PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation.

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin-proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.

Osteopontin is strongly expressed by alveolar macrophages in the lungs of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by an intense inflammatory response in the lung parenchyma. Recent studies suggest that excessive nitric oxide (NO) production mediated by inducible NO synthase (iNOS) in macrophages is partially involved in mediating acute lung injury in ARDS. On the other hand, osteopontin (OPN) is a cytokine which is capable of inhibiting NO production by suppressing iNOS mRNA expression in macrophages. In this study, we investigated the expression of OPN in the lungs of 10 patients with ARDS. In most patients, OPN is strongly expressed on alveolar macrophages. In addition, we produced a murine model for ARDS by intratracheal administration of lipopolysaccharide and investigated the expression of endogenous OPN and iNOS in the lungs of ARDS mice. Immunostaining demonstrated that in vivo OPN protein was coinduced with iNOS protein predominantly in the accumulating alveolar macrophages. OPN mRNA expression was also coinduced with iNOS mRNA, but was induced more slowly than iNOS mRNA in the lungs of ARDS mice. These results suggested that OPN, which may reduce NO production of macrophages by inhibiting iNOS expression, is significantly induced and expressed on alveolar macrophages in the lungs of ARDS. It is possible that OPN is partially involved in playing a protective role against excessive production of NO in ARDS.

Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth.

Angiogenesis, which is essential for tumor growth, is regulated by various angiogenic factors. Osteopontin (OPN) is expressed in various human tumors and is postulated to be involved in tumor progression. We have recently reported that culture medium with murine neuroblastoma C1300 cells transfected with OPN gene significantly stimulates human umbilical vein endothelial cell migration and induces neovascularization in mice by dorsal air sac assay. However, the effect of OPN on tumorigenesis as an angiogenic factor remains to be clarified. In this study, we injected the OPN-transfected C1300 cells and control cells into the nude mice subcutaneously. OPN-overexpressing C1300 cells significantly formed rapidly growing tumor as compared to the control cells in mice, although in vitro and in vivo cell growth rates were similar. In vivo tumorigenecity of these cells correlated with the amount of secreted OPN protein. In addition, neovascularization of OPN-transfected tumor was significantly increased in comparison with those of control cells by immunohistochemistry for CD31. In vitro chemoinvasiveness and gene expression of proteases including uPA, MMP2, 9, MT1-MMP, and cathepsin B, D, L, were not different between OPN-transfected and control cells determined with matrigel invasion assay and cDNA expression macroarray, respectively. Conclusively, these results strongly imply that OPN plays an important role in tumor growth through the enhancement of angiogenesis in vivo.

Quantification of elastin cross-linking amino acids, desmosine and isodesmosine, in hydrolysates of rat lung by ion-pair liquid chromatography-mass spectrometry.

The elastin cross-linking amino acids, desmosine (DES) and isodesmosine (IDE), in hydrolysates of rat lungs were quantified by ion-pair liquid chromatography-electrospray mass spectrometry. The column was a 2.0 mm i.d. x 150 mm Develosil UG3 (ODS) with a mobile phase A of 7 mM pentafluoropropionic anhydride (PFPA) using ultrapure water as the ion-pair reagent, and a mobile phase B of 7 mM PFPA in 80% methanol. The retention times of IDE and DES were 25.5 and 26.6 min, respectively. The mean concentrations of IDE and DES in the lung were 191.6+/-54.5 nmol/g lung (dry tissue) (+/-SD) and 184.0+/-39.3 nmol/g lung, respectively, and the IDE/DES ratio was 1.04, in Wistar Kyoto rats. Our results indicate that ion-pair liquid chromatography-mass spectrometry is a useful procedure for quantitation of DES and IDE in hydrolysates of rat lung.