Irisin Mediates Effects on Bone and Fat via αV Integrin Receptors.

Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/ß5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.

The Osteocyte: New Insights.

Osteocytes are an ancient cell, appearing in fossilized skeletal remains of early fish and dinosaurs. Despite its relative high abundance, even in the context of nonskeletal cells, the osteocyte is perhaps among the least studied cells in all of vertebrate biology. Osteocytes are cells embedded in bone, able to modify their surrounding extracellular matrix via specialized molecular remodeling mechanisms that are independent of the bone forming osteoblasts and bone-resorbing osteoclasts. Osteocytes communicate with osteoclasts and osteoblasts via distinct signaling molecules that include the RankL/OPG axis and the Sost/Dkk1/Wnt axis, among others. Osteocytes also extend their influence beyond the local bone environment by functioning as an endocrine cell that controls phosphate reabsorption in the kidney, insulin secretion in the pancreas, and skeletal muscle function. These cells are also finely tuned sensors of mechanical stimulation to coordinate with effector cells to adjust bone mass, size, and shape to conform to mechanical demands.

Metabolic Health and Disease: A Role of Osteokines?

Maintenance of skeletal health is tightly regulated by osteocytes, osteoblasts, and osteoclasts via coordinated secretion of bone-derived factors, termed osteokines. Disruption of this coordinated process due to aging and metabolic disease promotes loss of bone mass and increased risk of fracture. Indeed, growing evidence demonstrates that metabolic diseases, including type 2 diabetes, liver disease and cancer are accompanied by bone loss and altered osteokine levels. With the persistent prevalence of cancer and the growing epidemic of metabolic disorders, investigations into the role of inter-tissue communication during disease progression are on the rise. While osteokines are imperative for bone homeostasis, work from us and others have identified that osteokines possess endocrine functions, exerting effects on distant tissues including skeletal muscle and liver. In this review we first discuss the prevalence of bone loss and osteokine alterations in patients with type 2 diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, and cancer. We then discuss the effects of osteokines in mediating skeletal muscle and liver homeostasis, including RANKL, sclerostin, osteocalcin, FGF23, PGE2, TGF-ß, BMPs, IGF-1 and PTHrP. To better understand how inter-tissue communication contributes to disease progression, it is essential that we include the bone secretome and the systemic roles of osteokines.

Role of the Osteocyte in Musculoskeletal Disease.

PURPOSE OF THE REVIEW: The purpose of this review is to summarize the role of the osteocyte in muscle atrophy in cancer patients, sarcopenia, spinal cord injury, Duchenne's muscular dystrophy, and other conditions associated with muscle deterioration. RECENT FINDINGS: One type of bone cell, the osteocyte, appears to play a major role in muscle and bone crosstalk, whether physiological or pathological. Osteocytes are cells living within the bone-mineralized matrix. These cells are connected to each other by means of dendrites to create an intricately connected network. The osteocyte network has been shown to respond to different types of stimuli such as mechanical unloading, immobilization, aging, and cancer by producing osteocytes-derived factors. It is now becoming clear that some of these factors including sclerostin, RANKL, TGF-ß, and TNF-α have detrimental effects on skeletal muscle. Bone and muscle not only communicate mechanically but also biochemically. Osteocyte-derived factors appear to contribute to the pathogenesis of muscle disease and could be used as a cellular target for new therapeutic approaches.

Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

Podoplanin is dispensable for mineralized tissue formation and maintenance in the Swiss outbred mouse background.

Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

Sclerostin Directly Stimulates Osteocyte Synthesis of Fibroblast Growth Factor-23.

Osteocyte produced fibroblast growth factor 23 (FGF23) is the key regulator of serum phosphate (Pi) homeostasis. The interplay between parathyroid hormone (PTH), FGF23 and other proteins that regulate FGF23 production and serum Pi levels is complex and incompletely characterised. Evidence suggests that the protein product of the SOST gene, sclerostin (SCL), also a PTH target and also produced by osteocytes, plays a role in FGF23 expression, however the mechanism for this effect is unclear. Part of the problem of understanding the interplay of these mediators is the complex multi-organ system that achieves Pi homeostasis in vivo. In the current study, we sought to address this using a cell line model of the osteocyte, IDG-SW3, known to express FGF23 at both the mRNA and protein levels. In cultures of differentiated IDG-SW3 cells, both PTH1-34 and recombinant human (rh) SCL remarkably induced Fgf23 mRNA expression dose-dependently within 3 h. Both rhPTH1-34 and rhSCL also strongly induced C-terminal FGF23 protein secretion. Secreted intact FGF23 levels remained unchanged, consistent with constitutive post-translational cleavage of FGF23 in this cell model. Both rhPTH1-34 and rhSCL treatments significantly suppressed mRNA levels of Phex, Dmp1 and Enpp1 mRNA, encoding putative negative regulators of FGF23 levels, and induced Galnt3 mRNA expression, encoding N-acetylgalactosaminyl-transferase 3 (GalNAc-T3), which protects FGF23 from furin-like proprotein convertase-mediated cleavage. The effect of both rhPTH1-34 and rhSCL was antagonised by pre-treatment with the NF-κß signalling inhibitors, BAY11 and TPCK. RhSCL also stimulated FGF23 mRNA expression in ex vivo cultures of human bone. These findings provide evidence for the direct regulation of FGF23 expression by sclerostin. Locally expressed sclerostin via the induction of FGF23 in osteocytes thus has the potential to contribute to the regulation of Pi homeostasis.

Osteocytes and Cancer.

PURPOSE OF REVIEW: While the function of osteocytes under physiologic conditions is well defined, their role and involvement in cancer disease remains relatively unexplored, especially in a context of non-bone metastatic cancer. This review will focus on describing the more advanced knowledge regarding the interactions between osteocytes and cancer. RECENT FINDINGS: We will discuss the involvement of osteocytes in the onset and progression of osteosarcoma, with the common bone cancers, as well as the interaction that is established between osteocytes and multiple myeloma. Mechanisms responsible for cancer dissemination to bone, as frequently occur with advanced breast and prostate cancers, will be reviewed. While a role for osteocytes in the stimulation and proliferation of cancer cells has been reported, protective effects of osteocytes against bone colonization have been described as well, thus increasing ambiguity regarding the role of osteocytes in cancer progression and dissemination. Lastly, supporting the idea that skeletal defects can occur also in the absence of direct cancer dissemination or osteolytic lesions directly adjacent to the bone, our recent findings will be presented showing that in the absence of bone metastases, the bone microenvironment and, particularly, osteocytes, can manifest a clear and dramatic response to the distant, non-metastatic tumor. Our observations support new studies to clarify whether treatments designed to preserve the osteocytes can be combined with traditional anticancer therapies, even when bone is not directly affected by tumor growth.

The role of sphingosine-1-phosphate signaling pathway in cementocyte mechanotransduction.

Iatrogenic external root resorption can become a serious pathological condition with clinical tooth movement. Little is known regarding how cementum responds to mechanical loading in contrast to bone, especially under compressive stress. In the field of bone biology, several studies have established the contribution of sphingosine-1-phosphate (S1P) signaling in bone remodeling, mechanical transduction and homeostasis. As osteocytes and cementocytes share similar morphological and functional characteristics, this study aimed to investigate the mechanotransduction ability of cementocytes and to explore the contribution of S1P signaling under compressive stress induced mechanotransduction. We found that compressive stress inhibited major S1P signaling and promoted the expression of anabolic factors in IDG-CM6 cells, a novel immortalized murine cementocyte cell line. By inhibiting S1P signaling, we verified that S1P signaling played a vital role in regulating the expression of the mechanotransduction factors prostaglandin E2 (PGE2) and ß-catenin, as well as factors responsible for cementogenesis and cementoclastogenesis in IDG-CM6 cells. These results support the hypothesis that cementocytes act as key mechanically responsive cells in cementum, responding to compressive stress and directing local cementum metabolism.

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models.

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.

Multi-Staged Regulation of Lipid Signaling Mediators during Myogenesis by COX-1/2 Pathways.

Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis. Knocking down COXs, particularly COX-1, significantly reduced the release of PGs from muscle cells, especially PGE2 and PGF2α, as well as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA). Moreover, COXs could interplay with LOXs to regulate the signaling of hydroxyeicosatetraenoic acids (HETEs). The changes in LMs are associated with the expression of genes, such as Itrp1 (calcium signaling) and Myh7 (myogenic differentiation), in skeletal muscle. In conclusion, both COX-1 and -2 contribute to LMs production during myogenesis in vitro, and COXs could interact with LOXs during this process. These interactions and the fine-tuning of the levels of these LMs are most likely important for skeletal muscle myogenesis, and potentially, muscle repair and regeneration.

Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.

Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication.

Physiological and pathological osteocytic osteolysis.

Osteocytes, the most abundant bone cell in the adult skeleton, can function as mechanosensors directing osteoblast and osteoclast function in order to maintain optimal load bearing bone in addition to functioning as endocrine cells regulating phosphate metabolism. A controversial function, previously overlooked or denied, has been osteocytes as regulators of calcium metabolism. Early histologists upon observing enlarged osteocyte lacunae in bone sections proposed that mature osteocytes could remove their perilacunar matrix, a term called "osteocytic osteolysis". New insights into this process have occurred during the last decade using novel technology thereby providing a means to identify molecular mechanisms responsible for osteocytic osteolysis. As release of calcium from a mineralized matrix requires a more acidic pH and specialized enzymes, it was proposed that osteocytes may utilize similar molecular mechanisms as osteoclasts to remove mineral. The idea that a cell descended from mesenchymal progenitors (the osteocyte) could function similarly to a cell descended from hematopoietic progenitors (the osteoclast) was challenged as being improbable. Here we review the molecular mechanisms behind this osteocyte function, the role of osteocytic osteolysis in health and disease, and the capacity of the osteocyte to reverse the osteolytic process by replacing the removed matrix, a revived osteoblast function.

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers.

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of ß-aminoisobutyric acid (ß-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-ß-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 µM), an intermediate level of l-BAIBA (0.8 µM), and low but detectable levels (<0.2 µM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.

Myelopoiesis is regulated by osteocytes through Gsα-dependent signaling.

Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSF­neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.

Muscle-Bone Crosstalk in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a fatal neuromuscular disorder characterized by degeneration of motor neurons and by skeletal muscle atrophy. Although the death of motor neurons is a pathological hallmark of ALS, the potential role of other organs in disease progression remains to be elucidated. Skeletal muscle and bone are the two largest organs in the human body. They are responsible not only for locomotion but also for maintaining whole body normal metabolism and homeostasis. Patients with ALS display severe muscle atrophy, which may reflect intrinsic defects in mitochondrial respiratory function and calcium (Ca) signaling in muscle fibers, in addition to the role of axonal withdrawal associated with ALS progression. Incidence of fractures is high in ALS patients, indicating there are potential bone defects in individuals with this condition. There is a lifelong interaction between skeletal muscle and bone. The severe muscle degeneration that occurs during ALS progression may potentially have a significant impact on bone function, and the defective bone may also contribute significantly to neuromuscular degeneration in the course of the disease. Due to the nature of the rapid and severe neuromuscular symptoms, a majority of studies on ALS have focused on neurodegeneration. Just a few studies have explored the possible contribution of muscle defects, even fewer on bone defects, and fewer still on possible muscle-bone crosstalk in ALS. This review article discusses current studies on bone defects and potential defects in muscle-bone crosstalk in ALS.

Mechanical stress-activated integrin α5ß1 induces opening of connexin 43 hemichannels.

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5ß1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5ß1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5ß1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.