The guiding mission of Dr. Hankenson’s research is to elucidate cellular and molecular mechanisms regulating bone regeneration. This research has two long-term translational goals: (1) eliminating human osteoporosis by developing therapies to restore lost bone, and (2) enhancing bone regeneration in both humans and animals, particularly in populations with poor healing such as geriatric patients and those with compromised non-healing fractures.
Bone is formed by osteoblasts which develop from stem cells, termed mesenchymal stem cells (MSC). To this end, the Hankenson laboratory studies molecular and cellular mechanisms of MSC osteoblast differentiation (osteoblastogenesis).
Thrombospondins and bone regeneration
A group of specialized ECM proteins termed matricellular proteins (MP) are highly expressed in the skeleton by MSC. Furthermore, TSP2 is highly expressed in healing tissues and the impact of TSP2 deficiency is often more profound during injury. The work from my laboratory was the first to show a significant role for TSPs in bone regeneration. On-going studies explore the mechanism of TSP regulation of bone regeneration and determine whether inhibition of TSPs could be used therapeutically to promote ischemic fracture healing.
Taylor, D. K., Meganck, J. A., Terkhorn, S., Rajani, R., Naik, A., O'Keefe, R. J., Goldstein, S. A., and Hankenson, K. D. (2009) Thrombospondin-2 Influences the Proportion of Cartilage and Bone During Fracture Healing. J Bone Miner Res 24, 1043-1054
Miedel, E., Dishowitz, M. I., Myers, M. H., Dopkin, D., Yu, Y. Y., Miclau, T. S., Marcucio, R., Ahn, J., and Hankenson, K. D. (2013) Disruption of thrombospondin-2 accelerates ischemic fracture healing. J Orthop Res 31, 935-943
Burke, D., Dishowitz, M., Sweetwyne, M., Miedel, E., Hankenson, K. D., and Kelly, D. J. (2013) The role of oxygen as a regulator of stem cell fate during fracture repair in TSP2-null mice. J Orthop Res
R-spondins and Wnt signaling
More than a decade ago my laboratory collaborated with Ormond MacDougald here at Michigan to demonstrate that Wnt10b is an endogenous Wnt that could regulate bone mass. Next we showed that Wnt11also increases osteoblast differentiation (17). Our work with Wnt11 led to the discovery of R-spondin 2 (Rspo2) as a matricellular protein that regulates osteoblast differentiation. On-going studies explore the significance of Wnt11 and Rspo2 in genetically engineered mice. Most recently we have generated a Rspo2 floxed mice and charaterized a deficiency in bone formation in mice with out Rspo2 in osteoblasts (Ocn-Cre).
Kang, S., Bennett, C. N., Gerin, I., Rapp, L. A., Hankenson, K. D., and Macdougald, O. A. (2007) Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. J Biol Chem 282, 14515-14524
Bennett, C. N., Longo, K. A., Wright, W. S., Suva, L. J., Lane, T. F., Hankenson, K. D., and MacDougald, O. A. (2005) Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A 102, 3324-3329
Aslanidi, G., Kroutov, V., Philipsberg, G., Lamb, K., Campbell-Thompson, M., Walter, G. A., Kurenov, S., Ignacio Aguirre, J., Keller, P., Hankenson, K., Macdougald, O. A., and Zolotukhin, S. (2007) Ectopic expression of Wnt10b decreases adiposity and improves glucose homeostasis in obese rats. Am J Physiol Endocrinol Metab 293, E726-736
Bennett, C. N., Ouyang, H., Ma, Y. L., Zeng, Q., Gerin, I., Sousa, K. M., Lane, T. F., Krishnan, V., Hankenson, K. D., and MacDougald, O. A. (2007) Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation. J Bone Miner Res 22, 1924-1932
Friedman, M. S., Oyserman, S. M., and Hankenson, K. D. (2009) Wnt11 promotes osteoblast maturation and mineralization through R-spondin 2. J Biol Chem 284, 14117-14125
Knight MN, Karuppaiah K, Lowe M, Mohanty S, Zondervan RL, Bell S, Ahn J, Hankenson KD. R-spondin-2 is a Wnt agonist that regulates osteoblast activity and bone mass. Bone Res. 2018 Aug 14;6:24
My laboratory has been actively pursuing multiple and varied experiments related to Notch signaling in MSC and bone. We have recently published on the osteoinductive influences of Jagged-1 on human osteoblastogenesis, and future studies will focus on the mechanism(s) of Notch regulated osteoblast differentiation. As a translational extension of this work, we have become very interested in the role of Notch signaling in bone regeneration. We are now pursuing several lines of investigation to ask about the role of Notch signaling in bone healing, including working on the development of Jagged-1 delivery as an osteogenic molecule for bone regeneration.Zhu, F., Sweetwyne, M. T., and Hankenson, K. D. (2013) PKCdelta Is Required for Jagged-1 Induction of Human Mesenchymal Stem Cell Osteogenic Differentiation. Stem Cells 31, 1181-1192
Dishowitz, M. I., Mutyaba, P. L., Takacs, J. D., Barr, A. M., Engiles, J. B., Ahn, J., and Hankenson, K. D. (2013) Systemic inhibition of canonical Notch signaling results in sustained callus inflammation and alters multiple phases of fracture healing. PLoS One 8, e6872
Dishowitz, M. I., Terkhorn, S. P., Bostic, S. A., and Hankenson, K. D. (2012) Notch signaling components are upregulated during both endochondral and intramembranous bone regeneration. Journal of Orthopaedic Research 30, 296-303
Dishowitz, M. I., Zhu, F., Sundararaghavan, H. G., Ifkovits, J. L., Burdick, J. A., and Hankenson, K. D. (2013) Jagged1 immobilization to an osteoconductive polymer activates the notch signaling pathway and induces osteogenesis. J Biomed Mater Res A 102, 1558-1567
Youngstrom DW, Senos R, Zondervan RL, Brodeur JD, Lints AR, Young DR, Mitchell TL, Moore ME, Myers MH, Tseng WJ, Loomes KM, Hankenson KD. Intraoperative delivery of the Notch ligand Jagged-1 regenerates appendicular and craniofacialbone defects. NPJ Regen Med. 2017 Dec 15;2:32.
MSC transcriptional regulation and genomics
The Hankenson lab discovered that BMP6 is the most consistent and potent inducer of human osteoblast differentiation of the various osteogenic BMPs. A series of systems biology studies demonstrated novel pathways regulated by BMP6 signaling, including Notch signaling and the Swi/Snf chromatin remodeling complex. As well, we found that the transcription factor Osterix (SP7) is regulated by BMP6 and clusters with a set of unique ECM molecules. Next we demonstrated that Osterix is also essential for human osteoblastogenesis, yet is not sufficient. Interestingly, Osterix has been identified in a number of osteoporosis genome wide association studies (GWAS). Current studies in collaboration with Dr. Struan Grant, Children's Hospital of Philadelphia, seek to identify the promoterome of human osteoblasts and using chromatin analysis techniques such as 4C and RNAseq to identify novel osteoblastogenesis genes.
Friedman, M. S., Long, M. W., and Hankenson, K. D. (2006) Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6. J Cell Biochem 98, 538-554
Luo, W., Friedman, M., Shedden, K., Hankenson, K. D., and Woolf, P. (2009) GAGE: Generally Applicable Gene Set Enrichment for Pathway Inference. BMC Bioinformatics 10, 161
Luo, W., Hankenson, K. D., and Woolf, P. J. (2008) Learning transcriptional regulatory networks from high throughput gene expression data using continuous three-way mutual information. BMC Bioinformatics 9, 467
Luo, W., Friedman, M., Hankenson, K., and Woolf, P. (2011) Time Series Gene Expression Profiling and Temporal Regulatory Pathway Analysis of BMP6 Induced Osteoblast Differentiation and Mineralization. BMC Systems Biology 5, 82
Zhu, F., Friedman, M. S., Luo, W., Woolf, P., and Hankenson, K. D. (2011) The transcription factor osterix (SP7) regulates BMP6-induced human osteoblast differentiation. J Cell Physiol 227, 2677-2685