Molecular cloning, expression, and function ofosteoclastic calcineurin A - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:33 |只看该作者 |倒序浏览 |打印

作者：LiSun, Baljit S.Moonga, MinLu, NeehaZaidi, JameelIqbal, Harry C.Blair, SolomonEpstein, EtsukoAbe, Bruce R.Troen, Christopher L.-H.Huang,  MoneZaidi作者单位：1 Mount Sinai Bone Program and Division ofEndocrinology, Mount Sinai School of Medicine, New York, 1002 andEndocrine Division and The Geriatric Research Education andClinical Center, Veterans Affairs Medical Center, Bronx, New York10468; Department of Pathology, University ofPittsburgh, Pittsburgh,

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      【摘要】* [; w/ h" ?7 v* a8 q
   This study explores the role of thecalmodulin- and Ca 2  -sensitive phosphatase calcineurin A inthe control of bone resorption by mature osteoclasts. We first clonedfull-length calcineurin A and A cDNA from a rabbit osteoclastlibrary. Sequence analysis revealed an ~95 and 86% homology betweenthe amino acid and the nucleotide sequences, respectively, of the twoisoforms. The two rabbit isoforms also showed significant homology withthe mouse, rat, and human homologs. In situ RT-PCR showed evidence ofhigh 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 incells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 × 10 7 M) andFK506 (5 × 10 9 and 5 × 10 7 M).We then constructed a fusion protein comprising calcineurin A andTAT, a 12-amino acid-long arginine-rich sequence of the humanimmunodeficiency virus protein. Others have previously shown that thefusion of proteins to this sequence results in their receptor-lesstransduction into cells, including osteoclasts. Similarly, unfolding ofthe TAT-calcineurin A fusion protein by shocking with 8 M urearesulted in its rapid influx, within minutes, into as many as 90% ofall freshly isolated rat osteoclasts, as was evident on doubleimmunostaining with anti-calcineurin A and anti-TAT antibodies. Pitassays performed with TAT-calcineurin A -positive osteoclastsrevealed a concentration-dependent (10-200 nM) attenuation of boneresorption in the absence of cell cytotoxicity or changes in cellnumber. TAT-hemaglutinin did not produce significant effects on boneresorption or cell number. The study suggests the following: 1 ) the 61-kDa protein phosphatase calcineurin A can beeffectively tranduced into osteoclasts by using the TAT-based approach,and 2 ) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.
      【关键词】 calcium channel gene cloning osteoclast osteoporosis3 O0 \2 [+ u, v5 ]+ S' }$ b
   INTRODUCTION

MAINTENANCE OF SKELETAL INTEGRITY depends on a precise balance between bone formation andresorption. An absolute or relative increase in resorption overformation results in bone loss. Bone is removed by osteoclasts andis rebuilt by osteoblasts as part of the bone remodeling process. Theactivity of both osteoclasts and osteoblasts is regulated by precisemolecular signals, some of which are sensitive to changes in cytosolicCa 2  ( 50 ). The osteoclast in particular isexposed to high millimolar extracellular concentrations ofCa 2  during resorption ( 40 ). It has anextracellular Ca 2  sensor thought to be a type II ryanodinereceptor (RyR) expressed at the plasma membrane ( 30, 47, 49 ).
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Calcineurin is the only serine/threonine protein phosphatase sensitiveto both Ca 2  and calmodulin that plays a critical role incoupling Ca 2  signals to cellular responses ( 22, 23, 41 ). The calcineurin heterodimer comprises one catalytic and oneregulatory subunit (subunits A and B, respectively); the latter ishighly conserved from yeast to humans ( 16 ). The threeknown isoforms of mammalian calcineurin A (,, and ) areproducts 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, muscledevelopment, and cardiac valve formation. We recently providedpreliminary evidence that calcineurin A, expressed in osteoclasts,plays a role in the regulation of bone resorption ( 4 ).
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Here, we propose to understand more fully the function of calcineurin Ain bone resorption by using a complement of molecular and cellularapproaches. First, we cloned full-length cDNAs for the calcineurin Aisoforms and. We then utilized in situ RT-PCR to demonstratemRNA expression in freshly isolated mature osteoclasts. Using the sametechnique, we examined, in a semiquantitative manner, changes inexpression of calcineurin A in response to two known inhibitors ofcalcineurin activity, cyclosporin A and tacrolimus (FK506). Bothinhibitors are known to inhibit the phosphatase activity of calcineurinbut may have downstream effects on calcineurin A expression( 3, 37 ). Both cyclosporin A and FK506 also cause profoundbone loss in vivo both in animals and in humans ( 4, 7 ).However, the precise mechanism of their action on bone cells remains unclear.( J9 L2 |: a9 S: o1 k1 v4 b

To evaluate the function of calcineurin in osteoclastic boneresorption, we developed a method through which we were able totransduce most of the relatively sparse population of cells that weisolate fresh from neonatal rats. The technique involved creating afusion 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 thathave been unfolded by 8 M urea treatment, have been shown to rapidlytraverse cell membranes ( 46 ). At least two proteins, 3and 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 ofproteins is receptor independent ( 46 ).

We first detected high-efficiency transduction through the use ofan anti-TAT antibody. We then examined the function of freshly isolatedosteoclasts transduced with TAT-calcineurin A, with TAT-HA ascontrol, by using the traditional pit assay in which the resorption ofbone and number of resorbing cells are quantitated by simplemorphometry. We found that TAT-calcineurin A inhibited osteoclasticbone resorption, whereas TAT-HA did not. Both fusion proteins did notaffect cell number. Overall, therefore, the study provides compellingmolecular evidence for the following: 1 ) calcineurin Aisoforms and are expressed in osteoclasts, and 2 )calcineurin A inhibits bone resorption.
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MATERIAL AND METHODS  k( P& M0 H; R! x( l5 c
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Cloning of calcineurin A and A isoforms. A rabbit osteoclast cDNA library constructed in the ZAP IIexpression vector containing 1 × 10 10 independentclones, kindly provided by Prof. M. Kumegawa (Saitama, Japan), was usedfor PCR amplification ( 21, 44 ). Theoligonucleotide primers for calcineurin A and A were designed onthe basis of analyses of the nucleotide sequences of rat, mouse, human,and bovine cDNA as described previously ( 4 ). Their primersequences 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 × 10 7 independent clones), 1 µl of eacholigonucleotide (50 µM), and 1 µl (5 U) of Ampli Taq (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 for40 s. The PCR products were separated by agarose gelelectrophoresis. The 670- and 570-bp fragments of calcineurin A andA, respectively, were isolated from excised gel slices by using agel purification kit from Qiagen (Valencia, CA) and ligated into Eco RV-cut pBluescript II SK  vector (Stratagene, La Jolla,CA). The resulting plasmids, pBS-CNA 670 and pBS-CNA 570, were thentransformed into competent DH5 cells. The sequences of both insertswere confirmed by sequence analysis and used as probes for library screening.

To obtain full-length calcineurin A and A, the 670- and 570-bpfragments were labeled with [ - 32 P]dCTP (3,000 Ci/mmol;NEN Life Science, Boston, MA) by using the Red prime RandomPrime Labeling Kit (Amersham Pharmacia Biotech, Piscataway, NJ). DNAmanipulation was performed by using the standard protocol as describedby Sambrook et al. ( 36 ). Approximately 1 × 10 7 plaques of the rabbit osteoclast cDNA library werescreened initially with the probes of calcineurin A and A.Plating with independent clones made two replica filters. AfterSDS-alkali treatment, Tris neutralization, and cross-linking of nucleicacids to nylon membrane with a GS Gene Linker UV chamber (Bio-Rad,Hercules CA), the filters were hybridized overnight at 42°C withlabeled probe in a solution containing formamide (50% vol/vol), 6×SSC, 5× Denhardt's, SDS (0.5% wt/vol), and denatured fragmentedsalmon sperm DNA (0.1 mg/ml). After a high-stringency wash at 68°Cfor 1 h, the filters were subjected to autoradiography for 20 h at 70°C. Positive recombinant plaques were purified from phagelysates according to the ZAP II library's instruction (Stratagene).The cDNA clones were confirmed by Southern blot and PCR analysis. Thepositive clones were then sequenced and compared with those ofcalcineurin A and A 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 previouspublications that contain the primer sequences for cathepsin K andGAPDH ( 2, 43 ). Osteoclasts were isolated from neonatal ratlong bones and cultured on glass coverslips in Medium 199 with Earle'sbalanced salts (6.6 mM Na 2 CO 3, M199-E) for6 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 andwashed with diethyl pyrocarbonate-treated water (Sigma). Cellswere then treated with proteinase K (5 µg/ml in 10 mM-Tris · HCl, pH 8) for 15 min at 37°Cfollowed by cold paraformaldehyde (4% vol/vol) for 30 min at 4°C.Before being air dried, the cells were dehydrated by sequentialimmersions 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 wassynthesized by incubating cultures with 50 µl RT mixture (1 mM dNTP,0.01 M DTT, 400 nM reverse primer in diethyl pyrocarbonate-treatedwater, and 14 U/µl SuperscriptII) for 60 min at 4°C. The sampleswere then treated separately with 50 µl PCR mixture containing 0.2 mMdNTP, 1× PCR buffer, 2.5 mM MgCl 2, 0.1 U/µl Taq polymerase, 400 nM forward and reverse primers, and 10 µM digoxigenin (DIG)-labeled-11-dUTP (Boehringer Mannheim). Eachsample was then gently covered with an AmpliCover disk, ensuring theabsence 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 alkalinephosphatase-conjugated anti-DIG antiserum and alkaline phosphatasesubstrates, 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 thestaining 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 afrequency histogram. This allowed us to determine the proportion ofcells that lie in a certain intensity range. This analysis was utilizedto 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 geneexpression in separate experiments.
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Synthesis, purification, and transduction of TAT-calcineurinA. We inserted a 42-bp double-stranded oligomeric nucleotide encoding the12-amino acid TAT protein transduction domain flanked by glycineresidues (YGRKKRRQRRRG) and Bam HI and Xho Irestriction endonuclease recognition sites at the 5'- and 3'-ends,respectively, into pRSET A vector (Invitrogen, Carlsbad, CA). Thisgenerated the plasmid pTAT, which was then transformed into competentDH5 cells. Transformants were selected on LB agar plates containing 100 µg/ml ampicillin. Colonies expressing ampicillinresistance were screened for the presence of the pTAT recombinantplasmid by Bam HI/ Xho I restriction analysis, andthe sequence of insert was confirmed by sequencing analysis.Complementary DNA for full-length calcineurin A was inserted inframe into the Xho I/ Eco RI-cut pTAT expressionvector. The resulting plasmid, pTAT-CNA, contains a 6-histidine tagfollowed by the 12-amino acid TAT transduction domain. The pTAT-HAvector containing an 11-amino acid TAT domain was a kind gift fromProf. Y. Abou-Amer (Washington University, St. Louis, MO).

The expression constructs pTAT-CNA and pTAT-HA were transformed into Escherichia coli BL21 (DE3) pLysS cells and allowed to growat 30°C in 1 liter SOB medium containing 100 µg/ml ampicillin for4 h to midlog phase.Isopropyl- - D -thiogalactopyranoside was then added to afinal concentration of 1 mM. The incubation was continued for another3 h. Cells (1.2 g wet wt) were harvested by centrifugation at4,000 g for 20 min. The cell pellet was resuspended in 10 mlof buffer A (6 M guanidine hydrochloride, 0.1 MNaH 2 PO 4, and 0.01 M Tris, pH 8.0) and stirredfor 1 h at room temperature followed by sonication on ice untilturbid. After centrifugation at 12,000 g for 15 min, thesupernatant containing crude extracts was applied to a Ni-NTApurification column (Qiagen) and washed with 10 bed volumes of bufferA, 5 bed volumes of buffer B (8 M urea, 0.1 MNaH 2 PO 4, and 0.01 M Tris, pH 8.0), and 10 bedvolumes of buffer C (8 M urea, 0.1 M NaH 2 PO 4,and 0.01 M Tris, pH 6.3) plus 0.2 M NaCl. The resin-boundTAT-calcineurin A or TAT-HA fusion proteins were subsequently elutedwith buffer C containing 0.25 M imidazole. Urea was removed by dialysisagainst PBS in a volume of 2 liters for 8 h at 4°C. A total of 4 and 8 mg recombinant TAT-calcineurin A and TAT-HA proteins,respectively, were obtained and stored at 70°C.
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Immunocytochemistry and confocal imaging. Freshly isolated osteoclasts (as above) were incubated in -MEMcontaining 10% FBS for 24 h. Serum was removed and incubation wascontinued for a further 2 h. The cells were incubated with TAT-calcineurin A (200 nM) for 10 min at 37°C, fixed inparaformaldehyde (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 goatanti-calcineurin A antiserum (PP2BA, Santa Cruz, Santa Cruz, CA)or nonimmune goat IgG or antiserum PP2BA plus a mouse monoclonalanti-TAT antibody (ABI Advanced Biotechnologies, Columbia, MD) forcolocalization studies (all in DMEM, 1:100). After a 6-hincubation, the coverslips were rinsed gently with PBS, drained, andreincubated with donkey FITC-conjugated anti-goat IgG (green) or withboth anti-goat IgG and tetramethylrhodamine isothiocyanate-conjugatedanti-mouse IgM (red) for colocalization experiments in PBS for 60 min(Jackon ImmunoReserach Laboratories, West Grove, PA). The coverslipswere then washed gently and drained. An epifluorescence microscope (Olympus AX-700) was used to visualize the staining by using FITC andrhodamine filters, as appropriate.( Q+ ^+ q: B. ?8 T
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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 boneslices ( 6, 10, 13, 31 ). The cells were allowed to settlefor 30 min, after which each slice was washed in M199-E (with 10% FBSvol/vol) to remove contaminants. The bone-osteoclast cultures werefurther allowed to incubate in the same medium to enable attachment andthen incubated with various concentrations of TAT-calcineurin A orTAT-HA (10-200 nM) for 10 min at 37°C. The cells werewashed again and allowed to incubate overnight at 37°C in humidified5% CO 2 (pH 6.9), after which the slices were fixed in 10%glutaraldehyde and stained for tartrate-resistant acid phosphatase byusing a kit (386A, Sigma). Multinucleated osteoclasts were counted, andthe slices were then bleached with NaOCl (5 min) before air drying andstaining for toluidine blue to allow for visualization of theosteoclastic excavations (pits). The number of pits was determined bylight microscopy. Each experiment was performed with osteoclastsobtained from three animals with 5 or 6 bone slices/treatment. Thenumber of pits or osteoclasts per bone slice was expressed asmeans ± SE. Student's unpaired t -test withBonferroni's correction for inequality was used to analyze the effectof treatment, which was considered significant at P ( 10, 13 ).
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RESULTS
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Two positive cDNA clones were obtained by screening 1 × 10 7 clones of the rabbit osteoclast cDNA library by usingthe 670-bp calcineurin A and 570-bp calcineurin A cDNA codingregion of the respective PCR fragments as probes. Figure 1 shows thefull-length osteoclast calcineurin A and A cDNA coding region aswell as the predicted amino acid sequences. There was 68 and78% similarity between the cDNA and amino acid sequences ofcalcineurin A and A, respectively (GenBank accession nos. AF541960 and AF541961 ).
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Fig. 1. The cDNA and amino acid sequences of calcineurin A (CNA ) and A (CNA ) cloned from a cDNA library that wasconstructed previously from freshly isolated rabbit osteoclastpreparations. Gaps have been introduced to maximize homology.

In addition to the coding region sequence shown in Fig. 1, we have alsoobtained the full sequence of the cloned cDNA. Notably, nucleotidesequence analysis of the 1,836-bp cloned cDNA fragment of thecalcineurin 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'- and3'-untranslated regions, respectively. The 1,575-bp coding region ofthe 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.
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The cDNA sequence of osteoclastic calcineurin A was 94, 93, 93, and93% similar to corresponding full-length cDNA coding region sequencesof the human (GenBank accession no. BC025714 ), mouse ( J05479 ), rat( X57115 ), and bovine ( U33868 ) homologs, respectively. The cDNA sequenceof osteoclast calcineurin A was 97, 95, and 94% similar tocorresponding 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 foundbetween the sequence of the inserts and any other sequence in theGenBank database.

Having cloned full-length calcineurin A, we next explored whether itwas expressed in freshly isolated mature osteoclasts. The same primersas used above were employed in in situ RT-PCR experiments by using acytoimaging technique described previously ( 2, 43 ). Figure 2 A shows light micrographs ofhistostained osteoclasts after RT-PCR: a, an untreated osteoclast in anexperiment in which primers were omitted; b and c, intense brownstaining, demonstrating the expression of the two control genescathepsin K (cell-specific control) and GAPDH (housekeeping gene); d,an osteoclast staining for calcineurin A mRNA after vehicletreatment; and e-g, similarly intense calcineurin A mRNAhistostaining osteoclasts that had been treated with 8 × 10 7 M cyclosporin A, 5 × 10 9 M FK506,or 5 × 10 7 M FK506, respectively.# k7 d' R! i6 R

Fig. 2. A : in situ RT-PCR performed on freshly isolatedosteoclasts by using primers constructed for CNA, cathepsin K (CathK; cell-specific control gene), and GAPDH (housekeeping gene). Cellswere also treated with the calcineurin inhibitors cyclosporin A (CsA)or FK506. B : semiquantitative estimates of stainingintensity shown in frequency histograms. Staining intensity was gradedas described in MATERIALS AND METHODS by an independentblinded observer who scored the intensity from 0 (no staining) to 3 (intense staining) in 3 experiments. The data were analyzedstatistically for skews, and shifts were considered significant if P

On visual examination, we found no differences in the overall stainingpattern in treated osteoclasts compared with untreated cells. We thenperformed a semiquantitative analysis of staining intensity by using amethod modified from that reported by Adebanjo et al.( 2 ). Figure 2 B shows osteoclasts staining forcalcineurin A mRNA that were assessed by a blinded observer, whoassigned 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. 2 B, a; n = 16 cells) showed a normal(Gaussian) distribution of their assigned scores. Unlike what we haveseen before with our studies with interleukin-6 ( 2 ), thedata did not become significantly skewed when osteoclasts were treatedwith 8 × 10 7 M cyclosporin A (Fig. 2 B,b; n = 25 cells), 5 × 10 9 M FK506(c; n = 11 cells), or 5 × 10 7 MFK506 (d; n = 25 cells). This suggested that thecalcineurin activity inhibitors cyclosporin A and FK506 did notsignificantly alter calcineurin A gene expression.8 V( b( p" F, ~6 ^

We next explored whether calcineurin could affect the resorptivefunction of mature osteoclasts. It is difficult to transfect or virallyinfect mature resorbing osteoclasts with genes encoding proteins ofinterest, mainly because of the sparse number and limited lifespan of freshly isolated cells. We therefore utilized a novel TATtransduction method (see MATERIALS AND METHODS ) for delivering the calcineurin A protein into osteoclasts. Thisinvolved the initial synthesis of a TAT-calcineurin A fusion proteinin E. coli. We constructed a plasmid, pTAT-CNA, thatcontained the 12-amino acid TAT protein transduction domain(YGRKKRRQRRRG), the cloned calcineurin A gene, andNH 2 -terminal of the 6-histidine tag (Fig. 3 A ). The plasmid wastransformed into BL21 cells that were induced withisopropyl- - 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 oranti-HA antibody showed intense bands, molecular sizes ~63 and 12 kDa, which corresponded to the TAT-calcineurin A and TAT-HA fusionproteins, respectively.
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Fig. 3. Synthesis and purification of TAT-CNA fusion protein. A : construction of plasmid pTAT (2.95 kb) by inserting theTAT sequence and the CNA coding region cDNA. B : Westernimmunoblot of either a crude extract of BL21 (DE3) pLysS cellstransformed with pTAT-CNA (TAT-CNA ) or after its purification ona Ni-NTA column (Purified TAT-CNA ). Lane 1 : control cellsthat were not transformed. An anti-CNA antiserum (PP2BA ) was usedto immunostain the blots.& U( O. ?5 O" ^7 S
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Isolated osteoclasts were then transduced with 200 nM TAT-calcineurinA by incubation at 37°C for 10 min. The cells were then costainedwith a polyclonal anti-calcineurin A antiserum (green) and amonoclonal anti-TAT antibody (red). Without transduction, osteoclastswere found to immunostain only with the anti-calcineurin A antiserum(green only), not with anti-TAT antibody, confirming the presence ofcalcineurin A protein in untransduced osteoclasts (Fig. 4, A-C ). However, aftertransduction, an intense and mostly overlapping pattern (orange toyellow) of red and green staining was noted (Fig. 4, D-F ). Immunodetection by both anti-calcineurin A andanti-TAT antibodies strongly suggested influx of the applied TAT-calcineurin A fusion protein.8 G2 C1 E' u" e8 X2 U  ?" w

Fig. 4. Double immunostaining of isolated osteoclasts with a polyclonalgoat anti-CNA antiserum (PP2BA ) and monoclonal mouse anti-TATantibody. FITC-labeled anti-goat IgG (green) and tetramethylrhodamineisothiocyanate-conjugated anti-mouse IgM (red), respectively, were usedas secondary antibodies. A-C : osteoclast that wasincubated with vehicle, hence, the absence of anti-TAT (red) stainingin B. A : endogenous expression of CNA. D-F : cell that was incubated with 200 nM TAT-CNA for10 min at 37°C. Both anti-CNA (green; D ) and anti-TAT(red; E ) staining are superimposed on the merged image( F ).
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We next examined the bone resorptive function of osteoclasts transducedwith TAT-calcineurin A. Because we cannot visualize staining inosteoclasts settled on bone, we carried out immunostaining experiments(as above) in parallel with our resorption assays for consistency ofour methodology and to ensure the uptake of the TAT fusion protein.After a 10-min incubation at 37°C, osteoclasts previously settled ondevitalized 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. Incontrast, the control protein TAT-HA (10-200 nM) did notsignificantly inhibit bone resorption or change cell number. The latterindicates that the effect of TAT-calcineurin A on osteoclastresorptive function was not due to cell toxicity, although subtleeffects on cell viability cannot be excluded.1 {2 t7 \* L4 K& d6 }0 Z
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Fig. 5. Effect of incubation with TAT-hemaglutinin (HA) orTAT-CNA (10, 100, and 200 nM) for 10 min at 37°C on osteoclasticbone 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 withBonferroni's correction for inequality. P
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DISCUSSION

The expression of calcineurin isoforms in the osteoclast is notunexpected. First, osteoclasts are unique in handlinghigh-Ca 2  loads, both extracellularly and intracellularly( 50 ). One would therefore expect this cell to possess aphosphatase that was responsive to changes in cytosolicCa 2  : calcineurin fits that role perfectly. Second, andperhaps more important, is the likely critical role of calcineurin incontrolling gene transcription in the osteoclast, a cell that passesthrough several stages of differentiation before acquiring a boneresorptive phenotype ( 45 ). Finally, during resorption,osteoclasts actively secrete both acid and proteolytic enzymes( 48 ). The underlying process of vesicular trafficking hasbeen shown to be sensitive to calcineurin in other cells, such assynaptic neurons ( 26 ).

Our cloning and sequencing of two calcineurin isoforms, A and A,from a cDNA library constructed previously from pure rabbit osteoclastpreparations by Tezuka et al. ( 44 ) provides definitive evidence for their osteoclastic expression. Furthermore, using in situRT-PCR, we show that calcineurin A mRNA is expressed in matureosteoclasts that are capable of resorbing bone. We also show that byusing a specific anti-calcineurin A antibody, the protein isexpressed in osteoclasts.

Clearly, the expression of calcineurin in mature osteoclasts raises thequestion of its possible function in bone resorption. We provide directevidence that TAT-calcineurin A, the cell-permeant calcineurinconstruct, inhibited bone resorption by isolated osteoclasts, whereasthe control TAT-HA protein did not. Osteoclast number did not changewith either fusion protein, thereby excluding cytotoxicity andapoptosis. Previous apparently paradoxical observations on theinhibition of bone resorption by cyclosporin A, a calcineurin inhibitor, continue to exist ( 4 ). However, it is knownthat cyclosporin A interacts with other cellular targets independently of calcineurin; this may explain inhibition of bone resorption with thedrug ( 39 ).

Several mechanisms nevertheless underscore the antiresorptive action ofTAT-calcineurin A in the absence of cell toxicity. First, it ispossible that calcineurin might directly dephosphorylate proteinsinvolved in vesicular trafficking in response to changes in cytosolicCa 2  occurring as a result of bone resorption( 42 ). Proteins, such as dynamin, may be critical targetsin inhibiting acid secretion and enzyme release ( 26 ).Second, calcineurin may trigger the redistribution of osteoclastintegrins in response to Ca 2  transients through a directeffect, as has been shown for other cells ( 27, 34 ).Although this effect is particularly relevant to the osteoclast, adirect molecular interaction between v 3, the main osteoclast integrin, and calcineurin has not yet been established.4 K+ F8 e1 n- S0 S
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Third, a longer term and possibly more physiologically relevantresponse could occur through effects on gene transcription exertedthrough the traditional NFATc signaling pathway used by calcineurin inlymphocytes and cardiac cells ( 12 ). In lymphocytes, activation of calcineurin by calmodulin or Ca 2  results intransactivation of several critical genes, including thegranulocyte-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 transcriptionfactors NFATc1-4 ( 12 ), cardiac endothelial cellgrowth and hippocampal neuronal stimulation involve NFATc1 and NFATC4,respectively ( 17, 35 ). We are unclear as to which NFATcisoform is involved in calcineurin's effects on osteoclasts. Inaddition, from unpublished studies on myoblastic cells and otherpublished evidence, we can speculate that there are additionaldephosphorylation targets for calcineurin in the osteoclast, notablyI B, NF- B, and MeF ( 5, 14, 29 ).
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It is likely that critical genes for Ca 2  release channelsincluding IP 3 receptors and RyRs that are widely expressedin 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 stressresulting from mitochondrial DNA deletion, for example, is associatedwith the enhanced expression of both RyR-1 and calcineurin A ( 5 ). Thus there is a clear direct relationship between theexpression of calcineurin A and target RyR genes. In addition,inhibitors such as FK506 decouple the molecular interaction betweenRyRs and calcineurin ( 9, 20 ). Should the relationshipbetween 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 thissurface-expressed RyR-2 plays a critical role in extracellularCa 2  sensing, a process by which an osteoclast monitorschanges in ambient Ca 2  levels and transduces intracellularCa 2  signals during bone resorption ( 49, 50 ).A high intracellular Ca 2  level during bone resorptioncould potentially, through the activation of calcineurin, result inelevated RyR-2 expression. Admittedly speculative, this may be apositive feedback mechanism through which the sensitivity of theosteoclast to changes in extracellular Ca 2 , exerted viaRyR expression, is maintained during resorption.
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Although the physiological relevance of calcineurin in bone resorptionand the molecular mechanisms thereof remain issues for furtherinvestigation, this study clarifies that calcineurin does not alter itsown expression. From our in situ RT-PCR studies, albeitsemiquantitative, it is clear that the two inhibitors, cyclosporin Aand FK506, did not affect the expression of the calcineurin A gene.In other words, calcineurin gene expression is not regulated by itsphosphatase activity. Similarly, to our knowledge, such regulation hasnot been documented in other cells. It is also unlikely thatTAT-mediated transduction is affected by cyclosporin A or FK506, asthese drugs are not known to affect TAT delivery into cells.5 I. S6 d* c3 f( X) b: B
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An interesting clinical paradigm has emerged recently thatsignificantly enhances the importance of our discovery of calcineurin in the osteoclast. It is now known that the DSCR1 gene, apotent 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 indefects in the development of the brain, immune system, heart, andskeleton in these children. Localization of calcineurin in a skeletalcell, such as the osteoclast, and a prediction of its function inskeletal remodeling might therefore be a first step in understandingthe molecular pathophysiology of the skeletal defects in Down syndrome ( 12 ).
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Finally, this study further documents the use of the TAT-transductionsystem as a reliable means of transducing mature osteoclasts withproteins with high efficiency. At least two reports have similarly usedTAT to transduce the mutated form of I K and the small GTP-bindingprotein Rho, respectively, into osteoclasts ( 1, 11 ). Thesystem, developed initially by Vocero-Akbani et al. ( 46 ),establishes a new paradigm for protein transduction in sparsepopulations of cells, such as osteoclasts, without the need to infect,transfect, or microinject.
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In conclusion, we have documented the existence of theCa 2  /calmodulin phosphatase calcineurin in osteoclasts anddemonstrated its role as an inhibitor of bone resorption. We have alsoshown that calcineurin A can be effectively delivered intoosteoclasts as a functionally active protein through its fusion to TAT,an arginine-rich sequence derived from the human immunodeficiency virusprotein. The study paves the way for future investigations to studycalcineurin signal transduction in osteoclasts by using similarcell-permeant constructs of calcineurin and its signaling molecules,such as the NFATc isoforms.
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ACKNOWLEDGEMENTS. Z  R: K9 G7 G! ]( s4 P; T! B% M% P
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M. Zaidi acknowledges the support of the National Institute onAging (RO1-AG-14197-07) and Department of Veterans Affairs (MeritReview and Geriatric Research, Education, and Clinical Centers Program).
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