STING regulates peripheral nerve regeneration and colony stimulating factor 1 receptor (CSF1R) processing in microglia.

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.

ATP Citrate Lyase Regulates Myofiber Differentiation and Increases Regeneration by Altering Histone Acetylation.

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.

Blockade of activin type II receptors with a dual anti-ActRIIA/IIB antibody is critical to promote maximal skeletal muscle hypertrophy.

The TGF-ß 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 improves mitochondrial function in skeletal muscle.

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 acetyl-CoA. 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.

An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy.

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.

Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism.

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.

Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.

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/ß-catenin signaling. We found the extracellular ß-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.

Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo.

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.

Follicle-stimulating hormone does not impact male bone mass in vivo or human male osteoclasts in vitro.

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.

Bone protection by estrens occurs through non-tissue-selective activation of the androgen receptor.

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.

Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass.

UNLABELLED: Wnt/beta-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. INTRODUCTION: The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism. MATERIALS AND METHODS: In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1(+/-) animals. RESULTS: Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1(+/-) mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold. CONCLUSIONS: We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.

Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation and bone mass in mice.

One of the well characterized cell biologic actions of lithium is the inhibition of glycogen synthase kinase-3beta and the consequent activation of canonical Wnt signaling. Because deficient Wnt signaling has been implicated in disorders of reduced bone mass, we tested whether lithium could improve bone mass in mice. We gavage-fed lithium chloride to 8-week-old mice from three different strains (Lrp5(-/-), SAMP6, and C57BL/6) and assessed the effect on bone metabolism after 4 weeks of therapy. Lrp5(-/-) mice lack the Wnt coreceptor low-density lipoprotein receptor-related protein 5 and have markedly reduced bone mass. Lithium, which is predicted to act downstream of this receptor, restored bone metabolism and bone mass to near wild-type levels in these mice. SAMP6 mice have accelerated osteoporosis due to inadequate osteoblast renewal. Lithium significantly improved bone mass in these mice and in wild-type C57BL/6 mice. We found that lithium activated canonical Wnt signaling in cultured calvarial osteoblasts from Lrp5(-/-) mice ex vivo and that lithium-treated mice had increased expression of Wnt-responsive genes in their bone marrow cells in vivo. These data lead us to conclude that lithium enhances bone formation and improves bone mass in mice and that it may do so via activation of the canonical Wnt pathway. Lithium has been used safely and effectively for over half a century in the treatment of bipolar illness. Prospective studies in patients receiving lithium should determine whether it also improves bone mass in humans.

An engineered biopolymer prevents mucositis induced by 5-fluorouracil in hamsters.

Oral mucositis is a common, treatment-limiting, and costly side effect of cancer treatments whose biological underpinnings remain poorly understood. In this study, mucositis induced in hamsters by 5-fluorouracil (5-FU) was observed after cheek-pouch scarifications, with and without administration of RGTA (RG1503), a polymer engineered to mimic the protective effects of heparan sulfate. RG1503 had no effects on 5-FU-induced decreases in body weight, blood cell counts, or cheek-pouch and jejunum epithelium proliferation rates, suggesting absence of interference with the cytotoxic effects of 5-FU. Extensive mucositis occurred in all of the untreated animals, and consisted of severe damage to cheek pouch tissues (epithelium, underlying connective tissue, and muscle bundles). Only half of the RG1503-treated animals had mucositis, over a mean area 70% smaller than in the untreated animals. Basement membranes were almost completely destroyed in the untreated group but was preserved in the RG1503 group. RG1503 blunted or abolished the following 5-FU-induced effects: increases in matrix metalloproteinase (MMP)-2, MMP-9, and plasmin, and decreases in tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These data indicate that mucositis lesions are related to massive release of proteolytic enzymes and are improved by RG1503 treatment, this effect being ascribable in part to restoration of the MMP-TIMP balance. RG1503 given with cancer treatment might protect patients from mucositis.

RGTA modulates the healing pattern of a defect in a monolayer of osteoblastic cells by acting on both proliferation and migration.

A family of heparan-like polymers, RGTAs, was shown to promote repair of various tissues. Like heparin and heparan-sulfates, RGTAs potentiate in vitro the biological activities of heparin-binding growth factors (HBGFs) and protect them against proteolytic degradation. It was postulated that RGTAs stimulate bone healing by interacting with HBGFs released in the wound site and, subsequently, by promoting the proliferation of cells implicated in this process. In a previous report, we examined how RGTA can modulate the proliferation of MC3T3-E1 osteoblastic cells. To further complete this study and to support this hypothesis, we developed an in vitro model of bone repair and examined the effects of RGTA alone or in association with FGF2, BMP-2, and TGF-beta1 which are representative of HBGFs known to stimulate bone repair. The model consisted of a 6-mm reproducible defect created on a MC3T3-E1 cell monolayer. In the presence of the different products added to the medium, the process of wound repair was measured through the filling of the acellular defect. We show that in 8 days, RGTA slightly inhibits repair alone compared to the control (2% FBS), that it inhibits the mitogenic effect of FGF2, and that it amplifies the inhibitory effect of BMP-2 and TGF-beta1. Repair was realized by an association of cell migration and cell proliferation mechanisms. To determine the part played by each process, DNA synthesis was evaluated for cell proliferation using an immunodetection technique [to measure incorporation of 5-bromo-2-deoxyuridine (BrdU)], coupled with a computer-assisted image analysis. The results show that the presence of RGTA (1) amplified the number of labeled nuclei compared to the control, (2) added to FGF2 or TGF-beta1, it reduced the number of labeled nuclei compared to FGF2 or TGF-beta1 alone, and (3) in the presence of BMP-2, it amplified the number of labeled nuclei compared to BMP-2 alone. Proper interpretation of these data requires a better understanding of the mechanism of action of RGTA on bone healing.