HES Factors Regulate Specific Aspects of Chondrogenesis and Chondrocyte Hypertrophy During Cartilage Development
(2016) Journal of Cell Science 129(11): 2145-55
Rutkowski TP, Kohn A, Sharma D, Ren Y, Mirando AJ, and Hilton MJ.
This publication is the first to describe precisely how specific HES factors regulate cartilage development. We developed unique in vivo genetic models and in vitro approaches to demonstrate that the RBPjκ-dependent Notch targets HES1 and HES5 suppress chondrogenesis and promote the onset of chondrocyte hypertrophy. HES1 and HES5 might have some overlapping function in these processes, although only HES5 directly regulates Sox9 transcription to coordinate cartilage development. HEY1 and HEYL play no discernable role in regulating chondrogenesis or chondrocyte hypertrophy, whereas none of the HES or HEY factors appear to mediate Notch regulation of cartilage matrix catabolism during development. This work identifies important candidates that might function as downstream mediators of Notch signaling both during normal skeletal development and in Notch-related skeletal disorders.
NOTCH Signaling in Skeletal Progenitors is Critical for Fracture Repair
(2016) Journal of Clinical Investigation doi:10.1172/JCI80672
Wang C, Inzana JA, Mirando AJ, Ren Y, Liu Z, Shen J, O'Keefe RJ, Awad HA, and Hilton MJ.
This publication is the first to describe how NOTCH signaling within bone marrow stromal/stem cells (BMSCs) and BMSCs themselves are critical for normal fracture healing. Our data demonstrate that if NOTCH signaling is defective specifically within BMSCs, the BMSC population is depleted and/or defective resulting in severely compromised healing and fracture nonunions. Finally, our NOTCH defective BMSC animal model demonstrated an ability to produce a fracture nonunion irrespective of fracture stability and without any signs of altered vascular involvement, two very important and common causes for fracture nonunions. These data suggest that NOTCH signaling status within BMSCs and BMSC populations themselves could serve as predictors for those fractures that heal successfully and those that proceed to a nonunion.
A Dual Role for NOTCH Signaling in Joint Maintenance and Osteoarthritis.
(2015) Science Signaling 8(386): ra71.
Liu Z, Chen J, Mirando AJ, Wang C, Zuscik MJ, O'Keefe RJ, and Hilton MJ.
This publication describes how NOTCH signaling strength, duration, and context in joint cartilages mediates the balance between joint maintenance and osteoarthritis. We demonstrate that transient activation of NOTCH signaling in the joint cartilages favors normal physiology and maintenance and that sustained or pathological NOTCH signaling is capable of inducing osteoarthritis, independent of other external factors. This study is the first to reconcile how NOTCH signaling is both required for joint maintenance and capable of inducing joint disease. Furthermore, this work identified novel targets of the NOTCH signaling pathway in chondrocytes that mediate some of the destructive effects of sustained NOTCH signaling on joint tissues, and has therefore implicated these molecules as potential disease modifying osteoarthritis drug (DMOAD) targets.
Notch Signaling Controls Chondrocyte Hypertrophy via Indirect Regulation of Sox9.
(2015) Bone Research 3: 15021.
Kohn A, Rutkowski TP, Liu Z, Mirando AJ, Zuscik MJ,O'Keefe RJ, and Hilton MJ.
This publication describes the molecular mechanism by which the RBPjk-dependent Notch signaling pathway controls the onset of chondrocyte hypertrophy. We demonstrate that RBPjk-dependent Notch signaling indirectly suppresses Sox9 expression via secondary effectors, which are likely members of the HES/HEY transcription factor family. Furthermore, we demonstrate that this regulation coordinates the onset of chondrocyte maturation, but has no effect on terminal chondrocyte hypertrophy and cartilage matrix turnover.
Transient Gamma-Secretase Inhibition Accelerates and Enhances Fracture Repair Likely Via Notch Signaling Modulation.
(2015) Bone 73: 77-89.
Wang C, Shen J, Yukata K, Inzana JA, Mirando AJ, O'Keefe RJ, Awad HA, and Hilton MJ.
This publication describes a novel therapeutic strategy for acclerating and enhancing skeletal fracture repair. We demonstrate that transient, but not sustained, gamma-secretase and Notch inhibition promotes and acclerates fracture repair via the enhanced differentiation of skeletal progenitors. This study is the first to demonstrate that the gamma-secretase and Notch inhibitor, DAPT, can be utilized in the context of skeletal regeneration and repair and serves as a proof-of-concept for further exploration of these drugs in translational studies.
NOTCH-Mediated Maintenance and Expansion of Human Bone Marrow Stromal/Stem Cells: A Technology Designed for Orthopedic Regenerative Medicine.
(2014) Stem Cells Translational Medicine 3(12): 1456-1466.
Dong Y, Long T, Wang C, Mirando AJ, Chen J, O'Keefe RJ, and Hilton MJ.
This publication describes a novel biological method that entails selection of human BMSCs based on NOTCH2 expression and activation of the NOTCH signaling pathway in cultured BMSCs via a tissue culture plate coated with recombinant human JAGGED1 (JAG1) ligand. We demonstrate that transient JAG1-mediated NOTCH signaling promotes human BMSC maintenance and expansion while increasing their skeletogenic differentiation capacity, both ex vivo and in vivo. This study is the first of its kind to describe a NOTCH-mediated methodology for the maintenance and expansion of human BMSCs and will serve as a platform for future clinical or translational studies aimed at skeletal regeneration and repair.
Skeletal Development and Repair: Methods and Protocols
(2014) Methods in Molecular Biology
Editor: Matthew J. Hilton
This volume, Skeletal Development and Repair, in the Methods in Molecular Biology series is designed as a comprehensive laboratory manual for all levels of basic research scientists working in the broad fields of skeletal development and skeletal repair research. The protocols highlighted here not only encompass the most current and cutting-edge techniques in skeletal development and repair, but also showcase those protocols that have been modified and perfected over the course of several decades of skeletal research. Three chapters from this work were developed directly by the Hilton Laboratory.
RBPjκ-dependent Notch signaling is required for articular cartilage and joint maintenance.
(2013) Arthritis and Rheumatism. 65(10):2623-2633.
Mirando AJ*, Liu Z*, Moore T, Lang A, Jesse AM, Kohn A, O’Keefe RJ, Mooney RA, Zuscik MJ, and Hilton MJ.
This publication is the first to indicate that the RBP-Jκ–dependent Notch pathway is a novel pathway involved in joint maintenance and articular cartilage homeostasis, a critical regulator of articular cartilage ECM-related molecules, and a potentially important therapeutic target for OA-like joint disease.
Cartilage-specific RBPjκ-dependent and -independent Notch signals regulate cartilage and bone development.
(2012) Development. 139(6): 1198-212.
Kohn A, Dong Y, Mirando A, Jesse AM, Honjo T, Zuscik MJ, O’Keefe RJ, and Hilton MJ.
This publication was the first to demonstrate that Notch regulation of chondrocyte maturation is solely mediated via the RBPjκ-dependent pathway, and that the perichodrium or osteogenic lineage influences chondrocyte terminal maturation. Furthermore, we identified the cartilage-specific RBPjκ-independent pathway as a critical regulator of chondrocyte proliferation, survival, and columnar chondrocyte organization. We also identified RBPjk-independent signals as an important long-range cell non-autonomous regulator of perichondral bone formation that corrdinates chondrocyte and osteoblast differentiation during skeletal development.
RBPJk-dependent Notch Signaling Regulates Mesenchymal Progenitor Cell Proliferation and Differentiation During Skeletal Development.
(2010) Development. 137(9): 1461-1471.
Dong Y*, Jesse A*, Kohn A, Gunnell LM, Honjo T, Zuscik MJ, O’Keefe RJ, and Hilton MJ.
This publication demonstrates for the first time that the RBPjkappa-dependent Notch signaling pathway is a crucial regulator of mesenchymal progenitor cell (MPC) proliferation and differentiation during skeletal development. We also implicated the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjk-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis.
Notch Signaling Maintains Bone Marrow Mesenchymal Progenitors by Suppressing Osteoblast Differentiation.
(2008) Nature Medicine. 14 (3): 306-14.
Hilton MJ*, Tu X*, Bai S, Zhao H, Kobayashi T, Kronenberg HM, Teitelbaum SL, Ross FP, Kopan R, and Long F.
This publication was the first to demonstrate the importance of Notch signaling in bone marrow mesenchymal progenitor cells. Our data support a model wherein Notch signaling in bone marrow normally acts to maintain a pool of mesenchymal progenitors by suppressing osteoblast differentiation. Thus, mesenchymal progenitors may be expanded in vitro by activating the Notch pathway, whereas bone formation in vivo may be enhanced by transiently suppressing this pathway.