Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone

Osteocytes, >90% of the cells in bone, lie embedded within the mineralized matrix and coordinate osteoclast and osteoblast activity on bone surfaces by mechanisms still unclear. Bone anabolic stimuli activate Wnt signaling, and human mutations of components along this pathway underscore its crucial role in bone accrual and maintenance. However, the cell responsible for orchestrating Wnt anabolic actions has remained elusive. We show herein that activation of canonical Wnt signaling exclusively in osteocytes [dominant active (da)βcat(Ot) mice] induces bone anabolism and triggers Notch signaling without affecting survival. These features contrast with those of mice expressing the same daß-catenin in osteoblasts, which exhibit decreased resorption and perinatal death from leukemia. daßcat(Ot) mice exhibit increased bone mineral density in the axial and appendicular skeleton, and marked increase in bone volume in cancellous/trabecular and cortical compartments compared with littermate controls. daßcat(Ot) mice display increased resorption and formation markers, high number of osteoclasts and osteoblasts in cancellous and cortical bone, increased bone matrix production, and markedly elevated periosteal bone formation rate. Wnt and Notch signaling target genes, osteoblast and osteocyte markers, and proosteoclastogenic and antiosteoclastogenic cytokines are elevated in bones of daßcat(Ot) mice. Further, the increase in RANKL depends on Sost/sclerostin. Thus, activation of osteocytic β-catenin signaling increases both osteoclasts and osteoblasts, leading to bone gain, and is sufficient to activate the Notch pathway. These findings demonstrate disparate outcomes of β-catenin activation in osteocytes versus osteoblasts and identify osteocytes as central target cells of the anabolic actions of canonical Wnt/β-catenin signaling in bone

PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading

There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects

Mecanismos moleculares implicados en la mecanotransducción osteocítica. Alteraciones en la osteopatía diabética y efecto compensador de la proteína relacionada con la parathormona (PTHrP)

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-07-2016Esta tesis tiene embargado el acceso al texto completo hasta el 22-01-2018El esqueleto es capaz de adaptar su masa, estructura y microarquitectura a la estimulación mecánica recibida. Así, el aumento de actividad física conlleva un incremento de la formación ósea, mientras que la inmovilización aumenta la resorción. Los osteocitos son las principales células mecanorreceptoras del hueso; sin embargo, los mecanismos subyacentes a esta función no están suficientemente esclarecidos. La proteína relacionada con la parathormona (PTHrP) es un importante regulador del remodelado óseo y de la formación ósea. Debido a su acción osteogénica, se ha propuesto como posible agente terapéutico para paliar la pérdida de masa ósea en diferentes tipos de osteoporosis, incluyendo la asociada a la diabetes mellitus (DM). En la presente Tesis Doctoral, hemos explorado, in vitro, nuevos mecanismos involucrados en la mecanotransducción osteocítica como dianas para posibles alternativas terapéuticas en la osteopenia. Además, in vivo, en un modelo de ratón con osteopatía asociada a DM tipo 1 (DM1), hemos estudiado la respuesta osteoinductora de dos péptidos derivados de la PTHrP correspondientes a sus dominios N-terminal [PTHrP (1-37)] y C-terminal [PTHrP (107-111)], administrados solos o combinados con estimulación mecánica, una estrategia que podría ser interesante en esta patología. En primer lugar, hemos caracterizado dos sistemas de estimulación mecánica in vitro en células osteocíticas MLOY4: mediante la exposición a un medio hipotónico o a un flujo de fluido (FF). Hemos identificado el papel del receptor tipo 2 de VEGF (VEGFR2) y del receptor de PTH/PTHrP tipo 1 (PTH1R) como mecanorreceptores, así como los mecanismos moleculares implicados en la activación de ambos tras la estimulación mecánica. Además, hemos evaluado la acción citoprotectora de ambos péptidos, PTHrP (1-36) y PTHrP (107-111), en las células osteocíticas. Posteriormente, hemos estudiado el efecto deletéreo de un ambiente de alta glucosa (AG, 25 mM), simulando in vitro la situación de hiperglucemia, sobre la protección osteocítica inducida por la estimulación mecánica; este efecto fue revertido por ambos péptidos de PTHrP. Nuestros hallazgos también indican que tanto la estimulación mecánica como la AG alteran la comunicación osteocito-osteoclasto, aunque de manera diferente: la estimulación mecánica de los osteocitos impide la formación de osteoclastos, mientras la AG en el entorno osteocítico induce la formación de osteoclastos inactivos. In vivo, en el modelo de ratón con osteopatía asociada a la DM1, demostramos que un tratamiento agudo (3 días) con ambos péptidos de la PTHrP fue eficaz para contrarrestar la pérdida de masa y la fragilidad ósea inducidas por la DM1, a través de sus acciones anabólicas y anti-resortivas. Además, nuestros resultados demuestran que la DM1 interfiere con la capacidad osteoformadora de la estimulación mecánica y que el tratamiento combinado con los péptidos de la PTHrP y la estimulación mecánica ejerce un efecto aditivo sobre la formación ósea perióstica en esta situación. En conclusión, los hallazgos de esta Tesis Doctoral demuestran el papel crucial de los osteocitos en la osteopatía diabética y plantean la utilización de agentes anabólicos como estos péptidos de la PTHrP actuando estas células como un nuevo enfoque terapéutico en esta patología.Mechanical loading is an important regulator of bone mass. The skeleton can adapt to inputs represented by mechanical forces by changing its mass, shape and microarchitecture. Increased physical activity leads to increased bone formation, whereas immobilization increases bone resorption. Osteocytes are the main bone mechanosensitive cells, although the underlying mechanisms of their function are ill-defined. Parathyroid hormone related protein (PTHrP) is an important regulator of bone remodeling and bone formation. Due to its osteogenic actions, it has been proposed as a possible therapeutic agent in bone loss-related conditions such as diabetes mellitus (DM). In this Doctoral Thesis, using in vitro approaches, we explored new mechanisms involved in osteocytic mechanotransduction, which might be possible targets for osteoporosis treatment. Moreover, in vivo in a murine model of type 1 DM (DM1) with osteopathy, we evaluated the relative efficacy of two PTHrP-derived peptides –corresponding to its N-terminal [PTHrP (1- 37)] and C-terminal [PTHrP (107-111)] domains-, alone or in combination with mechanical loading, as potential therapies in DM-related osteopathy. First, we characterized two in vitro mechanical stimulation aproaches in MLO-Y4 osteocytic cells: exposure to either a hypotonic medium or fluid flow (FF). We then identified the role of vascular endothelial growth factor type 2 receptor (VEGFR2) and PTH type 1 receptor (PTH1R) as bone mechanoreceptors, and the underlying molecular mechanisms for their activation after mechanical stimulation. Moreover, we assessed the protective action of PTHrP (1-36) and PTHrP (107-111) in these osteocytic cells. Afterwards, we characterized the deleterious effect of a high glucose (HG; 25 mM) environment - which mimics hyperglucemia in vivo- on cell viability protection conferred by mechanical stimulation of MLO-Y4 osteocytes; an effect that was reversed by each PTHrP peptide. Our studies also show that both mechanical stimulation and HG affect osteocyte-osteoclast communication in a different manner: whereas mechanically stimulated osteocytes impaired osteoclast differentiation, HG promoted formation of inactive osteoclast-like cells. In vivo, in the diabetic mouse model with osteopathy, we demonstrated that a short treatment (3 days) with either PTHrP (1-37) or PTHrP (107-111) similarly restored in part bone loss and strength by targeting both bone formation and bone resorption. Moreover, our results showed that DM negatively affected the bone anabolic effect of mechanical stimulation, and treatment with the PTHrP peptides together with mechanical loading induced an additive effect to enhance periosteal bone formation in this diabetic scenario. In conclusion, the findings in this Thesis demonstrate the crucial role of osteocytes in diabetic osteopathy, and point to osteocyte targeting agents like these PTHrP peptides as a novel therapeutic approach in this pathology

Molecular mechanisms in bone mechanotransduction

Bone is one of the most adaptable tissues in the body as it is continuously subjected to load bearing. In fact, mechanical loading is an important regulator of bone mass. The skeleton adjusts to load by changing its mass, shape and microarchitecture, depending on the magnitude of the strain. Mechanical stimulation is necessary for the development of the skeleton, whereas in adults physiological levels of strain help maintain bone mass by reducing bone resorption. On the other hand, an excessive level of strain or bone disuse induces bone loss. Osteocytes are long-lived cells comprising more than 90% of bone cellularity, which are embedded in the bone matrix forming a functional syncytium extending to the bone surface. These cells are considered to be the main bone cells responsible for translating mechanical strain into regulatory signals for osteoblasts and osteoclasts, leading to adapting bone responses to environmental changes. In this review, we discuss the complexity and well-orchestrated events that occur in bone mechanotransduction, focusing on osteocyte viability as an important biological response in this respect. Elucidation of the molecular mechanisms of bone mechanotransduction and the key role of osteocytes is opening new avenues for the treatment of bone loss-related diseases