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The WWOX Tumor Suppressor Is Essential for Postnatal Survival and Normal Bone Metabolism

2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês

10.1074/jbc.m800855200

1083-351X

Rami I. Aqeilan, Mohammad Q. Hassan, Alain de Bruin, John P. Hagan, Stefano Volinia, Titziana Palumbo, Sadiq Hussain, Suk‐Hee Lee, Tripti Gaur, Gary S. Stein, Jane B. Lian, Carlo M. Croce,

Ubiquitin and proteasome pathways

The WW domain-containing oxidoreductase (WWOX) gene encodes a tumor suppressor. We have previously shown that targeted ablation of the Wwox gene in mouse increases the incidence of spontaneous and chemically induced tumors. To investigate WWOX function in vivo, we examined Wwox-deficient (Wwox-/-) mice for phenotypical abnormalities. Wwox-/- mice are significantly reduced in size, die at the age of 2-3 weeks, and suffer a metabolic disorder that affects the skeleton. Wwox-/- mice exhibit a delay in bone formation from a cell autonomous defect in differentiation beginning at the mineralization stage shown in calvarial osteoblasts ex vivo and supported by significantly decreased bone formation parameters in Wwox-/- mice by microcomputed tomography analyses. Wwox-/- mice develop metabolic bone disease, as a consequence of reduced serum calcium, hypoproteinuria, and hypoglycemia leading to increased osteoclast activity and bone resorption. Interestingly, we find WWOX physically associates with RUNX2, the principal transcriptional regulator of osteoblast differentiation, and on osteocalcin chromatin. We show WWOX functionally suppresses RUNX2 transactivation ability in osteoblasts. In breast cancer MDA-MB-242 cells that lack endogenous WWOX protein, restoration of WWOX expression inhibited Runx2 and RUNX2 target genes related to metastasis. Affymetrix mRNA profiling revealed common gene targets in multiple tissues. In Wwox-/- mice, genes related to nucleosome assembly and cell growth genes were down-regulated, and negative regulators of skeletal metabolism exhibited increased expression. Our results demonstrate an essential requirement for the WWOX tumor suppressor in postnatal survival, growth, and metabolism and suggest a central role for WWOX in regulation of bone tissue formation. The WW domain-containing oxidoreductase (WWOX) gene encodes a tumor suppressor. We have previously shown that targeted ablation of the Wwox gene in mouse increases the incidence of spontaneous and chemically induced tumors. To investigate WWOX function in vivo, we examined Wwox-deficient (Wwox-/-) mice for phenotypical abnormalities. Wwox-/- mice are significantly reduced in size, die at the age of 2-3 weeks, and suffer a metabolic disorder that affects the skeleton. Wwox-/- mice exhibit a delay in bone formation from a cell autonomous defect in differentiation beginning at the mineralization stage shown in calvarial osteoblasts ex vivo and supported by significantly decreased bone formation parameters in Wwox-/- mice by microcomputed tomography analyses. Wwox-/- mice develop metabolic bone disease, as a consequence of reduced serum calcium, hypoproteinuria, and hypoglycemia leading to increased osteoclast activity and bone resorption. Interestingly, we find WWOX physically associates with RUNX2, the principal transcriptional regulator of osteoblast differentiation, and on osteocalcin chromatin. We show WWOX functionally suppresses RUNX2 transactivation ability in osteoblasts. In breast cancer MDA-MB-242 cells that lack endogenous WWOX protein, restoration of WWOX expression inhibited Runx2 and RUNX2 target genes related to metastasis. Affymetrix mRNA profiling revealed common gene targets in multiple tissues. In Wwox-/- mice, genes related to nucleosome assembly and cell growth genes were down-regulated, and negative regulators of skeletal metabolism exhibited increased expression. Our results demonstrate an essential requirement for the WWOX tumor suppressor in postnatal survival, growth, and metabolism and suggest a central role for WWOX in regulation of bone tissue formation. WW domain-containing oxidoreductase (WWOX) 3The abbreviations used are: WWOX, WW domain-containing oxidoreductase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; KO, knock-out; WT, wild type; HET, heterozygous; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; RANKL, receptor activator of NF-κB ligand; μCT, microcomputed tomography; HA, hemagglutinin. 3The abbreviations used are: WWOX, WW domain-containing oxidoreductase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; KO, knock-out; WT, wild type; HET, heterozygous; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; RANKL, receptor activator of NF-κB ligand; μCT, microcomputed tomography; HA, hemagglutinin. is a 46-kDa protein that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain (1Bednarek A.K. Laflin K.J. Daniel R.L. Liao Q. Hawkins K.A. Aldaz C.M. Cancer Res. 2000; 60: 2140-2145PubMed Google Scholar, 2Ried K. Finnis M. Hobson L. Mangelsdorf M. Dayan S. Nancarrow J.K. Woollatt E. Kremmidiotis G. Gardner A. Venter D. Baker E. Richards R.I. Hum. Mol. Genet. 2000; 9: 1651-1663Crossref PubMed Scopus (246) Google Scholar). WWOX was identified as a putative tumor suppressor in cancer cells because it lies in a genomic region that is frequently altered in pre-neoplastic and neoplastic lesions (1Bednarek A.K. Laflin K.J. Daniel R.L. Liao Q. Hawkins K.A. Aldaz C.M. Cancer Res. 2000; 60: 2140-2145PubMed Google Scholar, 2Ried K. Finnis M. Hobson L. Mangelsdorf M. Dayan S. Nancarrow J.K. Woollatt E. Kremmidiotis G. Gardner A. Venter D. Baker E. Richards R.I. Hum. Mol. Genet. 2000; 9: 1651-1663Crossref PubMed Scopus (246) Google Scholar). Indeed, expression of WWOX is deregulated in several types of cancer, including breast, prostate, lung, stomach, and pancreatic carcinomas (3Paige A.J. Taylor K.J. Taylor C. Hillier S.G. Farrington S. Scott D. Porteous D.J. Smyth J.F. Gabra H. Watson J.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11417-11422Crossref PubMed Scopus (195) Google Scholar, 4Aqeilan R.I. Croce C.M. J. Cell Physiol. 2007; 212: 307-310Crossref PubMed Scopus (102) Google Scholar). Ectopic expression of WWOX in cancer cells lacking expression of endogenous WWOX results in significant growth inhibition and prevents the development of tumors in athymic nude mice (5Bednarek A.K. Keck-Waggoner C.L. Daniel R.L. Laflin K.J. Bergsagel P.L. Kiguchi K. Brenner A.J. Aldaz C.M. Cancer Res. 2001; 61: 8068-8073PubMed Google Scholar, 6Fabbri M. Iliopoulos D. Trapasso F. Aqeilan R.I. Cimmino A. Zanesi N. Yendamuri S. Han S.Y. Amadori D. Huebner K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15611-15616Crossref PubMed Scopus (114) Google Scholar). Recently, we generated a mouse carrying a targeted deletion of the Wwox gene (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar). We reported that loss of both alleles of Wwox resulted in the formation of frequent juvenile osteosarcomas, whereas loss of one allele increased the incidence of spontaneous and chemically induced tumors (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar, 8Aqeilan R.I. Hagan J.P. Aqeilan H.A. Pichiorri F. Fong L.Y. Croce C.M. Cancer Res. 2007; 67: 5606-5610Crossref PubMed Scopus (64) Google Scholar) thus confirming that Wwox is a bona fide tumor suppressor. The identification of WWOX-interacting proteins has provided insights into the potential roles of WWOX in cell signaling and its impact on cell fate. WWOX cytosolic interactions, through its first WW domain that binds PPXY ligand-containing proteins, regulate the transactivation activity of several transcription factors. For example, we recently showed that WWOX interacts with p73 and suppresses its transactivation activity (9Aqeilan R.I. Pekarsky Y. Herrero J.J. Palamarchuk A. Letofsky J. Druck T. Trapasso F. Han S.Y. Melino G. Huebner K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4401-4406Crossref PubMed Scopus (199) Google Scholar). However, this association contributes to the increased rate of WWOX pro-apoptotic activity. We also reported that the association of WWOX with the AP2γ transcription factor and the ErbB4 intracellular domain might contribute to breast carcinogenesis (10Aqeilan R.I. Palamarchuk A. Weigel R.J. Herrero J.J. Pekarsky Y. Croce C.M. Cancer Res. 2004; 64: 8256-8261Crossref PubMed Scopus (133) Google Scholar, 11Aqeilan R.I. Donati V. Palamarchuk A. Trapasso F. Kaou M. Pekarsky Y. Sudol M. Croce C.M. Cancer Res. 2005; 65: 6764-6772Crossref PubMed Scopus (174) Google Scholar). In addition, WWOX antagonizes the function of Yes-associated protein (YAP), another WW domain-containing protein, and suppresses its co-activation ability of ErbB4-dependent transcription. WWOX physically associates with c-Jun following ultraviolet radiation and functionally suppresses its transcriptional ability (12Gaudio E. Palamarchuk A. Palumbo T. Trapasso F. Pekarsky Y. Croce C.M. Aqeilan R.I. Cancer Res. 2006; 66: 11585-11589Crossref PubMed Scopus (68) Google Scholar). Other research groups have shown that WWOX associates with proline-rich motifs of SIMPLE (13Ludes-Meyers J.H. Kil H. Bednarek A.K. Drake J. Bedford M.T. Aldaz C.M. Oncogene. 2004; 23: 5049-5055Crossref PubMed Scopus (97) Google Scholar) and EZRIN (14Jin C. Ge L. Ding X. Chen Y. Zhu H. Ward T. Wu F. Cao X. Wang Q. Yao X. Biochem. Biophys. Res. Commun. 2006; 341: 784-791Crossref PubMed Scopus (50) Google Scholar) and with proteins lacking the PPXY motif such as p53 and c-Jun N-terminal kinase (JNK) (15Chang N.S. Hsu L.J. Lin Y.S. Lai F.J. Sheu H.M. Trends Mol. Med. 2007; 13: 12-22Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Thus, although WWOX is involved in specific protein complexes that define its function as an important suppressor of transactivator functions, its specific function in vivo is not clearly defined. To investigate in vivo requirements for WWOX, we examined the Wwox-null mice for phenotypical abnormalities. Wwox-deficient mice display many postnatal defects that include growth retardation, postnatal lethality, and abnormalities of bone metabolism. Mice—C57Bl/6J × 129/SvJ-F1,-F2,-F3,-F4, and -F5 mice (B6-129 F1-F5; (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar)) were produced at Ohio State University animal facility. Animals were sacrificed; tissues of all organs were removed, fixed in 10% buffered formalin, and examined for histological abnormalities by two pathologists after hematoxylin and eosin staining. Bones were cleaned by separation from soft tissue (skin, muscle) leaving the periosteum intact for radiograph and μCT studies. Histology and LacZ Staining—Tissue from different organs were processed, embedded, sectioned (4 μm), and hematoxylin and eosin-stained according to standard methods. Bones were dissected for fixation in 4% paraformaldehyde for 48 h and either demineralized in 18% EDTA for paraffin embedding or embedded in methyl methacrylate for examination of sections of mineralized tissues. Sections were stained for mineral and matrix using the von Kossa 3% silver nitrate stain combined with toluidine blue to distinguish cartilage and bone. Alkaline phosphatase and tartrate-resistant acid phosphatase enzyme detection for demonstrating osteoblast and osteoclast activities, respectively, was performed using reagent kits from Sigma. Because our targeting construct included the lacZ gene (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar), whole mount β-galactosidase staining was performed using standard procedures (16Kim I.S. Otto F. Zabel B. Mundlos S. Mech. Dev. 1999; 80: 159-170Crossref PubMed Scopus (393) Google Scholar). Prior to preparing frozen sections from E17.5 embryos, limbs (femur with tibia) were removed for separate embedment. Frozen sections of stained embryos and limbs were refixed in 0.5% glutaraldehyde and restained in X-gal solution and serial sections counterstained in 0.5% eosin after dehydration (50% alcohol). Immunochemistry for WWOX was performed using WT limb sections of newborn mice with WWOX polyclonal anti-rabbit antibody (a gift from Dr. Kay Huebner, Ohio State University) at 1:1000 dilution (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar). Bone Analyses—Skeletons were prepared for visualization of cartilage and bone using Alcian blue and alizarin red stains, respectively. Briefly, eviscerated embryos were fixed in 100% ethanol, stained overnight in a solution containing 4 parts ethanol, 1 part glacial acetic acid, and 0.3 mg/ml Alcian blue 8GX (Sigma). Soft tissues were dissolved for 6 h in a 2% KOH followed by an overnight staining in a 1% KOH solution with 75 μg/ml alizarin red S (Sigma). Skeletons are destained in 20% glycerol, 1% KOH for several days and stored in 50% glycerol, 50% ethanol. Radiography of dissected limbs at ages 7, 12, 17, and 20 days was performed after fixation in 4% paraformaldehyde at 4 °C under vacuum 2 days and rinsing in PBS using a Faxitron MX-20 specimen radiography system. Microcomputed tomography studies were performed on limbs fixed in 70% ethanol for scanning. Qualitative and quantitative three-dimensional analysis of femurs was carried out using micro-CT imaging (μCT 40, Scanco Medical AG, Bassersdorf, Switzerland) at the University of Connecticut Health Science Center μCT Facility by Dr. Douglas J. Adams. Calvarial Osteoblast Differentiation—Primary calvarial osteoblasts were isolated from postnatal day 3 wild-type and homozygous KO pups following collagenase P digestion, and cultured in α-minimal essential medium supplemented with 10% FBS, as described previously (17Pratap J. Galindo M. Zaidi S.K. Vradii D. Bhat B.M. Robinson J.A. Choi J.Y. Komori T. Stein J.L. Lian J.B. Stein G.S. van Wijnen A.J. Cancer Res. 2003; 63: 5357-5362PubMed Google Scholar). For osteogenic differentiation of both primary osteoblast and MC3T3 cells, stimulus was provided at confluence by subsequent feedings with the BGJb medium containing 10 mm β-glycerol phosphate (BGP04) and 50 μg/ml ascorbic acid. For histochemical staining, cell layers were fixed in 2% paraformaldehyde before staining, and reagents used were from Sigma. Briefly, cells were stained with 0.05% naphthol AS-Mx phosphate disodium salt, 2.8% dimethyl formamide,. and 0.1% Fast Red TR salt in 0.1 m Tris maleate buffer (pH 8.4) at 37 °C for 30 min. Osteoclast Differentiation—Bone marrow cells were prepared from the long bones of 7-9-week-old BALB/c mice. Marrow was flushed from bones with a 30-gauge syringe, and cells were dispersed through a metal filter. 1 × 105 cells were plated in each well of a 12-well plate and incubated at 37 °C in α-minimum Eagle's medium supplemented with 10% FBS, 2 mm l-glutamine, 1% (v/v) penicillin/streptomycin, and 25 ng/ml macrophage colony-stimulating factor (R & D Systems, Minneapolis, MN) for 3 days before addition of recombinant RANKL (5 ng/ml). RAW264.7 cells (a kind gift from Philip Osdoby (Washington University, St. Louis, MO) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2 mm l-glutamine, and 1% (v/v) penicillin/streptomycin and plated in 6-well (2-4 × 105 cells/well) or 100-mm plates (1 × 106 cells). Cells were allowed to settle 2 days after plating before addition of RANKL (5 ng/ml). After differentiated osteoclasts were observed, mononuclear cells were shaken off and cell layers rinsed in PBS. Mononuclear cells (controls in which no RANKL was added) and the enriched multinucleated osteoclasts on the plates were harvested in TRIzol for mRNA isolation according to manufacturers' procedure (Invitrogen). Cell Lines, Transfection, and Reporter Assays—NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium and MC3T3 in a growth medium of α-minimum Eagle's medium (Invitrogen) supplemented with 10% FBS (Sigma). Transient transfections were performed at subconfluency using FuGENE 6 transfection reagent (Roche Applied Science). The RUNX2 and WWOX expression vector (pCMV-MYC-WWOX; pCMV-MYC-WWOXY33R; pcDNA3.1-HA-RUNX2) were used in this study. For control of expression, vector pcDNA3.1 was transfected according to the experimental conditions. Previously described constructs (18McCabe L.R. Banerjee C. Kundu R. Harrison R.J. Dobner P.R. Stein J.L. Lian J.B. Stein G.S. Endocrinology. 1996; 137: 4398-4408Crossref PubMed Scopus (161) Google Scholar) containing the rat osteocalcin (Oc) (-1097/+23 or -208/+23) promoter fused to the chloramphenicol acetyltransferase (CAT), as well as the Oc promoter with all three RUNX sites mutated (-1.1-kb rOc mABC-CAT) were co-transfected with expression plasmids. Real Time PCR—RNA was isolated from the different tissues followed by homogenizing in TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Bones were cut at the mid-diaphysis and flushed free of marrow by PBS after removing the epiphyses from either end. The bone halves were placed and frozen in TRIzol for RNA isolation by homogenization. cDNA was synthesized with oligo(dT) primers using the Super-Script first strand synthesis kit (Invitrogen) according to the manufacturer's protocol. Gene expression was assessed by semi-quantitative and quantitative real time PCR (Cyber Green and TaqMan) using primers listed in Table 1.TABLE 1Nucleotide sequence of primers used for quantitative reverse transcription-PCR detectionGenePrimer SequenceRunx2For 5′ CGC CCC TCC CTG AAC TCT 3′Rev 5′ TGC CTG CCT GGG ATC TGT A 3′BSPFor 5′ GCA CTC CAA CTG CCC AAG A 3′Rev 5′ TTT TGG AGC CCT GCT TTC TG 3′Col IFor 5′ GTA TCT GCC ACA ATG GCA CG 3′Rev 5′ CTT CAT TGC ATT GCA CGT CAT 3′OsteocalcinFor 5′ CTG ACA AAG CCT TCA TGT CCA A 3′Rev 5′ GCG CCG GAG TCT GTT CAC TA 3′Alkaline phosphataseFor 5′ TTG TGC GAG AGA AAG GAG A 3′Rev 5′ GTT TCA GGG CAT TTT TCA AGG T 3′Histone H4For 5′ CCA GCT GGT GTT TCA GAT TAC A 3′Rev 5′ ACC CTT GCC TAG ACC CTT TC 3′WwoxFor 5′ TCA CAC TGA GGA GAA GAC CCA 3′Rev 5′ CCT ATT CCC GAA TTT GCT CCA 3′Osteocalcin (human)For 5′ GGC AGC GAG GTA GTG AAG AG 3′Rev 5′ CGA TAG GCC TCC TGA AAC TC 3′VEGF (human)For 5′ CCT TGC TGC TCT ACC TCC AC 3′Rev 5′ CCA TGA ACT TCA CCA CTT CG 3′RUNX2 (human)For 5′-CGG CCC TCC CTG AAC TCT-3′Rev 5′-TGC CTG CCT GGG GTC TGT A-3′ Open table in a new tab Immunoprecipitation and Western Blot Analysis—Immunoprecipitation was performed as described previously (9Aqeilan R.I. Pekarsky Y. Herrero J.J. Palamarchuk A. Letofsky J. Druck T. Trapasso F. Han S.Y. Melino G. Huebner K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4401-4406Crossref PubMed Scopus (199) Google Scholar). Antibodies used were monoclonal anti-WWOX, monoclonal or polyclonal anti-RUNX-2, anti-MYC and anti-HA (Santa Cruz Biotechnology), anti-actin (Santa Cruz), and Lamin B1 (Zymed). Chromatin IP (ChIP) Assay—For identifying association of RUNX2 and WWOX on chromatin of a RUNX2 target gene osteocalcin, the mouse MC3T3-E1 osteoblastic cells at 30 to 50% confluence were transfected using Oligofectamine (Invitrogen) with small interfering RNA (siRNA) duplexes specific for murine Runx2 gene-specific siRNA ((5′-r(UGC CUC UGC UGU UUG AAA) d(TT)-3′) and nonspecific siRNA (5′-r(UUC UCC GAA CGU GUC ACG U) dTdT-3′) duplexes for 48 h, obtained from Qiagen Inc. (Stanford, CA). Total proteins from the specific siRNA oligonucleotide-treated and nonspecific-oligonucleotide-treated cells were analyzed by Western blotting to evaluate the RUNX2 knockdown profile. After Runx2 siRNA treatment, cells were cross-linked with 1% form-aldehyde, and soluble chromatin fractions were immunoprecipitated with RUNX2 rabbit polyclonal (3 μg) and WWOX rabbit polyclonal (3 μg) antibody. The procedure for ChIP in osteoblasts has been described (19Hassan M.Q. Tare R.S. Lee S.H. Mandeville M. Morasso M.I. Javed A. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Biol. Chem. 2006; 281: 40515-40526Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). DNA fragments from immunoprecipitates were analyzed by quantitative real time PCR using 2× SYBR Green Master Mix (Eurogentec) and a two-stage cycling protocol (60 °C annealing and extension, 94 °C denaturation, 40 cycles). The following primers were used to amplify the proximal mouse osteocalcin (mOC) promoter (mOC-F1 5′-CCC TCA GGG AAG AGG TCT G-3′ and mOC-R1 5′-CTA ATT GGG GGT CAT GTG CT-3′). Affymetrix Chip Analysis—mRNAs from 26 samples (including kidney, spleen, brain, pituitary gland, femur, and calvarial bones) were hybridized with Affymetrix mouse gene-chip 430 2.0 arrays. Normalization was performed by using GC robust multiarray average. Genes showing minimal variation across the set of arrays were excluded from the analysis. Genes whose expression differed by at least 1.5-fold from the median in at least 20% of the arrays were retained. For class comparison, we identified genes that were differentially expressed between KO and WT mice by using a multivariate permutation test (www.linus.nci.nih). In comparing the genotypes we controlled for the analyzed tissues, we used the multivariate permutation test to provide 90% confidence that the false discovery rate was less than 10%. The false discovery rate is the proportion of the list of genes claimed to be differentially expressed that are false positives. The test statistics used are random variance F-statistics for the effect of genotype on each gene. Although F-statistics were used, the multivariate permutation test is nonparametric and does not require the assumption of a gaussian distribution. The summarized cDNAs presented in supplemental Table 4 represent those up- and down-regulated genes that were common to all analyzed tissues. Wwox Is Essential for Postnatal Survival—To characterize the role of the Wwox gene in vivo, B6-129 F1-F5 hybrid mice were examined for phenotypical abnormalities. Genotype analysis of newborns obtained from a Wwox+/- intercross demonstrated the presence of all three genotypes with ratios consistent with the Mendelian distribution (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar). Wwox heterozygous (HET) pups were indistinguishable from wild-type (WT) animals at all stages of development and postnatal life. At birth, homozygous Wwox-null pups were indistinguishable from their WT or HET littermates up to 3 days postpartum; subsequently, homozygous (KO) pups were easily recognizable by their smaller size (Fig. 1A). KO pups continued to grow more slowly than their littermates, and 100% of the KO mice died by 3 weeks after birth (Fig. 1B). Although homozygous mice were runted, they did not exhibit any abnormal behavior or impaired motor skills. All KO pups showed normal suckling behavior, and milk was found in their stomachs. At 2 or 3 days prior to their death, Wwox-null pups became lethargic and showed signs of wasting. Consistent with growth retardation, key organs, including spleen, thymus, brown adipose tissue, heart, and liver, weighed less in both female and male KO pups compared with wild-type littermates as measured on postnatal day 14 (supplemental Table 2). Interestingly, the brain, adrenal and the pituitary gland of KO pups were heavier compared with WT littermates. Macroscopic and histological examination of the organs confirmed the atrophy of many organs in KO animals without significant microscopic lesions, though KO mice are born with gonadal abnormalities and displayed impaired steroidogenesis. 4R. Aqeilan, unpublished data. Serum chemistry analysis showed marked hypoproteinemia, hypoalbuminemia, hypoglycemia, hypocalcemia, hypotriglyceridemia, and hypocholesterolemia (supplemental Table 3). Reduced serum levels of proteins, carbohydrates, and lipids concurrently without significant histological lesions in liver, pancreas, intestinal tract, and kidney indicate that KO pups most likely suffered from severe metabolic defect. To further determine the in vivo functions of Wwox, we studied the differential expression of mRNAs in the Wwox-deficient mice compared with their WT and HET littermates. mRNAs extracted from spleen, kidney, brain, pituitary, femur and calvarial bone were analyzed using mouse Affymetrix microarray gene-chip 430 2.0 arrays. Chromatin structure, cell cycle, and other related genes were among the most differentially expressed genes in KO mice tissues as compared with control mice (supplemental Table 4). A Bone Metabolic Disorder in Wwox-null Mice—Limbs of the KO mice revealed not only a size difference proportional to the animal weight, but radiography revealed that KO limbs exhibited decreased density of trabeculae bone and a thinner cortex compared with WT beginning at day 7 (data not shown). Fig. 1C shows the striking differences in bone density at 3 weeks age. To determine whether the observed osteopenia was a consequence of a metabolic disorder or related to cell autonomous defects in bone cell populations, we first examined skeletal development using alizarin red/Alcian blue staining of newborn pups. As shown in Fig. 1D, no apparent size differences or abnormalities related to skeletal patterning were observed. Some variability in ossification of the calvarium was found at birth among HET and KO Wwox mice, but radiography showed no differences in craniofacial bones on day 7 (data not shown). Because our targeting vector included the lacZ gene (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar), we next examined expression of Wwox promoter in the skeleton. In our previous report, lacZ staining of Wwox-/- mice revealed Wwox expression throughout the skeleton of whole embryos in craniofacial bones, vertebrae, and limb bones (7Aqeilan R.I. Trapasso F. Hussain S. Costinean S. Marshall D. Pekarsky Y. Hagan J.P. Zanesi N. Kaou M. Stein G.S. Lian J.B. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3949-3954Crossref PubMed Scopus (177) Google Scholar). To address the activity of Wwox promoter in specific cell populations of the skeleton, we performed in situ immunohistochemistry (Fig. 2A, newborn) and sectioning of X-gal-stained embryos at E17.5 (Fig. 2, B-E). In the embryo, WWOX protein is expressed in chondrocytes and osteoblasts of the developing WT limb (Fig. 2A, newborn). Wwox promoter activity was identified in limb sections in chondrocytes, osteoblasts, and tendon fibroblasts in long bone (Fig. 2B). Chondrocytes and osteoblasts in vertebral bodies of the spine were also positive (Fig. 2, C and D). In the developing calvarium (Fig. 2E), we found robust expression of Wwox promoter activity in early differentiating osteoblasts that are forming the bone matrix. To identify modifications in bone cell populations and tissue organization to account for the apparent reduced bone formation revealed by radiography (less dense bone) in the Wwox-deficient mice, histologic sectioning of limbs was performed at several ages. Normal organization of zones of chondrocyte maturation at the growth plate was observed, and mineralization of trabecular and cortical bone tissue appeared normal on day 1 (Fig. 3A), but the KO mouse bone size was slightly smaller (∼8-10%). By day 3 impaired bone growth was visually evident, and as shown at day 5, the KO mouse bones clearly have fewer trabeculae compared with WT. The diminished volume of trabecular bone in the KO mice continued until the mice died. The three-dimensional μCT images of femur metaphysis in mice at day 15 (Fig. 3B) show reduced trabecular member connectivity and bone surface area in both the HET and KO. However, mineral content (tissue density parameter) of the trabecular bone in HET is not significantly changed from WT. Given the reduced serum values of 50% lower calcium and 20% increased phosphate in the KO mouse (supplemental Table 3), as well as loss of trabecular bone, these findings suggest a metabolic bone disease contributing to the osteopenic phenotype in KO mouse. We therefore examined bone tissue for the number and activity of osteoclasts (bone resorbing cells) by tartrate-resistant acid phosphatase histochemical staining at several postnatal ages (Fig. 3C). Higher acid phosphatase activity was consistently observed in femur bone sections of KO mice compared with the WT by day 3 in the primary spongiosa (Fig. 3C, upper panel). The day 5 (Fig. 3C, lower panel) sections of diaphysis are shown to illustrate the less osteoclast activity around mature cortical and trabeculae bone (indicated by *) compared with the KO. We next addressed if the stimulated osteoclast activity was solely because of the physiologic response to low serum calcium in Wwox-deficient mice or if WWOX deficiency directly influenced stimulation of osteoclast activity. Because of organ wasting, it was not possible to isolate viable progenitors from bone marrow or spleen from the Wwox-/- mice for ex vivo osteoclast differentiation. Therefore, WWOX expression was examined in two in vitro osteoclast differentiation systems (Fig. 3D). Both the RAW264.