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In Vivo Analysis of Wnt Signaling in Bone

Medical Scientist Training Program (D.A.G.), Baylor College of Medicine, Houston, Texas 77030; and Department of Genetics and Development (G.K.), Columbia University Medical Center, New York, New York 10032

Address all correspondence and requests for reprints to: Gerard Karsenty, Department of Genetics and Development, Columbia University Medical Center, 701 West 168th Street, Room 1602A, New York, New York 10032. E-mail: gk2172{at}columbia.edu.

	Abstract

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
Bone remodeling requires osteoblasts and osteoclasts working in concert to maintain a constant bone mass. The dysregulation of signaling pathways that affect osteoblast or osteoclast differentiation or function leads to either osteopenia or high bone mass. The discovery that activating and inactivating mutations in low-density lipoprotein receptor-related protein 5, a putative Wnt coreceptor, led to high bone mass and low bone mass in human beings, respectively, generated a tremendous amount of interest in the possible role of the Wnt signaling pathway in the regulation of bone remodeling. A number of mouse models have been generated to study a collection of Wnt signaling molecules that have been identified as regulators of bone mass. These mouse models help establish the canonical Wnt signaling pathway as a major regulator of chondrogenesis, osteoblastogenesis, and osteoclastogenesis. This review will summarize these advances.

	Introduction

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
THE REGULATION OF bone mass, or bone remodeling, is an active and dynamic process orchestrated by bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoporosis, the most frequent bone remodeling disease, is an incapacitating condition observed most frequently in postmenopausal women that is caused most often by an increase in bone resorption not compensated for by a similar increase in bone formation. The finding that loss and gain-of-function mutations in the surface molecule low-density lipoprotein receptor-related protein 5 (Lrp5) led to both osteoporosis and high bone mass (HBM), respectively, triggered a great deal of interest because of the sequence homology between Lrp5 and arrow, a coreceptor for the fly homolog of Wnt proteins. Since then, several studies have addressed in mice the function of Lrp5 and of the canonical Wnt signaling pathway in bone. This review of these animal models presumes the reader has some familiarity with osteoblast and osteoclast differentiation and function, as well as with the canonical Wnt signaling pathway. Karsenty and Wagner (1) did a comprehensive review of the former, while Logan and Nusse (2) provide a broad and in-depth perspective on the latter.

	Lrp5

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
Lrp5 belongs to the family of Lrps. Lrp5 and the closely related protein Lrp6 have the highest sequence homology with arrow, a Drosophila protein that acts as a coreceptor for wingless, the fly homolog of the Wnt proteins (3). To date there are three mouse models available to study the loss of Lrp5. These mice display a phenotype reminiscent of osteoporosis-pseudoglioma syndrome, a syndrome characterized by early onset osteoporosis and blindness caused by Lrp5 inactivation (4, 5, 6).

Gain-of-function mutations within the Lrp5 gene lead to a HBM phenotype in human beings; the best characterized of these being a glycine to valine change at amino acid 171 (G171V) within exon 3 (7, 8, 9). A transgenic mouse model that overexpresses Lrp5 with the G171V mutation via the rat ₁(I) collagen promoter has been generated. These mice have increased bone density and bone strength without effects on bone morphology, which recapitulates the human phenotype (10). However, our attempts at reproducing these results by overexpressing either wild-type Lrp5 or Lrp5 with the G171V mutation in transgenic mice did not reveal any significant effects on bone mass (Glass, D. A., and G. Karsenty, unpublished data).

In situ hybridization reveals that Lrp5 is detected in cells of the osteoblast lineage (5, 8, 11). Accordingly and as found with patients, mutations in Lrp5 in mice affect bone mass only by altering bone formation, with no change in osteoclast parameters (5). In particular, Lrp5^–/– mice have a decreased number of osteoblasts and a decrease in the bone formation rate (BFR) and osteoblast proliferation rate without any effects on apoptosis (5). The fact that Wnt and Lef1-mediated signaling can still partially occur in Lrp5^–/– osteoblasts (5) establishes that Wnt-mediated signaling occurs in the absence of Lrp5. Further experiments, as discussed later, confirmed this.

	Lrp6

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
In contrast to Lrp5, there are no known human diseases or syndromes ascribed to mutations in the Lrp6 locus. Lrp6^–/– mice, generated via an insertional mutation leading to truncation of the protein after residue 321 (12), die at birth. However, during embryogenesis, their phenotype mimics a combination of various Wnt loss-of-function mutations, namely midbrain and hindbrain defects seen in Wnt1^–/– mice, axial skeletal truncation found in Wnt3a^–/– mice, and limb patterning defects observed in Wnt7a^–/– mice (13, 14, 15, 16). There is also a hypomorphic Lrp6 mutant mouse model that allows us to examine the protein’s function in adult mice. The ringelschwanz mutant mouse has a naturally occurring arginine to tryptophan mutation at amino acid 886 (R886W) that inhibits efficient transmission of canonical Wnt signaling (17). These mice have axial skeleton, digit, and neural tube defects similar to those observed in some of the Wnt-deficient mice. There is also a delay in the appearance of ossification centers during embryogenesis and a decreased bone mass detected in adult mice, both of which are also seen in Lrp5^–/– mice (5, 17). These data suggest that Lrp6 may be a more bona fide Wnt coreceptor than Lrp5.

What are the effects of combined deficiencies of Lrp5 and Lrp6 on bone mass? Lrp6^+/–;Lrp5^–/– mice were more osteopenic than Lrp6^+/+;Lrp5^–/– mice (4). Lrp6^+/–;Lrp5^+/– mice had a higher bone mass than Lrp6^+/+;Lrp5^–/– mice but had a decreased bone mass compared with wild-type mice or either of the single heterozygous mice alone (4).

	ß-Catenin

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
ß-Catenin is an intracellular molecule involved in cell adhesion via its interaction with E-cadherin and -catenin (18). In addition, seminal work performed by several laboratories showed that ß-catenin is also the molecular node of the canonical Wnt signaling pathway (19). The cytoplasmic stabilization and accumulation of ß-catenin, via canonical Wnt signaling, leads to ß-catenin entering the nucleus and heterodimerizing with Lef/Tcf transcription factors to regulate Wnt target genes (19). ß-Catenin plays a variety of important roles during skeletal development. Ectopic Wnt signaling in chondrocytes via ₁(II)-collagen-Wnt14 transgenic mice enhances ossification and suppresses chondrogenesis. Conversely, inactivation of ß-catenin using ₁(II) collagen-Cre, Dermo1-Cre, or Prx1-Cre mice leads to ectopic chondrocyte formation at the expense of osteoblast differentiation, during both endochondral and intramembranous ossification (20, 21, 22) (Fig. 1). These data establish the essential inhibitory role that Wnt signaling via ß-catenin plays during osteoblast differentiation.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Role of canonical Wnt signaling during osteoblast differentiation. Mesenchymal precursor cells have the ability to differentiate into adipocytes, chondrocytes, or osteoblasts, depending on the signaling pathways influencing the surrounding milieu. Wnt10b encourages these cells to differentiate into skeletal precursors and inhibits them from differentiating into preadipocytes. With regards to the osteochondroprogenitor cell, high levels of canonical Wnt signaling (as determined by levels of ß-catenin) with the presence of Runx2 promote osteoblastogenesis at the expense of chondrocyte differentiation. In contrast, low levels of ß-catenin along with the presence of Sox9 lead to chondrocyte differentiation without supporting osteoblastogenesis. PPAR, Peroxisome activated receptor .

Similar findings were observed using osteocalcin-Cre mice to create osteoblast-specific inactivation of ß-catenin or of APC, to inactivate or constitutively activate the canonical Wnt signaling, respectively (25). Although there are differences between the osteocalcin-Cre mice and the ₁(I) collagen-Cre mice, their phenotypes differ drastically from those seen with Lrp5 mutations, in which bone accrual is affected without changes in bone resorption (Fig. 2). This raises the prospect that Lrp5 may not be solely a Wnt coreceptor or that its connection to Wnt signaling is indirect.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Lrp5 vs. ß-catenin-dependent signaling in osteoblasts. In the absence of any perturbation of Wnt signaling (left), the rate of bone accrual is balanced by the rate of bone resorption, keeping bone mass constant. Lrp5 may be involved in signaling, via an as yet unknown ligand, independent of canonical Wnt signaling. The loss of Lrp5 (middle) leads to a decreased number of osteoblasts, as well as a decrease in osteoblast parameters, by methods still poorly understood. The bone accrual rate is diminished without a change in bone resorption, leading to a low bone mass phenotype. Conversely, activation of ß-catenin-dependent signaling leads to a HBM phenotype (right). Changes in bone accrual do not cause this increased mass. In these mice there is a decrease in the number of osteoclasts and a decrease in osteoclast parameters; these changes are the result of an increase in Opg expression within osteoblasts.

	Lef/Tcfs

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

	Wnts

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
The nature of the actual Wnts involved in skeletal development and bone remodeling is still not fully clarified. Wnt7b is expressed in osteoblasts, and its expression level increases during osteoblast differentiation, thus, it may be required in an autocrine manner for osteoblast differentiation (22, 26). Osteoblasts, chondrocytes, myocytes, and adipocytes are all derived from the same mesenchymal precursor, with adipogenesis being the default pathway. Wnt1 and Wnt10b are both able to suppress adipogenesis in preadipocyte cells (27). Mice deficient for Wnt10b have less bone and fewer trabeculae than wild-type mice (28). Bone accrual may solely be affected in these mice because bone resorption markers were unchanged. In contrast, transgenic Wnt10b mice, using the adipocyte-specific FABP4 promoter, have increased bone mass, more trabeculae, and less fat than wild-type mice (28). Thus, Wnt10b seems to be important in cell fate determination between osteoblastogenesis and adipogenesis (Fig. 1).

	Dkks

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
Dickkopfs were first identified in Xenopus as being necessary and sufficient for head induction (29). Dkk1 and Dkk4 are able to inhibit Wnt signaling, Dkk2 can serve as either an agonist or an antagonist, and Dkk3 has no effect on Wnt signaling (19).

Dkk1^–/– mice die in utero because of head induction and limb formation defects (30). Dkk1^+/– mice are viable and have an increased bone mass caused by an increase in bone accrual, with increased numbers of osteoblasts and BFR, without any effects on bone resorption (31). In addition, there are two transgenic models for the overexpression of Dkk1. Mice overexpressing Dkk1 using the more ubiquitous rat 3.6kb ₁(I) collagen promoter are osteopenic with forelimb defects and alopecia. Meanwhile, overexpressing Dkk1 only in osteoblasts using the rat 2.3kb ₁(I) collagen leads to severe osteopenia because of decreased osteoblast number and bone accrual, without limb defects or hair loss (32). Collectively, these mouse models support the hypothesis that Dkk1 functions as a potent negative regulator of bone formation.

Dkk2^–/– mice are osteopenic, with increased osteoid and mineralization defects in osteoblast cultures in vitro, suggesting that Dkk2 is a Wnt agonist affecting bone accrual in vivo (33). Dkk2^–/– mice also have an increased number of osteoclasts with Rankl, but not Opg, increased only in immature osteoblasts. Therefore, Dkk2 influences both osteoblast and osteoclast biology.

	Sfrps

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
Secreted frizzled receptor proteins (Sfrps) are another class of Wnt antagonists. Sfrp1 is highly expressed during the transition from preosteoblast to osteoblast (34). Sfrp1^–/– mice have an increase in trabecular but not cortical bone, and this increase is more elevated in female mice (35). Sfrp1^–/– mice also have shortened growth plates and increased calcification of the hypertrophic zone of chondrocytes, indicating increased chondrocyte differentiation and endochondral ossification (36). Therefore, Sfrp1 may serve as a negative regulator of both osteogenesis and chondrogenesis.

	GSK3ß

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
Mice deficient for GSK3ß are embryonic lethal at E13.5–14.5 dpc because of defective nuclear factor-B signaling and hepatocyte apoptosis (37). Because no limb patterning defects were seen and no other Wnt abnormalities were observed, GSK3, or some other kinase, must be able to substitute for GSK3ß until this time point.

Lithium, a known inhibitor of GSK3ß, is able to rescue the osteopenia of Lrp5^–/– mice to nearly wild-type levels and increases the bone mass of wild-type mice (38). The modulation of GSK3ß by lithium or some other agent may have therapeutic usage by manipulating the canonical Wnt signaling pathway to increase bone mass.

	Other Wnt Signaling Molecules

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
There are other mouse models, because of mutations in lesser known genes, that may prove instrumental in furthering our understanding of Wnt signaling and bone regulation. Sclerosteosis is an autosomal recessive disease, very similar to HBM diseases, that is caused by mutations in the SOST gene, which encodes sclerostin (39, 40). Sclerostin binds to Lrp5/6 and prevents Lrp-Fzd interaction (26, 40). Mice transgenic for SOST via the OG2 promoter are osteopenic, with decreased alkaline phosphatase activity and a decrease in their BFR, suggesting that sclerostin negatively influences bone formation (41). Axin2^–/– mice have skull malformations that resemble craniosynostosis, including premature closure of sutures (42). The loss of Axin2 enhances both osteoblast proliferation and differentiation in vitro and in vivo (42). Finally, Ror2 is an orphan receptor tyrosine kinase that interacts with Wnts and Fzds; mutations in Ror2 lead to Robinow syndrome and brachydactyly type B (19). Ror2^–/– mice have several skeletal abnormalities that are attributable to cartilage and growth plate defects (43).

	Conclusions

Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 
There are a variety of animal models that illustrate the complex roles that Wnt signaling, of various natures, plays in skeletogenesis and bone mass regulation. Many questions still remain unanswered. The main undertaking is to understand what the molecular mechanisms are that account for the differences between Lrp5 and canonical Wnt signaling within osteoblasts. There is also very little known about the role of canonical Wnt signaling in osteoclasts. To study Wnt signaling in osteoclast progenitors, Pu.1-Cre or NF-kB-Cre mice could be generated to activate or remove ß-catenin in osteoclasts. Similarly, TRAP-Cre and Cathepsin K-Cre mice, which have already been generated (44, 45), could be used to study canonical Wnt signaling in fully differentiated osteoclasts. As we continue to elucidate more about the roles of canonical Wnt signaling in bone remodeling, animal models will remain an instrumental method of acquiring greater insight into this aspect of bone biology.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online March 29, 2007

Abbreviations: BFR, Bone formation rate; dpc, days post coitum; HBM, high bone mass; Lrp, low-density lipoprotein receptor-related protein; Opg, osteoprotegerin; Sfrps, secreted frizzled receptor proteins.

Received October 6, 2006.

Accepted for publication November 20, 2006.

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Top Abstract Introduction Lrp5 Lrp6 ß-Catenin Lef/Tcfs Wnts Dkks Sfrps GSK3ß Other Wnt Signaling Molecules Conclusions References

 

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