Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes mellitus (T2DM) is a growing problem globally and is associated with increased fracture risk and delayed bone healing. Novel approaches are needed in the treatment of T2DM and the resulting diabetic osteopathy. Recent studies highlight the role of bone as an endocrine organ producing factors that communicate with distant tissues to modulate systemic glucose metabolism. Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is a potent regulator of whole-body energy metabolism, inflammation, bone remodeling and fracture healing. Genetic ablation of CaMKK2 protects from diet-induced obesity, insulin resistance and inflammation, while enhancing pancreatic β cell survival and insulin secretion. Deletion or inhibition of CaMKK2 promotes bone accrual by stimulating osteoblast-mediated bone formation and suppressing osteoclast-mediated bone resorption; however, its specific role in osteocytes, the master regulator of bone remodeling remains unknown. Here we demonstrate that conditional deletion of CaMKK2 from osteocytes enhances bone mass in 3-month-old female, but not male mice, due to suppression of osteoclasts. Conditioned media experiments and proteomics analysis revealed that female osteocytes lacking CaMKK2 suppressed osteoclast formation and function through enhanced secretion of calpastatin, a potent inhibitor of calpains, which are calciumdependent cysteine proteases that support osteoclasts. Further, to determine if CaMKK2- deficient osteocytes regulate whole-body glucose homeostasis, we placed these mice on a high-fat diet (HFD) for a period of 16 weeks. Although the diet did not significantly impact bone mass or strength, we found that conditional deletion of CaMKK2 in osteocytes enhanced bone microarchitecture in 6-month-old male and female mice. We also observed that conditional deletion of CaMKK2 from osteocytes protected male and female mice from HFD-induced obesity and insulin insensitivity. Taken together, these findings highlight CaMKK2 as a potent regulator of osteocyte-mediated modulation of bone remodeling and whole-body energy metabolism. / 2024-08-02

Osteocytes have been proposed as the mechanosensory cells during the process of bone adaption. In this thesis, a microfluidics chamber system (MCS) device was designed, fabricated and tested as a means to maximally simulate the in vivo osteocytic ultrastructure and reproduce the in vivo shear stress experienced by osteocyte, providing an ideal platform for in vitro study on osteocyte mechanotransduction. By employing a micropipette aspiration technique, single osteocyte adhesion and osteocytic process formation were achieved on PDMS with MCS structure. In this study, the involvement of sphingosine-1-phosphate (S1P) signaling pathway in osteocytes responding to oscillatory fluid flow (OFF) was also examined. Firstly, MLO-Y4 osteocytes like cells were demonstrated to express integrated and functional S1P cascade. By modulating S1P cascade components and testing a series of cellular outcomes, it was indicated that exogenous S1P, endogenous S1P and S1P receptor S1P2 were involved in the regulation of loading induced osteocytic responses.

Osteocytes have been proposed as the mechanosensory cells during the process of bone adaption. In this thesis, a microfluidics chamber system (MCS) device was designed, fabricated and tested as a means to maximally simulate the in vivo osteocytic ultrastructure and reproduce the in vivo shear stress experienced by osteocyte, providing an ideal platform for in vitro study on osteocyte mechanotransduction. By employing a micropipette aspiration technique, single osteocyte adhesion and osteocytic process formation were achieved on PDMS with MCS structure. In this study, the involvement of sphingosine-1-phosphate (S1P) signaling pathway in osteocytes responding to oscillatory fluid flow (OFF) was also examined. Firstly, MLO-Y4 osteocytes like cells were demonstrated to express integrated and functional S1P cascade. By modulating S1P cascade components and testing a series of cellular outcomes, it was indicated that exogenous S1P, endogenous S1P and S1P receptor S1P2 were involved in the regulation of loading induced osteocytic responses.

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Of the three major cell types in bone, osteocytes are considered the major mechanosensors, capable of detecting whole-bone mechanical forces at a cellular level and coordinating tissue-level bone formation and resorption responses. The pathology of age-induced bone loss, a major factor in the development of osteoporosis, is attributed to impaired osteocyte mechanosensing. However, real-time evidence of immediate osteocyte responses to mechanical load to support the blunted tissue-level responses that have been demonstrated thus far is lacking. A ubiquitous cellular response upstream of many functions in all cell types, intracellular calcium (Ca2+) is an early mechanosensitive signal in osteocytes, wherein the response characteristics studied in systems of multiple scales are related to mechanical stimuli. Thus, this phenomenon can be characterized as a real-time measure of osteocyte mechanosensitivity. The objective of this thesis was to utilize an ex vivo model of osteocyte Ca2+ signaling to investigate potentially altered mechanosensitivity of the osteocyte network in two clinical contexts: aging, and a recently-approved therapy for treatment of osteoporosis. Additionally, we aimed to enhance this ex vivo model to identify a functional consequence of this robust Ca2+ signaling response to mechanical load in the context of osteocyte mechanotransduction. We first sought to characterize and compare Ca2+ signaling responses to mechanical load in osteocytes from aged and young-adult mice using an ex vivo model to visualize cell networks in viable mouse tibiae. We found that fewer osteocytes responded to whole-bone cyclic mechanical loading in aged mice tibiae compared to those from young-adult mice and did so in a delayed manner, suggesting a diminished mechanosensitivity to load. Osteocytes from aged mice also lacked the well-correlated relationship between Ca2+ signaling synchrony and cell-cell distance exhibited by young-adult osteocyte networks. Taken together, we have demonstrated, for the first time, a real-time measure of the dampened mechanosensing and lack of signal coordination in aged osteocyte networks in situ, which may contribute to blunted long-term bone formation responses to load. Next, we utilized the ex vivo Ca2+ signaling model to investigate the effect of bone formation in response to treatment with sclerostin antibody (Scl-Ab) on osteocyte mechanosensing. Previous studies have identified two phases of bone formation response to Scl-Ab treatment: an initial period of rapid bone formation with short-term dosing and a return to a steady phase of bone formation response with long-term dosing. Thus, we treated mice according to three groups: vehicle, short-term Scl-Ab, and long-term Scl-Ab. Serum P1NP assays and biweekly micro-CT scans throughout the treatment period confirmed the two phases of bone formation response to Scl-Ab. At the conclusion of treatment, under ex vivo whole-bone loading matched at 10 N, there were no significant differences in osteocyte Ca2+ signaling parameters between treatment groups. However, under strain-matched loading, fewer osteocytes from the short-term group exhibited Ca2+ responses and the initiation of Ca2+ signaling was delayed. We interpreted this as reduced mechanosensing in osteocytes that have been newly-embedded in bone that has been rapidly formed in response to Scl-Ab, as confirmed by alizarin red intensity analysis in the osteocyte field of view ex vivo. This study provides real-time evidence of the cellular responses under the distinct phases of bone formation response to Scl-Ab and demonstrates that osteocyte mechanosensing is maintained with long-term treatment, suggesting that other mechanisms may be responsible for self-regulation of bone formation. Given the robust Ca2+ responses to load characterized in osteocytes by our group and others, we concluded this work by investigating a consequence of this mechanism that may contribute to osteocyte mechanotransduction. A common Ca2+-dependent mechanism that has been demonstrated in osteocytes in vitro with possible implications for cell-cell communication is contraction of the actin cytoskeleton. Therefore, we sought to confirm this mechanism in osteocytes maintained in their native 3D network and morphology using the ex vivo murine tibia model. We successfully enhanced the model to simultaneously image intracellular Ca2+ and the F-actin network of individual osteocytes in situ at high magnification using transgenic Lifeact mice paired with either Ca2+ dye or bred with Ca2+ indicator mice. In both models, using biochemical stimuli, we quantified actin network dynamics over time and identified Ca2+-dependent contractile events. Under mechanical loading, phasic actin network contractions corresponded to individual Ca2+ peaks in single osteocytes. The mechanosensitive nature of these contractions was demonstrated by comparing cellular dynamics in single cells under two paired mechanical loading levels; interestingly, mechanosensitivity was dependent on the order of application of these load magnitudes. In identifying this novel mechanosensitive Ca2+-dependent mechanism, we enhance the understanding of the mechanotransduction pathway in osteocytes and have provided a potential point of intervention in cases where osteocyte mechanotransduction is inhibited, such as in osteoporosis. Taken together, this body of work contributes to knowledge of how osteocytes are sensing mechanical forces in different contexts and transducing signals to effector cells. We provide novel, real-time, immediate measures of osteocyte mechanosensing in situ that may correspond to whole-bone responses, such as age-induced bone loss or the differential responses to Scl-Ab treatment. Future work will focus on ways to recover diminished osteocyte mechanosensing and further connect the cell responses we observe herein to long-term bone formation responses in clinical applications.

Muscle and bone are two tightly connected systems on both an anatomic and functional level. Bone and muscle diseases like osteoporosis and sarcopenia have been found to show an association with each other. These two organs form a complex musculoskeletal system and have been found to secrete hormone-like factors called myokines and osteokines that can influence and affect each other. Indeed, the crosstalk between bone and muscle plays an important role during development and aging. For example, myostatin, also known as growth differentiation factor 8 (GDF-8), a cytokine secreted by muscle cells, is a negative regulator of muscle and bone mass. Over expression or loss of function mutations of myostatin in mice have led to muscle atrophy and hypertrophy respectively. Interleukin-6 (IL-6) is expressed abundantly in muscle and is released during exercise and muscle contraction. It has been shown to increase osteoclast (bone cells that break down bone) formation. In the bone, osteocytes make up the majority of all cells and are terminally differentiated osteoblasts. Osteocytes control the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Osteocytes are also known to express receptors for various hormones, including parathyroid hormone (PTH) receptor. As osteocytes comprise more than 90% of all bone cells in adult bone, we hypothesize that osteocytes might secrete factors capable of controlling muscle cells and that PTH might control the expression of these factors. To test this hypothesis, we used an osteocytic cell line Ocy454-12H as well as C2C12 cells, which are a well-accepted model of myocyte differentiation. To investigate the effects of osteocyte-derived factors on myocytes, C2C12 cells were treated with conditioned medium (CM) from osteocytes during specific times. We found that during C2C12 proliferation, when compared to the αMEM control, mRNA expression of MSS51 was decreased for both cells that were treated with CM of osteocytes treated with PTH (PTH CM, p=0.00570) and cells that were treated with CM of osteocytes treated with vehicle only (CM control, p=0.0178). During C2C12 differentiation, mRNA expression of myostatin was significantly (p=0.0387) decreased in cells that were treated with PTH CM compared to cells that were treated with CM control. Considering the importance of mitochondrial respiration in cells, we next analyzed oxygen consumption and metabolism in C2C12 myocytes treated with CM from Ocy454-12H using a Seahorse XF Cell Mito Stress Test. Metabolic analysis revealed that during proliferation, PTH CM led to higher basal respiration, ATP production, and coupling efficiency in C2C12 cells while lowering spare respiratory capacity. In differentiation, there was a trend in which CM control would cause a decrease across all parameters compared to the control group and the PTH CM group. Interestingly, PTH CM-treated C2C12 cells were shown to have a higher oxygen consumption rate (OCR) than the CM-control treated group and would have similar values to that of the control group (C2C12 not treated with CM). Taken together these results suggest that osteocytes might control muscle cells differentiation and metabolism via a PTH-mediated signaling pathway.

INTRODUCTION: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone modeling and remodeling through direct contact with adjacent cells and via secreted factors that can reach cells in the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCRs) and mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes have abnormal myelopoiesis, skeletal abnormalities and reduced adipose tissue. This study aimed at evaluating the effects of osteocyte-secreted factors on myeloid cell proliferation and differentiation in vitro. To investigate cross-talk between osteocytes and the BMM, we established osteocytic cell lines lacking Gsα expression to study the molecular mechanisms by which osteocytes control myeloid cell proliferation and differentiation. METHODS: CRISPR/Cas9 was used to knockout Gsα in the osteocytic cell line Ocy454. Conditioned media (CM) from differentiated Ocy-GsαCtrl and Ocy-GsαKO cells were used to treat myeloid cells and bone marrow mononuclear cells (BMNCs) isolated from long bones of 6-8-week-old C57/BL6 mice. BMNCs were cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa β Ligand (RANKL) to induce osteoclast differentiation. Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring formation and mRNA expression were used to evaluate cell proliferation, differentiation and function of the induced osteoclasts. Proteomics analysis of CM was performed to identify osteocyte-secreted factors capable of controlling myelopoiesis and osteoclastogenesis. RESULTS: Myeloid cells treated with CM from Ocy-GsαKO showed a significant increase in cell proliferation compared to Ocy-GsαCtrl CM and non-treated control. BMNCs treated with CM from Ocy-GsαCtrl and Ocy-GsαKO showed a significant increase in cell proliferation as compared to non-treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-GsαCtrl and further suppressed by CM from Ocy-GsαKO compared to non-treated control. Osteoclasts exposed to CM from Ocy-GsαKO showed a significant defect in activity and function as compared to cells exposed to CM from Ocy-GsαCtrl and non-treated cells. Osteoclast apoptosis was significantly enhanced by Ocy-GsαCtrl and Ocy-GsαKO CM compared to non-treated control. Among osteocyte secreted factors, we identified neuropilin-1 (NRP-1) as a Gsα-dependent osteocytic factor capable of suppressing osteoclastogenesis. CM from Ocy-GsαKO in which M-CSF was reduced by shRNA demonstrated decrease in BMNCs proliferation, demonstrating that osteocytes are also important sources of this cytokine. CONCLUSIONS: Osteocytes produce several Gsα-dependent and -independent secreted factors capable of supporting myelopoiesis, promoting macrophage proliferation and suppressing osteoclast formation. We identified osteocyte-derived NRP-1 as a novel factor capable of decreasing osteoclastogenesis. In addition, we found that M-CSF secreted by osteocytes is responsible in part for BMNC proliferation. Future studies should focus on determining the role of osteocyte-mediated NRP-1 and other secreted factor(s) in control of myelopoiesis and osteoclastogenesis. / 2020-06-26T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts.

Osteocytes are widely regarded as mechanosensors, capable of detecting changes in the mechanical environment of the bone tissue and modifying cellular responses accordingly. Indeed, an intact osteocyte network is required for bone changes in response to unloading, and studies have shown that loading/unloading influences osteocyte expression of proteins that modulate bone turnover, such as sclerostin and receptor activator of nuclear factor kappa B ligand (RANKL). However, mechanisms underlying osteocyte mechanotransduction remain unclear. For instance, one of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular, or cytosolic, calcium (Ca2+cyt), but the mechanisms by which osteocytes generate or utilize Ca2+ signals to direct bone adaptation are largely unknown. In this thesis, I explored the mechanisms underlying the sustainment of Ca2+cyt oscillations in osteocytes as well as downstream consequences of these patterns. I discovered that Ca2+cyt oscillations are generated in osteocytes by Ca2+ release from the endoplasmic reticulum and that the predominant expression of T-Type voltage sensitive Ca2+ channels in these cells facilitates this behavior. I also explored the role of the actin cytoskeleton – another prominent feature in osteocytes – and found that actin dynamics are important for the generation of Ca2+cyt signals. Furthermore, I confirmed that Ca2+cyt transients subsequently activate actomyosin contractions in osteocytes by monitoring interactions of osteocytes exposed to Ca2+ agonists on micropillar substrates. With this information, I sought to relate Ca2+cyt signaling and actomyosin contractility in osteocytes to their roles as coordinators of bone adaptation. Ca2+-dependent contractions have been shown to facilitate the release of extracellular vesicles, small membrane-enclosed packages of proteins that cells use for communication, in other cell types. I found that mechanical stimulation increased the production and release of extracellular vesicles in osteocytes, and this was dependent on Ca2+ signaling. These extracellular vesicles contained key bone regulatory proteins and were small enough to plausibly transport through the lacunocanalicular system. Thus, I uncovered a novel mechanotransduction pathway by which osteocytes may coordinate tissue-level adaptation. As an extension of this work, I also characterized these behaviors in new osteocyte cell lines which may better reflect native cell physiology. The work in this thesis anchors Ca2+ signaling as a critical osteocyte response to mechanical loading and adds to the body of work exploring how and why these signals are generated. The results of these studies add new information to the still limited knowledge of this important bone cell and extend Ca2+ signaling research by connecting early mechanosensation events to subsequent protein responses to mechanical loading. Understanding the mechanisms behind the robust Ca2+cyt oscillations in osteocytes and how they relate to their roles as coordinators of bone adaptation may improve our ability to prevent or treat bone degeneration in diseases like osteoporosis where mechanosensitivity is impaired.