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1.
Nature ; 480(7378): 557-60, 2011 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-22121020

RESUMEN

Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA. These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes. However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.


Asunto(s)
Acetilglucosamina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Células HeLa , Histonas/química , Histonas/genética , Humanos , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitinación
2.
Nature ; 459(7245): 455-9, 2009 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-19377461

RESUMEN

The post-translational modifications of histone tails generate a 'histone code' that defines local and global chromatin states. The resultant regulation of gene function is thought to govern cell fate, proliferation and differentiation. Reversible histone modifications such as methylation are under mutual controls to organize chromosomal events. Among the histone modifications, methylation of specific lysine and arginine residues seems to be critical for chromatin configuration and control of gene expression. Methylation of histone H3 lysine 4 (H3K4) changes chromatin into a transcriptionally active state. Reversible modification of proteins by beta-N-acetylglucosamine (O-GlcNAc) in response to serum glucose levels regulates diverse cellular processes. However, the epigenetic impact of protein GlcNAcylation is unknown. Here we report that nuclear GlcNAcylation of a histone lysine methyltransferase (HKMT), MLL5, by O-GlcNAc transferase facilitates retinoic-acid-induced granulopoiesis in human HL60 promyelocytes through methylation of H3K4. MLL5 is biochemically identified in a GlcNAcylation-dependent multi-subunit complex associating with nuclear retinoic acid receptor RARalpha (also known as RARA), serving as a mono- and di-methyl transferase to H3K4. GlcNAcylation at Thr 440 in the MLL5 SET domain evokes its H3K4 HKMT activity and co-activates RARalpha in target gene promoters. Increased nuclear GlcNAcylation by means of O-GlcNAc transferase potentiates retinoic-acid-induced HL60 granulopoiesis and restores the retinoic acid response in the retinoic-acid-resistant HL60-R2 cell line. Thus, nuclear MLL5 GlcNAcylation triggers cell lineage determination of HL60 through activation of its HKMT activity.


Asunto(s)
Acetilglucosamina/metabolismo , Proteínas de Unión al ADN/metabolismo , Granulocitos/citología , Granulocitos/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Leucopoyesis/efectos de los fármacos , N-Acetilglucosaminiltransferasas/metabolismo , Tretinoina/farmacología , Linaje de la Célula , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HL-60 , N-Metiltransferasa de Histona-Lisina/química , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , N-Acetilglucosaminiltransferasas/química , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Treonina/metabolismo
3.
Genes Cells ; 15(8): 867-73, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20629981

RESUMEN

The methylation states of histone lysine residues are regarded as significant epigenetic marks governing transcriptional regulation. A number of histone demethylases containing a jumonji C (JmjC) domain have been recognized; however, their properties remain to be investigated. Here, we show that KIAA1718, a PHF2/PHF8 subfamily member, possesses histone demethylase activity specific for H3K9 and H3K27, transcriptionally repressive histone marks. Biochemical purification of the KIAA1718 interactants reveals that KIAA1718 forms complexes with several factors including KAP1, a transcriptional co-activator. Consistent with these findings, KIAA1718 shows a transcriptional activation function in the chromatin context. Thus, our study identifies KIAA1718 as a histone demethylase for repressive methyl marks and shows that it is involved in transcriptional activation.


Asunto(s)
Histona Demetilasas/metabolismo , Histonas/metabolismo , Animales , Células Cultivadas , Histona Demetilasas/química , Humanos , Lisina/metabolismo , Metilación , Ratones
4.
Biochem Biophys Res Commun ; 394(4): 865-70, 2010 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-20206135

RESUMEN

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1alpha and -1beta genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1alpha gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1alpha gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.


Asunto(s)
Quimiocina CCL3/genética , Epigénesis Genética , Glucosa/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Línea Celular , Quimiocina CCL3/metabolismo , Regulación de la Expresión Génica , Glucosa/farmacología , Glicosilación , Humanos , Monocitos/enzimología , N-Acetilglucosaminiltransferasas/genética , Regiones Promotoras Genéticas , Transcripción Genética
5.
Front Immunol ; 8: 1206, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28993781

RESUMEN

Current tumor therapies, including immunotherapies, focus on passive eradication or at least reduction of the tumor mass. However, cancer patients quite often suffer from tumor relapse or metastasis after such treatments. To overcome these problems, we have developed a natural killer T (NKT) cell-targeted immunotherapy focusing on active engagement of the patient's immune system, but not directly targeting the tumor cells themselves. NKT cells express an invariant antigen receptor α chain encoded by Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans and recognize glycolipid ligand in conjunction with a monomorphic CD1d molecule. The NKT cells play a pivotal role in the orchestration of antitumor immune responses by mediating adjuvant effects that activate various antitumor effector cells of both innate and adaptive immune systems and also aid in establishing a long-term memory response. Here, we established NKT cell-targeted therapy using a newly discovered NKT cell glycolipid ligand, RK, which has a stronger capacity to stimulate both human and mouse NKT cells compared to previous NKT cell ligand. Moreover, RK mediates strong adjuvant effects in activating various effector cell types and establishes long-term memory responses, resulting in the continuous attack on the tumor that confers long-lasting and potent antitumor effects. Since the NKT cell ligand presented by the monomorphic CD1d can be used for all humans irrespective of HLA types, and also because NKT cell-targeted therapy does not directly target tumor cells, this therapy can potentially be applied to all cancer patients and any tumor types.

7.
Stem Cells Transl Med ; 1(8): 615-26, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23197867

RESUMEN

Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-ß2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-ß2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD(3)) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD(3) upregulation of Wnt10b, ALPL, and TGF-ß2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD(3) significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD(3) may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Dermis/citología , Dermis/metabolismo , Folículo Piloso/citología , Regeneración/efectos de los fármacos , Vitamina D/análogos & derivados , Fosfatasa Alcalina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Dermis/efectos de los fármacos , Folículo Piloso/efectos de los fármacos , Folículo Piloso/metabolismo , Humanos , Técnicas para Inmunoenzimas , Queratolíticos/farmacología , Masculino , Ratones , ARN Mensajero/genética , Ratas , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Vitamina D/farmacología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
8.
Eur J Pharmacol ; 629(1-3): 118-24, 2010 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-20006961

RESUMEN

Renal fibrosis is the final common pathway of chronic kidney disease, and its progression predicts the degree of renal dysfunction. We investigated the renoprotective properties of pirfenidone in a remnant kidney model of chronic renal failure to determine its pharmacological potency compared to enalapril. Five-sixths nephrectomized rats were fed diet containing pirfenidone (approximately 700mg/kg/day) for 8weeks. Pirfenidone steadily inhibited the progression of proteinuria, but not to a significant degree. Pirfenidone prevented the elevation of plasma creatinine and blood urea nitrogen. At the end of the experiment, pirfenidone had reduced systolic blood pressure by means of its renoprotective effect. In a histological study, pirfenidone improved interstitial fibrosis in the renal cortex. These effects were supported by the suppression of the expression of TGF-beta and fibronectin in the mRNA of the kidney. In contrast, pirfenidone had little effect on the expression of alpha-smooth muscle actin, which is one of the proteins responsible for epithelial-mesenchymal transition. This property was confirmed by the TGF-beta-induced transdifferentiation observed in cultured normal rat kidney tubular epithelial NRK52E cells. These results suggest that pirfenidone improves the progression of chronic renal failure via its antifibrotic action, although pirfenidone has less effective TGF-beta-induced epithelial to mesenchymal transdifferentiation.


Asunto(s)
Riñón/efectos de los fármacos , Riñón/cirugía , Nefrectomía , Piridonas/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Enfermedad Crónica , Progresión de la Enfermedad , Enalapril/farmacología , Células Epiteliales/patología , Fibrosis/tratamiento farmacológico , Riñón/metabolismo , Riñón/patología , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/patología , Masculino , Mesodermo/patología , Proteinuria/metabolismo , Piridonas/uso terapéutico , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta/farmacología
9.
J Orthop Res ; 26(9): 1274-8, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18383135

RESUMEN

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.


Asunto(s)
Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Desplazamiento del Disco Intervertebral/diagnóstico por imagen , Desplazamiento del Disco Intervertebral/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Western Blotting , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ratas , Ultrasonografía/métodos , Cicatrización de Heridas/inmunología
10.
Eur J Biochem ; 271(4): 809-20, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14764098

RESUMEN

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.


Asunto(s)
Angiostatinas/química , Activadores Plasminogénicos/farmacología , Plasminógeno/metabolismo , Aminoácidos/análisis , Ácido Aminocaproico/farmacología , Angiostatinas/farmacología , Animales , Benzopiranos/química , Benzopiranos/farmacología , Sitios de Unión , Células CHO , Bovinos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Clorofenoles/química , Clorofenoles/farmacología , Cricetinae , Endotelio Vascular/citología , Fibrinolisina/metabolismo , Humanos , Espectrometría de Masas/métodos , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Plasminógeno/química , Activadores Plasminogénicos/química , Pirrolidinonas/química , Pirrolidinonas/farmacología , Análisis de Secuencia de Proteína/métodos , Venas Umbilicales
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