Inflammatory responses are crucial for regeneration following peripheral nerve injury (PNI). PNI triggers inflammatory responses at the site of injury. The DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream effector stimulator of interferon genes (STING) sense foreign and self-DNA and trigger type I interferon (IFN) immune responses. We demonstrate here that following PNI, the cGAS/STING pathway is upregulated in the sciatic nerve of naive rats and dysregulated in old rats. In a nerve crush mouse model where STING is knocked out, myelin content in sciatic nerve is increased resulting in accelerated functional axon recovery. STING KO mice have lower macrophage number in sciatic nerve and decreased microglia activation in spinal cord 1 week post injury. STING activation regulated processing of colony stimulating factor 1 receptor (CSF1R) and microglia survival in vitro. Taking together, these data highlight a previously unrecognized role of STING in the regulation of nerve regeneration.

The TGF-β family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc "trap," to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit.

ATP citrate lyase (ACL) plays a key role in regulating mitochondrial function, as well as glucose and lipid metabolism in skeletal muscle. We report here that ACL silencing impairs myoblast and satellite cell (SC) differentiation, and it is accompanied by a decrease in fast myosin heavy chain isoforms and MYOD. Conversely, overexpression of ACL enhances MYOD levels and promotes myogenesis. Myogenesis is dependent on transcriptional but also other mechanisms. We show that ACL regulates the net amount of acetyl groups available, leading to alterations in acetylation of H3(K9/14) and H3(K27) at the MYOD locus, thus increasing MYOD expression. ACL overexpression in murine skeletal muscle leads to improved regeneration after cardiotoxin-mediated damage. Thus, our findings suggest a mechanism for regulating SC differentiation and enhancing regeneration, which might be exploited for devising therapeutic approaches for treating skeletal muscle disease.

Mitochondrial dysfunction is associated with skeletal muscle pathology, including cachexia, sarcopenia, and the muscular dystrophies. ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes mitochondria-derived citrate into oxaloacetate and acetylCoA. Here we report that activation of ACL in skeletal muscle results in improved mitochondrial function. IGF1 induces activation of ACL in an AKT-dependent fashion. This results in an increase in cardiolipin, thus increasing critical mitochondrial complexes and supercomplex activity, and a resultant increase in oxygen consumption and cellular ATP levels. Conversely, knockdown of ACL in myotubes not only reduces mitochondrial complex I, IV, and V activity but also blocks IGF1-induced increases in oxygen consumption. In vivo, ACL activity is associated with increased ATP. Activation of this IGF1/ACL/cardiolipin pathway combines anabolic signaling with induction of mechanisms needed to provide required ATP.

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.

Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/β-catenin signaling. We found the extracellular β-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.

Olivier Leupin 1* , Elke Piters 2* , Christine Halleux 1 , Shouih Hu 1 , Ina Kramer 1 , Frederic Morvan 1 , Tewis Bouwmeester 3,4 , Markus Schirle 5 , Manuel Bueno-Lozano 6 , Feliciano J Ramos Fuentes 6 , Peter H Itin 7 , Eveline Boudin 2 , Fenna de Freitas 2 , Karen Jennes 2 , Barbara Brannetti 8 , Nadine Charara 9 , Hilmar Ebersbach 9 , Sabine Geisse 9 , Chris X. Lu 10 , Andreas Bauer 3,4 , Wim Van Hul 2 and Michaela Kneissel 1

Zfp521, a 30 C2H2 Kruppel-like zinc finger protein, is expressed at high levels at the periphery of early mesenchymal condensations prefiguring skeletal elements and in all developing bones in the perichondrium and periosteum, in osteoblast precursors and osteocytes, and in chondroblast precursors and growth plate prehypertrophic chondrocytes. Zfp521 expression in cultured mesenchymal cells is decreased by BMP-2 and increased by PTHrP, which promote and antagonize osteoblast differentiation, respectively. In vitro, Zfp521 overexpression reduces the expression of several downstream osteoblast marker genes and antagonizes osteoblast differentiation. Zfp521 binds Runx2 and represses its transcriptional activity, and Runx2 dose-dependently rescues Zfp521's inhibition of osteoblast differentiation. In contrast, osteocalcin promoter-targeted overexpression of Zfp521 in osteoblasts in vivo results in increased bone formation and bone mass. We propose that Zfp521 regulates the rate of osteoblast differentiation and bone formation during development and in the mature skeleton, in part by antagonizing Runx2.

The nuclear orphan receptor human estrogen receptor-related receptor (ERR)-alpha is implicated in bone metabolism. We studied the effect of ERRalpha silencing in human mesenchymal stem cells (hMSCs) during osteoblastogenesis. We found that ERRalpha silencing led to an increase of bone sialoprotein and a decrease of osteopontin mRNA levels, suggesting enhanced osteoblastic differentiation. This was confirmed by an increased ability of hMSCs to deposit calcium. Concomitantly, knockdown of ERRalpha inhibited adipogenesis, resulting in a decrease in adipocyte number and adipocyte marker gene expression. In line with a negative role of ERRalpha in bone metabolism, we found that adult female and male ERRalpha-deficient mice displayed a moderate increase in femoral cancellous bone volume and density. Osteoblast surface was increased and marrow fat volume decreased in these animals. Furthermore, ERRalpha-deficient osteoblasts displayed increased differentiation properties in vitro in line with our observations in hMSCs. In summary, we identified a role for ERRalpha in bone mass regulation by affecting osteoblastic differentiation.

Bone loss in the elderly is mainly caused by osteoclast-induced bone resorption thought to be causally linked to the decline in estrogen and testosterone levels in females and males. Recently, involvement of follicle stimulating-hormone (FSH) in this process has been suggested to explain in part the etiology of the disease in females, whereas its role in males has never been examined. In this study, the direct impact of FSH on bone mass of 16-week-old C57BL/6J male mice by either daily intermittent application of 6 or 60 mug/kg of FSH or continuous delivery via miniosmotic pump of a dose of 6 mug/kg over the course of a month was assessed. Femoral peripheral quantitative computed tomographic and microcomputed tomographic analyses at 0, 2, and 4 weeks of FSH-treated mice did not reveal any differences in cancellous and cortical bone compared to sham-treated mice. FSH functionality was verified by demonstrating cAMP induction and activation of a cAMP-response element-containing reporter cell line by FSH. Furthermore, osteoclastogenesis from human mononuclear cell precursors and from RAW 264.7 cells was not affected by FSH (3, 10, 30 ng/mL) compared to control. No direct effect of FSH on gene regulation was observed by Affymetrix Gene Array on RAW 264.7 cells. Lastly, no expression of FSH receptor (FSHR) mRNA or FSHR was observed by quantitative polymerase chain reaction and Western blot in either human male osteoclasts or RAW 264.7 cells. These data show that FSH does not appear to modulate male bone mass regulation in vivo and does not act directly on osteoclastogenesis in vitro.

Wnt/β‐catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation.

The use of estrogens and androgens to prevent bone loss is limited by their unwanted side effects, especially in reproductive organs and breast. Selective estrogen receptor modulators (SERMs) partially avoid such unwanted effects, but their efficacy on bone is only moderate compared with that of estradiol or androgens. Estrens have been suggested to not only prevent bone loss but also exert anabolic effects on bone while avoiding unwanted effects on reproductive organs. In this study, we compared the effects of a SERM (PSK3471) and 2 estrens (estren-alpha and estren-beta) on bone and reproductive organs to determine whether estrens are safe and act via the estrogen receptors and/or the androgen receptor (AR). Estrens and PSK3471 prevented gonadectomy-induced bone loss in male and female mice, but none showed true anabolic effects. Unlike SERMs, the estrens induced reproductive organ hypertrophy in both male and female mice and enhanced MCF-7 cell proliferation in vitro. Estrens directly activated transcription in several cell lines, albeit at much higher concentrations than estradiol or the SERM, and acted for the most part through the AR. We conclude that the estrens act mostly through the AR and, in mice, do not fulfill the preclinical efficacy or safety criteria required for the treatment or prevention of osteoporosis.