1 Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile
2 Institut Fédératif de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040 CNRS, 91198 Gif-sur-Yvette CEDEX, France
3 Laboratorio de Fisiología de La Conducta, Facultad de Medicina, Universidad de Los Andes, Mérida 5101, Venezuela
4 Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA
* Author for correspondence (e-mail: ejaimovi{at}med.uchile.cl)
Accepted 20 April 2005
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Summary |
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Key words: Skeletal muscle, Myonuclei, Inositol 1, 4, 5-trisphosphate receptors, Nuclear envelope, Transcription factors, Gene expression
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Introduction |
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IP3Rs are a family of Ca2+-permeable channels composed of three isoforms that differ in amino acid sequence, affinity for IP3 and modulation by Ca2+ (Joseph, 1996). The subcellular distribution of IP3R isoforms is also different in many cells, and the distribution of nuclear isoforms in particular remains unclear. The IP3Rs in the nucleus could be activated locally after generation of IP3 by the nuclear phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] present in the nuclear membrane (Irvine, 2000
). Moreover, the nucleus has other components needed for IP3 metabolism, as PtdIns 3-kinases, PtdIns 4-kinases, PtdIns(4)P 5-kinases and diacylglycerol kinases (Maraldi et al., 1999
). Thus, the nucleus has the capability to regulate nucleoplasmic Ca2+, independently of cytoplasmic Ca2+ levels.
Nuclear Ca2+ increases have been suggested to control the cAMP response element binding protein (CREB)-mediated gene expression in neurons (Hardingham et al., 1997; Hardingham et al., 2001
). However neither the source of Ca2+, nor the participation of nuclear channels is clear. It was recently reported that nuclear K+-ATP-dependent channels triggered by nuclear Ca2+ increases induced CREB phosphorylation in isolated nuclei (Quesada et al., 2002
). However, the possible participation of IP3Rs in modulating the CREB phosphorylation response in nuclei is unknown.
Our previous findings of Ca2+ signals mediated by IP3 with an important nuclear component, following depolarization of skeletal myotubes (Jaimovich et al., 2000; Powell et al., 2001
; Eltit et al., 2004
), or of different muscle cell lines (Estrada et al., 2001
) led us to consider the possibility that IP3Rs have a role in the generation of nuclear Ca2+ signals. It has been suggested that the IP3-mediated Ca2+ increase regulates early gene expression (Powell et al., 2001
; Araya et al., 2003
; Carrasco et al., 2003
).
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Materials and Methods |
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Cell culture and immunostaining
Muscle cells in primary culture and myoblasts of the immortalized dyspedic mouse cell line 1B5 (kindly provided by Paul Allen, Brigham and Women Hospital, Boston, MA) were prepared as described previously (Jaimovich et al., 2000; Moore et al., 1998
; Takeshima et al., 1994
). Immunocytochemistry of isolated nuclei and cultured cells was performed essentially as previously reported (Powell et al., 1996
). Briefly, cells and nuclear monolayers placed on coverslips were fixed with methanol at 20°C for 12 minutes, blocked in 1% BSA and incubated with primary antibodies overnight at 4°C. Cells or isolated nuclei were then washed and incubated with secondary antibody for 1 hour at room temperature. Coverslips were mounted in Vectashield (Vector Laboratories) for confocal microscopy and representative images were acquired (Carl Zeiss Axiovert 135, LSM Microsystems). Negative controls had only secondary antibodies applied. For type-3 IP3R, the pre-immune serum was also used. The images reproduced were manipulated in Adobe PhotoshopTM to improve clarity; no information was added or deleted by those adjustments.
Isolation of nuclei
Highly purified nuclei were obtained by the combined use of a hypotonic shock and mechanical disruption in a Dounce homogenizer as previously reported (Martelli et al., 1992). Briefly, cells (10 x106) were washed in saline phosphate buffer and incubated in 0.1 % trypsin (v/v) for 20 minutes at 37°C and later scraped off using a rubber policeman. Cells were precipitated at 4000 g to eliminate trypsin. The resulting cell pellet was suspended in hypotonic buffer (10 mM Tris-HCl, pH 7.8, 10 mM ß-mercaptoethanol, 0.5 mM PMSF, 1 µg/ml aprotinin, leupeptin and pepstatin). After 20 minutes on ice the swollen cells were broken in a Dounce homogenizer. The nuclear pellet was obtained by centrifugation at 500 g for 6 minutes at 4°C and was then washed in 10 mM Tris-HCl, pH 7.2, 2 mM MgCl2 plus the protease inhibitors. Finally the nuclear pellet was resuspended in 10 mM HEPES-Tris-HCl (pH 7.6), 110 mM KCl, 1 mM MgCl2 and protease inhibitors. Nuclear integrity was checked by electron and confocal microscopy. After the nuclear pellet was obtained, a `crude microsomal fraction' was obtained as described (Jaimovich et al., 2000
) and this fraction was used for the [3H]IP3 binding experiments.
Immunoelectron microscopy
Purified myonuclei were centrifuged for 10 minutes at 4000 g and the nuclear pellet was immediately fixed for 1 hour at room temperature with 4% formaldehyde and 0.1% glutaraldehyde (Polyscience) in phosphate buffer, pH 7.4. The nuclear pellet was dehydrated in a graded ethanol series and embedded in LR-White resin (Electron Microscopy Sciences, Hatfield, PA). All immunolabeling was carried out at room temperature. Silver-grey thin sections were treated with 0.1% sodium borohydride (Sigma Chemical, St Louis, MO) PBS solution for 15 minutes. Non-specific labeling was blocked with PBS solution containing 5% acetylated BSA, 5% normal goat serum and 0.1% coldwater fish skin gelatin (Sigma). Samples were incubated with the same polyclonal rabbit anti-type-1 or type-3 IP3R antibodies described above. Grids were then incubated for 2 hours with goat anti-rabbit IgGs conjugated to 10 nm colloidal gold particles (BioCell, Cardiff, UK). After thorough washing, samples were fixed with 2.5% glutaraldehyde (Polyscience) in PBS and post-fixed with 1% osmium tetroxide vapors. Finally, grids were counterstained with 2.5% uranyl acetate and 1% lead citrate and examined using a Philips CM-10 electron microscope.
[3H]IP3 binding
Radioligand binding assays for [3H]IP3 were performed as previously described (Liberona et al., 1998). Briefly, isolated nuclei from confluent plates of dyspedic mouse cell lines, 5-7 days after withdrawal of serum, were washed three times with PBS and homogenized with a Dounce homogenizer. They were then incubated in a medium that contained 50 mM Tris-HCl, pH 8.4, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 10-200 nM [3H]IP3 (D-[2-3H]-myo-inositol 1,4,5-trisphosphate), specific activity 21.0 Ci/mmol (DuPont, NEN), 800-1000 cpm/pmol at 4°C for 30 minutes. After incubation, the reaction was stopped by centrifugation at 10,000 g for 10 minutes, the supernatant was aspired and the pellets were washed with PBS and dissolved in 1M NaOH to measure radioactivity. Nonspecific binding was determined in the presence of 2 µM IP3 (Sigma).
Western blot analysis
Cells were lyzed in 60 µl ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5 mM Na3VO4, 20 mM NaF, 10 mM NaPP and a protease inhibitor cocktail (Calbiochem). Cell lysates were sonicated, incubated on ice for 20 minutes and centrifuged to remove debris. Protein concentration of the supernatants was determined with BSA as standard. Lysate proteins were suspended in Laemmli buffer, separated in 10% SDS-polyacrylamide gels and transferred to PDVF membranes (Millipore). Blocking was at room temperature for 1 hour in 3% fat-free milk, and the membranes were incubated overnight with the appropriate primary antibody. Immunoreactive proteins were detected using ECL reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. For CREB analysis, the films were scanned, and densitometry analysis of the bands was performed using the Scion Image program (http://www.scioncorp.com). To correct for loading, membranes were stripped in buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS and 50 mM ß-mercaptoethanol at 50°C for 30 minutes, and reprobed with the corresponding antibodies.
Confocal microscopy and fluorimetry for Ca2+
Nuclei were pre-incubated in a resting solution containing 125 mM KCl, 2 mM K2HPO4, 50 mM HEPES, 4 mM MgCl2 and 0.1 mM EGTA (with <5 nM free Ca2+), plus 5.4 µM fluo-3/AM (Molecular Probes, Eugene, OR) for 45 minutes at 4°C and then were incubated in a solution containing 140 mM KCl, 10 mM HEPES, 1 mM MgCl2, 100 µM EGTA, 75 µM CaCl2 with 200 nM free Ca2+ (estimated using the Winmaxc program, http://www.stanford.edu/~cpatton/winmaxc2.html) for loading. Loaded nuclei were washed and centrifuged, mounted in a 1 ml capacity perfusion chamber and placed in the microscope stage of a confocal laser-scanning system (Carl Zeiss Axiovert 135 M, LSM Microsystems) for fluorescence measurements. Nuclei were then stimulated with 10 µM IP3 and fluorescent images were collected every second and analyzed frame by frame with a data acquisition program. The same protocol was used when the nuclei were stimulated in the presence of 50 µM 2-APB an inhibitor of IP3 signals. Fluorimetric experiments were also performed. A suspension of nuclei was loaded with either mag-fluo-4 or fluo-4 dextran under the same conditions described for fluo-3/AM above. Fluorescence was measured using a multi-label reader Mithras LB 940 (Berthold technologies, Bad Wildbad, Germany).
Data analysis
All experiments were performed a minimum of three times. Results are expressed as mean±s.e.m. The significance of any differences among treatments was evaluated using the Student's t-test for paired data, or analysis of variance followed by Dunnett's post-test for multiple comparisons.
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Results |
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Type-1 IP3R colocalizes with a specific marker of the inner nuclear membrane
To examine whether IP3R localization in the nucleoplasm corresponds to invaginations of the inner nuclear membrane, as described for the nucleus of a glioma cell line (Lui et al., 1997), or are part of an independent structure, double immunostaining of IP3R was performed with LAP2, a specific inner nuclear membrane protein marker. LAP2 showed a continuous staining pattern around the nuclei (Fig. 2), and also in invaginations of the inner nuclear membrane towards the nucleoplasm. Superimposition of type-1 IP3R with LAP2 labels showed a high colocalization, suggesting that type-1 IP3R is located in the nuclear envelope (Fig. 2A, lower panel). As expected, type-2 IP3R did not colocalize with LAP2, confirming that type-2 IP3R is confined to the reticulum (Fig. 2B, lower panel). Type-3 IP3R generally showed no colocalization with LAP2 in the nuclear envelope and invaginations, but immunostained the nucleoplasm with a reticular pattern in areas not labeled with LAP2 (Fig. 2C, lower panel). However, in a few cells (not shown), partial colocalization with LAP2 was detected. This suggests that type-3 IP3R labels some unknown nucleoplasmic structure that may correspond to the recently described nucleoplasmic reticulum (Echevarria et al., 2003).
Characterization of isolated myonuclei
To further substantiate the observation that IP3Rs are present in the nucleus, we characterized a preparation of purified nuclei of cultured cells with minimal contamination from other organelles whilst conserving nuclear integrity. The isolation of nuclei was based on the application of a hypotonic shock combined with mechanical disruption (Martelli et al., 1992). The specific inner nuclear membrane marker LAP2 shows integrity and homogeneity of the preparation (Fig. 3A). In addition, immunoblot analyses were performed to determine the purity of the nuclear fractions. Two ER markers, triadin and calsequestrin were tested; these proteins were absent in the nuclear fraction. Furthermore, an important amount of the inner nuclear membrane protein LAP2, was evident in the nuclear fraction, whereas its presence in the cytosolic fraction was minimal (Fig. 3B). In all cases, the same amount of protein was loaded. Finally, integrity and purity of the nuclear fractions were examined by electron microscopy. The nuclear membrane integrity and the absence of detritus were evident in isolated myonuclei (Fig. 3C). The continuity of the nuclear membrane was only disrupted by the presence of nuclear pores (Fig. 3D). Other organelles like mitochondria or ER vesicles were absent from the preparation (data not shown).
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We also looked in the nuclear fraction of primary myotubes for the expression of RyRs, a well-known intracellular Ca2+ channel in skeletal muscle cells. Immunoblot and immunocytochemistry analyses revealed that RyRs are present in the nuclear fraction and are located in the nuclear envelope (data not shown).
Immunogold cytochemistry was carried out to determine the fine nuclear localization of IP3R isoforms. Type-1 IP3R reacting gold particles were found mainly in clusters of varying numbers (from 2 to 30 particles) (Fig. 5A). More than half of the gold particles found were in clusters of three or more particles, 72 % of particles being in the vicinity of one or more particles. They were preferentially located near the inner nuclear membrane with some associated with structures in the nucleoplasm. Surprisingly, a considerable number of particles appeared to be associated with the nucleolus. It was also possible to see a few particles associated with the outer nuclear membrane (not shown). When the number of particles in the different nuclear compartments was quantified, we found that approximately 47% were associated with the nucleoplasm, 21% to the nucleolus, 25% to the inner nuclear membrane and 7% to the outer nuclear membrane. Gold particles reacting with type-3 IP3Rs were also localized near the inner nuclear membrane (20%) (Fig. 5B) whereas their presence in nucleoplasm-associated structures was greater than that for type-1 IP3R, reaching 54%. Nearly 26% of particles accumulated in the nucleolus (Fig. 5B, inset). Here the particles were also found in clusters, reaching 78% compared to isolated particles; the mean number of particles within a given cluster was higher for type-3 than for type-1 receptors. The outer nuclear membrane was not labeled.
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In parallel control experiments without the primary antibody, virtually no gold particles were found (not shown), further demonstrating the specificity of the immunogold labeling.
Changes in nucleoplasmic Ca2+ induced by IP3Rs
Nuclei were loaded with Fluo-3/AM to determine whether IP3Rs present in the nucleus could directly induce nuclear Ca2+ increases. For each experiment, the nuclear envelope was initially loaded with Ca2+ in the presence of 1-2 mM ATP and 100-200 nM extranuclear Ca2+, as described (Gerasimenko et al., 1995); nuclei were then exposed to IP3 in the absence of Ca2+. Batch application of 10 µM IP3 elicited a quick and transient increase of Ca2+, as shown in the response of a nucleus (Fig. 6A, right panel), representative of 40 different nuclei analyzed from eight different preparations. In order to determine the specificity of this signal, nuclei from the same preparation were either stimulated with saline, or were pre-incubated with either 50 µM 2-APB, an inhibitor of the IP3-dependent signals, or 10 µM xestospongin B (XeB), a competitive blocker of IP3Rs (Jaimovich et al., 2005
). In all cases, no response was obtained (n=25 nuclei from different preparations, for both the vehicle and 2-APB; n=18 nuclei from two different preparations for XeB). In all studies using either saline or inhibitor, the response of the nuclei was first proved positive with IP3 (Fig. 6A, left panel). In addition, we used isolated nuclei from the 1B5 cell line, characterized by the lack of expression of RyR isoform, in which myotubes responded to depolarization with an increase in nuclear Ca2+ that was inhibited by 2-APB (Estrada et al., 2001
). Therefore, we stimulated isolated nuclei from IB5 cells with 10 µM IP3 and, as for primary cells; we obtained a similar, quick and transient response, which was inhibited by both 50 µM 2-APB and 10 µM XeB (Fig. 6B). Again, nuclei treated with saline showed no response (Fig. 6B, n=35 for IP3; n=25 for both the vehicle and 2-APB; n=15 for XeB).
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Discussion |
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Several reports indicate the presence of active IP3R in the nucleus. Electrophysiological measurements directly show IP3-dependent receptor channel activity in isolated nuclei from Xenopus laevis oocytes (Stehno-Bittel et al., 1995), or from the mammalian nuclear envelope (Boehning et al., 2001
). On the other hand, Gerasimenko and co-workers (Gerasimenko et al., 1995
), elegantly demonstrated using confocal microscopy that isolated nuclei from liver show an increase of nucleoplasm Ca2+ after IP3 stimulation, and determined that the source of Ca2+ was in the nuclear envelope. However, the source of nuclear Ca2+ remains a controversial issue. The presence of nuclear pores, with a diameter of 9 nm and a length of 15 nm, suggest that it should be permeable to all molecules up to 50 kDa (Stoffler et al., 1999
), suggesting that Ca2+ is able to equilibrate rapidly between the cytosolic and nuclear compartments by simple diffusion. In neuroendocrine pancreatic ß-cells it was demonstrated that cytosolic Ca2+ oscillations induced by glucose, potassium and carbachol enter the nucleus without restriction (Brown et al., 1997
). No significant differences between nuclear and cytosolic Ca2+ concentration were observed in HeLa cells when monitoring Ca2+ inside the nucleus using a chimeric cDNA encoding a fusion protein with the photoprotein aequorin, and a nuclear translocation signal derived from the rat glucocorticoid receptor (Brini et al., 1993
).
However, it has been demonstrated both in oocytes and in HeLa cells that it is possible to induce an IP3-dependent rise of nuclear Ca2+, independently of the cytoplasm (Lui et al., 1998; Santella et al., 1998
). The participation of IP3Rs in regulating nuclear Ca2+ in skeletal muscle has not yet been described.
As for the distribution of IP3Rs in the nucleus, not much is known about their precise topology. Humbert and colleagues (Humbert et al., 1996) demonstrated that IP3Rs are located in the inner nuclear membrane. However, it has been recently demonstrated that different IP3R isoforms have different locations in the nucleus; in HepG2 liver cells, type-2 IP3Rs are expressed in the nucleus, mainly in the nuclear envelope and occasionally in the nucleoplasm, whereas the type-3 IP3R was only expressed in the cytoplasm, and type-1 was absent in these cells (Leite et al., 2003
). In addition, different IP3-induced Ca2+ responses were measured in the nucleus and cytoplasm, which were attributed to different characteristics of the IP3R isoforms (Leite et al., 2003
). In endothelial cells, type-2 IP3R are distributed uniformly within the nucleus, but type-1 and type-3 IP3Rs were absent from the nucleus (Laflamme et al., 2002
). In neonatal rat cardiomyocytes, the distribution of receptors is similar to that presented here for myotubes (Ibarra et al., 2004
), whereas in adult cardiac cells, type 2 IP3R is present in the nuclear envelope (Bare et al., 2005
). It is interesting to note that even though there is continuity between the ER and nuclear envelope membranes, their protein composition appears to be different. The ER itself is not uniform with respect to channel, pump and protein distribution (reviewed by Petersen et al., 2001
); for example, calsequestrin was located in the ER lumen and was enriched within small vacuoles that were also equipped with SERCAs (sarco/endoplasmic reticular Ca2+-ATPases). Some, but not all, of these vacuoles (calciosomes) contained IP3Rs (Volpe et al., 1991
). Our results showing that isolated nuclei lack sarcoplasmic reticulum markers confirm this point.
The presence of IP3R in the nucleoplasm is a controversial point because it is assumed that the receptor will necessarily be associated with a membrane. The presence of tubular structures inside the nucleus was described in the C6 glioma cell line and in HeLa cells. These structures apparently correspond to extrusions of the nuclear envelope, enriched in IP3R (Lui et al., 1998; Lui et al., 2003
). In isolated nuclei from Xenopus oocytes, expression of lamin B1 and B2, was demonstrated to induce formation of intranuclear membranes that resemble ER cisternae (Ralle et al., 2004
).
The nucleoplasmic reticulum, a reticular network of nuclear Ca2+ store that is continuous with the ER and nuclear envelope was recently proposed in SKHep1 epithelial cells (Echevarria et al., 2003) to be enriched in type-2 IP3R and to a lesser extent in type-3 IP3R. Moreover IP3-induced Ca2+ release from the nucleoplasmic reticulum was detected by confocal laser-scanning microscopy examining the release of nitrophenylethyl ester-caged IP3 microinjected into the cell (Echevarria et al., 2003
). In the present study, we found that in skeletal muscle cells all three isoforms of IP3R are expressed with different intracellular distribution. Type-2 IP3R was distributed more or less homogeneously with a reticular pattern that probably represents sarcoplasmic reticulum membranes in undifferentiated myotubes. We have previously reported that type-1 IP3R is expressed in the nuclear envelope (as was now confirmed by its colocalization with LAP2, the inner nuclear membrane marker, and by the location of immunogold particles), as well as in the sarcoplasmic reticulum with a striated pattern (Powell et al., 2001
). Furthermore, type-3 IP3R was mainly distributed inside the nucleus, not exclusively associated with the nuclear membrane, as assessed both by minimal confocal colocalization using LAP2, and by electron microscopy using inmunogold. This suggests that most type-3 (and also a number of type-1) IP3Rs are probably located in a different structure; it is possible that some anti-IP3R antibodies would bind to nuclear proteins other than the receptor (Bare et al., 2005
). Indeed the western blot does show very faint bands in addition to that of
260 kDa, which corresponds to the receptor. An additional protein having the same molecular size of the receptor is also a possibility. Non-specific binding is unlikely as we tried four different antibodies raised against two different peptides and obtained similar results. We can conclude then that either a membrane structure, possibly the recently described nucleoplasmic reticulum (Echevarria et al., 2003
) is present in our nuclear preparation and we are unable to visualize it under EM, or that there is specific binding of the antibody to an unknown nuclear protein that we cannot identify. Analysis of [3H]IP3 binding experiments reflect the important component of total receptors located in the nuclear fraction (Fig. 4). Taken together, these data show that IP3R isoforms have a differential intracellular distribution in skeletal muscle cells, and that at least one of these isoforms functions as a Ca2+ release channel in nuclear compartments. This finding, together with the presence of Ca2+-ATPase activity associated to the nuclear fraction, suggests a role of the nuclear envelope and associated membrane structures in the regulation of nucleoplasmic Ca2+ concentration.
We also provide here direct evidence for the control of CREB phosphorylation by the increase in nuclear Ca2+. We found that IP3 induced quick CREB phosphorylation in isolated myonuclei. As for the protein responsible for CREB phosphorylation, we recently demonstrated that PKC- is present in the nucleus in basal conditions, which also translocates to the nucleus after depolarization of myotubes. We also showed that PKC-
is responsible for CREB phosphorylation induced by IP3 (Cardenas et al., 2004
). Thus, PKC appears to be a good candidate for directly or non-directly controlling CREB phosphorylation, although further studies are needed in order to confirm this point.
Local nuclear IP3 production and local Ca2+ release are likely to play a major role in regulating both transcription factors and the transcription process of many genes; isolated nuclei from cultured muscle cells constitute a good model system to unravel such mechanisms.
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Acknowledgments |
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