1 Mount Sinai Bone Program and Division of Endocrinology, Mount Sinai School of Medicine, New York, 10029, and Endocrine Division and The Geriatric Research Education and Clinical Center, Veterans Affairs Medical Center, Bronx, New York 10468; 2 Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; and 3 Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, United Kingdom
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ABSTRACT |
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This study explores the role of the
calmodulin- and Ca2+-sensitive phosphatase calcineurin A in
the control of bone resorption by mature osteoclasts. We first cloned
full-length calcineurin A and A
cDNA from a rabbit osteoclast
library. Sequence analysis revealed an ~95 and 86% homology between
the amino acid and the nucleotide sequences, respectively, of the two
isoforms. The two rabbit isoforms also showed significant homology with
the mouse, rat, and human homologs. In situ RT-PCR showed evidence of
high levels of expression of calcineurin A
mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin A
expression in
cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 × 10
7 M) and
FK506 (5 × 10
9 and 5 × 10
7 M).
We then constructed a fusion protein comprising calcineurin A
and
TAT, a 12-amino acid-long arginine-rich sequence of the human
immunodeficiency virus protein. Others have previously shown that the
fusion of proteins to this sequence results in their receptor-less
transduction into cells, including osteoclasts. Similarly, unfolding of
the TAT-calcineurin A
fusion protein by shocking with 8 M urea
resulted in its rapid influx, within minutes, into as many as 90% of
all freshly isolated rat osteoclasts, as was evident on double
immunostaining with anti-calcineurin A
and anti-TAT antibodies. Pit
assays performed with TAT-calcineurin A
-positive osteoclasts
revealed a concentration-dependent (10-200 nM) attenuation of bone
resorption in the absence of cell cytotoxicity or changes in cell
number. TAT-hemaglutinin did not produce significant effects on bone
resorption or cell number. The study suggests the following:
1) the 61-kDa protein phosphatase calcineurin A
can be
effectively tranduced into osteoclasts by using the TAT-based approach,
and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.
calcium channel; gene cloning; osteoclast; osteoporosis
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INTRODUCTION |
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MAINTENANCE OF SKELETAL INTEGRITY depends on a precise balance between bone formation and resorption. An absolute or relative increase in resorption over formation results in bone loss. Bone is removed by osteoclasts and is rebuilt by osteoblasts as part of the bone remodeling process. The activity of both osteoclasts and osteoblasts is regulated by precise molecular signals, some of which are sensitive to changes in cytosolic Ca2+ (50). The osteoclast in particular is exposed to high millimolar extracellular concentrations of Ca2+ during resorption (40). It has an extracellular Ca2+ sensor thought to be a type II ryanodine receptor (RyR) expressed at the plasma membrane (30, 47, 49).
Calcineurin is the only serine/threonine protein phosphatase sensitive
to both Ca2+ and calmodulin that plays a critical role in
coupling Ca2+ signals to cellular responses (22, 23,
41). The calcineurin heterodimer comprises one catalytic and one
regulatory subunit (subunits A and B, respectively); the latter is
highly conserved from yeast to humans (16). The three
known isoforms of mammalian calcineurin A (,
, and
) are
products of different genes and exhibit ~86% sequence homology
(GenBank accession no. J05479, M81483, and NM_008915, respectively).
Calcineurin A
is widely distributed (8, 18, 24, 25, 28)
and has established roles in T cell activation, vesicular trafficking,
cell growth, apoptosis, neuron depotentiation, muscle
development, and cardiac valve formation. We recently provided
preliminary evidence that calcineurin A
, expressed in osteoclasts,
plays a role in the regulation of bone resorption (4).
Here, we propose to understand more fully the function of calcineurin A
in bone resorption by using a complement of molecular and cellular
approaches. First, we cloned full-length cDNAs for the calcineurin A
isoforms and
. We then utilized in situ RT-PCR to demonstrate
mRNA expression in freshly isolated mature osteoclasts. Using the same
technique, we examined, in a semiquantitative manner, changes in
expression of calcineurin A
in response to two known inhibitors of
calcineurin activity, cyclosporin A and tacrolimus (FK506). Both
inhibitors are known to inhibit the phosphatase activity of calcineurin
but may have downstream effects on calcineurin A
expression
(3, 37). Both cyclosporin A and FK506 also cause profound
bone loss in vivo both in animals and in humans (4, 7).
However, the precise mechanism of their action on bone cells remains unclear.
To evaluate the function of calcineurin in osteoclastic bone
resorption, we developed a method through which we were able to
transduce most of the relatively sparse population of cells that we
isolate fresh from neonatal rats. The technique involved creating a
fusion protein comprising calcineurin A and TAT, a 12-amino acid,
arginine-rich sequence of the human immunodeficiency virus protein
(32). A control protein, TAT-HA, was also similarly synthesized and purified. TAT fusion proteins, particularly those that
have been unfolded by 8 M urea treatment, have been shown to rapidly
traverse cell membranes (46). At least two proteins,
3
and rho A, have been successfully transduced into osteoclasts (1,
11). The mechanism of the effect of TAT is unclear, although present evidence suggests that TAT-assisted cellular permeation of
proteins is receptor independent (46).
We first detected high-efficiency transduction through the use of
an anti-TAT antibody. We then examined the function of freshly isolated
osteoclasts transduced with TAT-calcineurin A, with TAT-HA as
control, by using the traditional pit assay in which the resorption of
bone and number of resorbing cells are quantitated by simple
morphometry. We found that TAT-calcineurin A
inhibited osteoclastic
bone resorption, whereas TAT-HA did not. Both fusion proteins did not
affect cell number. Overall, therefore, the study provides compelling
molecular evidence for the following: 1) calcineurin A
isoforms
and
are expressed in osteoclasts, and 2)
calcineurin A
inhibits bone resorption.
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MATERIAL AND METHODS |
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Cloning of calcineurin A and A
isoforms.
A rabbit osteoclast cDNA library constructed in the
ZAP II
expression vector containing 1 × 1010 independent
clones, kindly provided by Prof. M. Kumegawa (Saitama, Japan), was used
for PCR amplification (21, 44). The
oligonucleotide primers for calcineurin A
and A
were designed on
the basis of analyses of the nucleotide sequences of rat, mouse, human,
and bovine cDNA as described previously (4). Their primer
sequences were calcineurin A
, forward:
5'-CGACAGGAAAAAAACTTGCTGGAT-3' (424-447), reverse:
5'-GTTTGGCTTTTCCTGTACATG-3' (1094-1075; GenBank accession no.
D90035); and calcineurin A
, forward:
5'-AACCATGATAGAAGTAGAAGCT-3' (294-315),
reverse: 5'-CACACACTGCTGGATAGTTATAA-3' (865-843;
GenBank accession no. D90036). The coding regions of the calcineurin A
and A
cDNA fragments were then amplified by PCR in a final volume of 50 µl containing 0.1 µl of rabbit osteoclast cDNA library (1 × 107 independent clones), 1 µl of each
oligonucleotide (50 µM), and 1 µl (5 U) of AmpliTaq
(PerkinElmer, Foster City, CA). The GeneAmp PCR System 2400 (PerkinElmer) was programmed as follows: 94°C for 5 min and then 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for
40 s. The PCR products were separated by agarose gel
electrophoresis. The 670- and 570-bp fragments of calcineurin A
and
A
, respectively, were isolated from excised gel slices by using a
gel purification kit from Qiagen (Valencia, CA) and ligated into
EcoRV-cut pBluescript II SK+ vector (Stratagene, La Jolla,
CA). The resulting plasmids, pBS-CNA
670 and pBS-CNA
570, were then
transformed into competent DH5
cells. The sequences of both inserts
were confirmed by sequence analysis and used as probes for library screening.
In situ RT-PCR cytoimaging of freshly isolated osteoclasts. The method has been described in detail in our laboratory's previous publications that contain the primer sequences for cathepsin K and GAPDH (2, 43). Osteoclasts were isolated from neonatal rat long bones and cultured on glass coverslips in Medium 199 with Earle's balanced salts (6.6 mM Na2CO3, M199-E) for 6 h, after which they were fixed with paraformaldehyde (4% vol/vol) in PBS for 20 min at 4°C. After two washes with cold PBS, the fixed cells were treated with 0.2 N HCl for 20 min at 20°C and washed with diethyl pyrocarbonate-treated water (Sigma). Cells were then treated with proteinase K (5 µg/ml in 10 mM-Tris · HCl, pH 8) for 15 min at 37°C followed by cold paraformaldehyde (4% vol/vol) for 30 min at 4°C. Before being air dried, the cells were dehydrated by sequential immersions in ethanol solutions, 70, 80, 90, and 100% (vol/vol), for 1 min at each concentration. The samples were then incubated overnight (37°C) with 1,500 U/ml RNase-free DNase I (Boehringer Mannheim, Indianapolis, IN) to remove genomic DNA. First-strand cDNA was synthesized by incubating cultures with 50 µl RT mixture (1 mM dNTP, 0.01 M DTT, 400 nM reverse primer in diethyl pyrocarbonate-treated water, and 14 U/µl SuperscriptII) for 60 min at 4°C. The samples were then treated separately with 50 µl PCR mixture containing 0.2 mM dNTP, 1× PCR buffer, 2.5 mM MgCl2, 0.1 U/µl Taq polymerase, 400 nM forward and reverse primers, and 10 µM digoxigenin (DIG)-labeled-11-dUTP (Boehringer Mannheim). Each sample was then gently covered with an AmpliCover disk, ensuring the absence of air bubbles. The GeneAmp In Situ PCR System 1000 (PerkinElmer) was programmed as follows: 94°C for 4 min and then 40 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min.
Incorporated DIG-11-dUTP in the PCR product was detected by an alkaline phosphatase-conjugated anti-DIG antiserum and alkaline phosphatase substrates, 4-nitroblue tetrazolium chloride, and 5-bromo-4-chloro-3-indoyl-phosphate using a DIG Nucleic Acid Detection Kit (Boehringer Mannheim) per manufacturer's protocol. Negative controls, in which primers were omitted, were run in parallel. Messenger RNA-expressing cells stained dark purplish brown, whereas negative controls did not stain. We then performed an analysis of the staining intensity by using a blinded observer as described previously (2, 43). Osteoclasts were scored on a scale from 0 to 3 (no staining to intense staining). The results were then plotted as a frequency histogram. This allowed us to determine the proportion of cells that lie in a certain intensity range. This analysis was utilized to examine the effect of incubating osteoclasts with cyclosporin A (8 × 10Synthesis, purification, and transduction of TAT-calcineurin
A.
We inserted a 42-bp double-stranded oligomeric nucleotide encoding the
12-amino acid TAT protein transduction domain flanked by glycine
residues (YGRKKRRQRRRG) and BamHI and XhoI
restriction endonuclease recognition sites at the 5'- and 3'-ends,
respectively, into pRSET A vector (Invitrogen, Carlsbad, CA). This
generated the plasmid pTAT, which was then transformed into competent
DH5
cells. Transformants were selected on LB agar plates containing 100 µg/ml ampicillin. Colonies expressing ampicillin
resistance were screened for the presence of the pTAT recombinant
plasmid by BamHI/XhoI restriction analysis, and
the sequence of insert was confirmed by sequencing analysis.
Complementary DNA for full-length calcineurin A
was inserted in
frame into the XhoI/EcoRI-cut pTAT expression
vector. The resulting plasmid, pTAT-CNA
, contains a 6-histidine tag
followed by the 12-amino acid TAT transduction domain. The pTAT-HA
vector containing an 11-amino acid TAT domain was a kind gift from
Prof. Y. Abou-Amer (Washington University, St. Louis, MO).
Immunocytochemistry and confocal imaging.
Freshly isolated osteoclasts (as above) were incubated in -MEM
containing 10% FBS for 24 h. Serum was removed and incubation was
continued for a further 2 h. The cells were incubated with TAT-calcineurin A
(200 nM) for 10 min at 37°C, fixed in
paraformaldehyde (4% vol/vol, in PBS, pH 7.4) for 20 min at 20°C,
incubated with precooled ethanol/acetic acid (2:1), and washed with PBS
(GIBCO-BRL). The cells were then exposed to polyclonal goat
anti-calcineurin A
antiserum (PP2BA
, Santa Cruz, Santa Cruz, CA)
or nonimmune goat IgG or antiserum PP2BA
plus a mouse monoclonal
anti-TAT antibody (ABI Advanced Biotechnologies, Columbia, MD) for
colocalization studies (all in DMEM, 1:100). After a 6-h
incubation, the coverslips were rinsed gently with PBS, drained, and
reincubated with donkey FITC-conjugated anti-goat IgG (green) or with
both anti-goat IgG and tetramethylrhodamine isothiocyanate-conjugated
anti-mouse IgM (red) for colocalization experiments in PBS for 60 min
(Jackon ImmunoReserach Laboratories, West Grove, PA). The coverslips
were then washed gently and drained. An epifluorescence microscope (Olympus AX-700) was used to visualize the staining by using FITC and
rhodamine filters, as appropriate.
Bone resorption assay.
The crude osteoclast suspension isolated in M199-H from neonatal (24- to 48-h-old) rat long bone was dispersed directly on devitalized bone
slices (6, 10, 13, 31). The cells were allowed to settle
for 30 min, after which each slice was washed in M199-E (with 10% FBS
vol/vol) to remove contaminants. The bone-osteoclast cultures were
further allowed to incubate in the same medium to enable attachment and
then incubated with various concentrations of TAT-calcineurin A or
TAT-HA (10-200 nM) for 10 min at 37°C. The cells were
washed again and allowed to incubate overnight at 37°C in humidified
5% CO2 (pH 6.9), after which the slices were fixed in 10%
glutaraldehyde and stained for tartrate-resistant acid phosphatase by
using a kit (386A, Sigma). Multinucleated osteoclasts were counted, and
the slices were then bleached with NaOCl (5 min) before air drying and
staining for toluidine blue to allow for visualization of the
osteoclastic excavations (pits). The number of pits was determined by
light microscopy. Each experiment was performed with osteoclasts
obtained from three animals with 5 or 6 bone slices/treatment. The
number of pits or osteoclasts per bone slice was expressed as
means ± SE. Student's unpaired t-test with
Bonferroni's correction for inequality was used to analyze the effect
of treatment, which was considered significant at P < 0.05. Note that at pH 6.9, resorptive activity is maximal (10,
13).
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RESULTS |
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Two positive cDNA clones were obtained by screening 1 × 107 clones of the rabbit osteoclast cDNA library by using
the 670-bp calcineurin A and 570-bp calcineurin A
cDNA coding
region of the respective PCR fragments as probes. Figure
1 shows the
full-length osteoclast calcineurin A
and A
cDNA coding region as
well as the predicted amino acid sequences. There was 68 and
78% similarity between the cDNA and amino acid sequences of
calcineurin A
and A
, respectively (GenBank accession nos.
AF541960 and AF541961).
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In addition to the coding region sequence shown in Fig. 1, we have also
obtained the full sequence of the cloned cDNA. Notably, nucleotide
sequence analysis of the 1,836-bp cloned cDNA fragment of the
calcineurin A gene revealed a 1,536-nucleotide-long coding region.
This encoded a 511-amino acid protein (molecular ratio = 56 kDa) and contained 396 and 284 nucleotides representing the 5'- and
3'-untranslated regions, respectively. The 1,575-bp coding region of
the A
gene encoded a 525-amino acid protein (molecular ratio = 58 kDa) and contained 36 and 1,621 bp of 5'- and 3'-untranslated regions.
The cDNA sequence of osteoclastic calcineurin A was 94, 93, 93, and
93% similar to corresponding full-length cDNA coding region sequences
of the human (GenBank accession no. BC025714), mouse (J05479), rat
(X57115), and bovine (U33868) homologs, respectively. The cDNA sequence
of osteoclast calcineurin A
was 97, 95, and 94% similar to
corresponding full-length cDNA coding region sequences of the human
(NM_021132), mouse (M81483), and rat (NM_017042) homologs,
respectively. On the other hand, no significant homology was found
between the sequence of the inserts and any other sequence in the
GenBank database.
Having cloned full-length calcineurin A, we next explored whether it
was expressed in freshly isolated mature osteoclasts. The same primers
as used above were employed in in situ RT-PCR experiments by using a
cytoimaging technique described previously (2, 43). Figure
2A shows light micrographs of
histostained osteoclasts after RT-PCR: a, an untreated osteoclast in an
experiment in which primers were omitted; b and c, intense brown
staining, demonstrating the expression of the two control genes
cathepsin K (cell-specific control) and GAPDH (housekeeping gene); d,
an osteoclast staining for calcineurin A
mRNA after vehicle
treatment; and e-g, similarly intense calcineurin A
mRNA
histostaining osteoclasts that had been treated with 8 × 10
7 M cyclosporin A, 5 × 10
9 M FK506,
or 5 × 10
7 M FK506, respectively.
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On visual examination, we found no differences in the overall staining
pattern in treated osteoclasts compared with untreated cells. We then
performed a semiquantitative analysis of staining intensity by using a
method modified from that reported by Adebanjo et al.
(2). Figure 2B shows osteoclasts staining for
calcineurin A mRNA that were assessed by a blinded observer, who
assigned an intensity level to the staining as a number from 0 to 3 (no staining to intense staining). Osteoclasts that underwent in situ RT-PCR incubated with primers but without treatment (Fig.
2B, a; n = 16 cells) showed a normal
(Gaussian) distribution of their assigned scores. Unlike what we have
seen before with our studies with interleukin-6 (2), the
data did not become significantly skewed when osteoclasts were treated
with 8 × 10
7 M cyclosporin A (Fig. 2B,
b; n = 25 cells), 5 × 10
9 M FK506
(c; n = 11 cells), or 5 × 10
7 M
FK506 (d; n = 25 cells). This suggested that the
calcineurin activity inhibitors cyclosporin A and FK506 did not
significantly alter calcineurin A
gene expression.
We next explored whether calcineurin could affect the resorptive
function of mature osteoclasts. It is difficult to transfect or virally
infect mature resorbing osteoclasts with genes encoding proteins of
interest, mainly because of the sparse number and limited life
span of freshly isolated cells. We therefore utilized a novel TAT
transduction method (see MATERIALS AND METHODS) for delivering the calcineurin A protein into osteoclasts. This
involved the initial synthesis of a TAT-calcineurin A
fusion protein
in E. coli. We constructed a plasmid, pTAT-CNA
, that
contained the 12-amino acid TAT protein transduction domain
(YGRKKRRQRRRG), the cloned calcineurin A
gene, and
NH2-terminal of the 6-histidine tag (Fig.
3A). The plasmid was
transformed into BL21 cells that were induced with
isopropyl-
-D-thiogalactopyranoside. The resulting protein was purified with an Ni-NTA affinity column. We similarly synthesized and purified TAT-HA as a control fusion protein. Western blotting of supernatants with the anti-calcineurin A
antibody or
anti-HA antibody showed intense bands, molecular sizes ~63 and 12 kDa, which corresponded to the TAT-calcineurin A
and TAT-HA fusion
proteins, respectively.
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Isolated osteoclasts were then transduced with 200 nM TAT-calcineurin
A by incubation at 37°C for 10 min. The cells were then costained
with a polyclonal anti-calcineurin A
antiserum (green) and a
monoclonal anti-TAT antibody (red). Without transduction, osteoclasts
were found to immunostain only with the anti-calcineurin A
antiserum
(green only), not with anti-TAT antibody, confirming the presence of
calcineurin A
protein in untransduced osteoclasts (Fig. 4,
A-C). However, after
transduction, an intense and mostly overlapping pattern (orange to
yellow) of red and green staining was noted (Fig. 4,
D-F). Immunodetection by both anti-calcineurin A
and
anti-TAT antibodies strongly suggested influx of the applied TAT-calcineurin A
fusion protein.
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We next examined the bone resorptive function of osteoclasts transduced
with TAT-calcineurin A. Because we cannot visualize staining in
osteoclasts settled on bone, we carried out immunostaining experiments
(as above) in parallel with our resorption assays for consistency of
our methodology and to ensure the uptake of the TAT fusion protein.
After a 10-min incubation at 37°C, osteoclasts previously settled on
devitalized bone slices were allowed to incubate further for 18 h.
Figure 5 shows a highly significant, concentration-dependent (10-200 nM) reduction in the number of pits per slice, with no change in the number of cells per slice. In
contrast, the control protein TAT-HA (10-200 nM) did not
significantly inhibit bone resorption or change cell number. The latter
indicates that the effect of TAT-calcineurin A
on osteoclast
resorptive function was not due to cell toxicity, although subtle
effects on cell viability cannot be excluded.
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DISCUSSION |
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The expression of calcineurin isoforms in the osteoclast is not unexpected. First, osteoclasts are unique in handling high-Ca2+ loads, both extracellularly and intracellularly (50). One would therefore expect this cell to possess a phosphatase that was responsive to changes in cytosolic Ca2+: calcineurin fits that role perfectly. Second, and perhaps more important, is the likely critical role of calcineurin in controlling gene transcription in the osteoclast, a cell that passes through several stages of differentiation before acquiring a bone resorptive phenotype (45). Finally, during resorption, osteoclasts actively secrete both acid and proteolytic enzymes (48). The underlying process of vesicular trafficking has been shown to be sensitive to calcineurin in other cells, such as synaptic neurons (26).
Our cloning and sequencing of two calcineurin isoforms, A and A
,
from a cDNA library constructed previously from pure rabbit osteoclast
preparations by Tezuka et al. (44) provides definitive evidence for their osteoclastic expression. Furthermore, using in situ
RT-PCR, we show that calcineurin A
mRNA is expressed in mature
osteoclasts that are capable of resorbing bone. We also show that by
using a specific anti-calcineurin A
antibody, the protein is
expressed in osteoclasts.
Clearly, the expression of calcineurin in mature osteoclasts raises the
question of its possible function in bone resorption. We provide direct
evidence that TAT-calcineurin A, the cell-permeant calcineurin
construct, inhibited bone resorption by isolated osteoclasts, whereas
the control TAT-HA protein did not. Osteoclast number did not change
with either fusion protein, thereby excluding cytotoxicity and
apoptosis. Previous apparently paradoxical observations on the
inhibition of bone resorption by cyclosporin A, a calcineurin inhibitor, continue to exist (4). However, it is known
that cyclosporin A interacts with other cellular targets independently of calcineurin; this may explain inhibition of bone resorption with the
drug (39).
Several mechanisms nevertheless underscore the antiresorptive action of
TAT-calcineurin A in the absence of cell toxicity. First, it is
possible that calcineurin might directly dephosphorylate proteins
involved in vesicular trafficking in response to changes in cytosolic
Ca2+ occurring as a result of bone resorption
(42). Proteins, such as dynamin, may be critical targets
in inhibiting acid secretion and enzyme release (26).
Second, calcineurin may trigger the redistribution of osteoclast
integrins in response to Ca2+ transients through a direct
effect, as has been shown for other cells (27, 34).
Although this effect is particularly relevant to the osteoclast, a
direct molecular interaction between
v
3, the main osteoclast integrin, and calcineurin has not yet been established.
Third, a longer term and possibly more physiologically relevant
response could occur through effects on gene transcription exerted
through the traditional NFATc signaling pathway used by calcineurin in
lymphocytes and cardiac cells (12). In lymphocytes, activation of calcineurin by calmodulin or Ca2+ results in
transactivation of several critical genes, including the
granulocyte-macrophage colony-stimulating factor, interleukins-2, -3, -4, and -5, CD40, and the Fas genes (12, 19, 33,
38). Although these responses are mediated by the transcription
factors NFATc1-4 (12), cardiac endothelial cell
growth and hippocampal neuronal stimulation involve NFATc1 and NFATC4,
respectively (17, 35). We are unclear as to which NFATc
isoform is involved in calcineurin's effects on osteoclasts. In
addition, from unpublished studies on myoblastic cells and other
published evidence, we can speculate that there are additional
dephosphorylation targets for calcineurin in the osteoclast, notably
IB
, NF-
B, and MeF (5, 14, 29).
It is likely that critical genes for Ca2+ release channels
including IP3 receptors and RyRs that are widely expressed
in the osteoclast are potential targets for osteoclastic calcineurin. It has been shown that the overexpression of calcineurin A in myoblastic C2C12 cells results in dramatic increases in the expression of RyR-1 (5). In the same cell type oxidative stress
resulting from mitochondrial DNA deletion, for example, is associated
with the enhanced expression of both RyR-1 and calcineurin A
(5). Thus there is a clear direct relationship between the
expression of calcineurin A
and target RyR genes. In addition,
inhibitors such as FK506 decouple the molecular interaction between
RyRs and calcineurin (9, 20). Should the relationship
between calcineurin and RyR expression hold in the osteoclast, it would be of special relevance to the function of this cell. Osteoclasts express high levels of RyR-2 uniquely at their plasma membrane (49). We have provided evidence that this
surface-expressed RyR-2 plays a critical role in extracellular
Ca2+ sensing, a process by which an osteoclast monitors
changes in ambient Ca2+ levels and transduces intracellular
Ca2+ signals during bone resorption (49, 50).
A high intracellular Ca2+ level during bone resorption
could potentially, through the activation of calcineurin, result in
elevated RyR-2 expression. Admittedly speculative, this may be a
positive feedback mechanism through which the sensitivity of the
osteoclast to changes in extracellular Ca2+, exerted via
RyR expression, is maintained during resorption.
Although the physiological relevance of calcineurin in bone resorption
and the molecular mechanisms thereof remain issues for further
investigation, this study clarifies that calcineurin does not alter its
own expression. From our in situ RT-PCR studies, albeit
semiquantitative, it is clear that the two inhibitors, cyclosporin A
and FK506, did not affect the expression of the calcineurin A gene.
In other words, calcineurin gene expression is not regulated by its
phosphatase activity. Similarly, to our knowledge, such regulation has
not been documented in other cells. It is also unlikely that
TAT-mediated transduction is affected by cyclosporin A or FK506, as
these drugs are not known to affect TAT delivery into cells.
An interesting clinical paradigm has emerged recently that significantly enhances the importance of our discovery of calcineurin in the osteoclast. It is now known that the DSCR1 gene, a potent inhibitor of calcineurin activity located on human chromosome 21, is overexpressed in Down syndrome as a result of trisomy (15). It is believed that such overexpression results in defects in the development of the brain, immune system, heart, and skeleton in these children. Localization of calcineurin in a skeletal cell, such as the osteoclast, and a prediction of its function in skeletal remodeling might therefore be a first step in understanding the molecular pathophysiology of the skeletal defects in Down syndrome (12).
Finally, this study further documents the use of the TAT-transduction
system as a reliable means of transducing mature osteoclasts with
proteins with high efficiency. At least two reports have similarly used
TAT to transduce the mutated form of IK
and the small GTP-binding
protein Rho, respectively, into osteoclasts (1, 11). The
system, developed initially by Vocero-Akbani et al. (46),
establishes a new paradigm for protein transduction in sparse
populations of cells, such as osteoclasts, without the need to infect,
transfect, or microinject.
In conclusion, we have documented the existence of the
Ca2+/calmodulin phosphatase calcineurin in osteoclasts and
demonstrated its role as an inhibitor of bone resorption. We have also
shown that calcineurin A can be effectively delivered into
osteoclasts as a functionally active protein through its fusion to TAT,
an arginine-rich sequence derived from the human immunodeficiency virus
protein. The study paves the way for future investigations to study
calcineurin signal transduction in osteoclasts by using similar
cell-permeant constructs of calcineurin and its signaling molecules,
such as the NFATc isoforms.
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ACKNOWLEDGEMENTS |
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M. Zaidi acknowledges the support of the National Institute on Aging (RO1-AG-14197-07) and Department of Veterans Affairs (Merit Review and Geriatric Research, Education, and Clinical Centers Program).
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Sun, Box 1055, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029 (E-mail: li.sun{at}mssm.edu).
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. Section 1734 solely to indicate this fact.
First published November 5, 2002;10.1152/ajprenal.00084.2002
Received 11 March 2002; accepted in final form 2 November 2002.
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