Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha

Li Sun1, Baljit S. Moonga1, Min Lu1, Neeha Zaidi1, Jameel Iqbal1, Harry C. Blair2, Solomon Epstein1, Etsuko Abe1, Bruce R. Troen1, Christopher L.-H. Huang3, and Mone Zaidi1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Aalpha and Abeta 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 Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha 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 Aalpha 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 Aalpha 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 Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha -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 Aalpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta , and gamma ) are products of different genes and exhibit ~86% sequence homology (GenBank accession no. J05479, M81483, and NM_008915, respectively). Calcineurin Aalpha 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 Aalpha , 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 alpha  and beta . 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 Aalpha 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 Aalpha 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 Aalpha 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, beta 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 Aalpha , 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 Aalpha 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 alpha  and beta  are expressed in osteoclasts, and 2) calcineurin Aalpha inhibits bone resorption.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of calcineurin Aalpha and Abeta isoforms. A rabbit osteoclast cDNA library constructed in the lambda 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 Aalpha and Abeta 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 Aalpha , forward: 5'-CGACAGGAAAAAAACTTGCTGGAT-3' (424-447), reverse: 5'-GTTTGGCTTTTCCTGTACATG-3' (1094-1075; GenBank accession no. D90035); and calcineurin Abeta , forward: 5'-AACCATGATAGAAGTAGAAGCT-3' (294-315), reverse: 5'-CACACACTGCTGGATAGTTATAA-3' (865-843; GenBank accession no. D90036). The coding regions of the calcineurin Aalpha and Abeta 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 Aalpha and Abeta , 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-CNAalpha 670 and pBS-CNAbeta 570, were then transformed into competent DH5alpha cells. The sequences of both inserts were confirmed by sequence analysis and used as probes for library screening.

To obtain full-length calcineurin Aalpha and Abeta , the 670- and 570-bp fragments were labeled with [alpha -32P]dCTP (3,000 Ci/mmol; NEN Life Science, Boston, MA) by using the Redprime Random Prime Labeling Kit (Amersham Pharmacia Biotech, Piscataway, NJ). DNA manipulation was performed by using the standard protocol as described by Sambrook et al. (36). Approximately 1 × 107 plaques of the rabbit osteoclast cDNA library were screened initially with the probes of calcineurin Aalpha and Abeta . Plating with independent clones made two replica filters. After SDS-alkali treatment, Tris neutralization, and cross-linking of nucleic acids to nylon membrane with a GS Gene Linker UV chamber (Bio-Rad, Hercules CA), the filters were hybridized overnight at 42°C with labeled probe in a solution containing formamide (50% vol/vol), 6× SSC, 5× Denhardt's, SDS (0.5% wt/vol), and denatured fragmented salmon sperm DNA (0.1 mg/ml). After a high-stringency wash at 68°C for 1 h, the filters were subjected to autoradiography for 20 h at -70°C. Positive recombinant plaques were purified from phage lysates according to the lambda ZAP II library's instruction (Stratagene). The cDNA clones were confirmed by Southern blot and PCR analysis. The positive clones were then sequenced and compared with those of calcineurin Aalpha and Abeta from humans, mice, and rats.

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 × 10-7 M) or FK506 (5 × 10-9 and 5 × 10-7 M) or appropriate vehicle on gene expression in separate experiments.

Synthesis, purification, and transduction of TAT-calcineurin Aalpha . 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 DH5alpha 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 Aalpha was inserted in frame into the XhoI/EcoRI-cut pTAT expression vector. The resulting plasmid, pTAT-CNAalpha , 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).

The expression constructs pTAT-CNAalpha and pTAT-HA were transformed into Escherichia coli BL21 (DE3) pLysS cells and allowed to grow at 30°C in 1 liter SOB medium containing 100 µg/ml ampicillin for 4 h to midlog phase. Isopropyl-beta -D-thiogalactopyranoside was then added to a final concentration of 1 mM. The incubation was continued for another 3 h. Cells (1.2 g wet wt) were harvested by centrifugation at 4,000 g for 20 min. The cell pellet was resuspended in 10 ml of buffer A (6 M guanidine hydrochloride, 0.1 M NaH2PO4, and 0.01 M Tris, pH 8.0) and stirred for 1 h at room temperature followed by sonication on ice until turbid. After centrifugation at 12,000 g for 15 min, the supernatant containing crude extracts was applied to a Ni-NTA purification column (Qiagen) and washed with 10 bed volumes of buffer A, 5 bed volumes of buffer B (8 M urea, 0.1 M NaH2PO4, and 0.01 M Tris, pH 8.0), and 10 bed volumes of buffer C (8 M urea, 0.1 M NaH2PO4, and 0.01 M Tris, pH 6.3) plus 0.2 M NaCl. The resin-bound TAT-calcineurin Aalpha or TAT-HA fusion proteins were subsequently eluted with buffer C containing 0.25 M imidazole. Urea was removed by dialysis against PBS in a volume of 2 liters for 8 h at 4°C. A total of 4 and 8 mg recombinant TAT-calcineurin Aalpha and TAT-HA proteins, respectively, were obtained and stored at -70°C.

Immunocytochemistry and confocal imaging. Freshly isolated osteoclasts (as above) were incubated in alpha -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 Aalpha (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 Aalpha antiserum (PP2BAalpha , Santa Cruz, Santa Cruz, CA) or nonimmune goat IgG or antiserum PP2BAalpha 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 Aalpha 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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two positive cDNA clones were obtained by screening 1 × 107 clones of the rabbit osteoclast cDNA library by using the 670-bp calcineurin Aalpha and 570-bp calcineurin Abeta cDNA coding region of the respective PCR fragments as probes. Figure 1 shows the full-length osteoclast calcineurin Aalpha and Abeta 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 Aalpha and Abeta , respectively (GenBank accession nos. AF541960 and AF541961).


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Fig. 1.   The cDNA and amino acid sequences of calcineurin Aalpha (CNAalpha ) and Abeta (CNAbeta ) cloned from a cDNA library that was constructed previously from freshly isolated rabbit osteoclast preparations. Gaps have been introduced to maximize homology.

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 Aalpha 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 Abeta 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 Aalpha 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 Abeta 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 Aalpha , 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 Aalpha mRNA after vehicle treatment; and e-g, similarly intense calcineurin Aalpha 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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Fig. 2.   A: in situ RT-PCR performed on freshly isolated osteoclasts by using primers constructed for CNAalpha , cathepsin K (Cath K; cell-specific control gene), and GAPDH (housekeeping gene). Cells were also treated with the calcineurin inhibitors cyclosporin A (CsA) or FK506. B: semiquantitative estimates of staining intensity shown in frequency histograms. Staining intensity was graded as described in MATERIALS AND METHODS by an independent blinded observer who scored the intensity from 0 (no staining) to 3 (intense staining) in 3 experiments. The data were analyzed statistically for skews, and shifts were considered significant if P < 0.01.

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 Aalpha 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 Aalpha 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 Aalpha protein into osteoclasts. This involved the initial synthesis of a TAT-calcineurin Aalpha fusion protein in E. coli. We constructed a plasmid, pTAT-CNAalpha , that contained the 12-amino acid TAT protein transduction domain (YGRKKRRQRRRG), the cloned calcineurin Aalpha gene, and NH2-terminal of the 6-histidine tag (Fig. 3A). The plasmid was transformed into BL21 cells that were induced with isopropyl-beta -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 Aalpha antibody or anti-HA antibody showed intense bands, molecular sizes ~63 and 12 kDa, which corresponded to the TAT-calcineurin Aalpha and TAT-HA fusion proteins, respectively.


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Fig. 3.   Synthesis and purification of TAT-CNAalpha fusion protein. A: construction of plasmid pTAT (2.95 kb) by inserting the TAT sequence and the CNAalpha coding region cDNA. B: Western immunoblot of either a crude extract of BL21 (DE3) pLysS cells transformed with pTAT-CNAalpha (TAT-CNAalpha ) or after its purification on a Ni-NTA column (Purified TAT-CNAalpha ). Lane 1: control cells that were not transformed. An anti-CNAalpha antiserum (PP2BAalpha ) was used to immunostain the blots.

Isolated osteoclasts were then transduced with 200 nM TAT-calcineurin Aalpha by incubation at 37°C for 10 min. The cells were then costained with a polyclonal anti-calcineurin Aalpha antiserum (green) and a monoclonal anti-TAT antibody (red). Without transduction, osteoclasts were found to immunostain only with the anti-calcineurin Aalpha antiserum (green only), not with anti-TAT antibody, confirming the presence of calcineurin Aalpha 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 Aalpha and anti-TAT antibodies strongly suggested influx of the applied TAT-calcineurin Aalpha fusion protein.


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Fig. 4.   Double immunostaining of isolated osteoclasts with a polyclonal goat anti-CNAalpha antiserum (PP2BAalpha ) and monoclonal mouse anti-TAT antibody. FITC-labeled anti-goat IgG (green) and tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgM (red), respectively, were used as secondary antibodies. A-C: osteoclast that was incubated with vehicle, hence, the absence of anti-TAT (red) staining in B. A: endogenous expression of CNAalpha . D-F: cell that was incubated with 200 nM TAT-CNAalpha for 10 min at 37°C. Both anti-CNAalpha (green; D) and anti-TAT (red; E) staining are superimposed on the merged image (F).

We next examined the bone resorptive function of osteoclasts transduced with TAT-calcineurin Aalpha . 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 Aalpha on osteoclast resorptive function was not due to cell toxicity, although subtle effects on cell viability cannot be excluded.


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Fig. 5.   Effect of incubation with TAT-hemaglutinin (HA) or TAT-CNAalpha (10, 100, and 200 nM) for 10 min at 37°C on osteoclastic bone resorption (number of pits/slice; A and C) and osteoclast number (number of cells/slice; B and D). Values are by Student's t-test with Bonferroni's correction for inequality. P < 0.01, significant difference.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, Aalpha and Abeta , 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 Aalpha mRNA is expressed in mature osteoclasts that are capable of resorbing bone. We also show that by using a specific anti-calcineurin Aalpha 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 Aalpha , 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 Aalpha 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 alpha vbeta 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 Ikappa Bbeta , NF-kappa 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 Aalpha 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 Aalpha (5). Thus there is a clear direct relationship between the expression of calcineurin Aalpha 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 Aalpha 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 Ikappa Kbeta 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 Aalpha 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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