Regulation of osteoblastogenesis and bone mass by Wnt10b

Christina Bennett

Kenneth Longo

Wendy Wright

Larry Suva

Timothy Lane

Kurt Hankenson

Ormond 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}

^{To whom correspondence should be addressed. E-mail:}ude.hcimu@aguodcam.

Edited by M. Daniel Lane, Johns Hopkins University School of Medicine, Baltimore, MD

Received 2004 Nov 23; Accepted 2005 Jan 19.

Abstract

Wnts comprise a family of secreted signaling proteins that regulate diverse developmental processes. Activation of Wnt signaling by Wnt10b inhibits differentiation of preadipocytes and blocks adipose tissue development; however, the effect of Wnt10b on other mesenchymal lineages has not been defined. To explore the physiological role of Wnt signaling in bone development, we analyzed FABP4-Wnt10b mice, which express the Wnt10b transgene in marrow. Femurs from FABP4-Wnt10b mice have almost four times as much bone in the distal metaphyses and are mechanically stronger. These mice maintain elevated bone mass at least through 23 months of age. In addition, FABP4-Wnt10b mice are protected from the bone loss characteristic of estrogen deficiency. We used pharmacological and genetic approaches to demonstrate that canonical Wnt signaling stimulates osteoblastogenesis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage by induction of the osteoblastogenic transcription factors Runx2, Dlx5, and osterix and suppression of the adipogenic transcription factors C/EBPα and PPARγ. One mechanism whereby Wnt10b promotes osteoblastogenesis is suppression of PPARγ expression. Finally, Wnt10b-/- mice have decreased trabecular bone and serum osteocalcin, confirming that Wnt10b is an endogenous regulator of bone formation.

Keywords: adipogenesis, development, stem cells

Abstract

Mesenchymal progenitors can differentiate into a number of cell types, including adipocytes and osteoblasts (1). One factor that regulates the reciprocal relationship between these lineages is Wnt signaling, which inhibits adipogenesis and stimulates osteoblastogenesis. Activation of Wnt signaling blocks preadipocyte differentiation by inhibiting expression of the adipogenic transcription factors C/EBPα and PPARγ (2–5). The endogenous inhibitory Wnt signal may be initiated by Wnt10b, which is expressed in preadipocytes and stromal vascular cells but not in adipocytes (2, 3). Expression of Wnt10b from the FABP4 promoter decreases accumulation of white adipose tissue by ≈50% and completely blocks the development of brown fat (6, 7). Furthermore, FABP4-Wnt10b mice resist diet-induced obesity and the associated glucose intolerance.

The first indication that Wnt signaling plays a critical role in bone formation came from human studies where inactivating mutations in the Wnt coreceptor LRP5 were shown to cause osteoporosis (8). These findings were supported by the observation that LRP5-/- mice also have low bone mass (9). Furthermore, gain-of-function mutations in LRP5 that increase Wnt signaling result in higher bone density in humans and mice (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, an early osteoblast marker (12–14). Although these and other studies suggest that endogenous Wnt signaling regulates osteoblastogenesis and bone formation (15), a specific Wnt or Wnts responsible for activation of this pathway in marrow have not been identified.

We report herein that FABP4-Wnt10b mice have increased bone mass and strength and that they resist the loss of bone that occurs with aging or estrogen deficiency. The expression of Wnt10b in mesenchymal progenitors induces the expression of osteoblastogenic transcription factors Runx2, Dlx5, and osterix and strongly stimulates osteoblastogenesis. In addition, Wnt10b inhibits the adipogenic transcription factors C/EBPα and PPARγ and blocks adipogenesis. Suppression of PPARγ is one mechanism whereby Wnt10b induces osteoblastogenesis. Compelling evidence supportingWnt10b as an endogenous regulator of bone mass comes from analyses of Wnt10b-/- mice, which have decreased trabecular bone mass and serum osteocalcin.

Percent change in morphometric properties compared with 6 months of age. BV/TV, bone volume fraction; BMD, bone mineral density; Tb. n., trabecular number; Tb.Th., trabecular thickness; Tb.Sp., trabecular spacing.

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Acknowledgments

We thank Jacklyn Kriegl and David Fisher for μCT scans and Philip Leder (Harvard Medical School) for his considerable contribution to making Wnt10b-/- mice. This work was supported by grants from the National Institutes of Health [DK51563 and DK62876 (to O.A.M.) and RR00161 and AR49682 (to K.D.H.)]. Other support was from the Diabetes Research and Training Center (P60 DK20572), the Nathan Shock Mutant and Transgenic Rodent Core, and the Core Center for Musculoskeletal Disorders (P30AR046024). Fellowships to C.N.B. were from Tissue Engineering and Regeneration Training Grant DE07057 and the American Physiological Society Porter Fellowship. K.A.L. was supported by a mentor-based postdoctoral fellowship from the American Diabetes Association.

Acknowledgments

Notes

Author contributions: C.N.B., K.D.H., and O.A.M. designed research; C.N.B., K.A.L., W.S.W., L.J.S., and K.D.H. performed research; T.F.L. and K.D.H. contributed new reagents/analytic tools; C.N.B., K.A.L., W.S.W., L.J.S., K.D.H., and O.A.M. analyzed data; and C.N.B. and O.A.M. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: μCT, microcomputerized tomography.

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: μCT, microcomputerized tomography.

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