Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

2-(Trimethylammonium) Ethyl (R)-3-Methoxy-3-oxo-2-Stearamidopropyl Phosphate Suppresses Osteoclast Maturation and Bone Resorption by Targeting Macrophage-Colony Stimulating Factor Signaling

2-(Trimethylammonium) ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient megakaryopoiesis. Here, we show that (R)-TEMOSPho blocks osteoclast maturation from progenitor cells of hematopoietic origin, as well as blocking the resorptive function of mature osteoclasts. The inhibitory effect of (R)-TEMOSPho on osteoclasts was due to a disruption of the actin cytoskeleton, resulting from impaired downstream signaling of c-Fms, a receptor for macrophage-colony stimulating factor linked to c-Cbl, phosphoinositol-3-kinase (PI3K), Vav3, and Rac1. In addition, (R)-TEMOSPho blocked inflammation-induced bone destruction by reducing the numbers of osteoclasts produced in mice. Thus, (R)-TEMOSPho may represent a promising new class of antiresorptive drugs for the treatment of bone loss associated with increased osteoclast maturation and activity.     

1,25 Dihydroxyvitamin D3-dependent inhibition of growth or killing of Mycobacterium avium complex in human macrophages is mediated by TNF and GM-CSF

Vitamin D3 (D3) has been shown to activate several macrophage functions. To determine whether D3 could activate macrophages to kill or inhibit intracellular growth of Mycobacterium avium complex (MAC), human monocyte-derived macrophages were treated with D3 (10(-7), 10(-8), and 10(-9) M) 24 hr before or for 48 hr after MAC infection. All three concentrations were associated with inhibition of growth or killing of MAC in a dose-dependent fashion (28 +/- 4% and 22 +/- 3% of killing and inhibition of growth, respectively, at pharmacological concentrations) when added to the monolayer before injection or 60.4 +/- 6%, 50.4 +/- 3%, and 41.4 +/- 6%, respectively, when added to the monolayers after infection. We found that D3-treated macrophages produced increased concentrations of tumor necrosis factor (TNF) and granulocyte-monocyte colony stimulating factor (GM-CSF). Subsequently, macrophages were activated by D3 in the presence of anti-TNF or anti-GM-CSF antibody: At 10(-9) M of D3 there was no inhibition of D3-dependent macrophage activation by anti-TNF antibody, whereas anti-GM-CSF antibody was associated with 100% inhibition. At 10(-8) M of D3, anti-TNF antibody inhibited 35 +/- 6% of killing, and anti-GM-CSF antibody was associated with 100% inhibition. At 10(-7) M of D3, anti-TNF antibody inhibited 58 +/- 4% and anti-GM-CSF antibody 89 +/- 3% of killing. D3 treatment is associated with anti-MAC activity in human macrophages, and this activity appears to be mediated by both TNF and GM-CSF.     

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (⁷⁷⁴gpgfgssppGaeaapslRyvPY⁷⁹⁵) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X _2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.     

The Binaural Masking-Level Difference of Mandarin Tone Detection and the Binaural Intelligibility-Level Difference of Mandarin Tone Recognition in the Presence of Speech-Spectrum Noise

Binaural hearing involves using information relating to the differences between the signals that arrive at the two ears, and it can make it easier to detect and recognize signals in a noisy environment. This phenomenon of binaural hearing is quantified in laboratory studies as the binaural masking-level difference (BMLD). Mandarin is one of the most commonly used languages, but there are no publication values of BMLD or BILD based on Mandarin tones. Therefore, this study investigated the BMLD and BILD of Mandarin tones. The BMLDs of Mandarin tone detection were measured based on the detection threshold differences for the four tones of the voiced vowels /i/ (i.e., /i1/, /i2/, /i3/, and /i4/) and /u/ (i.e., /u1/, /u2/, /u3/, and /u4/) in the presence of speech-spectrum noise when presented interaurally in phase (S₀N₀) and interaurally in antiphase (S_πN₀). The BILDs of Mandarin tone recognition in speech-spectrum noise were determined as the differences in the target-to-masker ratio (TMR) required for 50% correct tone recognitions between the S₀N₀ and S_πN₀ conditions. The detection thresholds for the four tones of /i/ and /u/ differed significantly (p<0.001) between the S₀N₀ and S_πN₀ conditions. The average detection thresholds of Mandarin tones were all lower in the S_πN₀ condition than in the S₀N₀ condition, and the BMLDs ranged from 7.3 to 11.5 dB. The TMR for 50% correct Mandarin tone recognitions differed significantly (p<0.001) between the S₀N₀ and S_πN₀ conditions, at –13.4 and –18.0 dB, respectively, with a mean BILD of 4.6 dB. The study showed that the thresholds of Mandarin tone detection and recognition in the presence of speech-spectrum noise are improved when phase inversion is applied to the target speech. The average BILDs of Mandarin tones are smaller than the average BMLDs of Mandarin tones.