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, 283 (31), 21629-39

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

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

Rami I Aqeilan et al. J Biol Chem.

Abstract

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.

Figures

FIGURE 1.
FIGURE 1.
Phenotypic analysis of Wwox null mice. Impaired growth is shown in A. Three-week-old mice showing significant small size of KO mouse compared with WT and HET mice. B, growth curves of WT, HET, and KO mice. Newborn pups were weighed every day from day 1. Mice were then genotyped, and weights were plotted on growth curve. The weights of KO mice diverge after day 3 from that of the WT and HET mice. HT, heterozygous. C, radiography of femurs of 3-week-old mice showing smaller but also less dense bone of the homozygous (HO) Wwox-/- mouse. D, alizarin red/Alcian blue staining of newborn skeletons.
FIGURE 2.
FIGURE 2.
Wwox promoter activity in cartilage and bone of Wwox-null mice. A, in situ immunohistochemistry of newborn femur showing expression of WWOX protein in epiphysis, growth plates, chondrocytes, and osteoblasts in primary spongiosa. B, lacZ staining in bone section of KO femur at E17.5 (no counterstain). The four lower panels of B show higher magnification (×20) of tissues for visualization of Wwox-positive cells in tendon (a), chondrocytes (b), osteoblasts on the endosteal surface (c), and osteoblasts forming bone in the periosteal side of the limb (d). Muscle fibers (m) surrounding the bone do not show β-galactosidase activity. C, lacZ and eosin counterstain of E17.5 embryo section with higher magnifications in D showing vertebral body (VB) at ×20. E, calvarium at ×10 (top panel) and ×40 (lower panel). Intense staining of cells on the bone (Bo)-forming surfaces but not in mature osteocytes in the bone center. Br, brain tissue; Po, periosteum.
FIGURE 3.
FIGURE 3.
Metabolic bone disease phenotype of the Wwox null mouse. A, histologic sectioning of long bone of postnatal mouse day 1 (D1) and day 5 (D5) shows normal organization of the epiphysis and the growth plate and mineralization of bone tissue. By day 5 fewer trabeculae were in the KO mouse compared with WT and HT (bracket). Sections were stained with toluidine blue and von Kossa silver stain (for detection of mineralized tissues; black). B, μCT image of the metaphysis region of WT and KO femur at day 15 showing less volume of trabecular (Trab.) bone with accompanying quantitation of trabecular bone loss. HT, heterozygous; Conn Dens, connectivity density; Tiss Dens, tissue density. C, high rate of bone resorption in Wwox null mice is evident by tartrate-resistant acid phosphatase histochemical staining (red) with fast green counterstain of femurs from wild-type (WT) and homozygous (HO) mice. Note increased osteoclast activity in the primary spongiosa under the growth plate on day 3 and osteoclasts on nearly all trabeculae on day 5, consistent with decreased trabeculae number on day 15 in B. Inset in KO panel shows ×63 magnification of osteoclast. On day 5, asterisk shows bone spicules in WT without osteoclasts. D, WWOX expression/GAPDH by Q-PCR in the RAW274.6 monocyte cell line and bone marrow cells compared with osteoclasts (+Rank ligand-treated 4 days).
FIGURE 4.
FIGURE 4.
Delayed bone formation in the Wwox KO mouse. A, microcomputed tomography (μCT) three-dimensional images of WT, HET (HT), and KO genotypes at the indicated ages. Top panel, image from the midsection of femur that shows less trabecular bone and a delay in the secondary center of ossification in the KO; middle panel, cross-section at mid-diaphysis shows thin cortical bone of KO. B, quantitation of selected bone formation parameters, including bone volume, mineral density, area of bone formation surfaces, and cortical bone quality from μCT data analyses as indicated.
FIGURE 5.
FIGURE 5.
Gene expression markers of bone formation in Wwox WT and KO mice and during MC3T3 osteoblast differentiation. A, quantitative RT-PCR for expression of key genes in osteoblast-related genes in femur and calvarial bones from day 7 postnatal mice (n = 3). Expression of genes was normalized to Gapdh and plotted as relative expression in KO compared with WT set to 1.0. Alp, alkaline phosphatase; Bsp, bone sialoprotein; ColI, collagen-I; H4, histone (proliferation marker); Oc, osteocalcin. B, total RNA was prepared at the indicated day from differentiating MC3T3 calvarial osteoblasts (day 0, osteoprogenitors; day 7, growth; day 14, matrix maturation; and day 21, mineralization stages). C, Western blot analyses from cell lysates from the same experiment in which quantitative-PCR was performed (shown in Fig. 4B).
FIGURE 6.
FIGURE 6.
Cell autonomous defects in osteoblast differentiation in Wwox null mice. Ex vivo osteoblasts from day 3 calvaria postnatal of Wwox WT and KO mice are shown. A, phase contrast micrographs at the indicated days showing an increased proliferation with Wwox null cells reaching confluency sooner (day 5), but unable to form multilayered nodules (day 12). B, alkaline phosphatase (Alk Phos) and von Kossa histochemical staining for mineral shows cells reach the first stage of differentiation (alkaline phosphatase-positive), but have reduced mineralized nodules. C, quantitative reverse transcription-PCR expression of phenotypic genes on day 12. Alp, alkaline phosphatase; Bsp, bone sialoprotein; ColI, collagen-I; Oc, osteocalcin.
FIGURE 7.
FIGURE 7.
WWOX physically binds RUNX2 and together will associate with osteocalcin chromatin. A, physical interaction between RUNX2 and WWOX. Co-immunoprecipitation studies were carried out in non-osseous NIH3T3 cells transfected with expression vectors encoding MYC-WWOX and HA-RUNX2, followed by Western blot analysis using anti-HA and anti-Myc antibodies. B, co-immunoprecipitation of endogenous RUNX2 and WWOX by pulldown with anti-RUNX2 and anti-WWOX antibodies in ROS17/2.8 osteoblasts. The immunoprecipitates (IP) were detected by monoclonal anti-RUNX2 antibody. C, WWOX associates with RUNX2 via its first WW domain. HEK293 cells were co-transfected with HA-RUNX2 and MYC-WWOX or MYC-WWOXY33R plasmids. 24 h later, whole cell lysates were immunoprecipitated using anti-Myc and anti-HA antibodies followed by immunoblotting with anti-HA antibody. D, WWOX association with osteocalcin chromatin is dependent on RUNX2 occupancy of the Oc gene promoter. Chromatin immunoprecipitation was performed on endogenous mouse osteocalcin promoter in MC3T3 cells. Cells were treated with RUNX2 gene-specific and nonspecific siRNA duplexes for 48 h. Western blot with anti-RUNX2 and anti-WWOX antibodies demonstrate RUNX2 protein knockdown, but WWOX protein is not affected. Actin protein was used as loading control. ChIP-soluble chromatins from siRNA-treated cells were immunoprecipitated with anti-RUNX2 and anti-WWOX antibody. DNA fragments from immunoprecipitates were analyzed by quantitative real time.
FIGURE 8.
FIGURE 8.
WWOX suppresses RUNX2-transcriptional activity. A, dose-dependent suppression of RUNX2-mediated activation of the osteocalcin gene by WWOX is shown. NIH3T3 cells were transiently transfected with 1 μg of -1.1-kb rOc-CAT, with empty vector (100 ng of pcDNA, bar graph 1), WWOX (200 ng), RUNX2 (100 ng), and indicated increased amounts of WWOX. The Oc promoter activity (percent CAT conversion activity) is the average of six replicates. Values were calculated from the ratios of converted mono or diacetylchloramphenicol versus the total input chloramphenicol (S.E. of n = 6 wells). B, evidence that WWOX repression is mediated via RUNX2 regulatory elements. Comparison of inhibition on WT-Oc promoter and Oc promoter in which all three RUNX sites mutated (mABC-Oc). RUNX2 (100 ng) were co-transfected with WWOX (200 ng). C, WWOX suppresses RUNX2 target genes in metastatic breast cancer cells. MDA-MB-231 cells were transduced with adenovirus expressing green fluorescent protein (GFP) or WWOX. The relative expression of RUNX2, vascular endothelial growth factor (VEGF), and OC normalized to GAPDH is shown.

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