., 1997; Hardingham and Bading, 1999). The c-fos gene has two major calcium-responsive regulatory elements in its promoter region, the CRE (calcium/cAMP responsive element) and the SRE (serum response element). In neurons, Ca²⁺ influx, as result of membrane depolarization (or NMDA receptor activation), stimulates CREB phosphorylation, a response necessary for CRE-mediated transcription. The role of Ca²⁺is to enhance the activity of Ca²⁺-dependent kinases that phosphorylate CREB, as has been demonstrated in several neuronal and non-neuronal models (Hardingham and Bading, 1999; Shaywitz and Greenberg, 1999). Phosphorylated CREB recruits CBP (CREB binding protein), a transcriptional co-activator, and studies concerning the calcium requirement for both CREB phosphorylation and CBP activity have yielded very interesting insights to the mechanism of transcriptional regulation by calcium (Bading, 2000). Briefly, CREB phosphorylation was found to be necessary but not sufficient for transcription to occur (Chawla et al., 1998) and changes in nuclear rather than cytoplasmic Ca²⁺ were able to induce CBP activation, presumably by phosphorylation (Chawla et al., 1998; Hu et al., 1999). Serine 301 has been recently identified as the relevant CBP phosphorylation site (Impey et al., 2002). Since CBP interacts with many transcription factors in addition to CREB, CBP activation provides a mechanism by which nuclear Ca²⁺ stimulates a large number of transcription factors, including c-jun, and the expression of many genes (reviewed in Bading, 2000). SRE, the other calcium-responsive element in the c-fos promoter, contains two binding sites, one for the serum response factor (SRF) and another for ternary complex factors (TCF) of the Elk-1 family of ETS domain transcription factors. In this manner, SRE functions as an integrator of two calcium signaling pathways. Interestingly, SRE responds in neurons to cytoplasmic calcium , as opposed to CRE-mediated transcription which requires nuclear calcium (Hardingham and Bading, 1999).

In skeletal muscle cells, two major stimuli have been identified for growth and gene expression: electrical muscle activity (involving membrane depolarization) and growth factors. Muscle activity has long been known to stimulate (Brevet et al., 1976) or inhibit (Cohen and Fischbach, 1973) the production of extrajunctional muscle specific proteins, yet the series of molecular events linking muscle activity to cellular expression and accumulation of contractile proteins has not been defined. Activation of the ERK isoforms 1 and 2, as a consequence of muscle stimulation, has been widely reported (see for instance Goodyear et al., 1996; Sherwood et al., 1999; Ryder et al., 2000). However, a specific role for these MAP kinases in skeletal muscle gene expression was only recently demonstrated (Murgia et al., 2000). In regenerating muscle, transfection of constitutively active Ras and a Ras mutant that selectively activates ERKs, could mimic the effects of slow motor neuron activity on myosin gene expression. Several reports have shown that exercise or electrical activity increase early gene mRNA levels (Abu-Shakra et al., 1994; Michel et al., 1994: Osbaldeston et al., 1995; Aronson et al., 1997; Punschart et al., 1998). Since most immediate early gene products are transcription factors that bind to specific promoter sequences and regulate expression of downstream genes, they are likely to be involved in adaptative responses induced by neural activity and contractile work in skeletal muscle.

Intracellular calcium response in skeletal muscle cell cultures can be mediated by hormones and neurotransmitters

We have identified (Reyes and Jaimovich, 1996) the presence of functional muscarinic receptors in cultured muscle cells. Either muscarine or carbachol induced IP₃ increases that were associated with calcium transients in these cells, suggesting the presence of a metabotropic, G-protein mediated pathway.

Fast, non-genomic steroid actions in several cell types seem to be mediated by second messengers such as intracellular Ca²⁺ and IP₃. We have studied the effect of steroids on intracellular calcium by monitoring Fluo 3-AM loaded myotubes using either confocal or fluorescence microscopy. We observed (Estrada et al., 2000) intracellular calcium changes after either aldosterone (10-100 nM) or testosterone (10 nM) addition. A relatively fast (less than 2 min) calcium transient, frequently accompanied by oscillations, was observed with both hormones. A slow rise in intracellular Ca²⁺ that reached its maximum after 30 min was seen upon exposure to aldosterone. Calcium responses appear to be fairly specific for aldosterone and testosterone, as several other steroid hormones do not induce detectable changes in fluorescence even at 100-fold higher concentrations. The mass of IP₃ increased transiently to values 2-3 fold higher than the basal levels, 45 seconds after addition of either aldosterone or testosterone. The IP₃ transient was more rapid than the hormone induced fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor or cyproterone, an inhibitor of the testosterone receptor neither affected the fast Ca²⁺ signal, nor the increase in IP₃ mass (Estrada et. al., 2000, Estrada and Jaimovich, 2001). Taken together, these results indicate that distinct non-genomic signaling pathways exist for the action of these two steroids in skeletal muscle cells.

ACKNOWLEDGEMENTS

This work was supported by FONDECYT No. 8980010 and FONDAP 15010006.

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Corresponding author: E. Jaimovich, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile Casilla 70005, Santiago 6530499, Chile. Phone: (56-2) 678-6311; FAX: (56-2) 777-6916; e-mail: ejaimovi@machi.med.uchile.cl

Received: June 05, 2002. In revised form: July 03, 2002. Accepted: July 14, 2002