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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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Mechanisms by which muscle regulates bone are poorly understood., Results: Electrically stimulated muscle contraction reversed elevations in bone resorption and increased Wnt signaling in bone-derived cells after spinal cord transection., Conclusion: Muscle contraction reduced resorption of unloaded bone independently of the CNS, through mechanical effects and, potentially, nonmechanical signals (e.g. myokines)., Significance: The study provides new insights regarding muscle-bone interactions. Muscle and bone work as a functional unit. Cellular and molecular mechanisms underlying effects of muscle activity on bone mass are largely unknown. Spinal cord injury (SCI) causes muscle paralysis and extensive sublesional bone loss and disrupts neural connections between the central nervous system (CNS) and bone. Muscle contraction elicited by electrical stimulation (ES) of nerves partially protects against SCI-related bone loss. Thus, application of ES after SCI provides an opportunity to study the effects of muscle activity on bone and roles of the CNS in this interaction, as well as the underlying mechanisms. Using a rat model of SCI, the effects on bone of ES-induced muscle contraction were characterized. The SCI-mediated increase in serum C-terminal telopeptide of type I collagen (CTX) was completely reversed by ES. In ex vivo bone marrow cell cultures, SCI increased the number of osteoclasts and their expression of mRNA for several osteoclast differentiation markers, whereas ES significantly reduced these changes; SCI decreased osteoblast numbers, but increased expression in these cells of receptor activator of NF-κB ligand (RANKL) mRNA, whereas ES increased expression of osteoprotegerin (OPG) and the OPG/RANKL ratio. A microarray analysis revealed that ES partially reversed SCI-induced alterations in expression of genes involved in signaling through Wnt, FSH, parathyroid hormone (PTH), oxytocin, and calcineurin/nuclear factor of activated T-cells (NFAT) pathways. ES mitigated SCI-mediated increases in mRNA levels for the Wnt inhibitors DKK1, sFRP2, and sclerostin in ex vivo cultured osteoblasts. Our results demonstrate an anti-bone-resorptive activity of muscle contraction by ES that develops rapidly and is independent of the CNS. The pathways involved, particularly Wnt signaling, suggest future strategies to minimize bone loss after immobilization.

Cdc37 is a molecular chaperone closely associated with the folding of protein kinases. Results from studies using a yeast model system showed that it was also important for activation of the human androgen receptor (AR). Based on results from the yeast model system (Fliss, A. E., Fang, Y., Boschelli, F., and Caplan, A. J. (1997) Mol. Biol. Cell 8, 2501-2509), we initiated studies to address whether AR and Cdc37 interact with each other in animal cell systems. Our results show that Cdc37 binds to AR but not to glucocorticoid receptors (GR) synthesized in rabbit reticulocyte lysates. This binding occurs via the ligand-binding domain of the AR in a manner that is partially dependent on Hsp90 and the presence of hormone. Further studies using the yeast system showed that Cdc37 is not interchangeable with Hsp90, suggesting that it functions at a distinct step in the activation pathway. Expression of a dominant negative form of Cdc37 in animal cells down-regulates full-length AR but has very little effect on an AR truncation lacking the ligand-binding domain or full-length GR. These results reveal differences in the mechanisms by which AR and GR become active transcription factors and strengthen the notion that Cdc37 has a wider range of polypeptide clients than was realized previously.

The multicatalytic proteinase complex (MPC, proteasome) is assembled from 14 nonidentical protein subunits. It expresses five distinct proteolytic activities, including a chymotrypsin-like activity, cleaving after hydrophobic residues, and a branched chain amino acid-preferring component (BrAAP), cleaving preferentially after branched chain residues. Exposure of cells to interferons leads to replacement of the X, Y, and Z subunits by the LMP2, LMP7, and MECL1 subunits. This "immunoproteasome" is critical to processing of certain antigens. The enzymatic basis for enhanced antigen processing has not been determined. To gain insight into this question, we examined sites and relative rates of cleavage of bonds in denatured, reduced, carboxyamidomethylated lysozyme, a 129-amino acid protein, by MPC from bovine spleen, in which the X, Y, and Z subunits are replaced by LMP2, LMP7, and MECL1. We compared cleavages to those catalyzed by MPC from bovine pituitary, which contains only the X, Y, and Z subunits. We found marked increases in the rates and number of cleavages after branched chain residues in reduced, carboxyamidomethylated lysozyme by the spleen MPC. This was largely due to accelerated cleavages of bonds after a Phi-X-Br motif, where Phi is a hydrophobic residue, X is a small neutral or polar residue, and Br is a branched chain residue. Inhibitors with these structural properties were selective and potent inhibitors of the BrAAP activity of the spleen MPC. The above findings indicate that alterations in activity and substrate specificity of the BrAAP activity are important factors underlying the altered cleavages after hydrophobic residues associated with incorporation of interferon-inducible subunits. The potential relevance of the findings to antigen processing functions of MPC is discussed.

Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-gamma-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased Km, a highly increased catalytic efficiency (kcat), and a 80-180 times greater specificity constant (kcat/Km) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3 position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-gamma-inducible subunits.

Polyacrylamide gel electrophoresis and high performance liquid chromatography of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary, lung, and liver showed marked differences in the pattern of subunits. The concentrations of LMP7 in the lung and liver were 10 and 5 times, respectively, greater than those in the pituitary, whereas the chymotrypsin-like activity and the amount of a subunit (BO2), necessary for its expression, were markedly decreased in the lung and moderately decreased in the liver. Lower trypsin-like, small neutral amino acid preferring, and peptidyl-glutamyl-peptide hydrolyzing activities were also found in the lung and liver. The activity of the branched chain amino acid preferring component (BrAAP), predominantly latent in the pituitary, was highly activated in the lung and liver, as evidenced by a greatly decreased Km and a 20-fold increase of the specificity constant Vmax/Km, indicating facilitated substrate access to its active site and increased affinity toward substrates with branched chain amino acids in the P1 position. It is suggested that overexpression of LMP7 in the lung is related to increased exposure of the airways to foreign antigens. The possible association between amounts of LMP7 and the activation of the BrAAP component needs further examination.

Evidence indicates that a component of the multicatalytic proteinase complex (MPC) that preferentially cleaves bonds after branched chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the MPC. We report here the synthesis of substrate-related peptidyl aldehydes that inhibit the activity of this component toward both synthetic peptide substrates and proteins. The most potent of the inhibitors, Cbz-Gly-Pro-Phe-leucinal (Cbz-GPFL-CHO) inhibits competitively with a Ki of 1.5 microM. The peptidyl aldehydes also inhibit the small neutral amino acid preferring and the peptidylglutamyl-peptide hydrolyzing activities of the MPC. The chymotrypsin-like activity is only weakly inhibited, and the trypsin-like activity is moderately activated. The importance of a Pro residue in the P3 position and a leucinal residue in the P1 position for inhibition of the BrAAP component is indicated by the finding that replacement of these residues by a glycine or phenylalaninal, respectively, markedly increases the Ki. Cbz-GPFL-CHO inhibited the BrAAP activity with the same Ki both before and after activation of this component by exposure of the MPC to 3,4-dichloroisocoumarin, suggesting that the peptidyl aldehyde is an effective inhibitor of both the overt and latent proteolytic activities of the MPC. Incubation of a human breast cancer cell line (MCF-7) in culture with the inhibitors of the BrAAP component led to an accumulation of ubiquitin-protein conjugates, indicating inhibition of the ubiquitin-dependent proteolytic pathway.