Publications by authors named "Yoshiaki Azuma"

57 Publications

The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.

EMBO Rep 2021 Apr 14:e50684. Epub 2021 Apr 14.

Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR, USA.

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.
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http://dx.doi.org/10.15252/embr.202050684DOI Listing
April 2021

Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy.

J Biol Chem 2020 Dec 7. Epub 2020 Dec 7.

Molecular Biosciences, University of Kansas, United States.

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared to non-induced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wildtype EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the co-localization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase, and in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction, and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.
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http://dx.doi.org/10.1074/jbc.RA120.014328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857440PMC
December 2020

PICH regulates the abundance and localization of SUMOylated proteins on mitotic chromosomes.

Mol Biol Cell 2020 11 2;31(23):2537-2556. Epub 2020 Sep 2.

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045.

Proper chromosome segregation is essential for faithful cell division and if not maintained results in defective cell function caused by the abnormal distribution of genetic information. Polo-like kinase 1-interacting checkpoint helicase (PICH) is a DNA translocase essential for chromosome bridge resolution during mitosis. Its function in resolving chromosome bridges requires both DNA translocase activity and ability to bind chromosomal proteins modified by the small ubiquitin-like modifier (SUMO). However, it is unclear how these activities cooperate to resolve chromosome bridges. Here, we show that PICH specifically disperses SUMO2/3 foci on mitotic chromosomes. This PICH function is apparent toward SUMOylated topoisomerase IIα (TopoIIα) after inhibition of TopoIIα by ICRF-193. Conditional depletion of PICH using the auxin-inducible degron (AID) system resulted in the retention of SUMO2/3-modified chromosomal proteins, including TopoIIα, indicating that PICH functions to reduce the association of these proteins with chromosomes. Replacement of PICH with its translocase-deficient mutants led to increased SUMO2/3 foci on chromosomes, suggesting that the reduction of SUMO2/3 foci requires the remodeling activity of PICH. In vitro assays showed that PICH specifically attenuates SUMOylated TopoIIα activity using its SUMO-binding ability. Taking the results together, we propose a novel function of PICH in remodeling SUMOylated proteins to ensure faithful chromosome segregation.
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http://dx.doi.org/10.1091/mbc.E20-03-0180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851874PMC
November 2020

Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases.

J Cell Biol 2020 01;219(1)

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN.

Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.
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http://dx.doi.org/10.1083/jcb.201807189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039214PMC
January 2020

MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma.

Oncotarget 2018 Apr 20;9(30):21429-21443. Epub 2018 Apr 20.

Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.
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http://dx.doi.org/10.18632/oncotarget.25117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940416PMC
April 2018

Non-Catalytic Roles of the Topoisomerase IIα C-Terminal Domain.

Int J Mol Sci 2017 Nov 17;18(11). Epub 2017 Nov 17.

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.

DNA Topoisomerase IIα (Topo IIα) is a ubiquitous enzyme in eukaryotes that performs the strand passage reaction where a double helix of DNA is passed through a second double helix. This unique reaction is critical for numerous cellular processes. However, the enzyme also possesses a C-terminal domain (CTD) that is largely dispensable for the strand passage reaction but is nevertheless important for the fidelity of cell division. Recent studies have expanded our understanding of the roles of the Topo IIα CTD, in particular in mitotic mechanisms where the CTD is modified by Small Ubiquitin-like Modifier (SUMO), which in turn provides binding sites for key regulators of mitosis.
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http://dx.doi.org/10.3390/ijms18112438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713405PMC
November 2017

THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

J Bone Miner Metab 2018 Jul 2;36(4):410-419. Epub 2017 Aug 2.

Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.
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http://dx.doi.org/10.1007/s00774-017-0855-2DOI Listing
July 2018

Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy.

J Biol Chem 2017 06 28;292(24):10230-10238. Epub 2017 Apr 28.

From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.
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http://dx.doi.org/10.1074/jbc.M117.789982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473226PMC
June 2017

Mechanisms behind Topoisomerase II SUMOylation in chromosome segregation.

Cell Cycle 2016 12 2;15(23):3151-3152. Epub 2016 Aug 2.

a Department of Molecular Biosciences , University of Kansas , Lawrence , KS , USA.

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http://dx.doi.org/10.1080/15384101.2016.1216928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5176152PMC
December 2016

SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis.

J Cell Biol 2016 06;213(6):665-78

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045

DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres.
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http://dx.doi.org/10.1083/jcb.201511079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915188PMC
June 2016

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis.

Cell Cycle 2016 Aug 26;15(16):2135-2144. Epub 2016 May 26.

a Department of Molecular Biosciences , University of Kansas , Lawrence , KS , USA.

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.
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http://dx.doi.org/10.1080/15384101.2016.1191713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993536PMC
August 2016

SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin.

Cell Cycle 2015 1;14(17):2777-84. Epub 2015 Jul 1.

a Department of Molecular Biosciences ; University of Kansas ; Lawrence , KS USA.

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.
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http://dx.doi.org/10.1080/15384101.2015.1066537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614044PMC
June 2016

SUMOylation and Ubiquitylation Circuitry Controls Pregnane X Receptor Biology in Hepatocytes.

Drug Metab Dispos 2015 Sep 10;43(9):1316-25. Epub 2015 Jun 10.

Departments of Pharmacology and Toxicology (W.C., J.L.S.), Mass Spectrometry Laboratory (N.G., T.D.W.), and Molecular Biosciences, University of Kansas, Lawrence, Kansas (Y.A.); and Department of Medicine, University of California, San Diego, La Jolla, California (M.S.)

Several nuclear receptor (NR) superfamily members are known to be the molecular target of either the small ubiquitin-related modifier (SUMO) or ubiquitin-signaling pathways. However, little is currently known regarding how these two post-translational modifications interact to control NR biology. We show that SUMO and ubiquitin circuitry coordinately modifies the pregnane X receptor (PXR, NR1I2) to play a key role in regulating PXR protein stability, transactivation capacity, and transcriptional repression. The SUMOylation and ubiquitylation of PXR is increased in a ligand- and tumor necrosis factor alpha -: dependent manner in hepatocytes. The SUMO-E3 ligase enzymes protein inhibitor of activated signal transducer and activator of transcription-1 (STAT1) STAT-1 (PIAS1) and protein inhibitor of activated STAT Y (PIASy) drive high levels of PXR SUMOylation. Expression of protein inhibitor of activated stat 1 selectively increases SUMO(3)ylation as well as PXR-mediated induction of cytochrome P450, family 3, subfamily A and the xenobiotic response. The PIASy-mediated SUMO(1)ylation imparts a transcriptionally repressive function by ameliorating interaction of PXR with coactivator protein peroxisome proliferator-activated receptor gamma coactivator-1-alpha. The SUMO modification of PXR is effectively antagonized by the SUMO protease sentrin protease (SENP) 2, whereas SENP3 and SENP6 proteases are highly active in the removal of SUMO2/3 chains. The PIASy-mediated SUMO(1)ylation of PXR inhibits ubiquitin-mediated degradation of this important liver-enriched NR by the 26S proteasome. Our data reveal a working model that delineates the interactive role that these two post-translational modifications play in reconciling PXR-mediated gene activation of the xenobiotic response versus transcriptional repression of the proinflammatory response in hepatocytes. Taken together, our data reveal that the SUMOylation and ubiquitylation of the PXR interface in a fundamental manner directs its biologic function in the liver in response to xenobiotic or inflammatory stress.
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http://dx.doi.org/10.1124/dmd.115.065201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538856PMC
September 2015

SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

J Biol Chem 2015 Feb 6;290(6):3269-76. Epub 2015 Jan 6.

From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.
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http://dx.doi.org/10.1074/jbc.C114.601906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319000PMC
February 2015

SUMOylation of Psmd1 controls Adrm1 interaction with the proteasome.

Cell Rep 2014 Jun 5;7(6):1842-8. Epub 2014 Jun 5.

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

SUMOylation is the covalent conjugation of SUMO polypeptides to cellular target proteins. Psmd1 is a subunit of the proteasomal 19S regulatory particle that acts as a docking site for Adrm1, another proteasome subunit that recruits ubiquitinated substrates for proteolysis. Here, we show that the SUMO deconjugating enzyme xSENP1 specifically interacts with Psmd1 and that disruption of xSENP1 targeting delays mitotic exit. Psmd1 becomes SUMOylated through the action of the SUMO E3 enzyme PIASy. We mapped SUMOylation sites within Psmd1 and found that SUMOylation of a critical lysine immediately adjacent to the Adrm1-binding domain regulates the association of Adrm1 with Psmd1. Together, our findings suggest that the interaction of Psmd1 with Adrm1 is controlled by SUMOylation in a manner that may alter proteasome composition and function. These findings demonstrate a mechanism for regulation of ubiquitin-mediated protein degradation by ubiquitin-like proteins of the SUMO family.
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http://dx.doi.org/10.1016/j.celrep.2014.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104683PMC
June 2014

Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides.

Biochemistry 2014 Jul 30;53(27):4334-45. Epub 2014 Jun 30.

Department of Molecular Biosciences, University of Kansas , 2034 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, Kansas 66045, United States.

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.
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http://dx.doi.org/10.1021/bi500224tDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100786PMC
July 2014

Alfacalcidol enhances collagen quality in ovariectomized rat bones.

J Orthop Res 2014 Aug 9;32(8):1030-6. Epub 2014 May 9.

NC Oral Health Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D3 : ALF) on bone collagen employing an ovariectomized rat model. Thirty-five 16-week-old female Sprague-Dawley rats were divided into five groups: SHAM (sham-operated + vehicle), OVX (ovariectomy + vehicle), and three ALF-treated groups, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/kg/day ALF, and ovariectomy + 0.2 µg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized; however, with the ALF treatment, it was improved in a dose-dependent manner. Contents of collagen and pyridinoline cross-link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF-treated groups. The total aldehyde, that is, a sum of free and those involved cross-links, in the highest dose of ALF was significantly higher than the rest of the groups (p < 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF-treated groups compared with OVX (p < 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy-induced osteoporotic bones, which likely contributes to the improvement of bone quality.
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http://dx.doi.org/10.1002/jor.22642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071133PMC
August 2014

The SUMO ligase PIAS1 regulates UV-induced apoptosis by recruiting Daxx to SUMOylated foci.

J Cell Sci 2012 Dec 12;125(Pt 23):5819-29. Epub 2012 Sep 12.

Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Haworth Hall, Rm. 3037, Lawrence, KS 66045, USA.

The small ubiquitin-like modifier (SUMO) ligase PIAS1 (Protein Inhibitor of Activated Stat-1) has been shown to play a role in cellular stress response by SUMOylating several proteins that are involved in DNA repair, apoptosis and transcription. In this paper, we show that PIAS1 regulates ultraviolet (UV)-induced apoptosis by recruiting Death-associated protein 6 (Daxx) to PIAS1-generated SUMO-foci. Cells that ectopically express PIAS1, but not other PIASes, show increased sensitivity to UV irradiation, suggesting that PIAS1 has a distinct function in UV-dependent apoptosis. Domain analysis of PIAS1 indicates that both PIAS1 SUMO-ligase activity and the specific localization of PIAS1 through its N-terminal and C-terminal domains are essential for UV-induced cell death. Daxx colocalizes with PIAS1-generated SUMOylated foci, and the reduction of Daxx using RNAi alleviates UV-induced apoptosis in PIAS1-expressing cells. PIAS1-mediated recruitment of Daxx and apoptosis following UV irradiation are dependent upon the Daxx C-terminal SUMO-interacting motif (SIM). Overall, our data suggest that the pro-apoptotic protein Daxx specifically interacts with one or more substrates SUMOylated by PIAS1 and this interaction leads to apoptosis following UV irradiation.
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http://dx.doi.org/10.1242/jcs.110825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575712PMC
December 2012

PIASy-dependent SUMOylation regulates DNA topoisomerase IIalpha activity.

J Cell Biol 2010 Nov;191(4):783-94

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.

DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.
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http://dx.doi.org/10.1083/jcb.201004033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2983052PMC
November 2010

Rod/Zw10 complex is required for PIASy-dependent centromeric SUMOylation.

J Biol Chem 2010 Oct 9;285(42):32576-85. Epub 2010 Aug 9.

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.
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http://dx.doi.org/10.1074/jbc.M110.153817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952260PMC
October 2010

PIASy mediates SUMO-2/3 conjugation of poly(ADP-ribose) polymerase 1 (PARP1) on mitotic chromosomes.

J Biol Chem 2010 May 12;285(19):14415-23. Epub 2010 Mar 12.

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.
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http://dx.doi.org/10.1074/jbc.M109.074583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863168PMC
May 2010

Identification and developmental expression of Xenopus laevis SUMO proteases.

PLoS One 2009 Dec 24;4(12):e8462. Epub 2009 Dec 24.

Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, Maryland, United States of America.

SUMO proteins are small ubiquitin-related modifiers. All SUMOs are synthesized as propeptides that are post-translationally cleaved prior to conjugation. After processing, SUMOs become covalently conjugated to cellular targets through a pathway that is similar to ubiquitination. Ubiquitin like protein proteases/Sentrin specific proteases (Ulp/SENPs) mediate both processing and deconjugation of SUMOs. The action of Ulp/SENPs makes SUMOylation a highly dynamic post-translational modification. To investigate how Ulp/SENPs are regulated in a developmental context, we isolated and characterized all Ulp/SENPs in Xenopus laevis. Xenopus possess homologues of mammalian SENP3, 5, 6 and 7. All of these enzymes reacted with HA-tagged vinyl sulfone derivatives of SUMO-2 (HA-SU2-VS) but not SUMO-1 (HA-SU1-VS), suggesting that they act primarily on SUMO-2 and -3. In contrast, Xenopus possess a single member of the SENP1/SENP2 subfamily of Ulp/SENPs, most closely related to mammalian SENP1. Xenopus SENP1 reacted with HA-SU1-VS and HA-SU2-VS, suggesting that it acts on all SUMO paralogues. We analyzed the mRNA and protein levels for each of the Ulp/SENPs through development; we found that they show distinct patterns of expression that may involve both transcriptional and post-transcriptional regulation. Finally, we have characterized the developmental function of the most abundant Ulp/SENP found within Xenopus eggs, SENP3. Depletion of SENP3 using morpholino antisense oligonucleotides (morpholinos) caused accumulation of high molecular weight SUMO-2/3 conjugated species, defects in developing embryos and changes in the expression of some genes regulated by the transforming growth factor beta (TGF-beta) pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794540PMC
December 2009

Positive effect of alendronate on subchondral bone healing and subsequent cartilage repair in a rabbit osteochondral defect model.

Am J Sports Med 2009 Nov;37 Suppl 1:139S-47S

Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan.

Background: Cartilage and subchondral bone have recently been considered an osteochondral unit. The treatment of osteochondral lesions is still challenging, but better subchondral bone repair may result in higher quality repaired cartilage.

Hypotheses: Alendronate accelerates bone formation in osteochondral defects and affects the quality of the repaired cartilage.

Study Design: Controlled laboratory study.

Methods: Osteochondral defects were made on the left trochleas of 50 rabbits, which were assigned to 1 of 3 groups: control, ALN (weekly subcutaneous injection of 0.14 mg/mL alendronate), and ALN-S (alendronate injection in the first 8 weeks only). They were evaluated at 4, 8, 24, and 52 weeks. Bone repair was evaluated with microcomputed tomography and histologic evaluation. Cartilage repair was evaluated with ultrasound and histologic analyses.

Results: At 4 weeks, the defects were filled, and cartilage-like repair tissue was observed in the ALN group, whereas the defects were incompletely filled in the control group. Alendronate treatment enhanced early bone formation and mineralization in the osteochondral defect for the first 8 weeks. The continuous injection of alendronate for 24 weeks resulted in delayed bone remodeling, but the rabbits in the ALN-S group showed good integrity of the subchondral bone plate, without delayed remodeling. At 52 weeks, the ALN-S group had a columnar arrangement of chondrocytes that had less fibrillation and looked superior to those in the ALN and control groups. Ultrasound analysis showed better quality of repaired cartilage of the ALN and ALN-S group than the control group.

Conclusion: Alendronate accelerated bone formation without inhibiting its mineralization but thereafter inhibited bone remodeling in an osteochondral defect. The withdrawal of alendronate at 8 weeks avoided the delayed remodeling and showed better subchondral bone repair. At 52 weeks, better subchondral bone repair resulted in better cartilage quality.

Clinical Relevance: Alendronate administered in the early period accelerates bone formation and improves the quality of the repaired cartilage.
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http://dx.doi.org/10.1177/0363546509350984DOI Listing
November 2009

CDP/cut is an osteoblastic coactivator of the vitamin D receptor (VDR).

J Bone Miner Res 2010 May;25(5):1157-66

Teijin Institute for Bio-Medical Research, Teijin Pharma, Ltd., Tokyo, Japan.

Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.
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http://dx.doi.org/10.1359/jbmr.091105DOI Listing
May 2010

Novel association of APC with intermediate filaments identified using a new versatile APC antibody.

BMC Cell Biol 2009 Oct 21;10:75. Epub 2009 Oct 21.

Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA.

Background: As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.

Results: We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including beta-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.

Conclusion: We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.
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http://dx.doi.org/10.1186/1471-2121-10-75DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774295PMC
October 2009

Analysis of SUMOylation of topoisomerase IIalpha with Xenopus egg extracts.

Authors:
Yoshiaki Azuma

Methods Mol Biol 2009 ;582:221-31

Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA.

Posttranslational protein modification by the Small Ubiquitin-like MOdifiers (SUMO) is involved in many cellular functions including organization of nuclear structures and chromatin, transcriptional regulation, and nucleo-cytoplasmic transport. Both genetic and biochemical studies indicate that the SUMO modification pathway plays an important role in proper cell cycle control, especially in the normal progression of mitosis. DNA topoisomerase II has been shown to be modified by SUMO in budding yeast as well as in vertebrates. We have shown by biochemical analysis using the Xenopus egg extract (XEE) cell-free assay system that DNA topoisomerase IIalpha (Topo IIalpha) is modified by SUMO-2/3 on mitotic chromosomes in the early stages of mitosis. Inhibition of mitotic SUMOylation in the XEE assay system causes aberrant sister chromatid separation in anaphase and alters Topo IIalpha association with chromosomes.
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http://dx.doi.org/10.1007/978-1-60761-340-4_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703827PMC
January 2010

Stimulating parathyroid cell proliferation and PTH release with phosphate in organ cultures obtained from patients with primary and secondary hyperparathyroidism for a prolonged period.

J Bone Miner Metab 2009 6;27(2):224-33. Epub 2009 Feb 6.

Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical University, Kawada-cho 8-1, Shinjuku-ku, Tokyo 162-8666, Japan.

The pathogenesis of primary hyperparathyroidism (I degrees -HPT) and secondary hyperparathyroidism (II degrees -HPT) remains to be elucidated. To characterize their pathophysiology, we investigated the effects of calcium and phosphate on cell proliferation and PTH release in an organ culture of parathyroid tissues. Dissected parathyroid tissues obtained from patients with I degrees -HPT (adenoma) or II degrees -HPT (nodular hyperplasia) were precultured on a collagen-coated membrane for 1-4 week. After changing the medium for one containing various concentrations of phosphate, PTH release and [(3)H]thymidine incorporation were studied. In contrast to dispersed parathyroid cells cultured in a monolayer, calcium decreased PTH release in a concentration-dependent manner in parathyroid tissues. Furthermore, when parathyroid tissues obtained from II degrees -HPT were precultured for 1-4 weeks, PTH release and parathyroid cell proliferation were significantly increased in high-phosphate medium. These phosphate effects were also observed to a lesser extent in parathyroid tissues obtained from I degrees -HPT, but there was no significant difference between I degrees -HPT and II degrees -HPT. Microarray analyses revealed that mRNA levels of PTH, CaSR, and VDR were well preserved, and several growth factors (e.g. TGF-beta1-induced protein) were abundantly expressed in II degrees -HPT. Using organ cultures of hyperparathyroid tissues, in which PTH release and CaSR are well preserved for a prolonged period, we have demonstrated that phosphate stimulates parathyroid cell proliferation not only in II degrees -HPT but also in I degrees -HPT. Although the mechanism responsible for phosphate-induced cell proliferation remains to be elucidated, our in vitro findings suggest that both parathyroid tissues preserve to some extent a physiological response system to hyperphosphatemia as observed in normal parathyroid cells.
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http://dx.doi.org/10.1007/s00774-008-0032-8DOI Listing
April 2009

Interaction between tumor suppressor adenomatous polyposis coli and topoisomerase IIalpha: implication for the G2/M transition.

Mol Biol Cell 2008 Oct 16;19(10):4076-85. Epub 2008 Jul 16.

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.

The tumor suppressor adenomatous polyposis coli (APC) is implicated in regulating multiple stages of the cell cycle. APC participation in G1/S is attributed to its recognized role in Wnt signaling. APC function in the G2/M transition is less well established. To identify novel protein partners of APC that regulate the G2/M transition, APC was immunoprecipitated from colon cell lysates and associated proteins were analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). Topoisomerase IIalpha (topo IIalpha) was identified as a potential binding partner of APC. Topo IIalpha is a critical regulator of G2/M transition. Evidence supporting an interaction between endogenous APC and topo IIalpha was obtained by coimmunoprecipitation, colocalization, and Förster resonance energy transfer (FRET). The 15-amino acid repeat region of APC (M2-APC) interacted with topo IIalpha when expressed as a green fluorescent protein (GFP)-fusion protein in vivo. Although lacking defined nuclear localization signals (NLS) M2-APC predominantly localized to the nucleus. Furthermore, cells expressing M2-APC displayed condensed or fragmented nuclei, and they were arrested in the G2 phase of the cell cycle. Although M2-APC contains a beta-catenin binding domain, biochemical studies failed to implicate beta-catenin in the observed phenotype. Finally, purified recombinant M2-APC enhanced topo IIalpha activity in vitro. Together, these data support a novel role for APC in the G2/M transition, potentially through association with topo IIalpha.
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http://dx.doi.org/10.1091/mbc.e07-12-1296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2555924PMC
October 2008

Role of the tumor necrosis factor-alpha, cyclooxygenase-2, prostaglandin E2, and effect of low-intensity pulsed ultrasound in an in vitro herniated disc resorption model.

J Orthop Res 2008 Sep;26(9):1274-8

Biomedical Engineering Laboratories, Teijin Pharma Ltd., 4-3-2 Asahigaoka, Hino, Tokyo 191-8512, Japan. ac.jp

Spontaneous herniated disc resorption occurs via inflammatory reactions involving abundant neovascularization and macrophage phagocytotic activity. Nonthermal low-intensity pulsed ultrasound (LIPUS) treatment might be effective in shortening the duration of disc resorption. We developed a rat in vitro resorption model in which a coccygeal intervertebral disc and peritoneal macrophages were cocultured. Secretion of tumor necrosis factor-alpha (TNF-alpha) from macrophages was promoted by LIPUS, and the process of disc degeneration was thus accelerated. In this study, we further examined the effects of LIPUS using this in vitro model focusing on whether LIPUS affects cyclooxygenase-2 (COX-2) signaling pathways. We found that the levels of COX-2 and prostaglandin E2 (PGE2) secreted from macrophages were increased by LIPUS. However, these phenomena were not caused by LIPUS directly, as the levels of these substances were reduced by neutralizing TNF-alpha activity. Moreover, the wet weights of the disc samples were not changed by addition of PGE2, but were reduced by recombinant TNF-alpha. Our results suggest that the effects of LIPUS in enhancing the process of herniated disc resorption are caused mainly by TNF-alpha.
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http://dx.doi.org/10.1002/jor.20525DOI Listing
September 2008

Akt1 in osteoblasts and osteoclasts controls bone remodeling.

PLoS One 2007 Oct 24;2(10):e1058. Epub 2007 Oct 24.

Department of Sensory and Motor System Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001058PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2020440PMC
October 2007