New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Abstract\ud \ud \ud \ud Background\ud Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS.\ud \ud \ud \ud Results\ud From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied.\ud \ud \ud \ud Conclusions\ud Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.We would like to thank Marc Sylvester for his invaluable advices regarding quantitative proteomics, Marcella Nunes de Melo Braga (University of Southern Denmark) for helping to set up the dimethyl labeling, Giuseppe Palmisano (Harvard University) and Melanie Schultz (University of Southern Denmark) for advices regarding sample analysis in MS sample preparation and data analysis. The excellent technical assistance of Zizi de Mendonça, Debora Costa Lopes and Marluce C. Mantovani is also acknowledged. This project was supported by: BNDES, CAPES, CNPq, FAPESP (grant number 2008/53974-4),MCTI and MS-DECIT

Notch Signaling and Bone Fracture Healing

Bone fractures can exhibit delayed or non-union healing. Current treatments have well- documented limitations. Although morphological aspects of fracture healing are well- characterized, molecular mechanisms that regulate the complex progression of healing are poorly understood. Therefore, a need persists for the identification of novel pathways that regulate fracture healing, and for development of therapeutics targeting these pathways to enhance regeneration. Notch signaling regulates bone development, and many aspects of bone development are recapitulated during repair. Notch signaling is also required for repair of other tissues, and enhancing Notch signaling promotes regeneration. Therefore, the objective of this thesis was to determine the role of Notch signaling during bone fracture healing, and to create a translatable therapy targeting the pathway to enhance bone tissue formation. We hypothesized that (i) Notch signaling components are active during bone repair; (ii) inhibition of Notch signaling alters healing; (iii) expression of the Jagged1 ligand in mesenchymal cells regulates bone formation; and (iv) therapeutic delivery of Jagged1 will activate the Notch signaling pathway and promote osteogenesis. We first characterized activation of Notch signaling during tibial fracture and calvarial defect healing, and demonstrated that Notch signaling components are active during both methods of repair with Jagged1 the most highly upregulated ligand. Then we determined the importance of Notch signaling by using a temporally controlled inducible model (Mx1- Cre;dnMAMLf/-) to impair canonical signaling in all cells during tibial fracture and calvarial defect healing, and demonstrated that Notch inhibition alters the temporal progression of events required for healing, including inflammation, cartilage formation, callus vascularization and bone remodeling. Next we deleted Jagged1 in mesenchymal progenitors (Prx1-Cre;Jagged1f/f) or committed osteoblasts (Col2.3-Cre;Jagged1f/f), and determined that Jagged1 promotes bone formation during development. Finally, we developed a biomaterial construct comprised of Jagged1 and a poly(β-amino ester) scaffold, and demonstrated that it activates Notch signaling and enhances osteoblast differentiation. This thesis identified Notch signaling as an important regulator of fracture healing, developed a translatable therapeutic targeting the pathway to improve bone tissue formation. The study design outlined can also serve as a model for the discovery of novel pathways that regulate, and therefore could enhance, bone fracture healing

Skeletal Biomechanics and Response to Mechanical Load: A Comparative Approach in the Mouse and Chukar Partridge

Dynamic mechanical loading plays an important role in regulating bone geometry and strength. A healthy skeleton adapts to the bone tissue strain profile and magnitude of loads it experiences on a daily basis in order to maintain reasonable safety factors. In skeletal diseases, such as osteoporosis, a bone’s ability to adapt and maintain structural integrity in response to increased mechanical strains is apparently impaired, which allows skeletal resorption to progress unabated and could eventually lead to mechanical failure. In order to develop better treatments for bone wasting diseases, it is important to understand the mechanobiology of how the healthy skeleton responds to mechanical load. The non-invasive, axial compressive murine tibial loading model has been used extensively to study skeletal adaptation, but sole use of rodent models propagates a large gap in understanding skeletal sensitivity and response to load across terrestrial vertebrate groups. The avian skeleton exhibits several features that make it unique compared to the mammalian rodent skeleton, and these differences could affect how the avian skeleton responds to mechanical load relative to the rodent skeleton. To begin expanding our understanding of skeletal sensitivity across vertebrate species, we developed a novel non-invasive avian tibiotarsal (TBT) loading model using the chukar partridge to complement the use of the murine tibial loading model. For both the mouse and the bird, relatively similar increases in strain stimuli via experimentally applied loads were determined through a combination of in vivo strain gauging and finite element models. The cross-sectional strain distributions during locomotion and experimental loading were further characterized in the bird TBT after validating the use of planar strain theory for cortical bone loaded in bending. In response to several weeks of experimentally applied loading, the mouse tibia adapted its geometry and mass. In contrast, the birds adapted their cross-sectional geometry without complementary increases in bone mass while suppressing normal endocortical bone growth. Lastly, in order to study cortical bone’s response to mechanical load without the potentially confounding effects of varied systemic factors across species, we developed a novel isolated cortical bone culture model that can be mechanically loaded in vitro. We validated that osteocytes in a murine tibial bone segment maintained adequate survival over a five day culture period, and comprehensively characterized the load induced strain profile. Overall, this work takes novel steps to develop and validate comparative in vivo and in vitro models for comparatively assessing skeletal sensitivity across terrestrial vertebrate species. Continued work in this direction will enhance our understanding of how a healthy skeleton is regulated to maintain adequate bone strength