Prostaglandin E2 (PGE2), a prostanoid synthesized from arachidonic acid via the cyclooxygenase pathway, is a modulator of physiological responses including inflammation, fever, and muscle regeneration. Several patents have been filed that are related to PGE2, one of them being directly related to skeletal muscles. In this report, we first summarize the key patents describing inventions for the utilization of PGE2 for either diagnostic or therapeutic purposes, including skeletal muscle. In the second part of our work we present new and exciting data that demonstrates that PGE2 accelerates skeletal muscle myogenic differentiation. Our discovery resulted from our recent and novel concept of bone-muscle crosstalk. Bone and muscle are anatomically intimate endocrine organs and we aimed to determine whether this anatomical intimacy also translates into a biochemical communication from bone cells to muscle cells at the in vitro level. The effects of MLOY4 osteocyte-like cell conditioned medium (CM) and three osteocyte-secreted factors, PGE2, sclerostin and monocyte chemotactic protein (MCP-3), on C2C12 myogenic differentiation were evaluated using morphological analyses, a customized 96-gene PCR array, and measurements of intracellular calcium levels. MLO-Y4 CM and PGE2, but not sclerostin and MCP-3, induced acceleration of myogenesis of C2C12 myoblasts that was linked with significant modifications in intracellular calcium homeostasis. This finding should further stimulate the pursuit of new patents to explore the use of PGE2 and the new concept of bone-muscle crosstalk for the development and application of inventions designed to treat muscle diseases characterized by enhanced muscle wasting, such as sarcopenia.

Endometrial cancers (ECs) are the most common form of gynecologic malignancy. Recent studies have reported that ECs reveal distinct markers for molecular pathogenesis, which in turn is linked to the various histological types of ECs. To understand further the molecular events contributing to ECs and endometrial tumorigenesis in general, a more precise identification of cancer-associated molecules and signaling networks would be useful for the detection and monitoring of malignancy, improving clinical cancer therapy, and personalization of treatments.

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO‐Y4 osteocyte‐like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ∼25%. MLO‐Y4 and primary osteocyte CM (1% to 10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Because WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO‐Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5 ng/mL mirrored the effects of both primary osteocyte and MLO‐Y4 CM by inducing nuclear translocation of β‐catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO‐Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100 ng/mL) inhibited both the effects of MLO‐Y4 CM and WNT3a on C2C12 cell differentiation. RT‐PCR array results supported the activation of the Wnt/β‐catenin pathway by MLO‐Y4 CM and WNT3a. These results were confirmed by qPCR, showing upregulation of myogenic markers and two Wnt/β‐catenin downstream genes, Numb and Flh1. We postulated that MLO‐Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO‐Y4 CM and WNT3a increased caffeine‐induced Ca²⁺ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca²⁺ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca²⁺ signaling and the Wnt/ β‐Catenin pathway. © 2017 American Society for Bone and Mineral Research

Sarcopenia and osteoporosis are important public health problems that occur concurrently. A bivariate genome‐wide association study (GWAS) identified METTL21c as a suggestive pleiotropic gene for both bone and muscle. The METTL21 family of proteins methylates chaperones involved in the etiology of both myopathy and inclusion body myositis with Paget's disease. To validate these GWAS results, Mettl21c mRNA expression was reduced with siRNA in a mouse myogenic C2C12 cell line and the mouse osteocyte‐like cell line MLO‐Y4. At day 3, as C2C12 myoblasts start to differentiate into myotubes, a significant reduction in the number of myocytes aligning/organizing for fusion was observed in the siRNA‐treated cells. At day 5, both fewer and smaller myotubes were observed in the siRNA‐treated cells as confirmed by histomorphometric analyses and immunostaining with myosin heavy chain (MHC) antibody, which only stains myocytes/myotubes but not myoblasts. Intracellular calcium (Ca²⁺) measurements of the siRNA‐treated myotubes showed a decrease in maximal amplitude peak response to caffeine, suggesting that less Ca²⁺ is available for release due to the partial silencing of Mettl21c, correlating with impaired myogenesis. In siRNA‐treated MLO‐Y4 cells, 48 hours after treatment with dexamethasone there was a significant increase in cell death, suggesting a role of Mettl21c in osteocyte survival. To investigate the molecular signaling machinery induced by the partial silencing of Mettl21c, we used a real‐time PCR gene array to monitor the activity of 10 signaling pathways. We discovered that Mettl21c knockdown modulated only the NF‐κB signaling pathway (ie, Birc3, Ccl5, and Tnf). These results suggest that Mettl21c might exert its bone‐muscle pleiotropic function via the regulation of the NF‐κB signaling pathway, which is critical for bone and muscle homeostasis. These studies also provide rationale for cellular and molecular validation of GWAS, and warrant additional in vitro and in vivo studies to advance our understanding of role of METTL21C in musculoskeletal biology. © 2014 American Society for Bone and Mineral Research