Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondria-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondria matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca 2+ ) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis/mass spectrometry of Pro-Q Diamond staining while many more Pro-Q Diamond stained proteins were below mass spectrometry detection. Time dependent 32 P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondria matrix is extensive and dynamic. Ca 2+ has previously been shown to activate various dehydrogenases, promote reactive oxygen species (ROS) generation, and initiate apoptosis via cytochrome c release. To evaluate the Ca 2+ signaling network, the effects of a Ca 2+ challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca 2+ -induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca 2+ dose dependent dephosphorylation of MnSOD was associated with a ∼2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca 2+ . These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca 2+ modification of mitochondrial function at the level of MnSOD.Mitochondria are thought to be the result of an early interaction of two lines of cellular life, the bacterium and eukaryotic cell (1;2). At this point in time, mitochondria play a critical role in energy metabolism, apoptosis and cell signaling pathways in the cell. However, the acute and chronic regulatory mechanisms of this organelle remain poorly defined. One approach to assessing the function and regulation of the mitochondrion is an evaluation of the mitochondrial Address correspondence to: Robert S. Balaban, Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive Room B1D416, Bethesda, MD 20892-1061. Tel. 301 496-3658; Fax. 301 402-2389; E-mail: rsb@nih.gov. proteome. Estimates predict up to 3000 proteins (3;4) in mitochondria, however, recent largescale screening studies by Taylor (5) and Mootha (6) identified only about 600 distinct mitochondrial proteins. Many have used proteomic approaches to evaluate differential protein expr...

The increasing interest in nanoparticles for advanced technologies, consumer products, and biomedical applications has led to great excitement about potential benefits but also concern over the potential for adverse human health effects. The gastrointestinal tract represents a likely route of entry for many nanomaterials, both directly through intentional ingestion or indirectly via nanoparticle dissolution from food containers or by secondary ingestion of inhaled particles. Additionally, increased utilisation of nanoparticles may lead to increased environmental contamination and unintentional ingestion via water, food animals, or fish. The gastrointestinal tract is a site of complex, symbiotic interactions between host cells and the resident microbiome. Accordingly, evaluation of nanoparticles must take into consideration not only absorption and extraintestinal organ accumulation but also the potential for altered gut microbes and the effects of this perturbation on the host. The existing literature was evaluated for evidence of toxicity based on these considerations. Focus was placed on three categories of nanomaterials: nanometals and metal oxides, carbon-based nanoparticles, and polymer/dendrimers with emphasis on those particles of greatest relevance to gastrointestinal exposures.