Previously we reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondria͞cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. We also established a cell-free Bax degradation assay in which an in vitro-translated, 35 S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. Our studies strongly suggest that ubiquitin͞proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.
BackgroundGPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.Methods/Principal FindingsIn this study, we created and characterized the phenotype of GPRC6A −/− mice. We observed complex metabolic abnormalities in GPRC6A −/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A −/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A −/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A −/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.Conclusions/Significance GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.
Prostate cancer is the second leading cause of cancer death in the United States and, thus far, there has been no effective therapy for the treatment of hormonerefractory disease. Recently, the androgen receptor (AR) has been shown to play a critical role in the development and progression of the disease. In this report, we showed that knocking down the AR protein level by a small interfering RNA (siRNA) approach resulted in a significant apoptotic cell death as evidenced by an increased annexin V binding, reduced mitochondrial potential, caspase-3/6 activation, and DFF45 and poly(ADP-ribose) polymerase cleavage.
OBJECTIVEWe sought to assess the associations of testosterones and sex hormone–binding globulin (SHBG) with metabolic syndrome and insulin resistance in men.RESEARCH DESIGN AND METHODSWe defined metabolic syndrome according to the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Among men aged ≥20 years who participated in the Third National Health and Nutrition Examination Survey (n = 1,226), the Cox proportional hazards model was used to estimate the prevalence ratio and 95% CI of metabolic syndrome according to circulating concentrations of testosterones and SHBG.RESULTSAfter adjustment for age, race/ethnicity, smoking status, alcohol intake, physical activity level, LDL cholesterol, C-reactive protein, and insulin resistance, men in the first quartile (lowest) (prevalence ratio 2.16 [95% CI 1.53–3.06]) and second quartile of total testosterone (2.51 [1.86–3.37]) were more likely to have metabolic syndrome than men in the fourth quartile (highest, referent group) (P < 0.001 for linear trend). Similarly, men in the first quartile of SHBG (2.17 [1.32–3.56]) were more likely to have metabolic syndrome than men in the fourth quartile (P = 0.02 for linear trend). No significant associations of calculated free testosterone (P = 0.31 for linear trend) and bioavailable testosterone (P = 0.11 for linear trend) with metabolic syndrome were detected after adjustment for all possible confounders.CONCLUSIONSLow concentrations of total testosterone and SHBG were strongly associated with increased likelihood of having metabolic syndrome, independent of traditional cardiovascular risk factors and insulin resistance.
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