Multiple signaling pathways have been shown to regulate bone development and metabolism, and the WNT signaling pathway is emerging as one of the most crucial contributors. Several WNT ligands, receptors and WNT antagonists are expressed in bone and play a role in maintaining postnatal bone homeostasis. However, specific functions of individual WNT pathway members in bone are only beginning to be elucidated. Investigating the role of WNT signaling in bone development and metabolism will provide important implications for the treatment of fractures and bone thinning disorders such as osteoporosis and osteopenia. The focus of my thesis is to elucidate the functions of three out of nineteen WNT ligands and WNT co-receptors LRP5 and LRP6 in osteoblasts (bone forming cells).
In this thesis, I investigated the role of WNT ligands WNT3A, WNT5A and WNT16 in osteoblasts to identify the target genes regulated by these WNTs and to understand the molecular mechanism by which these WNTs regulate bone metabolism. Gene expression analysis of neonatal osteoblasts treated with recombinant WNTs identified more than 1000 genes regulated by WNT signaling in osteoblasts and suggested that WNT3A and WNT16 positively regulate early stages of osteoblast differentiation and inhibit osteoblast maturation/mineralization.
I also studied the role of WNT co-receptors LRP5 and LRP6 in mediating canonical WNT signaling. LRP5 and LRP6 are two WNT co-receptors that have been linked to bone development and metabolism. Both LRP5 and LRP6 are required for normal postnatal bone homeostasis. However, their specific roles are not well understood. To determine the roles of LRP5 and 6 in mediating canonical WNT signaling, osteoblasts lacking Lrp5, Lrp6 and both Lrp5 and 6 were treated with recombinant WNT3A. The RNA isolated from all WNT3A treated samples were sequenced and analyzed to identify genes regulated through LRP5 and LRP6 and genes that do not require LRP5/6 for WNT3A induced transcriptional regulation. This study revealed that LRP6 plays a dominant role in mediating WNT3A signaling in osteoblasts.
Canonical WNTs such as WNT3A regulate target gene expression by activating TCF/LEF family transcription factors. These transcription factors bind to promoters and/or enhancers of target genes to induce gene transcription. To identify direct targets of canonical WNT signaling, using ChIP-seq, I identified TCF/LEF binding sites near WNT3A targets. More than 80% WNT3A targets had TCF/LEF binding sites in their promoter and/or enhancers. This study also identified more than 500 putative WNT inducible enhancers in osteoblasts. A subset of predicted WNT inducible enhancers was validated experimentally to confirm WNT3A inducible enhancer activity.
My findings expand our current understanding of the role of WNT signaling pathway in regulating osteoblast differentiation and function, as well as contribute to the knowledge of the WNT signaling pathway itself. The WNT target genes identified in this study may be further explored for their therapeutic potential in treating osteoporosis and other bone disorders.
|2008||Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.|
|2010||Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Müller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. July 2010;25(7):1468-1486.|
|2005||Li X, Zhang Y, Kang H, Liu W, Liu P, Zhang J, Harris SE, Wu D. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem. May 20, 2005;280(20):19883-19887.|
|2002||Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor–related protein 5. NEJM. May 16, 2002;346(20):1513-1521.|
|2006||Sawakami K, Robling AG, Ai M, Pitner ND, Liu D, Warden SJ, Li J, Maye P, Rowe DW, Duncan RL, Warman ML, Turner CH. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem. August 18, 2006;281(33):23698-23711.|
|2004||van Bezooijen RL, Roelen BAJ, Visser A, van der Wee-Pals L, de Wilt E, Karperien M, Hamersma H, Papapoulos SE, ten Dijke P, Löwik CWGM. Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med. March 15, 2004;199(6):805-814.|
|2006||Hadjidakis DJ, Androulakis II. Bone remodeling. Annals NY Acad Sci. December 2006;1092(1):385-396.|
|2002||Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, Manning SP, Swain PM, Zhao S-C, Eustace B, Lappe MM, Spitzer L, Zweier S, Braunschweiger K, Benchekroun Y, Hu X, Adair R, Chee L, FitzGerald MG, Tulig C, Caruso A, Tzellas N, Bawa A, Franklin B, McGuire S, Nogues X, Gong G, Allen KM, Anisowicz A, Morales AJ, Lomedico PT, Recker SM, Van Eerdewegh P, Recker RR, Johnson ML. A mutation in the LDL receptor–related protein 5 gene results in the autosomal dominant high–bone-mass trait. Am J Hum Genet. January 2002;70(1):11-19.|
|2006||Johnell O, Kanis JA. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int. December 2006;17(12):1726-1733.|