Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Roberto Civitelli

Abstract

Skeletal development and post-natal bone homeostasis are dependent on the coordinated activity of bone-forming cells called osteoblasts and bone-resorbing cells called osteoclasts. Over 10 million people in the US currently suffer from osteoporosis, which increases the risk of low-trauma fractures and accounts for health care expenditures exceeding $14 billion per year. Osteoporosis is often treated with anti-resorptive compounds, primarily bisphosphonates, which inhibit osteoclast-mediated bone destruction. However, these drugs do not restore bone mass, which can only be accomplished by activation new bone formation, as with intermittent parathyroid hormone therapy. The development of additional bone "anabolic" therapies will require genetic and mechanistic information about other signaling pathways that can potently stimulate osteoblast differentiation and/or function. A wealth of genetic evidence in humans and mice clearly demonstrates that the canonical Wnt pathway is intimately involved in skeletal development and post-natal bone homeostasis. In fact, neutralizing antibodies to antagonists of the canonical Wnt pathway, such as Sclerostin and Dickkhopf1, are currently in clinical trials as potentially promising bone anabolic agents. However, it is still unclear how Wnt signaling provides such a strong osteogenic stimulus and how it interacts with BMP signaling, another strong pro-osteogenic pathway, to generate accurate and timely cues during the osteoblast differentiation process. Previous work in our lab showed that beta-catenin, an essential mediator of canonical Wnt signaling, synergizes with BMP2 to stimulate osteoblast differentiation and bone formation. My work was designed to conduct a full evaluation of how BMP and Wnt signals interact at discrete stages of the osteoblast lifecycle to regulate cell fate, establish a mitotic/post-mitotic boundary, stimulate production of the skeletal extracellular matrix, and prevent programmed cell death. Using an in vitro model of osteoblast differentiation, we find that beta-catenin acts downstream of BMP2 signaling to promote an osteoblast and suppress an adipocyte cell fate decision in multipotent progenitors, a lineage allocation mechanism requiring both Tcf/Lef-dependent and Tcf/Lef-independent mechanisms. We next examined BMP/Wnt interactions during proliferation of osteoprogenitors, by focusing on Smad4, a central mediator of the greater TGF-beta/BMP pathway. Acute, tamoxifen-induced deletion of Smad4 in otherwise normal osteoblasts in mice activates proliferation of predominantly post-mitotic Osterix+ cells on trabecular bone surfaces. Manipulation of Smad4 in osteoprogenitor cell lines indicates this effect is accomplished, at least in part, by the ability of BMP signals to stimulate physical recruitment of beta-catenin to the DNA-binding Smad4 transcription complex, occurring in a manner that antagonizes Tcf/Lef-dependent transcription and the pro-mitotic effects of canonical Wnts. This led us to hypothesize that deficiency of Smad4 could enhance the anabolic effects of anti-Dkk1 therapy. Using a genetic mouse model with constitutive embryonic deletion of Smad4 in Osterix+ cells, we instead find that long-term deficiency of Smad4 in Osterix+ cells causes a lethal impairment of post-natal skeletal development characterized by defective osteoblasts which are not sensitive to anabolic effects of BMP and are not rescued by anabolic Wnt therapy. Whereas this osteoblast defect can be explained by disruption of p38 MAPK signaling, the resistance to anabolic Wnt signaling can be linked to premature apoptosis and Caspase-3-mediated inactivation of beta-catenin. Thus, acute removal of Smad4 directly favors canonical Wnt signaling and mitosis, while long-term deficiency of Smad4 indirectly impairs Wnt signaling and osteoblast function. In summary, these studies provide evidence that multifaceted interactions between BMP and Wnt signaling regulate cell fate, proliferation, function, and survival of osteoblasts. Further work is needed to more carefully distinguish TGF-beta and BMP specific contributions to the role of Smad4 in skeletal development and post-natal bone homeostasis.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7QC01HP

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