Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs and RyRs in osteoblasts

Olugbenga A. Adebanjo1, Gopa Biswas2, Baljit S. Moonga1, Hindupur K. Anandatheerthavarada2, Li Sun1, Peter J. R. Bevis1, Bali R. Sodam1, F. Anthony Lai3, Narayan G. Avadhani2, and Mone Zaidi1

1 Division of Endocrinology and Metabolism, Mount Sinai School of Medicine, and Bronx Veterans Affairs Geriatric Research Education and Clinical Center, New York 10029; 2 School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 3 National Institute of Medical Research, London, NW7 1AA United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40 showed bands corresponding, respectively, to full-length RyR-1 (~500 kDa) and IP3R-1 (~250 kDa). Band intensity was reduced by just ~20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+ via Ca2+-ATPase activation (1 mM ATP and ~100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+]np elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+ pathway.

nuclear calcium channels; bone formation; osteoblasts; osteoporosis; ryanodine receptors; inositol 1,4,5-trisphosphate receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CELLULAR FUNCTIONS, such as differentiation, growth, and metabolism, are all regulated by cytoplasmic Ca2+ signals generated in response to membrane receptor activation. It has long been speculated, and upheld more recently, that such cytosolic Ca2+ changes are translated directly into nuclear Ca2+ changes through nuclear pores (2, 6). Nevertheless, it is unclear whether such passive Ca2+ influx could, in fact, regulate nuclear functions as critical as DNA repair, troposiomerase activation, polymerase unfolding, gene transcription, and apoptosis (41).

Several recent studies indicate that despite the presence of nuclear pores, there is an active Ca2+ gradient across the nuclear membrane (3, 9). Furthermore, nuclear pores can apparently exist in Ca2+-permeable and Ca2+-impermeable states depending on the perinuclear space Ca2+ concentration ([Ca2+]pns) (38). The latter appears to be monitored by an EF-hand-containing protein of the nuclear pore complex (42). Recent confocal microscopic studies have shown nuclear Ca2+ waves to emanate at the cytosol-nucleus border, indicating again, a relatively Ca2+-impermeant nuclear membrane (9). Indeed, it has been known for over three decades from studies demonstrating high impedance of the nuclear envelope, that its ionic permeability was tightly regulated (26).

Being an extension of the endoplasmic reticulum (ER), the nuclear envelope is now regarded as the equivalent of a "Ca2+ store." Thus most ER Ca2+ transporters appear in the nuclear membrane (41). Resident in the outer, and presumably in the inner, nuclear membrane is a Ca2+-ATPase (11, 15, 24, 35). The latter shares most properties with its ER counterpart (24). Conceivably, its function is to permit active nuclear envelope filling against a Ca2+ gradient. In addition, there is evidence for nuclear membrane Ca2+ release channels of the inositol 1,4,5-trisphosphate receptor (IP3R) type (11, 21, 27, 29, 36). Nevertheless, biochemical, immunological, and functional evidence for ryanodine receptors (RyRs) on the nuclear membrane is relatively scant. Furthermore, several IP3-generating molecules, including phosphatidylinositol (PI) kinases, PI transfer proteins, and phospholipases are located within distinct nuclear subcompartments. Although the latter findings are suggestive of intranuclear IP3 generation (5, 7, 37), it is unclear whether the RyR agonist cADP ribose (cADPr) can also be generated within the nucleoplasm.

Here we report studies on the osteoblast, a cell that is exquisitely sensitive not only to bone-forming hormones but also to Ca2+ to which it is exposed locally, presumably during active mineralization. The triggering of various hormone receptors, such as the parathyroid hormone receptor (17), results in IP3 generation and Ca2+ release through microsomal IP3Rs. It is therefore conceivable that the cytosolically generated IP3 also triggers putative nuclear membrane IP3Rs. Furthermore, as both IP3Rs and RyRs have high-affinity Ca2+-binding domains, any rise in cytosolic Ca2+ could, in principle, activate both channels. The possibility then, that nuclear Ca2+ might regulate gene expression, and hence, bone formation, prompted our present studies into osteoblastic nuclear Ca2+ transport. We provide complementary lines of evidence, biochemical and functional, for nucleoplasmic Ca2+ influx through nuclear membrane-resident, type I isoforms, of the IP3R and RyR families. This represents the first evidence for a nuclear Ca2+ channel in any bone cell.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials and buffers. Fura 2, fura 2-acetoxymethyl ester, and fura-dextran were purchased from Molecular Probes (Eugene, OR). Tissue culture materials, including heat-inactivated FBS were bought from GIBCO-BRL (Gaithersburg, MD). EGTA, ionomycin, ATP, IP3, and cADPr were all obtained from Sigma Chemical (St. Louis, MO).

Osteoblast culture and nuclear isolation. MC3T3-E1 osteoblastic cells were grown in HEPES-buffered RPMI-1640, supplemented with FBS (10%, vol/vol), glutamine (1%, wt/vol), pencillin (50 kU/l), and streptomycin (50 g/l). The cells were subcultured at confluence by washing in EDTA, followed by gentle trypsinization (0.025%, wt/vol; 2 min), addition of RPMI-1640 before centrifugation, and resuspension in medium. The cells were maintained in tissue culture flasks (37°C; Fisher Scientific, St. Louis MO) and harvested in their logarithmic growth phase.

For isolation of nuclei, the cells were scraped at confluence and suspended in cold TKM-sucrose solution (Tris · HCl, with 25 mM KCl, 5 mM MgCl2, and 0.25 mM sucrose; pH = 7.5). They were homogenized in a glass homogenizer (×9), filtered through three layers of sterile gauze, and then centrifuged (at 700 g, 10 min) to pellet the nuclei. The nuclear pellet was resuspended in TKM-sucrose solution (20 ml), rehomogenized (×5), and then recentrifuged (700 g, 10 min). The resulting pellet was finally suspended and centrifuged again (1,000 g, 5 min) to produce a highly purified preparation of intact nuclei (all procedures, 4°C). In separate experiments, higher nuclear purity was obtained by using the methods of Malviya et al. (29) and Humbert et al. (21), involving use of a higher sucrose concentration of 2.2 M.

Immunonucleochemical and confocal microscopic studies. Immunonucleochemical studies used two anti-receptor antisera: Ab34, raised to the consensus calmodulin-binding RyR sequence, and Ab40, raised to the purified IP3R-1 protein (Table 1) (44). Coverslips (360 mm2, Fisher, St. Louis, MO) containing freshly isolated osteoblastic nuclei were first incubated with normal goat serum in multiwell dishes (in 10 mM PBS, 1:10, pH = 7.4; 15 min). Excess serum was removed and replaced with Hanks' balanced salt solution (HBSS; GIBCO-BRL). The nuclei were then incubated without antibody, or with nonimmune rabbit serum (control), Ab34 , or Ab40 (in HBSS, 1:100, vol/vol, 1 h). The coverslips were then rinsed gently with HBSS, drained, incubated with goat anti-rabbit FITC (Sigma Chemical, in HBSS, 1:100; 1 h), washed gently, and drained. For epifluorescence microscopy, a 510-nm FITC filter was positioned in the emission path of the microspectrofluorimeter (Diaphot, Nikon, Tokyo, Japan). In addition, coverslips mounted onto glass slides were viewed in a laser confocal scanning microscope (Leica, Deerfield, IL). Fluorescent nuclei were visualized, using volume element imaging, as serial, 1-µm-thick optical sections in the coronal plane of each nucleus (44).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Isoform specificity of the panel of antibodies to the inositol 1,4,5-trisphosphate receptors and ryanodine receptors

Membrane isolation, protease protection assay, and Western blotting. For the isolation of ER membranes, intact osteoblasts were homogenized in sucrose-mannitol buffer (in mM) 20 HEPES, 70 sucrose, 220 mannitol, 2 EDTA, and 0.1 phenylmethylsulfanylfluoride, as well as 1.25 µg/ml each of antipain, chymostatin, leupeptin, and pepstatin. The homogenate was centrifuged (15,000 g, 20 min) to remove mitochondrial and nuclear membrane fractions. The supernatant was recentrifuged (10,000 g, 1 h, 4°C) to obtain the ER membrane fraction. The latter was suspended in homogenization buffer and repelleted (100,000 g, 1 h). The final pellet was resuspended in 500 µl sucrose-mannitol buffer (as described above) and stored at -70°C.

For isolation of purified nuclear membranes, the isolated nuclei (see above) were suspended in sucrose-mannitol buffer and centrifuged (100,000 g, 30 min). The pellet was then resuspended in the same buffer and sonicated (1 min). In separate experiments, nuclear and ER membranes were subjected to brief trypsin treatment (120 µg/mg protein) in a 500-µl reaction volume (on ice, 30 min) (4). The reaction was stopped by the addition of a trypsin inhibitor (1.2 mg/mg protein). Nuclear membranes were then isolated as described above. The trypsin-treated ER membranes were used as such.

The isolated ER and nuclear membranes were next incubated with 3 × Laemmli's sample buffer (37°C, 20 min). Two hundred micrograms of protein (estimated by Lowry's method) was then separated by SDS-PAGE (6%; Bio-Rad Minigels), followed by electroblotting onto nitrocellulose membranes. The blots were then air-dried and blocked with PBS-Tween 20 (PBST; 0.3%, vol/vol; 1 h), followed by incubation with Ab40, anti-RyR-1, Ab129, or Ab180 (in PBST 0.05%, vol/vol, 1:3,000; 1 h). The immunoblots were then developed with horseradish peroxidase-conjugated anti-rabbit antibodies (in PBST 0.05%, vol/vol; 1:30,000) by using Pierce's Supersignal Ultra substrate solution, per manufacturer's protocol. The blots were then quantitated by using the Bio-Rad Multianalyst program.

Nuclear Ca2+ measurements. The method used was modified from that used by Gerasimenko et al. (11). Isolated intact nuclei were incubated with fura-dextran (20 µM, 45 min, 4°C) in 12 ml "standard" solution comprising (in mM) 125 mM KCl, 2 mM K2HPO2, 50 mM HEPES, 4 mM MgCl2, 0.1 mM EGTA, and 1 mM ATP (pH = 7.4) ([Ca2+] < 5 nM, by fura-2-based cuvette measurements). The dye-loaded nuclei were washed in standard solution and centrifuged (1,000 g, 1 min). They were then seeded, at a low density, on glass coverslips (360 mm2, Fisher) for photometric studies. The nuclei were allowed to settle for 15 min in standard solution. The nuclear envelope was then loaded with Ca2+ in the same buffer (30 min, 37°C) at an extranuclear [Ca2+] of 200 nM (obtained by adding 107 µl of 1 M CaCl2 to 50 ml modified standard solution with 10 mM EGTA) (43). The relatively low extranuclear [Ca2+] in the presence of ATP permitted active nuclear envelope Ca2+ loading, presumably through an activated Ca2+-ATPase. Experiments performed at either higher (500 nM) or lower (< 5 nM) extranuclear [Ca2+] did not produce effective nuclear envelope Ca2+ loading (data not shown).

All photometric nucleoplasmic Ca2+ concentration ([Ca2+]np) measurements were done in the standard solution but without added ATP (see above). In addition, fast kinetic [Ca2+]np measurements were made every 250 ms by using an Ion Optix microspectrofluorimeter and fura-dextran as the fluorescent indicator (43). Nuclei were exposed to excitation lambda s of 340 and 380 nm, and the emitted fluorescence was monitored at 510 nm. Photon counts per second were used to calculate the emitted intensity ratio, I405/I480. Finally, the fluorochrome was calibrated (43) (see RESULTS).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear membrane immunolocalization of IP3R and RyR. We first examined whether IP3Rs and RyRs were detectable immunocytochemically by using our highly specific anti-receptor antisera Ab40 and Ab34. Note from Table 1 that Ab40 detects the most abundantly expressed IP3R-1 isoform (44). In contrast, having being raised against the consensus calmodulin-binding RyR sequence, Ab34 does not distinguish between the three RyR isoforms (44). Intact, nonpermeabilized, osteoblastic nuclei fluoresced intensely with both the antisera, Ab40 or Ab34 (Fig. 1, B and D). Nuclei incubated without antisera (Fig. 1A) or with nonimmune rabbit serum (Fig. 1C) did not stain.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Intense immunofluorescent staining of isolated, intact, nonpermeabilized osteoblastic nuclei with highly specific antibodies to inositol 1,4,5-trisphosphate receptor (IP3R; Ab40; B) or ryanodine receptor (RyR; Ab34; D). Control nuclei incubated without antiserum (A) or with nonimmune rabbit serum (C) showed no appreciable fluorescence. Experiments were repeated 5 times; each time up to 20 nuclei were visualized.

Confocal microscopy indicated that the IP3R and RyR fluorescence was almost strictly peripheral, with virtually no nucleoplasmic staining (Figs. 2, A and B). The confocal microscopic data were further analyzed by volume element imaging, which allowed a three-dimensional view of each confocal section. This again confirmed a peripheral nuclear membrane localization of the immunofluorescence.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Confocal microscopic localization of IP3Rs and RyRs to osteoblast nuclear membrane. A and B: intense peripheral immunofluorescent staining, respectively, with highly specific anti-IP3R (Ab40) and anti-RyR (Ab34 ) antisera. Serial sections were obtained in coronal plane of each nucleus at 1-µm intervals and analyzed by volume element imaging. There is a typically peripheral nuclear membrane fluorescence with minimal nucleoplasmic staining. Experiments were repeated 2 times; each time up to 20 nuclei were visualized.

Osteoblastic expression of type 1 RyR and type 1 IP3R isoforms. To examine for the presence of specific nuclear membrane IP3R and RyR isoforms, we performed SDS-PAGE and Western blotting by using our repertoire of isoform-specific, anti-IP3R and RyR antisera, namely, Ab40, anti-RyR-1, Ab129, and Ab180 (Table 1). Of note is that anti-RyR-1 was raised to the purified RyR-1 protein, whereas Ab129 and Ab180 were raised to unique peptide sequences of RyR-2 and RyR-3, respectively (Table 1). That these antisera specifically detect the respective proteins has been demonstrated previously (44). However, we again confirmed antibody specificity by using ER membranes from skeletal muscle (RyR-1 and RyR-3) and brain (RyR-2 and RyR-3) (Fig. 3). Similarly, immunoblotting of osteoblastic nuclear membranes revealed bands corresponding to the full-length IP3R-1 (~270 kDa) and RyR-1 (~500 kDa) (Fig. 3). No bands were observed with Ab129 and Ab180, excluding significant expression of RyR-2 and RyR-3. In parallel, osteoblastic ER membranes also expressed the same IP3R and RyR isoform, as did the nuclear membrane, notably the type 1 isoforms (Fig. 3). Although the presence of IP3Rs on the nuclear membrane is consistent with the studies of Guihard et al. (15), the group did not detect any RyR isoforms, including, RyR-1.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.   Osteoblast nuclear (nuclei) and endoplasmic reticular (ER) membranes, as well as ER membranes from skeletal muscle (SkM) and brain immunoblotted with a panel of isoform-specific antibodies to IP3R (~250 kDa) and RyR (~500 kDa). These include anti-IP3R-1 (Ab40; top left), anti-RyR-1 (top right), anti-RyR-2 (Ab129; bottom left), and anti-RyR-3 (Ab180; bottom right). Their specificities are detailed in Table 1 and Ref. 44. Experiments were repeated twice. Mr, marker.

A possibility arises, however, that our isolated nuclear preparations were contaminated with ER membranes and that these, rather than purely nuclear membranes, contributed to the detected signals. To check for such contamination, we used two complementary approaches. First, we blotted isolated nuclear membranes with an antibody to lamin-B, an inner nuclear membrane protein, as well as NADPH cytochrome P-450 reductase, an ER marker enzyme. We found lamin-B bands but no immunoreactivity for NADPH cytochrome P-450 reductase in our nuclear preparations. The converse was true for our ER preparations (not shown). These results suggested that the nuclear membrane preparations were mostly free from significant ER contamination.

Next, we incubated intact nuclei and ER membranes, in parallel, with trypsin (120 µg/mg protein, 30 min, on ice). The rationale of these experiments was that any exposed receptors, which would include those on attached ER fragments, would be amenable to proteolysis by trypsin. In contrast, receptors that were present intranuclearly, for example, in the inner nuclear membrane, would not be accessible to trypsin and hence would be insensitive to protease degradation. Thus, in the absence of ER contamination, nuclear membrane band intensity would be expected not to diminish significantly on proteolysis of intact nuclei. In contrast, trypsin should proteolyze any ER membrane fragments, resulting in a significant reduction in signal intensity. We found that nuclear membrane band intensity fell only by ~20% after brief tryptic proteolysis of intact nuclei (Fig. 4). However, when ER membranes were treated similarly, there was an expected reduction in band intensity by ~80% (Fig. 4). Taken together, the experiments confirm that our isolated nuclear preparations were mostly free of significant ER contamination; this makes it unlikely that the nuclear membrane IP3R-1 and RyR-1 bands were derived solely from contaminating ER fragments. The experiments also indicate a possible inner nuclear membrane of these receptors. A similar approach was used successfully by Anandatheerthavarada et al. (4).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Osteoblast nuclear and ER membranes immunoblotted with antibodies to type 1 isoforms of the RyR (RyR-1; ~500 kDa; anti-RyR-1) and IP3R (IP3R-1; ~250 kDa; anti- IP3R-1, Ab40). This followed a 30-min incubation of isolated nuclei or ER membranes with trypsin (120 µg/mg protein, on ice). (For details see, Protease protection assay.) Blots were quantitated by using the Bio-Rad Multianalyst program, and band intensities, expressed as a percentage of control means ± SD (n = 3), are plotted as histograms.

Taken together, the absence of an ER marker, NADPH cytochrome P-450 reductase, from our nuclear preparations together with the retention of IP3R-1 and RyR-1 bands on trypsin proteolysis of intact nuclei strongly suggest a lack of significant ER contamination and attest to the purity of these preparations.

Nucleoplasmic Ca2+ influx through RyRs and IP3Rs. Fura-dextran-based fast kinetic [Ca2+]np measurements were made photometrically (see MATERIALS AND METHODS). We initially calibrated fura 2 by using a modified "intracellular" protocol. Briefly, fura 2-loaded nuclei were bathed in a "calibration" solution consisting of (in mM) 125 KCl, 2 K2HPO2, 5 glucose, 10 HEPES, 0.8 MgCl2, and 1 CaCl2. The Ca2+ ionophore ionomycin (20 µM) was then applied to obtain the due to highest cytosolic [Ca2+] (Rmax) and the minimum fluorescence intensity at 380 nm (Fmin) (Fig. 5, A and B). Extranuclear Ca2+ was then chelated by adding 1.2 mM EGTA to obtain values of the minimum ratio (Rmin) and maximal fluorescence intensity at 380 nm (Fmax). The dissociation constant (Kd) for Ca2+ and fura 2 at a temperature of 20°C, an ionic strength of 0.1 M, and a pH of 6.85, is 224 nM. The values were substituted into the equation [Ca2+] = Kd × [(R - Rmin)/(Rmax - R)] × [(Fmax/Fmin)]. A calibration curve was essentially linear between Ca2+ concentrations of 150 and 1,000 nM and is unreliable beyond the latter concentration as fura 2 saturates.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Ca2+ measurement [counts/s (A); fluoresence ratio (F340/F380; B)] in fura-dextran-loaded nuclei using ionomycin (20 µM) in Ca2+-free, EGTA-containing medium, extranuclear Ca2+ concentration ([Ca2+]) < 5 nM to confirm Ca2+ loading. Experiments were repeated 5 times, and up to 20 nuclei were imaged on each occasion. Fmin and Fmax: minimum and maximum fluorescence intensity, respectively; Rmin and Rmax: minimum ratio and maximum ratio, respectively.

For each experiment, the nuclear envelope was again initially Ca2+ loaded in incubation solution in the presence of ATP and ~100 nM extranuclear Ca2+ (see MATERIALS AND METHODS). To further ensure adequacy of the Ca2+ loading, isolated nuclei were exposed to ionomycin (5 µM) in Ca2+-free, EGTA-containing, i.e., modified, incubation solution (extranuclear [Ca2+] < 5 nM) (also see above). In all instances, a large, but transient, increase of [Ca2+]np was noted, confirming a [Ca2+]pns-to-[Ca2+]np gradient (data not shown). Such Ca2+-loaded nuclei were then exposed to IP3 (10 µM) or cADPr (10 µM) in modified incubation solution in the presence or absence of a highly specific cADPr antagonist, 8-amino-cADPr (18 µM). A rapid sustained elevation of [Ca2+]np in fura-dextran loaded nuclei suggested nucleoplasmic Ca2+ influx, respectively, through IP3Rs and RyRs (Fig. 6). Notably, the response to cADPr was completely abolished with 8-amino-cADPr pretreatment, suggesting that cADPr specifically interacted with its binding site on the RyR. However, expectedly, the response to IP3 application remained intact even in the presence of 8-amino-cADPr, again suggesting response specificity. In contrast to the results by Gerasimenko et al. (11), the observed rather than transient responses were sustained. This could be due to either the absence or inactivation of Ca2+ efflux mechanisms in our specific experimental conditions. One such mechanism may involve the activation of the IP4 receptor (28).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6.   Representative traces from single nuclei showing effects of extranuclearly applied IP3 (10 µM) or cADP ribose (cADPr; 10 µM) on nucleoplasmic Ca2+ concentration ([Ca2+]np; nM) in absence (A and C) or presence (B and D) of RyR antagonist 8-amino-cADPr, respectively. Extranuclear [Ca2+] was < 5 nM. Each of the 4 protocols, in which between 4 and 6 fura-dextran-loaded nuclei were sampled, was repeated 3 times. Note that nuclear envelope was initially Ca2+ loaded in standard solution in presence of 1 mM ATP and ~200 nM extranuclear Ca2+ (see MATERIALS AND METHODS).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We provide biochemical and immunochemical evidence for the existence of the type 1 isoforms of both IP3R and RyR in osteoblast nuclear membranes. We also show that both Ca2+ channels allow nucleoplasmic Ca2+ influx triggered, respectively, by IP3 and cADPr. Furthermore, Ca2+ triggers either channel (33); likewise, our demonstration of Ca2+ -induced Ca2+ influx. Such second messenger gating of nuclear Ca2+ influx is not only of general biological interest but is also directly relevant to the physiology of the bone-forming osteoblast.

Several groups have identified IP3 receptors on a variety of eukaryotic cell nuclear membranes. Nevertheless, evidence for the presence of RyRs on cellular nuclear membranes is somewhat more tenuous, and in this respect, our study provides further biochemical and functional documentation. In fact, RyRs comprise a family of related isoforms, namely types 1, 2, and 3 (34). Of these, the type 1 isoform is expressed almost exclusively in skeletal muscle wherein it couples electrically to the dihydropyridine receptor and gates depolarization-induced Ca2+ release (34). It is therefore surprising that RyR-1, rather than the more ubiquitous RyR-2 or RyR-3, is the isoform we identify in osteoblastic cells. It is also notable also that although the presence of IP3Rs on the nuclear membrane was also documented by Guihard et al. (15), the group did not detect any RyR isoforms, including, RyR-1 (15).

A key issue thus arises, What is the precise function of these receptors in nuclear Ca2+ homeostasis in general, and in the osteoblast, in particular? Indeed, despite vigorous debate, the dogma of how nuclear Ca2+ is regulated still remains unsettled (27, 40). For example, it is unclear whether and, if so, under what circumstances, does the nuclear pore complex become permeable to Ca2+ (38). Most electrophysiological studies in isolated nuclei appear to oppose the concept of unrestricted cytosol-to-nucleoplasm Ca2+ exchange through the nuclear pore (26, 29). However, if the nuclear pore does become permeable to Ca2+, nuclear membrane IP3Rs and RyRs could still conceivably regulate [Ca2+]np in response to elevations in cytosolic IP3 and Ca2+. In the osteoblast, the latter events will arise from hormone and growth factor receptor activation (17), integrin-matrix interactions (33), and Ca2+ transfer through gap junctions (8).

Not much is known about the precise topology of the IP3Rs and RyRs in the nuclear membrane/s. Humbert et al. (21) have demonstrated quite elegantly that IP3Rs are located in the inner nuclear membrane. The movement of Ca2+ from the perinuclear space to the nucleoplasm also indicates that the large NH2-terminus-containing portion of the molecule is nucleoplasmic. Furthermore, we show that IP3R and RyR immunoreactivity are preserved when intact nuclei are treated with trypsin; this also indicates an intranuclear location of the two molecules.

Thus, in the presence of a nucleoplasmic IP3-binding site, for example, the proposed regulation of nuclear Ca2+ by cytosolic IP3 must assume that IP3 diffuses freely through the nuclear pore to activate an inner nuclear membrane IP3R. This is by no means certain. In Xenopus laevis oocytes, intracytoplasmic IP3 injection triggers intranuclear Ca2+ elevation, suggesting a cytosol-to-nucleoplasmic movement of IP3 (18). Our demonstration of nuclear Ca2+ gating by extranuclear IP3 and cADPr provides additional evidence for the inner nuclear membrane permeation of second messengers. Gerasimenko et al. (11) have also used extranuclei second messenger to activate nucleoplasmic Ca2+ release in isolated nuclei. In starfish oocytes, however, the cytosolic photolysis of "caged" IP3 does not trigger a nuclear Ca2+ signal, and intranuclearly injected cADPr exhibits only a limited outward diffusion. Both findings suggest that the second messenger permeability of the nuclear membrane may indeed be restricted (41).

In the absence of second messenger permeation into the nucleus, cytosolic IP3 must first release Ca2+ into the cytosol from the nuclear envelope. A consequent reduction in [Ca2+]pns, might then, through a positive feedback, trigger Ca2+ influx across the inner nuclear membrane. This is unlikely, as, thus far, IP3Rs have been documented not to exist on the outer nuclear membrane (21). It thus becomes challenging, on the basis of presumed topology of IP3Rs (and RyRs) and the questionable permeability of the inner nuclear membrane to IP3, to ascribe a role for cytosolic IP3 in the regulation of nuclear Ca2+. In fact, the generation of both IP3 and cADPr is possible within the nucleus (2). Namely, phospholipase Cbeta and Cdelta , PI transfer protein, and PI-4- and PI-5-kinases are all present within defined nuclear subcompartments (5, 7, 36). We have recently demonstrated that CD38, a cADPr-producing ribosyl cyclase, is also located in the inner nuclear membrane, with its catalytic site facing nucleoplasmically (1). Nevertheless, their functional significance remains unclear, as do the cellular signals that might conceivably trigger nuclear PI kinase activation.

Despite the paucity of mechanistic information, there is general agreement that nucleoplasmic Ca2+ controls at least two critical nuclear processes, gene transcription and apoptosis (28, 41). Ca2+ can regulate gene expression either directly, through Ca2+-response elements, or indirectly, via protein kinase C (39, 16). High nuclear Ca2+ enhances the expression of several early response genes, including c-fos, c-jun, jun B, fos B, nur-77, and zif-268, as well as certain late genes, such as the interleukin-2, -3, and -6 genes (12, 14, 15, 19, 39). Notably, both c-fos and interleukin-6 genes are critical to osteoblastic bone formation (15, 30, 31) and should, in principle, be regulated by changes in nuclear Ca2+. Nevertheless, a direct relationship between nuclear Ca2+ influx and the regulation of osteoblastic genes remains speculative.

Finally, enhanced osteoblast apoptosis is thought to underlie the bone loss associated with senescence (22). Cellular senescence, at least in vitro, is paralleled by an increased RyR expression (20). Furthermore, Ca2+ permits apoptosis by activating certain nuclear proteases, endonucleases, and members of the bcl-2 gene family (23, 32). Thus potential age-related increases in RyR, by enhancing nuclear Ca2+ gating, might result in an elevated [Ca2+]np and hence reduced osteoblast survival. Careful mechanistic studies into the permissive effects of Ca2+ on osteoblast apoptosis are therefore urgently warranted.


    ACKNOWLEDGEMENTS

The authors are grateful to Iain MacIntyre for encouragement and support; Qinwu Lin for help with confocal microscopy; Jerry Rosenzweig (Geriatrics Service) for assistance in grant management; and Stacy Marshall and Scot Teti (Medical Media) for illustrations.


    FOOTNOTES

The work was supported by National Institute on Aging Grant RO1 AG -14917 (to M. Zaidi) and Department of Veterans Affairs Merit Review Award (to M. Zaidi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Zaidi, Div. of Endocrinology and Metabolism, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029 (E-mail: mone.zaidi{at}mssm.edu).

Received 5 November 1998; accepted in final form 18 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adebanjo, OA, Anandateerthwardha HK, Koval AP, Moonga BS, Biswas G, Sun L, Sodam BR, Bevis PJ, Huang CL, Epstein S, Lai FA, Avadhani NG, and Zaidi M. A new function for CD38/ADP-Ribosyl cyclase in nuclear Ca2+ homeostasis. Nature Cell Biol 1: 409-414, 1999[ISI][Medline].

2.   Allbritton, NL, Oancea E, Kuhn MA, and Meyer T. Source of nuclear calcium signals. Proc Natl Acad Sci USA 91: 12458-12462, 1995[Abstract/Free Full Text].

3.   Al-Mohanna, FA, Caddy KWT, and Bolsover SR. The nucleus is insulated from large cytosolic calcium ion changes. Nature (Lond) 367: 745-750, 1994[ISI][Medline].

4.   Anandatheerthvardha, HK, Addya S, Dwivedi RS, Biswas G, Mullick J, and Avadhani NG. Localization of multiple forms of inducible cytochromes P450 in rat liver mitochondria: immunological characteristics and patterns of xenobiotic substrate metabolism. Arch Biochem Biophys 339: 136-150, 1997[ISI][Medline].

5.   Asano, M, Taniya-Koizumi K, Homms Y, Takenuma T, Nimura Y, Kojima K, and Yosihda S. Purification and characterization of nuclear phospholipase C specific for phosphoinositides. J Biol Chem 269: 12360-12366, 1994[Abstract/Free Full Text].

6.   Brini, M, Murgia L, Pasti L, Picard D, Pozzan T, and Rizzuto R. Nuclear calcium concentration measured with specifically targeted recombinant aequorin. EMBO J 12: 4813-4819, 1993[Abstract].

7.   Capitani, S, Holms B, Mazzoni M, Previati M, Bertagnolo V, Wirtz KWA, and Manzoli FA. Uptake and phosphorylation of phosphatidyl inositol by rat liver nuclei: role of phosphatidyl inositol transfer protein. Biochim Biophys Acta 1044: 193-200, 1990[ISI][Medline].

8.   Civitelli, R. Cell-cell communication in bone. In: Advances in Organ Biology. Molecular and Cell Biology of Bone, edited by Zaidi M., Adebanjo O. A., and Huang C. L.-H.. Stamford, CT: JAI Press/Elsevier, 1998, vol. 5B, p. 543-564.

9.   Fox, JL, Burgstahler AD, and Nathanson MH. Mechanism of long-range Ca2+ signaling in the nucleus of isolated rat hepatocytes. Biochem J 326: 491-495, 1997[ISI][Medline].

10.   Franchimont, N, Rydziel S, and Canalis E. Interleukin-6 is autoregulated by transcriptional mechanisms in cultures of rat osteoblastic cells. J Clin Invest 100: 1797-1803, 1997[Abstract/Free Full Text].

11.   Gerasimenko, OV, Gerasimenko JV, Tepikin AV, and Peterson OH. ATP-dependent accumulation and inositol trisphosphate or cyclic ADP ribose-mediated release of calcium from the nuclear envelope. Cell 80: 439-444, 1995[ISI][Medline].

12.   Gilchrist, JS, and Pierce GN. Identification and purification of a calcium-binding protein in hepatic nuclear membranes. J Biol Chem 268: 4291-4299, 1993[Abstract/Free Full Text].

13.   Ginty, DD, Bonni A, and Greenberg ME. Nerve growth factor activates a ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77: 713-725, 1994[ISI][Medline].

14.   Gregoriadis, AE, Schellander K, Wang ZQ, and Wagner EF. Osteoblasts are target cells for transciption in c-fos-transgenic mice. J Cell Biol 122: 685-701, 1993[Abstract].

15.   Guilhard, G, Proteau S, and Rousseau E. Does the nuclear envelope contain two types of ligand-gated Ca2+ release channels? FEBS Lett 414: 89-94, 1997[ISI][Medline].

16.   Hardingham, GE, Chawle S, Johnson CM, and Bading H. Distinct functions of nuclear and cytoplasmic Ca2+ in the control of gene expression. Nature (Lond) 285: 260, 1996.

17.   Henderson, JE, and Goltzman D. Osteoblastic receptors. In: Advances in Organ Biology. Molecular and Cell Biology of Bone, edited by Zaidi M., Adebanjo O. A., and Huang C. L.-H.. Stamford, CT: JAI Press/Elsevier, 1998, p. 499-512, 1998.

18.   Hennager, DJ, Welsh MJ, and DeLisle S. Changes in either cytosolic or nuleoplasmic inositol 1,4,5-trisphosphate levels can control nuclear calcium concentration. J Biol Chem 270: 4959-4962, 1995[Abstract/Free Full Text].

19.   Hill, CS, and Treisman R. Transcription regulation by extracellular signals: mechanisms and specificity. Cell 80: 199-211, 1995[ISI][Medline].

20.   Huang, M-S, Adebanjo OA, Moonga BS, Goldstein S, Lai FA, Lipschitz DA, and Zaidi M. Upregulation of functional ryanodine receptors during in vitro aging of human diploid fibroblasts. Biochem Biophys Res Commun 245: 50-52, 1998[ISI][Medline].

21.   Humbert, JP, Matter N, Artault JC, Koppler P, and Malviya AN. Inositol 1,4,5,-trisphosphate receptor is located to the inner nuclear membrane vindicating regulation of nuclear Ca2+ signaling by inositol 1,4,5-trisphosphate. J Biol Chem 271: 478-485, 1996[Abstract/Free Full Text].

22.   Jilka, RL, Weinstein RS, Bellido T, Parfitt AM, and Manolagas SC. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Min Res 13: 793-802, 1998[ISI][Medline].

23.   Jones, DP, McConkey DJ, Nicotera P, and Orrenius S. Calcium-activated DNA fragmentation in rat liver nuclei. J Biol Chem 264: 6398-6403, 1989[Abstract/Free Full Text].

24.   Lanini, L, Bachs O., and Carafoli E. The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum. J Biol Chem 267: 11548-11552, 1992[Abstract/Free Full Text].

25.   Larea, LS, and McNamara OJ. Ionotropic glutamate receptor subtypes activate c-fos transcription by distinct calcium-requiring intracellular signaling pathways. Neuron 10: 31-41, 1993[ISI][Medline].

26.   Loewenstein, WR, and Kanno Y. Some electrical properties of a nuclear membrane examined with a microelectrode. J Gen Physiol 46: 1123-1140, 1963[Abstract/Free Full Text].

27.   Mak, DOD, and Foskett JK. Single-channel inositol 1,4,5-trisphosphate receptor currents revealed by patch clamp of isolated Xenopus oocyte nuclei. J Biol Chem 269: 29375-29378, 1994[Abstract/Free Full Text].

28.   Malviya, A, and Rogue PJ. "Tell me where is calcium bred": clarifying the roles of nuclear calcium. Cell 92: 17-23, 1998[ISI][Medline].

29.   Malviya, AN, Rogue P, and Vincendon G. Sterospecific inositol 1,4,5-[32P] trisphosphate binding to isolated rat liver nuclei. Evidence for inositol trisphosphate receptor mediated calcium release from the nucleus. Proc Natl Acad Sci USA 87: 9270-9274, 1990[Abstract].

30.   Manolagas, SC, and Jilka RL. Bone Marrow, cytokines and bone remodeling. N Engl J Med 332: 305-311, 1995[Free Full Text].

31.   Marie, P. Osteoblastic bone formation. In: Advances in Organ Biology. Molecular and Cell Biology of Bone, edited by Zaidi M., Adebanjo O. A., and Huang C. L.-H... Stamford, CT: JAI Press/Elsevier, 1998, p. 445-473.

32.   Marin, M, Fernandez A, Bick RJ, Brisbay S, Buja LM, Snuggs M, McConkey DJ, vanEschenbach SC, Keating MJ, and McDonald TJ. Apoptosis suppression by bcl-2 is correlated with regulation of nuclear and cytosolic Ca2+. Oncogene 12: 2259-2266, 1996[ISI][Medline].

33.   McCann, TJ, Mason WT, Meikle MC, and McDonald F. A collagen peptide motif activates tyrosine kinase-dependent calcium signaling pathways in human osteoblast-like cells. Matrix Biol 16: 273-283, 1997[ISI][Medline].

34.   Meissner, G. The ryanodine receptor: structure and function. Ann Rev Physiol 56: 485-508, 1994[ISI][Medline].

35.   Nicotera, P, McConkey DJ, Jones DP, and Orrenius S. ATP stimulates Ca2+ uptake and increases the free Ca2+ concentration in isolated liver nuclei. Proc Natl Acad Sci USA 86: 453-457, 1989[Abstract].

36.   Nicotera, P, Orrenius S, Nilsson T, and Berggren PO. An inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in liver nuclei. Proc Natl Acad Sci USA 87: 6858-6862, 1990[Abstract].

37.   Payraste, B, Nievers M, Boonstra J., Breton M, Verkleij AJ, and Van Bargen Henegouwen PMP A differential localization of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. J Biol Chem 267: 5078-5084, 1992[Abstract/Free Full Text].

38.   Perez-Terzic, C, Stehno-Bittel L, and Clapham DE. Nucleoplasmic and cytoplasmic differences in the fluorescent properties of calcium indicator fluo-3. Cell Calcium 21: 275-282, 1997[ISI][Medline].

39.   Rosen, LB, Ginty DD, and Greenberg ME. Calcium regulation of gene expression. In: Advances in Second Messenger and Phosphoprotein Research, edited by Means AR.. New York: Raven, 1995, vol. 30, p. 225-253.

40.   Santella, L, and Carafoli E. Calcium signaling in the cell nucleus. FASEB J 11: 1091-1109, 1997[Abstract/Free Full Text].

41.   Santella, L, and Kyozuka K. Calcium release into the nucleus by 1,4,5-trisphosphate and cyclic ADP-ribose gated channels induced the resumption of meiosis in starfish oocytes. Cell Calcium 22: 1-10, 1997[ISI][Medline].

42.   Stehno-Bittel, L, Perez-Terzic C, and Clapham DE. Diffusion across the nuclear envelope inhibited by depletion of the nuclear Ca2+ store. Science 270: 1835-1838, 1995[Abstract].

43.   Zaidi, M, Alam ASMT, Bax C, Shankar V, Bevis PJR, Huang CL-H, Pazianas M, and Moonga BS. Cytosolic free calcium measurements in single cells using calcium-sensitive fluorochromes. In: Methods in Molecular Biology. Biomembrane Protocols: II. Architecture and Function, edited by Graham JM, and Higgins JA.. Totowa NJ: Humana, 1994, vol. 27, p. 279-293.

44.   Zaidi, M, Shankar VS, Tunwell R, Adebanjo OA, MacKrill J, Pazianas M, O'Connell D, Simon BJ, Rifkin BR, Ventikaraman AR, Huang CL-H, and Lai FA. A ryanodine receptor-like molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing. J Clin Invest 96: 1582-1590, 1995[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 278(5):F784-F791
0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society