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1.
Mol Cell ; 74(4): 742-757.e8, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30979586

RESUMO

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Doenças por Armazenamento dos Lisossomos/genética , Doenças Musculares/genética , Proteínas Serina-Treonina Quinases/genética , Proteína com Valosina/genética , Adenosina Trifosfatases/genética , Animais , Autofagia/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Camundongos , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Fosforilação/genética , Estresse Fisiológico/genética , Ubiquitina/genética
2.
Mol Cell ; 62(4): 491-506, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27203176

RESUMO

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Encéfalo/enzimologia , Retículo Endoplasmático/enzimologia , Fibroblastos/enzimologia , Complexo de Golgi/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Autofagia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Encéfalo/patologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/patologia , Feminino , Genótipo , Complexo de Golgi/patologia , Células HEK293 , Homeostase , Humanos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Interferência de RNA , Fatores de Tempo , Transfecção , Resposta a Proteínas não Dobradas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
Mol Cell ; 43(4): 572-85, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21855797

RESUMO

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Autofagia/fisiologia , Proteínas de Ciclo Celular/fisiologia , Chaperoninas/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Chaperoninas/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
5.
Exp Cell Res ; 316(4): 507-16, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20025870

RESUMO

In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor alpha are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCalpha. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.


Assuntos
Angiopoietinas/metabolismo , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Northern Blotting , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia
6.
Cancer Res ; 67(16): 7929-36, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17699800

RESUMO

Farnesol (FOH) and other isoprenoid alcohols induce apoptosis in various carcinoma cells and inhibit tumorigenesis in several in vivo models. However, the mechanisms by which they mediate their effects are not yet fully understood. In this study, we show that FOH is an effective inducer of apoptosis in several lung carcinoma cells, including H460. This induction is associated with activation of several caspases and cleavage of poly(ADP-ribose) polymerase (PARP). To obtain insight into the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and control H460 cells by microarray analysis. This analysis revealed that many genes implicated in endoplasmic reticulum (ER) stress signaling, including ATF3, DDIT3, HERPUD1, HSPA5, XBP1, PDIA4, and PHLDA1, were highly up-regulated within 4 h of FOH treatment, suggesting that FOH-induced apoptosis involves an ER stress response. This was supported by observations showing that treatment with FOH induces splicing of XBP1 mRNA and phosphorylation of eIF2alpha. FOH induces activation of several mitogen-activated protein kinase (MAPK) pathways, including p38, MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK, and c-jun NH2-terminal kinase (JNK). Inhibition of MEK1/2 by U0126 inhibited the induction of ER stress response genes. In addition, knockdown of the MEK1/2 and JNK1/2 expression by short interfering RNA (siRNA) effectively inhibited the cleavage of caspase-3 and PARP and apoptosis induced by FOH. However, only MEK1/2 siRNAs inhibited the induction of ER stress-related genes, XBP1 mRNA splicing, and eIF2alpha phosphorylation. Our results show that FOH-induced apoptosis is coupled to ER stress and that activation of MEK1/2 is an early upstream event in the FOH-induced ER stress signaling cascade.


Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Farneseno Álcool/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Interferente Pequeno/genética
7.
J Cell Biochem ; 103(4): 1183-97, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17721932

RESUMO

S100A6 (calcyclin) is a small calcium-binding protein which has been implicated in several cellular processes such as cell cycle progression, cytoskeleton rearrangement, and exocytosis. Also the upregulation of S100A6 has been reported in a variety of tumors and linked to metastasis. However, exact intracellular roles of S100A6 related with apoptosis have not been clarified yet. Here we demonstrated that the upregulation of S100A6 enhances the cell death rate compared to the control under the apoptotic conditions. In exogenously S100A6 induced Hep3B cells, cell viability was significantly decreased compared with mock and S100A6-knockdown cells under calcium ionophore A23187 treatment. The exogenously introduced S100A6 significantly affected the caspase-3-like activity in programmed cell death through the enhanced caspase-3 expression, which was verified by promoter assay in wild or mutant S100A6-transfected Hep3B cells. Next, the promoter activity of caspase-3 was increased by 2.5-folds in wild-type S100A6-transfected cells compared to mutant 2 (E67K, mutant of EF-hand motif) or control. Our results suggest that S100A6 might be involved in the processing of apoptosis by modulating the transcriptional regulation of caspase-3.


Assuntos
Apoptose/fisiologia , Calcimicina/farmacologia , Caspase 3/biossíntese , Proteínas de Ciclo Celular/fisiologia , Proteínas S100/fisiologia , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Ionóforos/farmacologia , Mutação , Regiões Promotoras Genéticas , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/genética , Regulação para Cima
8.
Autophagy ; 14(5): 796-811, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29099309

RESUMO

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1)+ or ubiquitin+ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Orientação de Axônios , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteínas Relacionadas à Autofagia , Axônios/metabolismo , Axônios/ultraestrutura , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Córtex Somatossensorial/metabolismo , Proteínas Ubiquitinadas/metabolismo
9.
J Clin Invest ; 128(8): 3319-3332, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29939162

RESUMO

SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A-D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss of SEC24C. Conditional KO of Sec24c in neural progenitors during embryogenesis caused perinatal mortality and microcephaly, with activation of the unfolded protein response and apoptotic cell death of postmitotic neurons in the murine cerebral cortex. The cell-autonomous function of SEC24C in postmitotic neurons was further highlighted by the loss of cell viability caused by disrupting Sec24c expression in forebrain neurons of mice postnatally and in differentiated neurons derived from human induced pluripotent stem cells. The neuronal cell death associated with Sec24c deficiency was rescued in knockin mice expressing Sec24d in place of Sec24c. These data suggest that SEC24C is a major cargo adapter for COPII-dependent transport in postmitotic neurons in developing and adult brains and that its functions overlap at least partially with those of SEC24D in mammals.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Homeostase , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Prosencéfalo/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Neurônios/citologia , Prosencéfalo/citologia , Proteínas de Transporte Vesicular/genética
10.
Biochem Pharmacol ; 97(3): 256-68, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26275811

RESUMO

In this study, we demonstrate that treatment of T lymphoblastic leukemic Molt4 cells with farnesol activates the apoptosome via the intrinsic pathway of apoptosis. This induction was associated with changes in the level of intracellular potassium and calcium, the dissipation of the mitochondrial and plasma membrane potential, release of cytochrome c, activation of several caspases, and PARP cleavage. The induction of apoptosis by farnesol was inhibited by the addition of the pan-caspase inhibitor Z-VAD-fmk and by the exogenous expression of the anti-apoptotic protein Bcl2. Analysis of the gene expression profiles by microarray analysis revealed that farnesol increased the expression of several genes related to the unfolded protein response (UPR), including CHOP and CHAC1. This induction was associated with the activation of the PERK-eIF2α-ATF3/4 cascade, but not the XBP-1 branch of the UPR. Although farnesol induced activation of the ERK1/2, p38, and JNK pathways, inhibition of these MAPKs had little effect on farnesol-induced apoptosis or the induction of UPR-related genes. Our data indicate that the induction of apoptosis in leukemic cells by farnesol is mediated through a pathway that involves activation of the apoptosome via the intrinsic pathway and induction of the PERK-eIF2α-ATF3/4 cascade in a manner that is independent of the farnesol-induced activation of MAPKs.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Farneseno Álcool/farmacologia , Fator de Transcrição CHOP/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/efeitos dos fármacos , Transfecção
11.
Cancer Lett ; 203(2): 217-24, 2004 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-14732230

RESUMO

To clarify the roles of Bmi-1 in colorectal carcinoma, we examined the expression of Bmi-1 in 41 samples out of 46 colorectal carcinomas by reverse transcription-PCR, whereas all 46 were analyzed by immunostaining. In addition, we analyzed the expression patterns of Bmi-1 in association with p16INK4a and p14ARF (in mouse p19ARF) in a series of colorectal carcinomas. The level of Bmi-1 mRNA in the carcinoma tissues was significantly higher than those of the adjacent non-neoplastic colonic mucosal tissues. Immunohistochemistry for Bmi-1 showed moderate or strong expression levels in 65% (30/46) of colorectal carcinomas. Colorectal carcinomas with moderate or strong Bmi-1 expression were more likely to have low levels of the INK4 locus proteins (p16INK4a/p14ARF) (P<0.07). These results suggested that modulation of Bmi-1 protein might be involved in human colorectal carcinogenesis by repressing the INK4a/ARF proteins.


Assuntos
Carcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras , Proteína Supressora de Tumor p14ARF/biossíntese , Regulação para Cima , Adulto , Diferenciação Celular , Colo/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mucosa/patologia , Neoplasias/patologia , Complexo Repressor Polycomb 1 , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dedos de Zinco
12.
Breast ; 13(5): 383-8, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15454193

RESUMO

The modulation of Bmi-1 is observed in several tumor tissues, and its heightened protein level is suspected to be involved in tumorigenesis by acting as a transcriptional repressor in the INK4a/ARF locus. To elucidate the modulation of Bmi-1 in invasive ductal breast cancers, we examined its transcript and protein levels. The bmi-1 mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) showed that it was significantly up-regulated in 28 specimens out of 33 breast carcinoma tissues compared with those of non-neoplastic tissues just adjusted to tested specimens. Immunohistochemical staining for Bmi-1 also showed that 44 specimens out of 71 breast carcinoma tissues (62%) had strong positive signals with a more intense staining pattern in the invading fronts than in the central portions of primary invasive breast cancers. Univariate and multivariate analyses showed that a high level of Bmi-1 expression was significantly correlated with axillary lymph node metastases and positive estrogen receptor status. These findings suggested that Bmi-1 might be involved in the tumor progression and metastasis of invasive ductal breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras/biossíntese , Axila , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Proteínas Oncogênicas/biossíntese , Complexo Repressor Polycomb 1 , Receptores de Esteroides/biossíntese
13.
Cancer Lett ; 287(2): 123-35, 2010 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-19520495

RESUMO

The isoprenoid alcohol farnesol is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, farnesol has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventative and anti-tumor agent in vivo. A number of different biochemical and cellular processes have been implicated in the growth-inhibitory and apoptosis-inducing effects of farnesol. These include regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and CTP:phosphocholine cytidylyltransferase alpha (CCTalpha), rate-limiting enzymes in the mevalonate pathway and phosphatidylcholine biosynthesis, respectively, and the generation of reactive oxygen species. In some cell types the action of farnesol is mediated through nuclear receptors, including activation of farnesoid X receptor (FXR) and peroxisome proliferator-activated receptors (PPARs). Recent studies have revealed that induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) play a critical role in the induction of apoptosis by farnesol in lung carcinoma cells. This induction was found to be dependent on the activation of the MEK1/2-ERK1/2 pathway. In addition, farnesol induces activation of the NF-kappaB signaling pathway and a number of NF-kappaB target genes. Optimal activation of NF-kappaB was reported to depend on the phosphorylation of p65/RelA by the MEK1/2-MSK1 signaling pathway. In a number of cells farnesol-induced apoptosis was found to be linked to activation of the apoptosome. This review provides an overview of the biochemical and cellular processes regulated by farnesol in relationship to its growth-inhibitory, apoptosis-promoting, and anti-tumor effects.


Assuntos
Apoptose , Farneseno Álcool/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptossomas/metabolismo , Proliferação de Células , Citidina Difosfato Colina/metabolismo , Retículo Endoplasmático/metabolismo , Farneseno Álcool/farmacologia , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Neoplasias/patologia , Estresse Oxidativo , PPAR gama/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores beta dos Hormônios Tireóideos/metabolismo , Resposta a Proteínas não Dobradas
14.
J Biol Chem ; 283(24): 16391-9, 2008 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-18424438

RESUMO

In this study, we demonstrate that treatment of human lung adenocarcinoma H460 cells with farnesol induces the expression of a number of immune response and inflammatory genes, including IL-6, CXCL3, IL-1alpha, and COX-2. This response was dependent on the activation of the NF-kappaB signaling pathway. Farnesol treatment reduces the level of IkappaBalpha and induces translocation of p65/RelA to the nucleus, its phosphorylation at Ser(276), and transactivation of NF-kappaB-dependent transcription. Moreover, overexpression of IkappaBalpha or treatment with the NF-kappaB inhibitor caffeic acid phenethyl ester greatly diminishes the induction of inflammatory gene expression by farnesol. We provide evidence indicating that the farnesol-induced phosphorylation of p65/RelA at Ser(276) is important for optimal transcriptional activity of NF-kappaB. The MEK1/2 inhibitor U0126 and knockdown of MEK1/2 expression with small interfering RNAs effectively blocked the phosphorylation of p65/RelA(Ser(276)) but not that of Ser(536), suggesting that this phosphorylation is dependent on the activation of the MEK1/2-ERK1/2 pathway. We further show that inhibition of MSK1, a kinase acting downstream of MEK1/2-ERK1/2, by H89 or knockdown of MSK1 expression also inhibited phosphorylation of p65/RelA(Ser(276)), suggesting that this phosphorylation is dependent on MSK1. Knockdown of MEK1/2 or MSK1 expression inhibits farnesol-induced expression of CXCL3, IL-1alpha, and COX-2 mRNA. Our results indicate that the induction of inflammatory genes by farnesol is mediated by the activation of the NF-kappaB pathway and involves MEK1/2-ERK1/2-MSK1-dependent phosphorylation of p65/RelA(Ser(276)). The activation of the NF-kappaB pathway by farnesol might be part of a prosurvival response during farnesol-induced ER stress.


Assuntos
Farneseno Álcool/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 1/metabolismo , NF-kappa B/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Transcrição RelA/metabolismo , Ativação Transcricional , Linhagem Celular Tumoral , Humanos , Proteínas I-kappa B/metabolismo , Neoplasias Pulmonares/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , Transdução de Sinais
15.
Biochem Biophys Res Commun ; 362(1): 132-138, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17698038

RESUMO

RAP80, a nuclear protein with two functional ubiquitin-interaction motifs (UIMs) at its N-terminus, plays a critical role in the regulation of estrogen receptor alpha and DNA damage response signaling. A yeast two-hybrid screen identified the SUMO-conjugating enzyme UBC9 as a protein interacting with RAP80. The interaction of RAP80 with UBC9 was confirmed by co-immunoprecipitation and GST pull-down analyses. The region between aa 122-204 was critical for the interaction of RAP80 with UBC9. In addition, we demonstrate that RAP80 is a target for SUMO-1 modification in intact cells. Expression of UBC9 enhanced RAP80 mono-sumoylation and also induced multi-sumoylation of RAP80. In addition to SUMO-1, RAP80 was efficiently conjugated to SUMO-3 but was only a weak substrate for SUMO-2 conjugation. These findings suggest that sumoylation plays a role in the regulation of RAP80 functions.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas Nucleares/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA , Receptor alfa de Estrogênio/metabolismo , Células HeLa , Chaperonas de Histonas , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Radiação Ionizante , Proteína SUMO-1/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/química , Ubiquitina/metabolismo
16.
Adv Dev Biol ; 16: 313-355, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-18418469

RESUMO

Retinoid-related orphan receptors RORalpha, -beta, and -gamma are transcription factors belonging to the steroid hormone receptor superfamily. During embryonic development RORs are expressed in a spatial and temporal manner and are critical in the regulation of cellular differentiation and the development of several tissues. RORalpha plays a key role in the development of the cerebellum particularly in the regulation of the maturation and survival of Purkinje cells. In RORalpha-deficient mice, the reduced production of sonic hedgehog by these cells appears to be the major cause of the decreased proliferation of granule cell precursors and the observed cerebellar atrophy. RORalpha has been implicated in the regulation of a number of other physiological processes, including bone formation. RORbeta expression is largely restricted to several regions of the brain, the retina, and pineal gland. Mice deficient in RORbeta develop retinal degeneration that results in blindness. RORgamma is essential for lymph node organogenesis. In the intestine RORgamma is required for the formation of several other lymphoid tissues: Peyer's patches, cryptopatches, and isolated lymphoid follicles. RORgamma plays a key role in the generation of lymphoid tissue inducer (LTi) cells that are essential for the development of these lymphoid tissues. In addition, RORgamma is a critical regulator of thymopoiesis. It controls the differentiation of immature single-positive thymocytes into double-positive thymocytes and promotes the survival of double-positive thymocytes by inducing the expression of the anti-apoptotic gene Bcl-X(L). Interestingly, all three ROR receptors appear to play a role in the control of circadian rhythms. RORalpha positively regulates the expression of Bmal1, a transcription factor that is critical in the control of the circadian clock. This review intends to provide an overview of the current status of the functions RORs have in these biological processes.

17.
Biochem Biophys Res Commun ; 326(1): 7-17, 2005 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-15567145

RESUMO

Many fundamental processes, including oncogenesis, have implicated HECT domain proteins with ubiquitin ligase activity. The protein human upstream regulatory element binding protein 1 (hUREB1) is a HECT domain protein whose function is not defined yet. Here, we investigate the function of hUREB1 as a ubiquitin-protein ligase in human colorectal cells. Ectopic expression of the HECT domain of hUREB1 reduces the protein level and transcriptional activity of the p53 tumor suppressor, which is abrogated by the deletion in the HECT domain or point mutations in the essential residues of the HECT domain. The ubiquitination and destabilization of p53 is observed in cells treated with the protease inhibitor MG132, implying that the HECT domain of hUREB1 suppresses the transcriptional activity of p53 through a ubiquitin-dependent degradation pathway. Based on the results of Northern blot analysis, RT-PCR, and immunohistochemical analyses, the over-expression of hUREB1 is associated with colorectal carcinoma. Moreover, protein levels of hUREB1 and p53 were inversely correlated. These findings suggest that hUREB1 can function, at least in part, as a negative regulator of p53 during the colorectal carcinoma progression through the ubiquitination pathway mediated by the HECT domain.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfoproteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfoproteínas/química , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases
18.
Biochem Biophys Res Commun ; 307(2): 274-80, 2003 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-12859951

RESUMO

S100A6 (calcyclin) is an acidic calcium binding protein with two EF-hand motifs and overexpressed in several tumors including intrahepatic carcinoma. TNFalpha, a strong NF-kappaB activator required for hepatocyte proliferation during liver regeneration, triggered the expression of S100A6 mRNA in human hepatoblastoma cell line HepG2. Transient expression of NF-kappaB (p65) increased S100A6 promoter activity and expression of inhibitor of NF-kappaB (IkappaBalpha) decreased TNFalpha-induced S100A6 promoter activity. To confirm the involvement of NF-kappaB in S100A6 promoter activation, we analyzed serially deleted promoter constructs of the S100A6 gene by luciferase reporter assay and found a NF-kappaB-responsive DNA fragment at the position between -584 and -361. Electrophoretic mobility shift assays showed that TNFalpha induced p65 binding to a potential NF-kappaB binding site at -460/-451. Furthermore, treatment of cells with CAPE (caffeic acid phenethyl ester), a specific NF-kappaB (p65) inhibitor, decreased NF-kappaB binding and promoter activity. These results suggest that NF-kappaB transcription factor contributes to the activation of S100A6 gene expression in response to TNFalpha in HepG2 cells.


Assuntos
Proteínas de Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , NF-kappa B/metabolismo , Proteínas S100/genética , Sítios de Ligação , Hepatoblastoma/genética , Humanos , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo
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