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1.
Biochim Biophys Acta ; 1813(1): 1-13, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21056597

RESUMEN

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.


Asunto(s)
Apoptosis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/patología , Mitocondrias/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Western Blotting , Caspasas/metabolismo , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión al GTP rab/genética
2.
Cell Biol Toxicol ; 27(1): 69-81, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20680429

RESUMEN

Epithelia in lung, skin, and kidney are often exposed to fluoride, and tissue damage in lung and kidney due to fluoride is well documented. Nevertheless, the biological effects of fluoride on epithelia are poorly investigated. In the present study, we report effects of sodium fluoride (NaF) on the differentiation of a human epithelial cell line, HaCaT. These cells may serve as a keratinocyte model, because they express a wide spectrum of keratins (Ks), and they associate into stratified tissue-like arrangements along with changes in their keratin pattern. NaF was added to the culture medium at concentrations of 0.5 and 5 mM. Cell proliferation remained intact, but cell functions were altered at high dose, and HSP70 was induced. Reverse transcription-polymerase chain reaction and Western blotting revealed that keratin (K) 15 mRNA and protein expression, associated with stratification of epithelia, were inhibited. Also, expression of keratins typical for terminal differentiation, K1 and K10, was decreased and so was the expression of the K1/10 regulating enhancer binding protein c/EBP alpha. Stratification of HaCaT cells was abolished at high fluoride dose, as assessed by electron microscopy. The changes in keratin expression were not reversed by withdrawal of fluoride. Taken together, NaF at high dose blocked terminal differentiation of HaCaT cells, visible by keratin expression and failing stratification. This effect may disturb tissue formation due to altered cell interactions.


Asunto(s)
Queratinocitos/efectos de los fármacos , Queratinas/biosíntesis , Fluoruro de Sodio/farmacología , Células Cultivadas , Expresión Génica , Humanos , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Queratinas/genética
3.
Eur J Oral Sci ; 118(1): 39-46, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20156263

RESUMEN

Tooth resorption occurs in 20-75% of cats (Felis catus). The aetiology is not known, but vitamin D is suggested to be involved. Vitamin D acts through a nuclear receptor (VDR) and increases the expression of receptor activator of nuclear factor-kappaB ligand (rankl) and muscle segment homeobox 2 (msx2) genes. Mice lacking the muscle segment homeobox 2 (msx2) gene show decreased levels of rankl, suggesting an interaction among VDR, MSX2, and RANKL. Here, we investigated the expression of VDR, MSX2, and RANKL proteins, and the activity of the VDR-mediated signalling pathway (using the quantitative polymerase chain reaction on VDR target genes), in tooth resorption, and measured the serum levels of vitamin D metabolites in cats. Tooth resorption was categorized into either resorptive or reparative stages. In the resorptive stage, odontoclasts expressed MSX2 and RANKL (100% and 88%, respectively) and fibroblasts expressed VDR and MSX2 (both at 100%), whereas fibroblasts expressed RANKL in only 29% of the sites analysed. In the reparative stage, cementoblasts expressed VDR, MSX2, and RANKL, whereas fibroblasts expressed VDR and MSX2, but not RANKL. The vitamin D status did not differ between the groups, based on the serum levels of 25-hydroxycholecalciferol. However, increased expression of VDR protein, and the relative gene expression levels of 1alpha-hydroxylase and the VDR-target gene, 24-hydroxylase, indicated the involvement of an active vitamin D signalling in the pathophysiology of tooth resorption in cats.


Asunto(s)
Proteínas de Homeodominio/biosíntesis , Ligando RANK/biosíntesis , Receptores de Calcitriol/biosíntesis , Resorción Dentaria/metabolismo , Vitamina D/fisiología , Animales , Gatos , Cemento Dental/metabolismo , Femenino , Fibroblastos/metabolismo , Proteínas de Homeodominio/genética , Hidroxicolecalciferoles/sangre , Técnicas para Inmunoenzimas , Inmunohistoquímica , Masculino , Osteoclastos/metabolismo , Ligando RANK/genética , Receptores de Calcitriol/genética , Transducción de Señal , Resorción Dentaria/patología
4.
Hum Pathol ; 37(3): 256-63, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16613320

RESUMEN

In a large-scale analysis of gene expression in pancreatic cancer, we isolated the homologue of the mouse Rab20. The mouse protein was previously identified during a search for novel Rab proteins, a family of small GTP-binding proteins involved in the regulation of intracellular vesicular transport. The Rab20 protein has no close relationship to any member of the Rab protein subfamily. In contrast to other members, it contains an insertion of 40 amino acids of unknown function and an inversion of 3 amino acids at the position corresponding to codon 61 in p21ras proteins. Using immunofluorescence and immunoelectron microscopy, we localized the Rab20 protein in the vicinity of the Golgi apparatus. Rab20 expression was detected by Western blot analysis in 11 of 11 pancreatic tumor cell lines and 7 of 8 primary pancreatic carcinomas. Absent or very faint expression was observed in normal pancreas cell extracts. Immunohistochemical analysis of Rab20 in tissues showed low or absent expression in normal pancreas and stronger expression in 15 of 18 exocrine pancreatic adenocarcinomas. Rab20 was also detected in preneoplastic pancreatic intraductal neoplasia lesions, suggesting that its up-regulation may be an early event in pancreatic carcinogenesis.


Asunto(s)
Adenocarcinoma/genética , Regulación Neoplásica de la Expresión Génica , Páncreas Exocrino/metabolismo , Neoplasias Pancreáticas/genética , Proteínas de Unión al GTP rab/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Secuencia de Aminoácidos , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Páncreas Exocrino/anatomía & histología , Páncreas Exocrino/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Homología de Secuencia de Aminoácido
5.
Cancer Genet Cytogenet ; 133(1): 55-60, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11890990

RESUMEN

We have identified a novel human cDNA overexpressed in a colon carcinoma cell line, TC7, established from a tumor with a normal karyotype arising in a patient with a hereditary nonpolyposis colorectal carcinoma. The OCC-1 (overexpressed in colon carcinoma-1) gene is composed of six exons and located in the q24.1 region of chromosome 12. It is transcribed as two mRNA species that differ in their 5'- and 3'-terminal ends. Abundant accumulation of both transcripts was found in placenta, skeletal muscle, kidney, and pancreas tissues. Absent or very faint expression was observed in heart, brain, lung and liver tissues. Overexpressed in colon carcinoma-1 cDNA direct in vitro translation of several polypeptides whose size is shorter than 9 kDa. Attempts to produce antibodies against these synthesized polypeptides in Escherichia coli failed. The absence of sequences at the mRNA and DNA levels hybridizing with mouse sequences together with the absence of a large open reading frame raise the possibility that OCC-1 sequences appeared recently in evolution and are transcribed as two noncoding regulatory RNA. Elevated levels of OCC-1 mRNA were observed in three of eight colon carcinomas as compared to normal mucosa of the same patient. Since these tumors shared the same characteristics of having a near diploid karyotype, OCC-1 overexpression may be a hallmark of this subset of colon carcinomas.


Asunto(s)
Cromosomas Humanos Par 12 , Neoplasias del Colon/genética , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , ARN Mensajero/genética , Adulto , Animales , Mapeo Cromosómico , ADN Complementario/aislamiento & purificación , Femenino , Humanos , Ratones , Sistemas de Lectura Abierta/genética , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas
6.
J Bone Miner Res ; 29(6): 1446-55, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24967458

RESUMEN

Microarray analysis of odontoblastic cells treated with sodium fluoride has identified the asporin gene as a fluoride target. Asporin is a member of the small leucine-rich repeat proteoglycan/protein (SLRP) family that is believed to be important in the mineralization process. In this study, asporin expression and distribution were investigated by systematic analysis of dentin and enamel, with and without fluoride treatment. Specific attention was focused on a major difference between the two mineralized tissues: the presence of a collagenous scaffold in dentin, and its absence in enamel. Normal and fluorotic, continually growing incisors from Wistar rats treated with 2.5 to 7.5 mM sodium fluoride (NaF) were studied by immunochemistry, in situ hybridization, Western blotting, and RT-qPCR. Asporin was continuously expressed in odontoblasts throughout dentin formation as expected. Asporin was also found, for the first time, in dental epithelial cells, particularly in maturation-stage ameloblasts. NaF decreased asporin expression in odontoblasts and enhanced it in ameloblasts, both in vivo and in vitro. The inverse response in the two cell types suggests that the effector, fluoride, is a trigger that elicits a cell-type-specific reaction. Confocal and ultrastructural immunohistochemistry evidenced an association between asporin and type 1 collagen in the pericellular nonmineralized compartments of both bone and dentin. In addition, transmission electron microscopy revealed asporin in the microenvironment of all cells observed. Thus, asporin is produced by collagen-matrix-forming and non-collagen-matrix-forming cells but may have different effects on the mineralization process. A model is proposed that predicts impaired mineral formation associated with the deficiency and excess of asporin.


Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Fluoruro de Sodio/farmacología , Ameloblastos/efectos de los fármacos , Ameloblastos/metabolismo , Animales , Línea Celular , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Proteínas de la Matriz Extracelular/genética , Fluorosis Dental/genética , Fluorosis Dental/patología , Regulación de la Expresión Génica/efectos de los fármacos , Incisivo/efectos de los fármacos , Incisivo/metabolismo , Incisivo/ultraestructura , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Odontoblastos/efectos de los fármacos , Odontoblastos/metabolismo , Odontoblastos/ultraestructura , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar
7.
PLoS One ; 8(11): e80054, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24278237

RESUMEN

The MSX2 homeoprotein is implicated in all aspects of craniofacial skeletal development. During postnatal growth, MSX2 is expressed in all cells involved in mineralized tissue formation and plays a role in their differentiation and function. Msx2 null (Msx2 (-/-)) mice display complex craniofacial skeleton abnormalities with bone and tooth defects. A moderate form osteopetrotic phenotype is observed, along with decreased expression of RANKL (TNFSF11), the main osteoclast-differentiating factor. In order to elucidate the role of such an osteopetrosis in the Msx2 (-/-) mouse dental phenotype, a bone resorption rescue was performed by mating Msx2 (-/-) mice with a transgenic mouse line overexpressing Rank (Tnfrsf11a). Msx2 (-/-) Rank(Tg) mice had significant improvement in the molar phenotype, while incisor epithelium defects were exacerbated in the enamel area, with formation of massive osteolytic tumors. Although compensation for RANKL loss of function could have potential as a therapy for osteopetrosis, but in Msx2 (-/-) mice, this approach via RANK overexpression in monocyte-derived lineages, amplified latent epithelial tumor development in the peculiar continuously growing incisor.


Asunto(s)
Proteínas de Homeodominio/fisiología , Osteopetrosis/fisiopatología , Ligando RANK/fisiología , Diente , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microtomografía por Rayos X
8.
J Histochem Cytochem ; 60(8): 603-10, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22614360

RESUMEN

Amelogenesis involves the coordinated expression of a set of molecules that includes enamel matrix proteins and calcium-binding proteins. Msx2 is a member of the divergent homeobox gene family and is instrumental in dental morphogenesis and biomineralization. This study focused on an EF-hand calcium-binding protein, calbindin-D(28k), which is highly expressed in dental epithelium. In vivo data showed that calbindin-D(28k) levels were higher in ameloblasts from Msx2(+/-) mice than Msx2(+/+) mice. Consistent with this finding, calbindin-D(28k) distribution was affected in transgenic mice with ectopic expression in root epithelium in rests of Malassez in Msx2(+/-) and more clearly in Msx2(-/-) mice. In accordance with these in vivo data, calbindin-D(28k) protein and mRNA levels were decreased in LS8 ameloblast-like cells by exogenous Msx2 overexpression. Furthermore, calbindin-D(28k) promoter activity (nt-1075/+34) was specifically diminished in the presence of Msx2 overexpression, showing that Msx2 behave as a transcriptional repressor for calbindin-D(28k) gene expression. In conclusion, Msx2 may control the spatiotemporally restricted frame of calbindin-D(28k) production in the dental epithelium in relation to enamel mineralization, as previously shown for amelogenin.


Asunto(s)
Células Epiteliales/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Ameloblastos/citología , Ameloblastos/metabolismo , Animales , Calbindinas , Línea Celular , Técnicas de Sustitución del Gen , Proteínas de Homeodominio/genética , Inmunohistoquímica , Incisivo/metabolismo , Ratones , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteína G de Unión al Calcio S100/genética , Raíz del Diente/metabolismo
9.
Mol Cell Biochem ; 271(1-2): 215-23, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15881673

RESUMEN

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1-6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína SUMO-1/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Factores de Transcripción de Tipo Kruppel , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Represoras/química , Factores de Transcripción/química , Enzimas Ubiquitina-Conjugadoras/genética , Dedos de Zinc
10.
J Cell Biochem ; 95(4): 763-8, 2005 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-15838871

RESUMEN

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We found an interaction between OZF and the telomeric hRap1 protein with a yeast two-hybrid screen. hRap1 (TERF2IP) is an ortholog of the yeast telomeric protein, scRap1 originally identified as a regulator of telomere length. In HeLa cells, it interacts with TRF2, a telomere repeat binding factor whose inactivation causes a dysregulation of telomere length and structure. Immunoprecipitation with anti-hRap1 antibodies in conditions that allow the purification of proteins associated with hRap1, demonstrated that OZF binds to hRap1 in HeLa cells. Using deletion mutants, we mapped the interacting domain of each protein. The three zinc fingers at the C-terminus of OZF interact with a region of hRap1 located downstream of the coil domain. It involves a stretch of at least 25 amino acids at the C-terminus of hRap1 that interact with TRF2. This suggests that OZF overexpression in tumours may alter the balance between hRap1 and other telomeric proteins and therefore that OZF function may be linked to telomere regulation.


Asunto(s)
Neoplasias del Colon/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Dedos de Zinc , Neoplasias del Colon/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Inmunoprecipitación , Factores de Transcripción de Tipo Kruppel , Unión Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Técnicas del Sistema de Dos Híbridos
11.
J Pathol ; 200(2): 177-82, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12754738

RESUMEN

Overexpression of the OZF gene has previously been demonstrated in the majority of pancreatic cancers. However, because the stages of tumour progression in this disease are poorly defined, no conclusion could be drawn concerning the relationship between OZF overexpression and the course of tumour progression. In contrast, initiation and progression steps are well defined in colorectal cancer. Most colon cancers are believed to arise from polypoid adenomas as a result of the gradual accumulation of genetic alterations, allowing the study of genetic events in the early stages of neoplasia. Accordingly, we wanted to assess the frequency of OZF overexpression in this tumour type and the relationship between overexpression and disease stage. Twenty-five colon carcinoma specimens from different sites and at various stages were analysed by immunoblotting using a highly specific mouse monoclonal antibody. Each sample was compared with adjacent normal colonic mucosa. Complementary immunohistochemical analysis was also carried out on a commercially available tissue array to identify cells expressing OZF. Of the 25 tumours analysed by immunoblotting, 20 expressed higher levels of OZF protein than their adjacent normal mucosa. Immunohistochemistry revealed OZF expression in tumour cells of 56/59 carcinomas and occasionally in infiltrating lymphocytes but at lower levels. Little or no staining was observed in cells taken from normal or inflammatory colon specimens. In both immunoblot and immunohistochemistry experiments, no correlation was found between OZF expression and clinical parameters such as TNM classification, stage, localization and age. Immunostaining was observed in low-grade adenomas, indicating that OZF expression occurs very early in the course of tumour progression. OZF expression, infrequent or absent in normal colonic mucosa, is present in more than 80% of colorectal cancers. Dysregulation of the OZF gene is an early event that may be implicated in the genesis of colonic carcinoma and may therefore provide a potential therapeutic target.


Asunto(s)
Adenoma/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenoma/patología , Anciano , Anciano de 80 o más Años , Western Blotting , Diferenciación Celular , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Dedos de Zinc
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