No abstract

Based on our findings, we suggest that the leptin present in the articular joint fluid protects articular chondrocytes against cumulative mechanical load and detrimental stresses throughout a lifetime, delaying the onset of degenerative changes in chondrocytes. We can further hypothesize that leptin protects articular chondrocytes against destructive stimuli even in the joints of osteoarthritis (OA) patients.

We investigated the induction and underlying mechanism of apoptosis in retinal pigment epithelial cells by the inhibition of proteasome activity using lactacystin. Rat retinal pigment epithelial cell line retinal pigment epithelial (RPE)-J was used in this study. Apoptosis was evaluated by light and electron microscopies, DNA electrophoresis, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The apoptosis-related proteins were localized in the cells by immunofluorescent microscopy, and the changes of their protein contents and the enzyme activation were monitored by Western blot. Mitochondrial membrane potential was quantified by measuring J aggregate (5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazol carbocyanine iodide) fluorescence. To measure changes in intracellular pH, cells were loaded with 2Ј,7Ј-bis(carboxyethyl)-5(6Ј)-carboxyfluorescein and assayed by flow cytometry. To elucidate the type of transport system involving intracellular pH regulation, several transporter inhibitors were used, and their effect on pH and membrane potential was assayed as described above. Lactacystin treatment significantly induced apoptosis in RPE-J cells. During the RPE cell apoptosis, 1) cytochrome c and Smac/DIABLO were released into cytosol from mitochondria, 2) translocation of apoptosis-inducing factor to the nucleus was evident, 3) Bax protein seemed to translocate to mitochondria, 4) procaspase-3 and poly(ADP-ribose) polymerase were cleaved, and 5) nuclear condensation and DNA fragmentation were clearly observed. Noticeably, a transient increase of mitochondrial membrane potential was coincidentally detected with the intracellular alkalinization after lactacystin administration. Furthermore, the lactacystin-induced early alkalinization was inhibited by 4-acetamido-4Ј-isothiocyanostilbene-2,2Ј-disulfonate, an inhibitor of Cl Ϫ /HCO 3 Ϫ anion exchanger, which also prevented early mitochondrial hyperpolarization and apoptosis. Lactacystin-induced apoptosis in RPE-J cells is closely associated with an early mitochondrial hyperpolarization induced by intracellular alkalinization.Apoptosis is an evolutionarily conserved, innate process by which cells systemically inactivate, disassemble, and degrade their own structural and functional components to complete their own demise (Wyllie et al., 1980). In this highly regulated process, a cascade of molecular and biochemical events leading to cell death is activated.Caspase activation is a central process in the execution of dying cells. The activation of an effector caspase, such as caspase-3, is stimulated by activated initiator caspases, caspase-8 or -9. Once activated, the effector caspases are responsible for the proteolytic degradation of a broad spectrum of cellular targets that ultimately leads to cell death (Thornberry and Lazebnik, 1998). However, the activation of effector caspases can be suppressed in the presence of inhibitors of apoptosis proteins (Roberts et al., 2001). Mitochondria play an important role in the regulation of apopto...

In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

In the present study, we identified a missense mutation (G199V) in KAT-18 cell line established from primary cultures of anaplastic thyroid cancer (ATC). Notably, knockdown of this mutant (mt) p53 reduced cell viability and exerted antitumor activity equivalent to high doses of several chemotherapeutic agents. We showed that p53 knockdown had an antitumor effect via the induction of apoptosis. We further examined the underlying mechanism by which mt p53 (G199V) gains antiapoptotic function in KAT-18 cells. Microarray analysis revealed that p53 knockdown modified the expression of numerous apoptosis-related genes. Importantly, p53 knockdown led to downregulation of signal transducer and activator of transcription-3 (STAT3) gene expression. We further observed that p53 knockdown induced the downregulation of STAT3 protein. We also observed that a STAT3 inhibitor augmented the reduction of cell viability induced by p53 knockdown, whereas interleukin-6 treatment alleviated this effect. In addition, overexpression of STAT3 protected ATC cells against cell death induced by p53 knockdown. Taken together, these data show that mt p53 (G199V) gains antiapoptotic function mediated by STAT3 in ATC cells. Inhibition of the function of mt p53 (G199V) could be a novel and useful therapeutic strategy for decreasing the extent and severity of toxicity due to chemotherapeutic agents. (Mol Cancer Res 2009; 7(10):1645-54)