Biomedical subjects

Zhao-Hui Wu

Publications and source records attributed to Zhao-Hui Wu.

6 recordsLinked to original sources

PubMed2006

Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli.

The mechanisms involved in activation of the transcription factor NF-kappaB by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IkappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO)/IKKgamma, is a component of a nuclear signal that is generated after DNA damage to mediate NF-kappaB activation. Here, we found that etoposide (VP16) and camptothecin induced increases in intracellular free calcium levels at 60 min after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-kappaB activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-kappaB activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16-treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclear exporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of a plausible calcium-dependent nuclear export receptor, calreticulin, failed to prevent NF-kappaB activation by VP16. However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell line harboring a temperature-sensitive mutant of RCC1 blocked NF-kappaB activation induced by genotoxic stimuli. Overexpression of Ran in this cell model showed that DNA damage stimuli induced formation of a complex between Ran and NEMO, suggesting that RCC1 regulated NF-kappaB activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of glutathione S-transferase (GST) pull-down assays using GST fused to the Ran binding domain of RanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter these interactions, suggesting that calcium is a necessary step beyond the formation of a Ran-GTP-NEMO complex in the nucleus. These results suggest that calcium has a unique role in genotoxic stress-induced NF-kappaB signaling by regulating nuclear export of NEMO subsequent to the formation of a nuclear export complex composed of Ran-GTP, NEMO, and presumably, an undefined nuclear export receptor.

Active Transport, Cell Nucleus↗

PubMed2006

Molecular linkage between the kinase ATM and NF-kappaB signaling in response to genotoxic stimuli.

The transcription factor NF-kappaB modulates apoptotic responses induced by genotoxic stress. We show that NF-kappaB essential modulator (NEMO), the regulatory subunit of IkappaB kinase (IKK) (which phosphorylates the NF-kappaB inhibitor IkappaB), associates with activated ataxia telangiectasia mutated (ATM) after the induction of DNA double-strand breaks. ATM phosphorylates serine-85 of NEMO to promote its ubiquitin-dependent nuclear export. ATM is also exported in a NEMO-dependent manner to the cytoplasm, where it associates with and causes the activation of IKK in a manner dependent on another IKK regulator, a protein rich in glutamate, leucine, lysine, and serine (ELKS). Thus, regulated nuclear shuttling of NEMO links two signaling kinases, ATM and IKK, to activate NF-kappaB by genotoxic signals.

Active Transport, Cell Nucleus↗

PubMed2006

[Research on the influence of compatibility on fingerprint of Glycyrrhiza uralensis in mahuang decoctions by HPLC].

OBJECTIVE: To set up the HPLC fingerprint of Glycyrrhiza uralensis in decoctions prepared from various combinations of the recipe named Mahuang decoctions and study the influence of compatibility on fingerprint of G. wralensis in decoctions. METHOD: The RP-HPLC method was used, chromatography conditions were Zorbax SB-C18 column (4.6 mm x 250 mm, 5 microm), the acetonitrile-1.2% acetic acid (gradient elution) as mobile phase and detective wavelength at 254 nm. RESULT: The method is stable and reliable with a good reproducibility and provides a reference standard for the quality control of G. uralensis, it can be used to determine the fingerprint of G. aralensis in Mahuang decoctions. The result showed that 8 peaks were common in the fingerprint of G. uralensis in Mahauang decoctions. The area changes of 8 common peaks in decoctions prepared from various combinations of Mahuang decoctions were studied. CONCLUSION: The fingerprint of G. uralensis in Mahuang decoctions is markedly influenced by herba ephedrae and ramulus cinnamomi and semen armeniacae amarum. The areas of 7 common peaks were reduced obviously, while the relative areas of 6 common peaks were increased remarkably.

Chromatography, High Pressure Liquid↗

PubMed2005

PIDD: a switch hitter.

In response to genotoxic stress, the PIDD protein promotes apoptosis downstream of the tumor-suppressor protein p53. In this issue of Cell, Janssens et al. (2005) demonstrate that, in response to such stress, PIDD forms a nuclear complex that enhances sumoylation of NEMO. This modification is important for the activation of the antiapoptotic transcription factor NF-kappaB.

Apoptosis↗

PubMed2003

Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress.

The transcription factor NF-kappaB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-kappaB signaling pathways is NEMO/IKKgamma, the regulatory subunit of the cytoplasmic IkappaB kinase (IKK) complex. While NF-kappaB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Here, we show that genotoxic stress causes nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-kappaB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets.

Active Transport, Cell Nucleus↗

PubMed2002

Rat MYH, a glycosylase for repair of oxidatively damaged DNA, has brain-specific isoforms that localize to neuronal mitochondria.

Mitochondrial genomes are exposed to a heavy load of reactive oxygen species (ROS) that damage DNA. Since in neurons, mitochondrial DNA integrity must be maintained over the entire mammalian life span, neuronal mitochondria most likely repair oxidatively damaged DNA. We show that the Escherichia coli MutY DNA glycosylase homolog (MYH) in rat (rMYH) involved in repair of oxidative damage is abundantly expressed in the rat brain, with isoforms that are exclusive to brain tissue. Confocal microscopy and western analyses reveal localization of rMYH in neuronal mitochondria. To assess involvement of MYH in the neuronal response to oxidative DNA damage, we used a rat model of respiratory hypoxia, in which acutely reduced blood oxygenation leads to generation of superoxide, and formation and subsequent removal of 8-hydroxy-2'-deoxyguanosine (8OHdG). Removal of 8OHdG is accompanied by a spatial increase in rMYH immunoreactivity in the brain and an increase in levels of one of the three mitochondrial MYH isoforms, suggesting that inducible and non-inducible MYH isoforms exist in the brain. The mitochondrial localization of oxidative DNA damage repair enzymes in neurons may represent a specialized neuronal mechanism that safeguards mitochondrial genomes in the face of routine and accidental exposures to heavy loads of injurious ROS.

8-Hydroxy-2'-Deoxyguanosine↗