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1.
Am J Physiol Gastrointest Liver Physiol ; 315(6): G980-G990, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30285465

RESUMEN

An impaired nitrergic system and altered redox signaling contribute to gastric dysmotility in diabetics. Our earlier studies show that NF-E2-related factor 2 (NRF2) and phase II antioxidant enzymes play a vital role in gastric neuronal nitric oxide synthase (nNOS) function. This study aims to investigate whether supplementation of sepiapterin (SEP), a precursor for tetrahydrobiopterin (BH4) (a cofactor of NOS) via the salvage pathway, restores altered nitrergic systems and redox balance in spontaneous diabetic (DB) female rats. Twelve-week spontaneous DB and age-matched, non-DB rats, with and without dietary SEP (daily 20 mg/kg body wt for 10 days) treatment, were used in this study. Gastric antrum muscular tissues were excised to investigate the effects of SEP in nitrergic relaxation and the nNOS-nitric oxide (NO)-NRF2 pathway(s). Dietary SEP supplementation significantly ( P < 0.05) reverted diabetes-induced changes in nNOS dimerization and function; nitric oxide (NO) downstream signaling molecules; HSP-90, a key regulator of nNOSα activity and dimerization; miRNA-28 that targets NRF2 messenger RNA (mRNA), and levels of microRNA (miRNA) biogenesis pathway components, such as DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and TRBP (HIV1-1 transactivating response RNA-binding protein). These findings emphasize the importance of the BH4 pathway in regulating gastric motility functions in DB animals by modulating nNOSα dimerization in association with changes in enteric NRF2 and NO downstream signaling. Our results also identify a new pathway, wherein SEP regulates NRF2 mRNA turnover by suppressing elevated miRNA-28, which could be related to alterations in miRNA biogenesis pathway components. NEW & NOTEWORTHY This study is the first to show a causal link between NF-E2-related factor 2 (NRF2) and neuronal nitric oxide synthase (nNOS) in gastric motility function. Our data demonstrate that critical regulators of the miRNA biosynthetic pathway are upregulated in the diabetic (DB) setting; these regulators were rescued by sepiapterin (SEP) treatment. Finally, we show that low dihydrofolate reductase expression may lead to impaired nNOS dimerization/function-reduced nitric oxide downstream signaling and elevate oxidative stress by suppressing the NRF2/phase II pathway through miRNA; SEP treatment restored all of the above in DB gastric muscular tissue. We suggest that tetrahydrobiopterin supplementation may be a useful therapy for patients with diabetes, as well as women with idiopathic gastroparesis.


Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Motilidad Gastrointestinal , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Pterinas/uso terapéutico , Píloro/efectos de los fármacos , Animales , Diabetes Mellitus/fisiopatología , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Relajación Muscular , Factor 2 Relacionado con NF-E2/genética , Pterinas/farmacología , Píloro/metabolismo , Píloro/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Transducción de Señal
2.
Gut ; 66(5): 852-862, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28389570

RESUMEN

OBJECTIVE: Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. DESIGN: We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. RESULTS: BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. CONCLUSION: Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.


Asunto(s)
Carcinogénesis/genética , Moléculas de Adhesión Celular/genética , Colitis Ulcerosa/metabolismo , Neoplasias del Colon/metabolismo , Proteínas de la Membrana/genética , Proteínas Musculares/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Biomarcadores de Tumor/genética , Células CACO-2 , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colitis Ulcerosa/genética , Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Metilación de ADN , Sulfato de Dextran , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , Vía de Señalización Wnt
3.
Stem Cells ; 34(6): 1626-36, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26891025

RESUMEN

Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Rayos gamma , Intestinos/citología , Proteínas Musculares/metabolismo , Células Madre/citología , Animales , Moléculas de Adhesión Celular/genética , Supervivencia Celular/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Femenino , Eliminación de Gen , Homeostasis/efectos de la radiación , Masculino , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Tolerancia a Radiación/efectos de la radiación , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de la radiación , Células Madre/metabolismo , Células Madre/efectos de la radiación , Vía de Señalización Wnt/efectos de la radiación
4.
Am J Physiol Gastrointest Liver Physiol ; 308(6): G562-71, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25573176

RESUMEN

Myeloid translocation genes (MTGs) are transcriptional corepressors implicated in development, malignancy, differentiation, and stem cell function. While MTG16 loss renders mice sensitive to chemical colitis, the role of MTG16 in the small intestine is unknown. Histological examination revealed that Mtg16(-/-) mice have increased enterocyte proliferation and goblet cell deficiency. After exposure to radiation, Mtg16(-/-) mice exhibited increased crypt viability and decreased apoptosis compared with wild-type (WT) mice. Flow cytometric and immunofluorescence analysis of intestinal epithelial cells for phospho-histone H2A.X also indicated decreased DNA damage and apoptosis in Mtg16(-/-) intestines. To determine if Mtg16 deletion affected epithelial cells in a cell-autonomous fashion, intestinal crypts were isolated from Mtg16(-/-) mice. Mtg16(-/-) and WT intestinal crypts showed similar enterosphere forming efficiencies when cultured in the presence of EGF, Noggin, and R-spondin. However, when Mtg16(-/-) crypts were cultured in the presence of Wnt3a, they demonstrated higher enterosphere forming efficiencies and delayed progression to mature enteroids. Mtg16(-/-) intestinal crypts isolated from irradiated mice exhibited increased survival compared with WT intestinal crypts. Interestingly, Mtg16 expression was reduced in a stem cell-enriched population at the time of crypt regeneration. This is consistent with MTG16 negatively regulating regeneration in vivo. Taken together, our data demonstrate that MTG16 loss promotes radioresistance and impacts intestinal stem cell function, possibly due to shifting cellular response away from DNA damage-induced apoptosis and towards DNA repair after injury.


Asunto(s)
Proliferación Celular , Rayos gamma , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Proteínas Nucleares/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Regeneración , Factores de Transcripción/metabolismo , Animales , Apoptosis , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Daño del ADN , Femenino , Regulación de la Expresión Génica , Células Caliciformes/metabolismo , Células Caliciformes/patología , Histonas/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fenotipo , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/patología , Tolerancia a Radiación , Regeneración/efectos de los fármacos , Proteínas Represoras , Transducción de Señal , Células Madre/metabolismo , Células Madre/patología , Técnicas de Cultivo de Tejidos , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteína Wnt3A/farmacología
5.
J Biol Chem ; 287(23): 19472-86, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-22496452

RESUMEN

One of highly pathogenic breast cancer cell types are the triple negative (negative in the expression of estrogen, progesterone, and ERBB2 receptors) breast cancer cells. These cells are highly motile and metastatic and are characterized by high levels of the metastasis regulator protein SLUG. Using isogenic breast cancer cell systems we have shown here that high motility of these cells is directly correlated with the levels of the SLUG in these cells. Because epithelial/mesenchymal cell motility is known to be negatively regulated by the catenin protein plakoglobin, we postulated that the transcriptional repressor protein SLUG increases the motility of the aggressive breast cancer cells through the knockdown of the transcription of the plakoglobin gene. We found that SLUG inhibits the expression of plakoglobin gene directly in these cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Blocking of SLUG function with a double-stranded DNA decoy that competes with the E2-box binding of SLUG also increased the levels of plakoglobin mRNA, protein, and promoter activity in the SLUG-high triple negative breast cancer cells. Overexpression of SLUG in the SLUG-deficient cells elevated the motility of these cells. Knockdown of plakoglobin in these low motility non-invasive breast cancer cells rearranged the actin filaments and increased the motility of these cells. Forced expression of plakoglobin in SLUG-high cells had the reverse effects on cellular motility. This study thus implicates SLUG-induced repression of plakoglobin as a motility determinant in highly disseminating breast cancer.


Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , Desmoplaquinas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Desmoplaquinas/genética , Femenino , Humanos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , gamma Catenina
6.
Synapse ; 67(5): 245-57, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23280858

RESUMEN

Methamphetamine (METH) is a highly addictive and neurotoxic psychostimulant. Its use in humans is often associated with neurocognitive impairment. Whether this is due to long-term deficits in short-term memory and/or hippocampal plasticity remains unclear. Recently, we reported that METH increases baseline synaptic transmission and reduces LTP in an ex vivo preparation of the hippocampal CA1 region from young mice. In the current study, we tested the hypothesis that a repeated neurotoxic regimen of METH exposure in adolescent mice decreases hippocampal synaptic plasticity and produces a deficit in short-term memory. Contrary to our prediction, there was no change in the hippocampal plasticity or short-term memory when measured after 14 days of METH exposure. However, we found that at 7, 14, and 21 days of drug abstinence, METH-exposed mice exhibited a deficit in spatial memory, which was accompanied by a decrease in hippocampal plasticity. Our results support the interpretation that the deleterious cognitive consequences of neurotoxic levels of METH exposure may manifest and persist after drug abstinence. Therefore, therapeutic strategies should consider short-term as well as long-term consequences of methamphetamine exposure.


Asunto(s)
Dopaminérgicos/toxicidad , Memoria a Corto Plazo/efectos de los fármacos , Metanfetamina/toxicidad , Factores de Edad , Animales , Hipocampo/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de Tiempo
7.
J Biol Chem ; 286(1): 469-79, 2011 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-21044962

RESUMEN

UbcH5c, a member of the UbcH5 family of protein ubiquitin conjugase E2 enzymes, is a critical component of biological processes in human cells, being the initial ubiquitinating enzyme of substrates like IκB, TP53, and cyclin D1. We report here that the metastasis regulator protein SLUG inhibits the expression of UbcH5c directly through chromatin remodeling and thus, among other downstream effects, elevates the level of cyclin D1, thus enhancing the growth rates of breast cancer cells. Overexpression of SLUG in the SLUG-deficient breast cancer cells significantly decreased the levels of mRNA and protein of UbcH5c but only elevated the protein levels of cyclin D1. On the contrary, knockdown of SLUG in SLUG-high breast cancer cells elevated the levels of UbcH5c while decreasing the level of cyclin D1 protein. SLUG is recruited at the E2-box sequence at the UbcH5c gene promoter along with the corepressor CtBP1 and the effector HDAC1 to silence the expression of this gene. Knockdown of UbcH5c in the SLUG-deficient human breast cells elevated the level of cyclin D1 as well as the rates of proliferation and invasiveness of these cells. Whereas the growth rates of the cells are enhanced due to overexpression of SLUG or knockdown of UbcH5c in the breast cancer cells tested, ER(+) cells also acquire resistance to the anti-estrogen 4-hydroxytamoxifen due to the rise of cyclin D1 levels in these cells. This study thus implicates high levels of SLUG and low levels of UbcH5c as a determinant in the progression of metastatic breast cancer.


Asunto(s)
Neoplasias de la Mama/patología , Ciclina D1/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Ensamble y Desensamble de Cromatina , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Invasividad Neoplásica/genética , Regiones Promotoras Genéticas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción de la Familia Snail , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Enzimas Ubiquitina-Conjugadoras/deficiencia , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/genética
8.
J Biol Chem ; 286(8): 6614-26, 2011 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-21149457

RESUMEN

The parasitic protozoan Leishmania invades mammalian macrophages to establish infection. We reported previously that Leishmania manipulates the expression of several non-coding RNA genes (e.g. Alu RNA, B1 RNA, and signal recognition particle RNA) in macrophages to favor the establishment of their infection in the phagolysosomes of these cells (Ueda, Y., and Chaudhuri, G. (2000) J. Biol. Chem. 275, 19428-19432; Misra, S., Tripathi, M. K., and Chaudhuri, G. (2005) J. Biol. Chem. 280, 29364-29373). We report here the mechanism of this down-regulation. We found that the non-coding RNA (ncRNA) genes that are repressed by Leishmania infection in macrophages contain a "B-box" in their promoters and thus require the polymerase III transcription factor TFIIIC for their expression. We also found that Leishmania promastigotes through their surface protease (leishmanolysin or gp63) activate the thrombin receptor PAR1 in the macrophages. This activation of PAR1 raised the cytosolic concentration of Ca(2+) into the micromolar range, thereby activating the Ca(2+)-dependent protease µ-calpain. µ-Calpain then degraded TFIIIC110 to inhibit the expression of the selected ncRNA genes. Avirulent stocks of Leishmania not expressing surface gp63 failed to down-regulate ncRNAs in the exposed macrophages. Inhibition of PAR1 or calpain 1 in macrophages made them resistant to Leishmania infection. These data suggest that macrophage PAR1 and calpain 1 are potential drug targets against leishmaniasis.


Asunto(s)
Regulación hacia Abajo , Leishmania major/metabolismo , Leishmaniasis Cutánea/metabolismo , Macrófagos/metabolismo , ARN Polimerasa III/metabolismo , ARN no Traducido/biosíntesis , Animales , Calcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Línea Celular Tumoral , Humanos , Leishmania major/genética , Leishmania major/patogenicidad , Leishmaniasis Cutánea/genética , Macrófagos/parasitología , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones , ARN Polimerasa III/genética , ARN no Traducido/genética , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Factores de Transcripción TFIII/genética , Factores de Transcripción TFIII/metabolismo
9.
Mol Cell Biochem ; 350(1-2): 47-57, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21165676

RESUMEN

Leishmania is a group of parasitic protozoa that infect blood and tissue phagocytes including macrophages. We hypothesize that Leishmania is capable of establishing infection inside the macrophages because (a) they infect a subpopulation of macrophages; and (b) they "renovate" the macrophages before the establishment of infection. We found that only alternatively activated polarized M2 macrophages support Leishmania growth. Exposure of M2 macrophages to Leishmania promastigotes represses several selected RNA polymerase III (PolIII)-transcribed non-coding RNA (ncRNA) genes including those of 7SL RNA, vault RNA, and B2 RNA which have B-box element at their promoters. The B-box-binding transcription factor TFIIIC110 is down-regulated in Leishmania-exposed macrophages. Both the surface protease gp63 and the surface glycolipid LPG are required for the down-regulation of the ncRNAs in the M2 macrophages. We conclude that Leishmania surface gp63 collaborates with LPG to down-regulate TFIIIC110 in M2 macrophages to repress B-box containing ncRNA gene promoters.


Asunto(s)
Polaridad Celular , Leishmania/fisiología , Macrófagos/metabolismo , Regiones Promotoras Genéticas , ARN no Traducido/genética , Elementos Reguladores de la Transcripción , Animales , Secuencia de Bases , Polaridad Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/fisiología , Regulación de la Expresión Génica , Humanos , Activación de Macrófagos/genética , Activación de Macrófagos/fisiología , Macrófagos/fisiología , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN Citoplasmático Pequeño/química , ARN Citoplasmático Pequeño/genética , Elementos Reguladores de la Transcripción/fisiología , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genética , Células U937
10.
Mol Cancer ; 9: 50, 2010 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-20202217

RESUMEN

BACKGROUND: BRCA2 gene expression is tightly regulated during the cell cycle in human breast cells. The expression of BRCA2 gene is silenced at the G0/G1 phase of cell growth and is de-silenced at the S/G2 phase. While studying the activity of BRCA2 gene promoter in breast cancer cells, we discovered that this promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter at the G0/G1 phase of the cell. Standard techniques like cell synchronization by serum starvation, flow cytometry, N-terminal or C-terminal FLAG epitope-tagged protein expression, immunofluorescence confocal microscopy, dual luciferase assay for promoter evaluation, and chromatin immunoprecipitation assay were employed during this study. RESULTS: Human BRCA2 gene promoter is active in both the forward and the reverse orientations. This promoter is 8-20 fold more active in the reverse orientation than in the forward orientation when the cells are in the non-dividing stage (G0/G1). When the cells are in the dividing state (S/G2), the forward activity of the promoter is 5-8 folds higher than the reverse activity. The reverse activity transcribes the ZAR2 mRNA with 966 nt coding sequence which codes for a 321 amino acid protein. ZAR2 has two C4 type zinc fingers at the carboxyl terminus. In the G0/G1 growth phase ZAR2 is predominantly located inside the nucleus of the breast cells, binds to the BRCA2 promoter and inhibits the expression of BRCA2. In the dividing cells, ZAR2 is trapped in the cytoplasm. CONCLUSIONS: BRCA2 gene promoter has bi-directional activity, expressing BRCA2 and a novel C4-type zinc finger containing transcription factor ZAR2. Subcellular location of ZAR2 and its expression from the reverse promoter of the BRCA2 gene are stringently regulated in a cell cycle dependent manner. ZAR2 binds to BRCA2/ZAR2 bi-directional promoter in vivo and is responsible, at least in part, for the silencing of BRCA2 gene expression in the G0/G1 phase in human breast cells.


Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Regiones no Traducidas 5'/genética , Animales , Proteína BRCA2/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Codón/genética , Secuencia Conservada , Citosol/metabolismo , Exones/genética , Femenino , Fase G1 , Técnicas de Silenciamiento del Gen , Reordenamiento Génico/genética , Humanos , Intrones/genética , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fase de Descanso del Ciclo Celular , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Vertebrados/genética
11.
Mol Biol Rep ; 37(3): 1221-7, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19277896

RESUMEN

SNAI1P, a protein coded by a retrogene, is a member of the SNAI family of E2-box binding transcriptional repressors. To evaluate whether the mode of action of SNAI1P is similar to those of the other predominant members of the SNAI family, we studied its action on human claudin 7 (CLDN7) gene promoter which has seven E2-boxes. We over-expressed FLAG-tagged SNAI1P in MCF7 and MDA-MB-468 cells. SNAI1P inhibited the expression of CLDN7 in these recombinant cells. SNAI1P also inhibited cloned CLDN7 gene promoter activity in human breast cancer cells. ChIP assays revealed that SNAI1P is recruited on the CLDN7 gene promoter along with the co-repressor CtBP1 and the effector HDAC1. Treatment of the cells with trichostatin A, an inhibitor of HDAC1, abrogated the repressor activity of SNAI1P. These data suggest that SNAI1P inhibits CLDN7 gene promoter epigenetically in breast cancer cells through chromatin remodeling.


Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Epigénesis Genética , Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina , Claudinas , Regulación de la Expresión Génica/genética , Humanos , Ácidos Hidroxámicos , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción de la Familia Snail
12.
Biochem Biophys Res Commun ; 372(1): 30-4, 2008 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-18485278

RESUMEN

The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628bp fragment (-613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.


Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Calcitriol/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Femenino , Histona Desacetilasa 1 , Histona Desacetilasas/metabolismo , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética
13.
Mucosal Immunol ; 11(5): 1363-1374, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29907869

RESUMEN

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves-/- mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves-/- mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves-/- mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.


Asunto(s)
Colitis Ulcerosa/metabolismo , Colon/metabolismo , Células Epiteliales/metabolismo , Absorción Intestinal/fisiología , Proteínas de la Membrana/metabolismo , Adulto , Animales , Células CACO-2 , Moléculas de Adhesión Celular , Línea Celular , Línea Celular Tumoral , Citrobacter rodentium/patogenicidad , Técnicas de Cocultivo , Colon/efectos de los fármacos , Sulfato de Dextran/farmacología , Células Epiteliales/efectos de los fármacos , Escherichia coli/metabolismo , Femenino , Células HEK293 , Humanos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas Musculares , Permeabilidad/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo
14.
Am J Trop Med Hyg ; 76(4): 681-8, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17426170

RESUMEN

Clinical resistance to pentavalent antimonial compounds has long been recognized as a major problem in the treatment of visceral leishmaniasis in India. However, mechanisms of natural resistance are unclear. In this study, we observed that Leishmania donovani clinical isolates not responsive to sodium stibogluconate showed resistance to antimony treatment in both in vitro and in vivo laboratory conditions. The resistant isolates have increased levels of intracellular thiols. This increase in thiol levels was not mediated by the amplification of gamma-glutamylcysteine synthetase, but was accompanied by amplification of trypanothione reductase and an intracellular ATP-binding cassette transporter gene MRPA. The resistance of parasites to antimony could be reversed by the glutathione biosynthesis-specific inhibitor, buthionine sulfoximine, which resulted in increased drug susceptibility. These results suggest the possible role of thiols and MRPA in antimony resistance in field isolates.


Asunto(s)
Antimonio/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos/fisiología , Leishmania donovani/efectos de los fármacos , Animales , Butionina Sulfoximina/farmacología , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Leishmania donovani/fisiología , Leishmaniasis Visceral/parasitología , Compuestos de Sulfhidrilo/metabolismo
15.
JCI Insight ; 2(16)2017 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-28814670

RESUMEN

MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16-/- mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16-/- mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16-/- mice failed to rescue the Mtg16-/- protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.

16.
Biochem Biophys Res Commun ; 353(3): 661-4, 2007 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-17194444

RESUMEN

SLUG is a transcriptional repressor protein implicated to have major role in the oncogenesis and metastasis of human breast cells. We previously have shown by chromatin immunoprecipitation assay that human SLUG (hSLUG) is co-localized with the co-repressor protein CtBP1 as bound to the BRCA2 gene silencer [M.K. Tripathi, S. Misra, S.V. Khedkar, N. Hamilton, C. Irvin-Wilson,, C. Sharan, L. Sealy, G. Chaudhuri, J. Biol. Chem. 280 (2005) 17163-17171]. hSLUG was predicted to be binding directly to CtBP1 because of an apparent presence of CtBP1 binding site in its amino acid sequences. Here, we provide evidence through yeast two-hybrid and in vitro co-immunoprecipitation analyses that hSLUG does not directly interacts with hCtBP1. This observation will help in the study of the mode of action of hSLUG in human cells.


Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Secuencia de Consenso , Humanos , Inmunoprecipitación , Factores de Transcripción de la Familia Snail , Técnicas del Sistema de Dos Híbridos
17.
Protein Expr Purif ; 40(2): 279-86, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15766869

RESUMEN

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.


Asunto(s)
Glutatión/análogos & derivados , Leishmania donovani/enzimología , NADH NADPH Oxidorreductasas/genética , Espermidina/análogos & derivados , Secuencia de Aminoácidos , Animales , Clonación Molecular/métodos , Dimerización , Escherichia coli/genética , Glutatión/metabolismo , Cinética , Peso Molecular , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/aislamiento & purificación , Oxidación-Reducción , Filogenia , Alineación de Secuencia , Espermidina/metabolismo
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