Biomedical subjects

Shigeki Miyamoto

Publications and source records attributed to Shigeki Miyamoto.

At least 19 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

PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress.

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes. SUMO-1 modification of NEMO (NF-kappaB essential modulator), the IkappaB kinase (IKK) regulatory subunit, is critical for activation of NF-kappaB by genotoxic agents. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-kappaB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO-3, in vitro. PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy-NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.

Active Transport, Cell Nucleus↗

PubMed2006

Cytochrome p450 expression of cultured rat small hepatocytes after long-term cryopreservation.

Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study, we investigated whether cryopreserved SHs could express cytochromes P450 (P450s), whether P450 expression was induced by appropriate inducers, and whether P450 activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and ethanol were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3-MC and PCN, respectively. Although Matrigel did not up-regulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs, although the activities in both were approximately 30% of that of MHs. Profiles of (14)C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by high-performance liquid chromatography. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of P450s after long-term cryopreservation.

Animals↗

PubMed2006

NFKB1 is a direct target of the TAL1 oncoprotein in human T leukemia cells.

We recently showed that a subset of human T acute lymphoblastic leukemia (T-ALL) cell lines expresses low basal levels of p50, a nuclear factor-kappaB (NF-kappaB)/Rel family member, resulting in their capacity to activate the atypical p65:cRel complex rather than the classic p50:p65 dimer. Here, we show that the transcription factor TAL1 (also known as SCL) binds to the promoter of the NFKB1 gene that encodes p50 and represses its transcription to set up this unique response in T-ALL cells. When TAL1 expression is reduced in CEM T leukemia cells, basal NFKB1 expression is increased, and the levels of p65:cRel complex and transcription of its target gene, such as intercellular adhesion molecule-1 (ICAM-1), are reduced in response to etoposide treatment. Moreover, a significant negative correlation between NFKB1 and TAL1 or LMO1 was found in primary human TAL1/LMO1 double-positive T-ALL samples previously described by Ferrando et al. Thus, TAL1 modulates NFKB1 expression and an NF-kappaB-dependent transcriptional program in a subset of human T-cell leukemia cells.

Animals↗

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

Galphaq expression activates EGFR and induces Akt mediated cardiomyocyte survival: dissociation from Galphaq mediated hypertrophy.

Our laboratory has previously shown that adenoviral-mediated overexpression of Galphaq in neonatal rat ventricular cardiomyocytes increases the phosphorylation of Akt, a well-established anti-apoptotic effector. As demonstrated here, Galphaq expression protects cardiomyocytes against apoptosis induced by treatment with 2-deoxyglucose (2DOG) and this protection is lost when Akt activation is prevented by treatment with LY294002 (an inhibitor of PI3K). Galphaq-induced Akt phosphorylation is not caused by increased Gbetagamma signaling and does not appear to involve PKC activation. Rather studies using the EGF receptor inhibitor AG1478 and the Src inhibitor PP2 implicate these tyrosine kinases in the pathway inducing Akt phosphorylation. EGFR phosphorylation is increased in cells expressing Galphaq and this effect is inhibited by PP2, placing Src upstream of EGFR phosphorylation. EGFR activation appears to be required for Galphaq-mediated protection since inhibition of Src or EGFR rendered cells susceptible to 2DOG-induced apoptosis. In contrast to the requirement for EGFR mediated Akt activation in cardioprotection, neither EGFR nor Akt activation are necessary for the hypertrophic increases in cell size or ANF content elicited by Galphaq overexpression. These data demonstrate that increased Galphaq activity can provide anti-apoptotic signals by eliciting EGFR phosphorylation and subsequent Akt activation, independent of the well-known ability of Galphaq signaling to elicit hypertrophy.

Animals↗

PubMed2006

Nuclear factor-kappaB dimer exchange promotes a p21(waf1/cip1) superinduction response in human T leukemic cells.

The nuclear factor-kappaB (NF-kappaB)/Rel transcription factors are recognized as critical apoptosis regulators. We reported previously that NF-kappaB contributes to chemoresistance of CEM human T leukemic cells in part through its ability to induce p21(waf1/cip1). Here, we provide evidence that sequential NF-kappaB-activating signals induce heightened NF-kappaB DNA binding and p21(waf1/cip1) induction in CEM and additional T leukemic cell lines. This response arises from exceedingly low basal expression of the p105/p50 NF-kappaB subunit encoded by the NFKB1 gene in these cell lines. An initial NF-kappaB activation event enhances the recruitment of p65 and ELF1 to the NFKB1 promoter, leading to p65- and ELF1-dependent synthesis of p105/p50, which promotes an exchange of NF-kappaB complexes to p50-containing complexes with an increased DNA-binding activity to certain NF-kappaB target elements. Subsequent stimulation of these cells with an anticancer agent, etoposide, results in augmented NF-kappaB-dependent p21(waf1/cip1) induction and increased chemoresistance of the leukemia cells. Thus, we propose that low basal NFKB1 expression coupled with sequential NF-kappaB activation events can promote increased chemoresistance in certain T leukemic cells.

Antineoplastic Agents, Phytogenic↗

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↗

PubMed2005

Perillyl alcohol inhibits a calcium-dependent constitutive nuclear factor-kappaB pathway.

The cell death induced by the monoterpene anticancer agent perillyl alcohol correlates to the increased expression of certain proapoptotic genes known to influence cell survival. Whereas sequence-specific DNA-binding factors dictate the expression patterns of genes through transcriptional regulation, those transcriptional factors influencing constitutive cell survival with perillyl alcohol treatment are not well studied. Here, we investigated whether the monoterpenes can regulate the activity of nuclear factor-kappaB (NF-kappaB), a calcium-dependent transcription factor necessary for survival in the WEHI-231 B-lymphoma cells. Unique among the monoterpenes, perillyl alcohol short-term treatment induced a persistent decrease of calcium levels, whereas other various monoterpenes caused transient reductions in calcium levels. Perillyl alcohol treatment also rapidly elicited reductions of NF-kappaB DNA-binding activity and target gene induction, which was associated with an increase in apoptosis in these B-lymphoma cells. This apoptosis was directly due to NF-kappaB because its prior activation abolished the cell killing effects of perillyl alcohol treatment. Our findings suggest that perillyl alcohol can inhibit NF-kappaB function to modulate gene expression patterns and cell survival of certain B-lymphoma cells. The effects of perillyl alcohol were not limited to these B-lymphoma cells but were also observed in MDA-MB 468 cells, an estrogen receptor-negative breast cancer cell line. These results identify a calcium-dependent NF-kappaB pathway as a molecular target of perillyl alcohol activity in different cancer cell types.

Antineoplastic Agents↗

PubMed2005

Ca2+ dysregulation induces mitochondrial depolarization and apoptosis: role of Na+/Ca2+ exchanger and AKT.

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.

Adenoviridae↗

PubMed2005

Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells.

Constitutive NF-kappaB activity has emerged as an important cell survival regulator. Canonical inducible NF-kappaB activation involves IkappaB kinase (IKK)-dependent dual phosphorylation of Ser 32 and 36 of IkappaBalpha to cause its beta-TrCP-dependent ubiquitylation and proteasomal degradation. We recently reported that constitutive NF-kappaB (p50/c-Rel) activity in WEHI231 B cells is maintained through proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in a manner that requires Ser 32 and 36, without the requirement of a direct interaction with beta-TrCP. Here we specifically examined whether dual phosphorylation of Ser 32 and 36 was required for PIR degradation. Through mutagenesis studies, we found that dual replacement of Ser 32 and 36 with Glu permitted beta-TrCP and proteasome-dependent, but not PIR, degradation. Moreover, single replacement of either Ser residue with Leu permitted PIR degradation in WEHI231 B cells. These results indicate that PIR degradation occurs in the absence of dual phosphorylation, thereby explaining the beta-TrCP-independent nature of the PIR pathway. Additionally, we found evidence that PIR IkappaBalpha degradation controls constitutive NF-kappaB activation in certain multiple myeloma cells. These results suggest that B lineage cells can differentiate between PIR and canonical IkappaBalpha degradation through the absence or presence of dually phosphorylated IkappaBalpha.

Amino Acid Substitution↗

PubMed2005

Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes.

BACKGROUND AND AIMS: Small hepatocytes (SH), which are hepatic progenitor cells, were isolated from an adult rat liver. SH in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The morphological changes of SH may be correlated with hepatic maturation. Cytochrome P450s (CYP) are drug-metabolizing enzymes and the expression is one of hepatic differentiated functions. However, it is well known that the re-expression and maintenance of CYP activity are very difficult in cultured hepatocytes. We investigated the expression of CYP and the enzymatic activities in long-term cultured SH. METHODS: SH were isolated from adult rat livers and SH colonies were collected, replated on new dishes, and then cultured. CYP1A1/2, CYP2B1, CYP3A2, CYP4A1, and CYP2E1 were induced by the addition of 3-methylcholanthrene, phenobarbital, pregnenolone-16alpha-carbonitrile, clofibric acid, and ethanol, respectively. Immunocytochemistry, immunoblots, and enzyme activities were examined. RESULTS: SH could differentiate into mature hepatocytes by the addition of Matrigel and re-express constitutive CYPs. The expression of CYP1A1/2, CYP2B1, CYP3A2, and CYP4A1 dose-dependently increased and the amounts gradually increased with time in culture, especially in the cells treated with Matrigel. Activities of CYP1A, CYP2B, CYP3A and CYP2E in SH treated with Matrigel induced by each of the inducers were approximately 120-fold, 2.8-fold, 6.4-fold and 0.8-fold higher than in the control. CONCLUSION: The matured SH could re-express the constitutive CYP and recover inducibility, not only of protein expression but also of enzyme activities.

Animals↗

PubMed2005

Enhanced G2-M arrest by nuclear factor-{kappa}B-dependent p21waf1/cip1 induction.

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates cell survival pathways, but the molecular mechanisms involved are not completely understood. Here, we developed a NF-kappaB reporter cell system derived from CEM T leukemic cells to monitor the consequences of NF-kappaB activation following DNA damage insults. Cells that activated NF-kappaB in response to ionizing radiation or etoposide arrested in the G2-M phase for a prolonged time, which was followed by increased cell cycle reentry and survival. In contrast, those that failed to activate NF-kappaB underwent transient G2-M arrest and extensive cell death. Importantly, p21waf1/cip1 was induced in S-G2-M phases in a NF-kappaB-dependent manner, and RNA interference of this cell cycle regulator reduced the observed NF-kappaB-dependent phenotypes. Thus, cell cycle-coupled induction of p21waf1/cip1 by NF-kappaB represents a resistance mechanism in certain cancer cells.

Antibiotics, Antineoplastic↗

PubMed2005

Inhibition of IkappaBalpha nuclear export as an approach to abrogate nuclear factor-kappaB-dependent cancer cell survival.

Deregulation of the transcription factor nuclear factor-kappaB (NF-kappaB) leading to its constitutive activation is frequently observed in human cancer. Because altered NF-kappaB activities often promote the survival of malignant cells, its inhibition is regarded as a promising anticancer strategy. Because activation of the latent cytoplasmic NF-kappaB complex can be induced by a wide variety of different stimuli, its deregulation may occur by an equally large number of distinct mechanisms. This diversity raises a conundrum in conceptualizing general approaches to attenuate NF-kappaB activity in cancer. Here, we provide evidence that inhibition of IkappaBalpha nuclear export is a viable target to generally abrogate constitutive NF-kappaB activity in different cancer cell types. We show that inhibition of IkappaBalpha nuclear export has an important course of events in cancer cells harboring constitutive NF-kappaB activity-an initial increase in the pool of stable nuclear NF-kappaB/IkappaBalpha complexes that leads to a reduction of constitutive NF-kappaB activity and subsequent induction of apoptosis. Importantly, similar effects on multiple different cancer cell types indicate that inhibition of nuclear export of IkappaBalpha leads to broad inhibition of constitutive NF-kappaB activation regardless of various deregulated, upstream events involved.

Active Transport, Cell Nucleus↗

PubMed2004

Effects of SEA0400, a Na+/Ca2+ exchange inhibitor, on ventricular arrhythmias in the in vivo dogs.

SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline), a novel and selective inhibitor of Na+/Ca2+ exchanger, was investigated for its possible antiarrhythmic effects on arrhythmias of Ca2+ overload induced by coronary ligation/reperfusion and by digitalis in the dog. SEA0400 (1.0 mg/kg) did not change the hemodynamics but slightly prolonged the QRS duration (P<0.05). Pre-ischemic administration (10 min before coronary occlusion) of SEA0400 (1.0 mg/kg) and post-ischemic administration (1 min before reperfusion) of SEA0400 (0.3, 1.0 and 3.0 mg/kg) had no effects on the incidence of ventricular fibrillation induced by coronary ligation/reperfusion. On the other hand, SEA0400 (3.0 mg/kg) decreased the arrhythmic ratio in the digitalis arrhythmias (P<0.01). However, atrioventricular block and cardiac standstill were induced in two digitalized dogs. In conclusion, SEA0400 has no significant antiarrhythmic effect on arrhythmias induced by coronary ligation/reperfusion, but has an obvious suppressing effect on tachyarrhythmias induced by digitalis in in vivo canine models.

Aniline Compounds↗

PubMed2004

Expanded nuclear roles for IkappaBs.

I kappa B (IkappaB) was initially identified as a factor that inhibits DNA binding and nuclear translocation of the transcription factor nuclear factor kappa B (NF-kappaB). Recently, however, IkappaB family members have demonstrated direct nuclear roles in regulating NF-kappaB-dependent transcription. Some IkappaB proteins, including IkappaBalpha and IkappaBbeta, can regulate transcription by modulating the concentration of active NF-kappaB complexes within the nucleus. Others, such as IkappaBzeta and Bcl-3, can directly activate transcription by forming transcriptional complexes at gene promoters. Thus, IkappaB proteins play important nuclear roles in regulating NF-kappaB-dependent transcription after stimulation with various extracellular signals.

Animals↗

PubMed2004

A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells.

Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway.

Animals↗

PubMed2004

RelA life and death decisions.

RelA is generally regarded as an activator of prosurvival gene transcription. However, in this issue of Molecular Cell, demonstrate that anticancer drugs turn RelA into an accomplice of apoptosis by instructing it to dominantly repress survival gene transcription.

Antineoplastic Agents↗