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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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).
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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.
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 ![]() |
RESULTS |
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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.
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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.
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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.
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DISCUSSION |
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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 C
and C
, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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