Browsing by Author "Witcher, Phillip C."

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    Improving bone properties and fracture susceptibility: experimental models of genetic manipulation, pharmacologic intervention, and cellular perturbation reveal new approaches for improving bone health
    (Office of the Vice Chancellor for Research, 2016-04-08) Witcher, Phillip C.; Lee, Joowon; Assaf, Noor; Mertz, Susan; Singh, Karan; Thompson, William R.; Robling, Alexander G.
    Bone, a crucial support structure in the human body, is often taken for granted for its lightweight properties and unparalleled strength. Skeletal fracture is a major clinical condition affecting millions of Americans, which results from abnormal aging, hormonal imbalance, genetic conditions, and lifestyle choices (e.g., exercise). Because fractures are caused by a number of different factors, reducing fracture incidence requires a multifactorial approach to unraveling the underlying biology of bone metabolism, in order to discover new ways to improve bone properties and prevent fractures. We have taken such an approach by conducting (1) genetic manipulation experiments in mice, where genes predicted to be involved in bone mass regulation were mutated; (2) pharmacologic experiments to quantify the dose-response effect of an agent that inhibits bone loss, and (3) cell culture experiments, aimed at revealing molecular pathways activated by mechanical stimulation. METHODS: Mice with mutations in two genes, likely to regulate bone mass (SOST, DKK1) were generated and subjected to in vivo dual energy x-ray absorptiometry (DEXA) scans at 6-wk old. Whole body scans were analyzed for bone mineral density (BMD) using Lunar Piximus II v2.10 software. Mice (6-wk) were also dosed (0, 1, 10, 100, or 1000 mg/kg) with daily alendronate HCl, a bisphosphonate that inhibits osteoclast activity. Six wks later, the mice were sacrificed, and the femurs were dissected and sectioned for histological analysis of bone formation parameters, including mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS). To understand the cellular signaling events in response to mechanical loading, bone marrow mesenchymal stem cells (MSCs) were treated with 10, 20, 30, or 40μM PF7408671, an S6 kinase inhibitor. Cells then were subject to 100 cycles of biaxial mechanical strain (2%, 10 cycles/min). Protein lysates were separated by electrophoresis and probed for phosphorylation of Rictor and Akt by Western blot. RESULTS: Mice harboring mutations in either the SOST gene or the DKK1gene exhibited significantly increased BMD compared to wild-type control mice, though the SOST mutation had a stronger effect on BMD than DKK1. Mice with compound mutations (SOST and DKK1 mutations) had significantly greater BMD than mice with either single mutation, suggesting that inhibition of SOST and DKK1 might be an effective means to increase bone mass in patients susceptible to fracture. Mice treated with high-dose alendronate (100 or 1,000 mg/kg) exhibited significant decreases in bone formation parameters (MS/BS, MAR, and BFR/BS) compared to untreated (0 mg) mice, suggesting that while this compound might be beneficial for inhibiting bone loss, it also inhibits bone formation. The signaling hub, mTORC2, is a critical regulator of mechanical force in MSC progenitors. Our data demonstrate that S6 kinase is an upstream activator of mTORC2 in response to mechanical strain. CONCLUSION: Our experiments suggest that genetic manipulation of mice reveal viable protein targets (e.g., SOST, DKK1) that could ultimately be manipulated pharmacologically to improve bone mass. We also found that an FDA-approved class of drugs inhibits bone formation even at very low doses, suggesting that additional pro-anabolic compounds might benefit patients taking bisphosphonates. On a cell signaling level, we found that the mTORC2 pathway shows considerable promise for pharmacologic manipulation to simulate the effects of exercise. Taken together, these experiments highlight the utility of a broad approach to solving bone metabolism challenges that can affect fracture susceptibility.
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    Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition
    (American Society for Clinical Investigation, 2018-06-07) Witcher, Phillip C.; Miner, Sara E.; Horan, Daniel J.; Bullock, Whitney A.; Lim, Kyung-Eun; Kang, Kyung Shin; Adaniya, Alison L.; Ross, Ryan D.; Loots, Gabriela G.; Robling, Alexander G.; Anatomy and Cell Biology, IU School of Medicine
    The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.