Nitric oxide regulates cartilage destruction by causing dedifferentiation and apoptosis of chondrocytes. We investigated the role of the mitogen-activated protein kinase subtypes, extracellular signal-regulated protein kinase (ERK)-1/2, and p38 kinase in NO-induced apoptosis of rabbit articular chondrocytes and their involvement in dedifferentiation. Generation of NO with sodium nitroprusside (SNP) caused dedifferentiation, as indicated by the inhibition of type II collagen expression and proteoglycan synthesis. NO additionally caused apoptosis, accompanied by p53 accumulation and caspase-3 activation. SNP treatment stimulated activation of ERK-1/2 and p38 kinase. Inhibition of ERK-1/2 with PD98059 rescued SNP-induced dedifferentiation but enhanced apoptosis up to 2-fold, whereas inhibition of p38 kinase with SB203580 enhanced dedifferentiation, with significant blockage of apoptosis. The stimulation of apoptosis by ERK inhibition was accompanied by increased p53 accumulation and caspase-3 activity, whereas the inhibitory effect of p38 kinase blockade was associated with reduced p53 accumulation and caspase-3 activity. Our results indicate that NO-induced p38 kinase functions as an induction signal for apoptosis and in the maintenance of chondrocyte phenotype, whereas ERK activity causes dedifferentiation and operates as an anti-apoptotic signal. NO generation is less proapoptotic in chondrocytes that are dedifferentiated by serial monolayer culture or phorbol ester treatment. NO-induced p38 kinase activity is low in dedifferentiated cells compared with that in differentiated chondrocytes, with lower levels of p53 accumulation and caspase-3 activity. Our findings collectively suggest that ERK-1/2 and p38 kinase oppositely regulate NO-induced apoptosis of chondrocytes, in association with p53 accumulation, caspase-3 activation, and differentiation status.

Ubiquitin-dependent proteolysis plays an essential role in the regulation of a variety of cellular processes, including cell proliferation, differentiation, and apoptosis (1-3). Ubiquitin (Ub) 3 is covalently attached to target proteins by a cascade enzyme system consisting of Ub-activating (E1), conjugating (E2), and ligating (E3) enzymes (1, 4). Ub E3 ligases that confer the substrate specificity have been grouped into two families; the HECT-domain family that is defined by its homology to E6-associated protein (E6AP) and the RING family carrying RING-finger domain that is essential for the Ub ligase activity (5, 6). One of the well defined RING E3 ligases is the Skp1/Cul1/F-box protein complex, in which Cul1 serves as a scaffold molecule that interacts with Skp1 and a small RING-finger protein Roc1, also known as Hrt1 and Rbx1 (7-9). F-box proteins are recruited to the complex by binding to the Skp1 adaptor protein.At least six Cul members have been identified: Cul1, Cul2, Cul3, Cul4A, Cul4B, and Cul5 (10). Of these, Cul3 is known to mediate the degradation of several proteins, such as cyclin E (11), but the molecular composition of Cul3-based Ub ligase was unknown. Recently, a large family of proteins having BTB (Bric-a-brac/Tramtrack/Broad complex) domain has been identified as novel Cul3-interacting proteins (12). Most BTB proteins, but not all, have additional domains for proteinprotein interaction, such as zinc fingers, Kelch repeats, and MATH motifs. Furthermore, a subset of proteins containing BTB domain has been identified to function as substrate-specific adaptors that bind to Cul3. Specifically, MEL-26, a homolog of human SPOP (speckle-type POZ protein) in Caenorhabditis elegans, was first identified as a BTB protein that serves as a specific adaptor of MEI-1 for the ubiquitination by Cul3-based Ub ligase and subsequent degradation by the proteasome (13). MEI-1 is a subunit of the katanin-like microtubule severing heterodimer MEI-1/MEI-2 that localizes to the spindles and the chromosomes during meiosis (14). SPOP BTB protein has also been shown to mediate the ubiquitination of the Polycomb group BMI and the variant histone MacroH2A (15). In addition, Keap1 BTB protein was shown to recruit Nrf2 to . Nrf2 is a transcription factor that regulates the expression of anti-oxidant genes upon oxidative stress. In Schizosaccharomyces pombe, Btb1p, Btb2p, and Btb3p interact with Cul3, but their functions remain unknown (20). Therefore, so far only a few protein substrates have been shown to interact with BTB proteins for their ubiquitination by Cul3-based Ub ligases.Daxx was originally identified as a protein that binds to the death domain of Fas receptor by yeast two-hybrid screening (21). Daxx interacts with the apoptosis signal-regulating kinase 1 (ASK1) and promotes Fas-mediated apoptosis through the activation of Jun N-terminal kinase (22). Subsequent studies have also shown that Daxx behaves as a proapoptotic protein under various stress conditions (21,(23)(24)(25). On the contrary, homozygous deletio...

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

Background and Purpose-Stroke is one of the leading causes of adult disability and death in developing countries.However, early diagnosis is difficult and no reliable biomarker is currently available. Thus, we applied a 1 H-NMR metabolomics approach to investigate the altered metabolic pattern in plasma and urine from patients with cerebral infarctions and sought to identify metabolic biomarkers associated with stroke. Methods-Metabolic profiles of plasma and urine from patients with cerebral infarctions, especially small vessel occlusion, were investigated using 1 H-NMR spectroscopy coupled with multivariate statistical analysis, such as principal components analysis and orthogonal partial least-squares discriminant analysis. Results-Multivariate statistical analysis showed a significant separation between patients and healthy individuals. The plasma of stroke patients was characterized by the increased excretion of lactate, pyruvate, glycolate, and formate, and by the decreased excretion of glutamine and methanol; the urine of stroke patients was characterized by decreased levels of citrate, hippurate, and glycine. These metabolites detected from plasma and urine of patients with cerebral infarctions were associated with anaerobic glycolysis, folic acid deficiency, and hyperhomocysteinemia. Furthermore, the presence of cerebral infarction in the external validation model was predicted with high accuracy. Conclusions-These data demonstrate that a metabolomics approach may be useful for the effective diagnosis of cerebral infarction and for the further understanding of stroke pathogenesis.