Regulation of Osteoblastogenesis and Bone Mass by Wnt10b

Regulation of osteoblastogenesis and bone mass by Wnt10b

Christina N. Bennett*, Kenneth A. Longo*, Wendy S. Wright*, Larry J. Suva†, Timothy F. Lane‡, Kurt D. Hankenson§, and Ormond A. MacDougald*¶

Departments of *Molecular and Integrative Physiology and §Orthopedic Surgery, University of Michigan Medical School, Ann Arbor, MI 48109-0622; †Department of Orthopaedic Surgery, Center for Orthopaedic Research, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and ‡Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095

Edited by M. Daniel Lane, Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 19, 2005 (received for review November 23, 2004) Wnts comprise a family of secreted signaling proteins that regulate responsible for activation of this pathway in marrow have not diverse developmental processes. Activation of Wnt signaling by been identified. Wnt10b inhibits differentiation of preadipocytes and blocks adi- We report herein that FABP4-Wnt10b mice have increased pose tissue development; however, the effect of Wnt10b on other bone mass and strength and that they resist the loss of bone that mesenchymal lineages has not been defined. To explore the occurs with aging or estrogen deficiency. The expression of physiological role of Wnt signaling in bone development, we Wnt10b in mesenchymal progenitors induces the expression of analyzed FABP4-Wnt10b mice, which express the Wnt10b trans- osteoblastogenic transcription factors Runx2, Dlx5, and osterix gene in marrow. Femurs from FABP4-Wnt10b mice have almost and strongly stimulates osteoblastogenesis. In addition, Wnt10b four times as much bone in the distal metaphyses and are me- inhibits the adipogenic transcription factors C͞EBP␣ and chanically stronger. These mice maintain elevated bone mass at PPAR␥ and blocks adipogenesis. Suppression of PPAR␥ is one least through 23 months of age. In addition, FABP4-Wnt10b mice mechanism whereby Wnt10b induces osteoblastogenesis. Com- are protected from the bone loss characteristic of estrogen defi- pelling evidence supportingWnt10b as an endogenous regulator ciency. We used pharmacological and genetic approaches to dem- of bone mass comes from analyses of Wnt10bϪ͞Ϫ mice, which onstrate that canonical Wnt signaling stimulates osteoblastogen- have decreased trabecular bone mass and serum osteocalcin. esis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage Methods by induction of the osteoblastogenic transcription factors Runx2, Animals. Animal studies were approved by the University Com- Dlx5, and osterix and suppression of the adipogenic transcription mittee on the Use and Care of Animals, and mice were cared for factors C͞EBP␣ and PPAR␥. One mechanism whereby Wnt10b by the Unit for Laboratory Animal Medicine. FABP4-Wnt10b promotes osteoblastogenesis is suppression of PPAR␥ expression. mice were generated and characterized as described (6, 7). Ϫ͞Ϫ Finally, Wnt10b؊͞؊ mice have decreased trabecular bone and Wnt10b mice were created by Timothy Lane (University of serum osteocalcin, confirming that Wnt10b is an endogenous California, Los Angeles) and Philip Leder (Harvard University, regulator of bone formation. Cambridge, MA). ␮ adipogenesis ͉ development ͉ stem cells Radiography, Microcomputerized Tomography ( CT), and Mechanical Testing. For microradiographic analysis, anesthetized mice were analyzed with Faxitron Specimen Radiography System Model esenchymal progenitors can differentiate into a number of MX-20 (Faxitron, Wheeling, IL). Femoral ␮CT was measured as Mcell types, including adipocytes and osteoblasts (1). One described (16) by using the Stereology function of GE Medical factor that regulates the reciprocal relationship between these Systems MICROVIEW software. Material properties of femurs lineages is Wnt signaling, which inhibits adipogenesis and stim- were evaluated by using the 810 Material Test System (Servo- ulates osteoblastogenesis. Activation of Wnt signaling blocks hydraulic, Eden Prairie, MN), as described (16). preadipocyte differentiation by inhibiting expression of the ͞ ␣ ␥ adipogenic transcription factors C EBP and PPAR (2–5). Ovariectomy. Wild-type and FABP4-Wnt10b mice were ovariec- The endogenous inhibitory Wnt signal may be initiated by tomized or sham-operated at 3 months of age, as described (16). Wnt10b, which is expressed in preadipocytes and stromal vas- Mice were killed 4 weeks later, and hind limbs were processed cular cells but not in adipocytes (2, 3). Expression of Wnt10b for ␮CT analysis. from the FABP4 promoter decreases accumulation of white adipose tissue by Ϸ50% and completely blocks the development Serology. Blood was collected at time of kill, and serum was of brown fat (6, 7). Furthermore, FABP4-Wnt10b mice resist prepared. ELISAs for osteocalcin (Biomedical Technologies, diet-induced obesity and the associated glucose intolerance. Stoughton, MA) and TRAP5b (SBA Sciences, Turku, Finland) The first indication that Wnt signaling plays a critical role in were performed according to the manufacturer’s protocols. bone formation came from human studies where inactivating mutations in the Wnt coreceptor LRP5 were shown to cause Cell Culture. Marrow-derived ST2 cells were incubated at 37°C osteoporosis (8). These findings were supported by the obser- and 5% CO2 in ␣-MEM supplemented with 10% FCS (Bio- vation that LRP5Ϫ͞Ϫ mice also have low bone mass (9). Whittaker) and 25 ␮g͞ml ascorbic acid (Sigma–Aldrich). For Furthermore, gain-of-function mutations in LRP5 that increase osteoblastogenesis, cells were cultured on 0.1% gelatin-coated Wnt signaling result in higher bone density in humans and mice plates in the same medium supplemented with 10 mM ␤- (10, 11). Consistent with the effects of LRP5 on bone mass being mediated through canonical Wnt signaling, activation of this pathway in vitro results in the expression of alkaline phosphatase, This paper was submitted directly (Track II) to the PNAS office. an early osteoblast marker (12–14). Although these and other Abbreviation: ␮CT, microcomputerized tomography. studies suggest that endogenous Wnt signaling regulates osteo- ¶To whom correspondence should be addressed. E-mail: [email protected]. blastogenesis and bone formation (15), a specific Wnt or Wnts © 2005 by The National Academy of Sciences of the USA

3324–3329 ͉ PNAS ͉ March 1, 2005 ͉ vol. 102 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408742102 Downloaded by guest on September 29, 2021 Fig. 1. Increased bone mass and strength in FABP4- Wnt10b mice. (A) ␮CT of femurs from wild-type and FABP4-Wnt10b mice (Upper). Three-dimensional re- constructions of metaphyseal trabeculae from regions highlighted (Lower). Morphometric properties of 1 mm3 of distal femur from wild-type (■) and FABP4- Wnt10b (p) mice at 6 months of age were analyzed for bone volume fraction (B;BV͞TV, %), bone mineral density (C; BMD), trabecular number (D; Tr. N.), trabecular thickness (E; Tb. Th.), trabecular spacing (F; Tb. Sp.), ultimate load (G), stiffness (H), and yield load (I). Sta- tistical signiﬁcance for each measurement was evaluated with Student’s t test: P Ͻ 0.05 (*), Ͻ 0.01 (#), Ͻ 0.001 (†), with n ϭ 6 males for each genotype.

glycerophosphate (Sigma–Aldrich). Where indicated, troglita- of age. Three-dimensional analyses of a 1-mm3 region of the zone (5 ␮M) was added to activate PPAR␥. Induction of distal femoral metaphyses (Fig. 1A) revealed a 4-fold increase in adipogenesis and staining with Oil red O was as described (17, bone volume͞total volume (Fig. 1B) and a similar rise in bone 18). The GSK3 inhibitor, CHIR99021, was a generous gift from mineral density (Fig. 1C). This increase in bone is the result of Chiron. Alkaline phosphatase activity was evaluated by using a a 3-fold increase in trabecular number (Fig. 1D) and an increase kit (86-C, Sigma–Aldrich). Mineralization of ␤-glycerophos- in trabecular thickness (Fig. 1E), with a corresponding decrease phate-treated cells was assessed by staining with 1% Alizarin red in trabecular spacing (Fig. 1F). Analysis of a 3-mm midcortical after fixation in 50% ethanol (19). Mineralized calcium was segment revealed an increase in bone cross-sectional area, evaluated by von Kossa staining (20). cortical thickness, and bending moments; however, diaphyseal analysis was complicated by high trabecular content (data not BIOLOGY

Retroviral Infection. Delivery of genes by retrovirus was as de- shown). DEVELOPMENTAL scribed (18). Retroviral vectors for Wnt1, Wnt10b, ␤-catenin, To assess the quality of bone in Wnt10b mice, femurs from dnTCF4, and Wnt5a have been described (2, 21). pMSCV and control and FABP4-Wnt10b mice were subjected to mechanical pMSCV-PPAR␥ were provided by Evan Rosen (Harvard Med- testing by four-point bending analyses. FABP4-Wnt10b mice ical School) (22). have increased ultimate load (Fig. 1G) and stiffness (Fig. 1H) without a significant change in other properties, including yield Expression of RNA. Expression of mRNAs was estimated by load (Fig. 1I) and energy and displacement ratio (data not quantitative PCR as described (6). All primers were verified by shown). In addition, material properties between genotypes were using ST2 cDNA, and sequences are available upon request. not different. Thus, Wnt10b signaling increases trabecular bone mass, which in turn enhances bending strength without adversely Results impacting bone material properties. Wnt10b Increases Bone Mass and Strength. Initial characterization of FABP4-Wnt10b mice revealed expression of Wnt10b from the FABP4-Wnt10b Mice Resist Loss of Bone Associated with Aging. To FABP4 promoter not only in white and brown adipose tissue but evaluate whether FABP4-Wnt10b mice resist the changes in also in bone marrow (6). To determine whether expression of bone architecture that occur with aging, femurs from mice at 23 Wnt10b in marrow alters bone, skeletons of FABP4-Wnt10b months of age were analyzed by ␮CT. Quantification of a 1-mm3 mice were evaluated. Analysis of femurs from FABP4-Wnt10b region of trabecular bone in distal femur indicated that bone mice with ␮CT at 18-␮m resolution showed increased trabecular volume fraction in wild-type mice was only slightly decreased bone throughout the endocortical compartment (Fig. 1A), with- with age (Table 1). Although the number of trabeculi was out substantial changes in cortical bone. Effects of Wnt10b on decreased by Ϸ60%, there was an increase in thickness of bone mass are not restricted to femur, because FABP4-Wnt10b remaining trabeculi that compensated for this loss. In contrast, mice have increased bone at the tibia, hip, and vertebrae (Fig. 5, the bone volume fraction of FABP4-Wnt10b mice increased which is published as supporting information on the PNAS web Ͼ2-fold from 6 to 23 months, mostly because of increased site) and the humerus (data not shown). This increase in bone trabecular thickness (Table 1). Visualization of bone architec- mass is observed in both sexes and is present as early as 8 weeks ture by ␮CT and histology further supports the observation that

Bennett et al. PNAS ͉ March 1, 2005 ͉ vol. 102 ͉ no. 9 ͉ 3325 Downloaded by guest on September 29, 2021 (Table 1. ␮CT of femurs from WT and FABP4-Wnt10b mice at 23 mo of age (mean ؎ SD Properties WT % change (from 6 mo) FABP4⅐Wnt10b % change (from 6 mo)

BV͞TV; % 2.9 Ϯ 2.4 Ϫ21.6 34.8 Ϯ 20* ϩ121.6† Tb. n. 0.57 Ϯ 0.44 Ϫ60.1† 5.2 Ϯ .9* ϩ10.6 Tb.Th., mm 0.045 Ϯ 0.017 ϩ84.4† 0.066 Ϯ 0.037 ϩ101 Tb.Sp.; mm 5.7 Ϯ 5.2 ϩ500† 0.15 Ϯ 0.09* Ϫ20.2 BMD; mg͞cc 100 Ϯ 32 Ϫ6.4 424 Ϯ 191* ϩ45.2

Percent change in morphometric properties compared with 6 months of age. BV͞TV, bone volume fraction; BMD, bone mineral density; Tb. n., trabecular number; Tb.Th., trabecular thickness; Tb.Sp., trabecular spacing. *Difference between genotype at 23 months P Ͻ 0.05. †Difference between 6 and 23 months of age within a genotype P Ͻ 0.06.

aged FABP4-Wnt10b mice maintain elevated levels of trabec- change bone volume͞total volume, bone mineral density, or ular bone compared with age-matched controls (Fig. 6, which is trabecular number (Fig. 2 A–C). In female mice, trabecular published as supporting information on the PNAS web site). thickness was unaltered by genotype or by surgery (Fig. 2D). As Thus, the elevated bone mass of FABP4-Wnt10b mice is main- expected, uteri of wild-type and FABP4-Wnt10b mice were tained throughout life. atrophied after ovariectomy (Fig. 2E). Although there may be some bone loss in FABP4-Wnt10b mice after ovariectomy, Wnt10b Protects Against Bone Loss Due to Ovariectomy. To deter- increased trabecular bone mass protects these mice against mine whether Wnt10b protects against bone loss associated with osteopenia associated with 4 weeks of estrogen deficiency. estrogen deficiency, wild-type and transgenic females at 9 weeks of age were sham-operated or ovariectomized. Four weeks later, Wnt Signaling Stimulates Osteoblastogenesis and Inhibits Adipogen- mice were killed, and effects on femoral architecture were esis of Bipotential Marrow Stromal Cells. Increased trabecular bone analyzed by ␮CT. Female FABP4-Wnt10b mice have a similar mass in FABP4-Wnt10b mice could be due to a direct effect of phenotype to that observed in male mice (Fig. 1), with increased Wnt10b in marrow. Alternatively, increased trabecular number bone volume fraction, bone mineral density, and trabecular could be an indirect effect of reduced serum leptin (6), which number (Fig. 2 A–C). Ovariectomy of wild-type mice caused a acts through a hypothalamic circuit to influence bone formation decrease in bone volume͞total volume and bone mineral density, (23, 24). To assess whether Wnt signaling directly stimulates primarily because of a loss of trabecular number. In contrast, osteoblastogenesis, bipotential ST2 cells were treated with ovariectomy of FABP4-Wnt10b mice did not significantly CHIR99021, an inhibitor of GSK3 that stabilizes cytosolic ␤-catenin in cultured cells (3). Exposure of ST2 cells to CHIR99021 induces osteoblastogenesis, as indicated by elevated alkaline phosphatase activity (Fig. 7A, which is published as supporting information on the PNAS web site), and increases mineralization, as assessed by Alizarin red staining. CHIR99021 also inhibits adipogenesis, as visualized by Oil red O staining (Fig. 7B). Although the effects of CHIR99021 are consistent with canonical Wnt signaling directly inducing osteoblast differentiation, the central role of GSK3 in several growth factor pathways leaves open the possibility that CHIR99021 stimulates osteoblastogenesis through another mechanism. To address this issue, activation of canonical Wnt signaling was initiated by infection of ST2 cells with retroviruses that express Wnt1, Wnt10b, or dominant stable ␤-catenin. When compared with controls, these cell lines had increased alkaline phosphatase activity at conflu- ent density and underwent rapid osteoblastogenesis and mineralization when cultured in osteogenic media (Fig. 8 A and B, which is published as supporting information on the PNAS web site). Control ST2 cells have exceedingly low levels of alkaline phosphatase and mineralization under these conditions, and this background osteoblastogenesis was influenced neither by dnTCF4 nor by activation of noncanonical signaling with Wnt5a. Furthermore, activation of noncanonical Wnt signaling with Wnt5a had no effect on adipogenesis of ST2 cells, whereas activation of the canonical pathway with Wnt10b or Wnt1 completely inhibited adipocyte differentiation (Fig. 8C). Similar results are observed in C3H10T1͞2 cells (data not shown). Taken together, these results demonstrate that canonical Wnt Fig. 2. Wnt10b expression maintains bone in ovarectomized mice. (A) ␮CT signaling promotes osteoblastogenesis and inhibits adipogenesis, of femurs from 4-month wild-type and FABP4-Wnt10b mice were either further supporting the hypothesis that expression of Wnt10b in sham-operated or ovariectomized at 3 months of age. Morphometric variables analyzed include bone volume͞total volume (B;BV͞TV), trabecular marrow directly increases bone mass in FABP4-Wnt10b mice. number (C; Tb. N.), trabecular thickness (D; Tb. Th.), trabecular spacing (E; To assess mechanisms whereby Wnt10b promotes osteoblas- Tb.Sp.), and uterine weight (F). Statistical signiﬁcance for each measurement togenesis, RNA was isolated from control and Wnt10b- was evaluated with Student’s t test: P Ͻ 0.05 (*), Ͻ 0.01 (#), with n ϭ 6–8 mice expressing ST2 cells 2 days after confluence. Consistent with the for each treatment. increase in alkaline phosphatase activity observed in Fig. 8A,

3326 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408742102 Bennett et al. Downloaded by guest on September 29, 2021 cells that expressed Wnt10b and PPAR␥ had greatly reduced osteoblastogenesis (Fig. 3C), and some cells were converted into adipocytes (data not shown). Thus, suppression of PPAR␥ is required for Wnt10b to induce osteoblastogenesis.

-Reduced Bone Mass in Femurs of Wnt10b؊͞؊ Mice. Although dif ferences in bone mass observed in mice and humans with mutations in LRP5 suggest that at least one of the 19 Wnts signals in marrow to regulate trabecular bone mass, the identity of the specific Wnt involved in this process remains unknown. One possibility is Wnt10b, which is expressed in marrow and activates canonical Wnt signaling (2, 29). To determine whether signaling by endogenous Wnt10b regulates bone mass, we analyzed mice with a deletion of the Wnt10b ORF. Femurs from 8-week Wnt10bϪ͞Ϫ females were analyzed by ␮CT. Three- dimensional analysis of the distal metaphyses revealed a 30% reduction in bone volume͞total volume and bone mineral density in these mice (Fig. 4 A–C). This loss is attributed to a decrease in trabecular number with a concomitant increase in trabecular spacing (Fig. 4 D and E). Quantitative analyses of age-matched wild-type and Wnt10bϪ͞Ϫ femora by histomor- phometry provided independent support for these observations (data not shown). In addition to decreased bone mass in the femur, Wnt10bϪ͞Ϫ mice also have decreased bone volume fraction in proximal tibia (data not shown). The comparable reduction in bone volume fraction and other bone variables in Wnt10bϪ͞Ϫ and LRP5Ϫ͞Ϫ mice suggests that the bone phe- Fig. 3. Wnt10b promotes osteoblastogenesis of pluripotent mesenchymal notype in LRP5Ϫ͞Ϫ mice may reflect a loss of endogenous precursors. ST2 cells were infected with control or Wnt10b retroviruses. Two Wnt10b signaling (29). Reduced levels of serum osteocalcin days postconﬂuence, RNA was isolated for quantitative PCR analysis of osteo- Ϫ͞Ϫ blast genes: alkaline phosphatase (Alk. Phos.); Twist, Runx2, Msx2, Dlx5, without changes in serum TRAP5b suggest that Wnt10b osterix (Osx); and type I collagen (Coll I) (A) and adipocyte genes: C͞EBP␤, mice have decreased osteoblast number or function rather than C͞EBP␣, PPAR␥, and FABP4 (B). Statistical differences from controls were increased number of osteoclasts or bone resorption (Fig. 4 G and evaluated with Student’s t test: P Ͻ 0.05 (*), Ͻ 0.01 (#), Ͻ 0.001 (†), with n ϭ H). Taken together, these experiments suggest that Wnt10b is an 3 mice. Results are representative of at least four independent experiments. endogenous regulator of bone mass. (C) Control and Wnt10b cells were infected with control and PPAR␥ retroviruses. Conﬂuent cells were incubated in osteogenic media supplemented with Discussion ␮ troglitazone (5 M). Mineral deposition was assessed on day 6 with von Kossa A relationship between bone and fat is established at both stain. developmental and physiological levels. During development, mesenchymal precursor cells can differentiate into adipocytes, quantitative PCR revealed that Wnt10b cells express high levels osteoblasts, and other cell types in vitro, and a reciprocal of alkaline phosphatase mRNA (Fig. 3A). Moreover, Wnt10b relationship exists such that increased osteoblast differentiation is associated with decreased adipocyte differentiation (30). This

induced expression of osteoblast transcription factors, Runx2, BIOLOGY Dlx5, and osterix, but Wnt10b did not consistently increase Msx2. relationship is also observed in vivo, where osteopetrosis is Type I collagen, a major component of the extracellular matrix associated with fewer marrow adipocytes, and osteopenia or DEVELOPMENTAL of active osteoblasts, is also elevated by Wnt10b (Fig. 3A). osteoporosis due to aging or disease is accompanied by increased marrow adipocytes (30, 31). Canonical Wnt signaling is one of Although Wnt signaling increases expression of twist in a the mechanisms controlling the development of precursor cells number of cell types (21, 25), induction of twist in ST2 cells is into osteoblasts or adipocytes (Figs. 7 and 8). Other investigators expected to oppose osteoblastogenic effects of Wnt10b (26). As have reported that activation of Wnt signaling with lithium predicted, expression of Wnt10b in ST2 cells did not suppress chloride or Wnt3a induces early stages of osteoblastogenesis, C͞EBP␤, but it inhibited expression of the major adipogenic ͞ ␣ ␥ including expression of alkaline phosphatase (12, 14, 32). How- transcription factors, C EBP , and PPAR , as well as the ever, we have observed that activation of canonical Wnt signaling adipocyte gene, FABP4 (Fig. 3B). Thus, Wnt10b signaling alters by pharmacological or genetic means is sufficient to stimulate cell fate of pluripotent mesenchymal precursors by shifting the the entire program of osteoblastogenesis, including expression of balance of transcription factors to favor osteoblastogenesis. osteoblast genes and matrix mineralization. ␥ Reduced PPAR expression and activity is associated with The discovery that obese mice have increased bone provided increased osteoblast mineralization and bone formation (27, 28). direct evidence for a physiological relationship between adi- ␥ To assess whether suppression of PPAR by Wnt10b is a pose tissue and bone (23). This connection is established mechanism whereby Wnt signaling promotes osteoblastogenesis, through leptin, which is released by adipocytes and signals we infected control and Wnt10b cells with control or PPAR␥- through the hypothalamus and sympathetic nervous system to expressing retroviruses and treated these cells for 6 days with inhibit bone formation. FABP4-Wnt10b mice have an Ϸ50% osteogenic media supplemented with the PPAR␥ ligand, trogli- decrease in whole body fat and a proportional decline in leptin tazone. As assessed by von Kossa staining, ST2 cells with empty (6). However, this decrease in serum leptin is unlikely to retroviral vectors failed to mineralize (Fig. 3C). Likewise, con- account for the 4-fold increase in bone mass, because complete trols cells infected with a retrovirus expressing PPAR␥ did not loss of leptin in ob͞ob mice causes only a 2-fold increase in undergo osteoblastogenesis but instead differentiated into adi- bone volume fraction (23). Further support for a direct effect pocytes (data not shown). Interestingly, although cells infected of Wnt10b on bone formation comes from Wnt10bϪ͞Ϫ mice, with Wnt10b and control vectors underwent mineralization, ST2 which have serum leptin levels indistinguishable from wild-

Bennett et al. PNAS ͉ March 1, 2005 ͉ vol. 102 ͉ no. 9 ͉ 3327 Downloaded by guest on September 29, 2021 Fig. 4. Reduced trabecular bone in femurs of Wnt10bϪ͞Ϫ mice. (A) ␮CT of femurs from wild-type and Wnt10bϪ͞Ϫ mice (Upper). Three-dimensional re- constructions of metaphyseal trabeculae from regions highlighted (Lower). Morphometric properties of distal femur (1 mm3) from wild-type (■) and Wnt10b null (p) mice. Properties analyzed include bone volume fraction (B;BV͞TV, %), bone mineral density (C; BMD), trabecular number (D; Tr. N.), trabecular thickness (E; Tb. Th.), and trabecular spacing (F; Tb. Sp.). Serum levels of osteocalcin (G) and TRAP5b activity (H)in wild-type (■) and Wnt10b null (p) mice. Statistical signiﬁcance for each measurement was evaluated with Student’s t test: P Ͻ 0.05 (*), Ͻ 0.01 (#), and Ͻ0.001 (†), with n ϭ 6 mice for each genotype.

type mice yet a 25–30% decrease in bone volume fraction, bone We have also found that expression of Wnt10b in marrow mineral density, and trabecular number compared with wild- increases trabecular bone mass and strength (Fig. 1). FABP4- type controls (Fig. 4). Thus, although there may be some effect Wnt10b mice resist loss of bone due to estrogen deficiency (Fig. of leptin deficiency on bone formation in FABP4-Wnt10b 2). Furthermore, they have increased bone volume͞total volume mice, it is likely that Wnt10b increases bone mass primarily and trabecular parameters throughout life (Table 1 and Fig. 6). through local effects in marrow. Decreased trabecular bone and serum osteocalcin in ␥ Our experiments indicate that suppression of PPAR by Wnt Wnt10bϪ͞Ϫ mice provide strong evidence that Wnt10b is an signaling is part of the mechanism whereby Wnt10b inhibits adi- endogenous regulator of bone formation (Fig. 4). Although pogenesis and stimulates osteoblastogenesis. (Fig. 3). In addition to Wnt10b may influence bone mass through increased osteoblast an integral role in adipogenesis and adipose tissue development (33, ͞ ␥ differentiation, replication, and or activity, it could also de- 34), PPAR inhibits osteoblastogenesis in vitro and bone formation crease osteoblast apoptosis (21). Based upon prior work in in vivo, perhaps by decreasing Wnt10b expression and inhibiting LRP5Ϫ͞Ϫ, LRP5 G171V, and sFRP1Ϫ͞Ϫ mice, Wnt10b is Runx2 activity (35–38). Furthermore, PPAR␥Ϫ͞Ϫ embryonic likely to influence osteoblast differentiation, replication, and stem cells and PPAR␥ϩ͞Ϫ mice have increased matrix mineral- apoptosis (9, 11, 15). Uncovering mechanisms whereby Wnt ization and bone formation, respectively (27), suggesting that suppression of PPAR␥ is sufficient to promote osteoblastogenesis. signaling influences development of bone and adipose tissue is However, in our experiments with ST2 cells, inhibition of PPAR␥ important for our understanding and potential treatment of with pharmacological inhibitors was not sufficient to stimulate osteoporosis and other human diseases. differentiation (data not shown), suggesting that induction of one ␮ or more osteoblastogenic transcription factors (e.g., Runx2, Dlx5, or We thank Jacklyn Kriegl and David Fisher for CT scans and Philip Leder (Harvard Medical School) for his considerable contribution to osterix; Fig. 3) is also required for the actions of Wnt10b. For Ϫ͞Ϫ example, induction of collagen type I in Wnt10b-expressing cells making Wnt10b mice. This work was supported by grants from the National Institutes of Health [DK51563 and DK62876 (to O.A.M.) and may be due to the increase in Dlx5 (Fig. 3), which has been shown RR00161 and AR49682 (to K.D.H.)]. Other support was from the to activate the collagen type I promoter (39). Interestingly, Wnt10b Diabetes Research and Training Center (P60 DK20572), the Nathan does not consistently induce Msx2, even though Msx2 inhibits Shock Mutant and Transgenic Rodent Core, and the Core Center for adipogenesis and promotes osteoblastogenesis (40, 41). Taken Musculoskeletal Disorders (P30AR046024). Fellowships to C.N.B. were together, these data suggest that Wnt signaling influences the from Tissue Engineering and Regeneration Training Grant DE07057 differentiation potential of mesenchymal precursors by repressing and the American Physiological Society Porter Fellowship. K.A.L. was PPAR␥ and increasing expression of key osteoblastogenic tran- supported by a mentor-based postdoctoral fellowship from the American scription factors. Diabetes Association.

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