Functional interaction between Wnt and Bmp signaling in periosteal bone growth

Wnt and Bmp proteins are well known to regulate bone development and homeostasis. Although both signals are extensively studied, their potential interaction in vivo is less well understood. Previous studies have shown that deletion of Bmpr1a, a type I receptor for Bmp signaling, results in excessive trabecular bone formation while diminishing periosteal bone growth. Moreover, forced-expression of the Wnt antagonist Sost suppresses the overgrowth of trabecular bone caused by Bmpr1a deletion, thus implicating hyperactive Wnt signaling in the excessive trabecular bone formation. However, it remains uncertain whether Wnt and Bmp signaling interacts in regulating the periosteal bone growth. Here we show that multiple Wnt genes are markedly suppressed in the cortical bone without Bmpr1a. Importantly, overexpression of Wnt7b fully rescues periosteal bone growth in the Bmpr1a-deficient mice. Thus, pharmacological activation of Wnt signaling can restore normal bone size without intact Bmp signaling.

In Bmp signaling, dimeric Bmp proteins bind to a hetero-tetramer of serine/threonine kinase receptors composed of two type I (Bmpr1a, Bmpr1b, Acvrl1, Acvr1) and two type II receptors (Bmpr2, Acvr2a, Acvr2b), leading to phosphorylation and activation of the type I receptor by the type II receptor which has constitutively active kinase activity 11,12 . The activated type I receptors then activate multiple downstream mechanisms including the Smad pathway, TAK1-p38 or PI3K-Akt signaling axis 3,[12][13][14][15][16] . More recently, Bmp signaling has been shown to activate both Smad and mTORC1 signaling to regulate osteoblast differentiation and bone formation 10,17 . Thus, depending on the cellular context, Bmp may employ different effectors to control various biological processes.
Wnt proteins, a family of secreted glycoproteins, have been shown to stimulate bone formation. In the best studied mechanism, Wnt proteins signal through frizzled proteins and the low-density lipoprotein receptorrelated protein Lrp5 or Lrp6 to stabilize ß-catenin which in turn activates gene expression 18 . Wnt signaling is tempered by various secreted antagonists including sclerostin (encoded by the SOST gene) that compete with Wnt proteins for binding to Lrp5/6 19 . In humans, inhibitory mutations of LRP5 cause osteoporosis, whereas activating mutations of LRP5 or loss of SOST results in osteosclerosis [20][21][22][23][24] . An antibody against sclerostin was recently approved by FDA for treatment of severe postmenopausal osteoporosis.
The role of Lrp5 in bone formation is conserved between mice and human, as Lrp5 knockout mice develop osteopenia whereas mice carrying the Lrp5 hyperactive mutations (e.g., A214V) exhibit osteosclerosis [25][26][27] . Consistent with the paradigm that Lrp5 and Lrp6 mediates Wnt signaling to stabilize β-catenin, numerous genetic studies have demonstrated that β-catenin critically controls osteoblast differentiation in the mouse embryo [28][29][30][31] . Postnatal deletion of β-catenin in osteoblast-lineage cells suppresses osteoblast differentiation, diminishes osteoblast activity and reduces osteoblast lifespan 32,33 . Besides β-catenin signaling, Wnt proteins also activate mTORC1 signaling to stimulate bone formation 34 . Upon activation of mTORC1 signaling, Wnt has been shown to stimulate glutaminolysis to meet the anabolic needs of osteoblasts 35 . In addition, Wnt acutely activates glycolysis through mTORC2 signaling 36 . Thus, Wnt signaling exerts the bone anabolic function through multiple intracellular mechanisms. Despite the extensive studies of Wnt signaling in bone, how Wnt interacts with Bmp in osteogenic regulation is less well explored. Previous experiments regarding the two signals in osteoblast differentiation in vitro have yielded disparate results, likely due to differences in the cellular context 17,37 .
To address the relationship between Bmp and Wnt signaling in vivo, we have previously shown that deletion of Bmpr1a by Dmp1-Cre leads to downregulation of Sost expression in osteocytes, and that forced-expression of Sost in the Bmpr1a-deficient mice partially corrected the hyperproliferation of preosteoblasts in trabecular bone 38 . Thus, Bmp signaling through Bmpr1a normally limits Wnt signaling via the induction of Sost to restrict preosteoblast proliferation during trabecular bone formation.
Here we report that multiple Wnt ligands are notably reduced in the cortical bone of Bmpr1a-deficient mice. Importantly, overexpression of Wnt7b restores the normal bone size by boosting osteoblast activity in the face of Bmpr1a deficiency. The results support a model wherein Bmp signaling via Bmpr1a induces Wnt expression to promote periosteal bone growth.

Results
Loss of Bmpr1a diminishes bone anabolic Wnt expression in cortical bone. To assess the potential effect of Bmp signaling on Wnt expression, we surveyed the mRNA levels of major Wnt ligands in the long bones of wild type versus Dmp1-Cre; Bmpr1a (Bmpr1a CKO) littermate mice. We selected the Wnt ligands with RPKM > 1 in a previous RNA-seq study of the normal cortical bone, but also included Wnt1 and Wnt7b known to stimulate bone growth 34,[39][40][41][42][43] . RT-qPCR experiments with RNA extracted from the tibial and femoral diaphysis showed that Wnt1, Wnt2b, Wnt5b and Wnt16 were significantly reduced in the Bmpr1a CKO versus wild type samples, whereas Wnt4 and Wnt10b were not affected and Wnt7b was below the detection level in both samples (Ct > 40) (Fig. 1). As both Wnt1 and Wnt16 have been shown to promote bone formation, the data indicate that downregulation of the bone anabolic Wnt ligands may contribute to the cortical bone defect in the Bmpr1a deficient mice 41,44,45 . Overexpression of Wnt7b rescues bone size in Bmpr1a-deficient mice. Although Wnt7b is normally expressed at a low level in the cortical bone, overexpression of the protein has been shown to stimulate bone growth at the periosteal surface 46 . Considering that Wnt1 and Wnt16, both physiological regulators of the cortical bone, were significantly reduced in the Bmpr1a CKO mice, we reasoned that overexpression of Wnt7b might compensate for the loss of the endogenous ligands and thus normalize the bone size. To test this hypothesis, we created compound mutant animals with the genotype of Dmp1-Cre; Bmpr1a f/f ; R26-Wnt7b (CKO, Wnt7b) to compare with the Dmp1-Cre; Bmpr1a f/f littermates (CKO) ( Fig. 2A). The same cross also produced normal mice with the genotype of Bmpr1a f/f or Bmpr1a f/+ (WT), and Wnt7b-overexpressing mice with the genotype of Dmp1-Cre; Bmpr1a f/+ ; R26-Wnt7b (Wnt7b). Mice of all genotypes appeared healthy without any overt abnormality.
The mice were subjected to X-ray-based imaging at 33 days of age. As expected from earlier studies, contact radiography revealed a marked increase in bone density in the Wnt7b-overexpressing mice (Wnt7b) compared to the normal control (WT) (Fig. 2B, left two images) 34 . Also confirming previous results, the Bmpr1a CKO mice exhibited a marked increase in trabecular bone, but a clear reduction in the cross-sectional size of the tibia and the proximal femur (Fig. 2B, CKO vs WT) 10 . Overexpression of Wnt7b in the background of CKO further increased trabecular bone mass (Fig. 2B, CKO; Wnt7b vs CKO, asterisks). µCT imaging of the distal femur trabecular bone confirmed that Wnt7b overexpression markedly increased the trabecular bone mass in both normal and CKO background (Fig. 2C). Quantification of the µCT data showed that overexpression of Wnt7b essentially maximized the trabecular bone mass regardless of Bmpr1a deletion (Fig. 2D). µCT quantification www.nature.com/scientificreports/ also revealed that in the normal background Wnt7b increased both trabecular number and thickness whereas deletion of Bmpr1a (CKO) increased trabecular number only (Fig. 2E,F). In the CKO background, however, Wnt7b increased only trabecular thickness likely due to the already high trabecular number (Fig. 2E,F). In keeping with the increase in trabecular number and thickness, trabecular separation was decreased in all mutant mice compared to the wild type (Fig. 2G). Thus, Wnt7b greatly enhances trabecular bone formation regardless of intact Bmp signaling. X-ray imaging also detected the impact of Wnt7b overexpression on bone size. X-ray contact radiography revealed that Wnt7b increased both tibial and femoral width in CKO (Fig. 2B, arrows). Further analyses of the proximal femur by µCT confirmed that the overall cross-sectional size was notably increased in the CKO; Wnt7b mice compared to CKO (Fig. 3A). Quantification of the µCT data confirmed that the total area (Tt. Ar) across the proximal femur was fully recovered to the normal size (WT or Wnt7b) in the CKO; Wnt7b mice from the deficit seen in CKO (Fig. 3B). Wnt7b also markedly increased the cortical bone area (Ct. Ar) and all but eliminated the marrow area (Ma. Ar) in both normal (WT) and CKO background (Fig. 3C,D). Similarly, µCT analysis of the tibia at the tibiofibular junction showed that Wnt7b restored the cross-sectional size (Tt. Ar) in CKO; Wnt7b mice to the normal level (WT) (Fig. 3E,F). However, here Wnt7b overexpression increased the cortical bone size above normal although it did not do so in the proximal femur, thus highlighting regional differences in the anabolic response (Fig. 3F). Like in the proximal femur, Wnt7b markedly increased the cortical bone area (Ct. Ar) and essentially eliminated the marrow space (Ma. Ar) in the tibia (Fig. 3G,H). Thus, Wnt7b overexpression not only increases trabecular and endosteal bone but also fully restores periosteal bone growth in the absence of Bmpr1a. www.nature.com/scientificreports/ Wnt7b overexpression restricts cell proliferation in trabecular bone region. We next examined the trabecular bone in more detail. H&E staining showed that Wnt7b overexpression in either normal or CKO background (Wnt7b or CKO; Wnt7b, respectively) caused massive bone buildup and complete preclusion of the hematopoietic marrow cells in the distal femur (Fig. 4A). Deletion of Bmpr1a, as previously reported, also caused severe osteosclerosis leaving only residual marrow cells present in the region (Fig. 4A, CKO) 10,38 . Thus, histology confirms that Wnt7b overexpression intensifies the osteosclerotic phenotype caused by Bmpr1a deletion. www.nature.com/scientificreports/ As previous studies have shown that Bmpr1a deletion increases preosteoblast proliferation as a main cause for osteosclerosis, we set out to determine whether Wnt7b has a similar effect by performing EdU labeling experiments. Remarkably, Wnt7b overexpression in the normal background greatly reduced the proliferation rate within the chondro-osseous junction where preosteoblasts were highly enriched (Fig. 4B,C, Wnt7b vs WT). Moreover, Wnt7b completely neutralized the hyperproliferation caused by Bmpr1a deletion. (Fig. 4B,C, CKO; Wnt7b vs CKO). Therefore, Wnt7b increases trabecular bone formation through a mechanism distinct from stimulation of cell proliferation, likely via increased osteoblast differentiation and activity.

Wnt7b stimulates periosteal bone growth in the absence of Bmpr1a.
To gain more insight about the effect of Wnt7b on the cortical bone, we performed histomorphometry with mice of the different genotypes. By H&E staining of longitudinal sections through the femur, Wnt7b overexpression in the normal background did not cause an increase in osteocyte density as seen in the CKO mouse (Fig. 5A,B). Wnt7b appeared to moderate the high osteocyte density in the CKO; Wnt7b compound mutant compared to CKO, but quantification showed that the downward trend did not reach statistical significance (Fig. 5A,B). The bone formation activity was monitored by double labeling with calcein followed by alizarin red. Wnt7b notably increased whereas Bmpr1a deletion (CKO) decreased the mineral apposition rate (MAR) at the periosteal bone surface when compared to normal (WT) (Fig. 5C,D). Importantly, MAR in the CKO; Wnt7b mouse was markedly higher than that in the CKO or WT mice (Fig. 5C,D). Overall, pharmacological overexpression of Wnt7b fully restored periosteal bone growth in the face of Bmpr1a deletion.

Discussion
We have shown that deletion of Bmpr1a in osteoblasts and osteocytes notably suppressed the expression of multiple bone anabolic Wnt genes in the cortical bone. Furthermore, overexpression of Wnt7b in the same cells fully rescued the defect in periosteal bone growth caused by Bmpr1a deletion. We have previously shown that a hyperactive form of Lrp5 (A214V) failed to correct the bone size deficit in the Bmpr1a-deficient background, raising the possibility that Bmp signaling might regulate periosteal growth independent of Wnt 38 . However, as signaling by Lrp5 (A214V) requires the binding of Wnt proteins, its activity is likely to be muted when the endogenous Wnt levels are diminished in the Bmpr1a CKO bones as shown here. Thus, the current study lends support to the alternative model that Bmp signaling stimulates periosteal bone growth partly through induction of Wnt expression in the cortical bone.
This and our previous study have shed light on the complexity of interaction between Wnt and Bmp in trabecular versus cortical bone 38 . In both trabecular and cortical bone, Bmpr1a deletion reduces the expression of the Wnt antagonist Sost, but a bone overgrowth phenotype is only seen with the trabecular bone which is at least partially due to increased preosteoblast proliferation and can be rescued by forced-expression of Sost 10,38 . In the cortical bone, the reduced Sost expression paradoxically concurs with diminished periosteal growth. In the current study, we show that Bmpr1a deletion causes a notable decrease in the expression of several Wnt ligands in the cortical bone although they are not examined in the trabecular bone. The functional contribution of those ligands is not tested here, but pharmacological overexpression of Wnt7b is sufficient to overcome the bone size deficit in the Bmpr1a CKO mice. The exact mechanism for Wnt7b to rescue periosteal bone growth remains to be   10,17 . Although we previously showed that the bone phenotype in Bmpr1a CKO was not replicated by Smad4 deletion, potential activation of Smad signaling upon Wnt7b overexpression could still contribute to the rescuing effect 10 . Finally, the identity of the Wnt molecules promoting preosteoblast proliferation in the trabecular bone is unknown at present, but it is clearly distinct from those with similar properties to Wnt7b, as Wnt7b overexpression had the opposite effect on cell proliferation in the trabecular region. Due to its low expression normally in the cortical bone, Wnt7b is unlikely to be the endogenous Wnt ligand responsible for the bone size defect in the Bmpr1a CKO mice. Wnt1 on the other hand is a probable candidate as it was clearly downregulated by Bmpr1a deletion, and has been shown to promote cortical bone growth in both lossand gain-of-function experiments 41 . Mechanistically, Wnt1, like Wnt7b, has been shown to activate mTORC1 signaling in osteoblasts 34,41 . Wnt16 was also reduced in bone upon Bmpr1a deletion, but it has been implicated mainly in suppressing osteoclastogenesis at the endosteal surface, even though overexpression of Wnt16 also stimulated bone formation while suppressing bone resorption 45,47 . The effect of Wnt16 on bone resorption also appears to diverge from that of Wnt1 or Wnt7b, as overexpression of either molecule increased overall bone resorption in the mouse 34,41 . Future experiments are necessary to determine whether Wnt1 is indeed the physiological ligand whose downregulation by Bmpr1a deletion is compensated by the forced-expression of Wnt7b.

Materials and methods
Mouse strains. Dmp1-Cre 48 , Bmpr1a f/f49 , R26-Wnt7b 34 mouse strains as previously described were maintained in a mixed genetic background of predominantly C57BL6/J. All analyses were conducted with sex-matched littermates including both males and females at 33 days of age (P33). Both males and females were analyzed for all parameters and they showed the same phenotype. The animals were group housed in a specific pathogen free (SPF) barrier facility with a 12-h light cycle (6 am-6 pm) and fed standard chow (PicoLab mouse diet 20, #5058). The Animal Studies Committee at Washington University in St. Louis School of Medicine approved the study. All methods were performed in compliance with relevant guidelines and regulations. The studies were carried out according to the ARRIVE guidelines.
Bone morphological analyses. X-ray contact radiography was performed with Faxitron (Faxitron X-ray Corp) for 20-s exposures at 25 kV. Micro-computed tomography was conducted with μCT 40 (Scanco Medical AG) and with key parameters as follows: voxel size 10 μm 3 , X-ray tube potential 55 kVp, X-ray intensity 145 μA, integration time 300 ms 50 . Quantitative trabecular bone parameters were assessed with 100 μCT slices (1.6 mm) immediately below the growth plate, with a threshold set at 240. For cortical bone parameters, 50 μCT slices (0.8 mm) at indicated locations were analyzed with a threshold of 260.