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.
Highlights
The identification of WWOX-interacting proteins has provided insights into the potential roles of WWOX in cell signaling and its impact on cell fate
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 [7, 8] confirming that Wwox is a bona fide tumor suppressor
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
Summary
Mice—C57Bl/6J ϫ 129/SvJ-F1, -F2, -F3, -F4, and -F5 mice (B6 –129 F1–F5; [7]) were produced at Ohio State University animal facility. 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 g/ml alizarin red S (Sigma). 1 ϫ 105 cells were plated in each well of a 12-well plate and incubated at 37 °C in ␣-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). 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).
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