RESUMEN
High-dose-radiation exposure in a short period of time leads to radiation syndromes characterized by severe acute and delayed organ-specific injury accompanied by elevated organismal morbidity and mortality. Radiation biodosimetry based on gene expression analysis of peripheral blood is a valuable tool to detect exposure to radiation after a radiological/nuclear incident and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation, can potentially obscure the predictive power of the method. GADD45A (Growth arrest and DNA damage-inducible gene a) plays important roles in cell growth control, differentiation, DNA repair, and apoptosis. GADD45A-deficient mice develop an autoimmune disease, similar to human systemic lupus erythematosus, characterized by severe hematological disorders, kidney disease, and premature death. The goal of this study was to elucidate how pre-existing inflammation in mice, induced by GADD45A ablation, can affect radiation biodosimetry. We exposed wild-type and GADD45A knockout male C57BL/6J mice to 7 Gy of X rays and 24 h later RNA was isolated from whole blood and subjected to whole genome microarray and gene ontology analyses. Dose reconstruction analysis using a gene signature trained on gene expression data from irradiated wild-type male mice showed accurate reconstruction of either a 0 Gy or 7 Gy dose with root mean square error of ± 1.05 Gy (R^2 = 1.00) in GADD45A knockout mice. Gene ontology analysis revealed that irradiation of both wild-type and GADD45A-null mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. However, based on their z-score, these pathways were predicted to be more significantly overrepresented in GADD45A-null mice, implying that GADD45A deletion may exacerbate the deleterious effects of radiation on blood cells. Numerous immune cell functions and quantities were predicted to be underrepresented in both genotypes; however, differentially expressed genes from irradiated GADD45A knockout mice predicted an increased deterioration in the numbers of T lymphocytes, as well as myeloid cells, compared with wild-type mice. Furthermore, an overrepresentation of genes associated with radiation-induced hematological malignancies was associated with GADD45A knockout mice, whereas hematopoietic and progenitor cell functions were predicted to be downregulated in irradiated GADD45A knockout mice. In conclusion, despite the significant differences in gene expression between wild-type and GADD45A knockout mice, it is still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of pre-existing inflammation status.
Asunto(s)
Proteínas de Ciclo Celular , Inflamación , Animales , Humanos , Masculino , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inflamación/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Rayos XRESUMEN
Gastrointestinal (GI) cancer risk among astronauts after encountering galactic cosmic radiation (GCR) is predicted to exceed safe permissible limits in long duration deep-space missions. Current predictions are based on relative biological effectiveness (RBE) values derived from in-vivo studies using single-ion beams, while GCR is essentially a mixed radiation field composed of protons (H), helium (He), and heavy ions. Therefore, a sequentially delivered proton (H) â Helium (He) â Oxygen (O) â Silicon (Si) beam was designed to simulate simplified-mixed-field GCR (Smf-GCR), and Apc1638N/+ mice were total-body irradiated to sham or γ (157Cs) or Smf-GCR followed by assessment of GI-tumorigenesis at 150 days post-exposure. Further, GI-tumor data from equivalent doses of heavy-ions (i.e., 0.05 Gy of O and Si) in 0.5 Gy of Smf-GCR were compared to understand the contributions of heavy-ions in GI-tumorigenesis. The Smf-GCR-induced tumor and carcinoma count were significantly greater than γ-rays, and male preponderance for GI-tumorigenesis was consistent with our earlier findings. Comparison of tumor data from Smf-GCR and equivalent doses of heavy ions revealed an association between higher GI-tumorigenesis where dose received from heavy-ions contributed to > 95% of the total GI-tumorigenic effect observed after Smf-GCR. This study provides the first experimental evidence that cancer risk after GCR exposure could largely depend on doses received from constituent heavy-ions.
Asunto(s)
Radiación Cósmica , Iones Pesados , Neoplasias Inducidas por Radiación , Exposición a la Radiación , Vuelo Espacial , Ratones , Masculino , Animales , Iones Pesados/efectos adversos , Helio , Radiación Cósmica/efectos adversos , Neoplasias Inducidas por Radiación/etiología , Neoplasias Inducidas por Radiación/patología , Carcinogénesis , ProtonesRESUMEN
Several diagnostic biodosimetry tools have been in development that may aid in radiological/nuclear emergency responses. Of these, correlating changes in non-invasive biofluid small-molecule signatures to tissue damage from ionizing radiation exposure show promise for inclusion in predictive biodosimetry models. Integral to dose reconstruction has been determining how genotypic variation in the general population will affect model performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway [p38αßY323F, double knock-in (DKI) mice] to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. We exposed adult male DKI mice (8-10 weeks old) to 2 and 7 Gy in parallel with wild-type mice and assessed perturbations in urine (days 1, 3, 7) and serum (day 1) using a global metabolomics approach. A multidimensional scaling plot showed excellent separation of radiation-exposed groups in wild-type mice with slightly dampened responses in DKI mice. Validated metabolite panels were developed for urine [N6,N6,N6-trimethyllysine (TML), N1-acetylspermidine, spermidine, carnitine, acylcarnitine C21H35NO5, aminohippuric acid] and serum [phenylalanine, glutamine, propionylcarnitine, lysophosphatidylcholine (LysoPC 14:0), LysoPC (22:5)] to determine the area under the receiver operating characteristic curve (AUROC). For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤2 Gy vs. 7 Gy mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes after irradiation. Overall, these results suggest immunosuppression should not compromise small molecule multiplex panels used in dose reconstruction for biodosimetry.
Asunto(s)
Metabolómica , Irradiación Corporal Total , Animales , Humanos , Terapia de Inmunosupresión , Masculino , Metabolómica/métodos , Ratones , Curva ROC , Linfocitos T , Irradiación Corporal Total/efectos adversosRESUMEN
Inevitable exposure to high-LET ionizing radiation (IR) present in galactic cosmic radiation (GCR) could enhance gastrointestinal (GI) cancer incidence among astronauts undertaking deep space exploration and GI-cancer mortality has been predicted to far exceed NASA's limit of < 3% REID (Radiation exposure-induced death) from cancer. Therefore, the development of countermeasure agents against high-LET radiation-induced GI cancer is needed to safeguard astronauts during and after an outer space mission. The cyclooxygenase-2/prostaglandin E2 (COX2/PGE2) mediated activation of pro-inflammatory and oncogenic signaling has been reported to play an important role in persistent inflammation and GI-tumorigenesis after high-LET radiation exposure. Therefore, aspirin, a well-known inhibitor of the COX/PGE2 pathway, was evaluated as a potential countermeasure against 28Si-induced PGE2 and tumorigenesis in Apc1638N/+, a murine model of human GI-cancer. Animals were fed either standard or aspirin supplemented diet (75, 150, or 300 mg/day of human equivalent dose) starting at the age of 4 weeks and continued till the end of the study, while mice were exposed to 28Si-ions (300 MeV/n; 69 keV/µm) at the age of 8 weeks. Serum PGE2 level, GI tumor size (>2mm2), number, and cluster (>5 adjoining tumors) were analyzed at 150 days post-exposure. Aspirin led to a significant reduction in PGE2 in a dose-dependent manner but did not reduce 28Si-induced GI tumorigenesis even at the highest (300 mg/day) dose. In summary, this study suggests that aspirin could reduce high-LET IR-induced pro-inflammatory PGE2 levels, however, lacks the ability to reduce high-LET IR-induced GI tumorigenesis in Apc1638N/+ mice.
Asunto(s)
Radiación Cósmica , Dinoprostona , Animales , Aspirina , Carcinogénesis , Dieta , RatonesRESUMEN
The incidence of both nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been increasing at an alarming rate. Little is known about NAFLD without cirrhosis as a risk for HCC. Here we report, for the first time, generation of a mouse model with a defect in long-chain 3-hydoxy acyl-CoA dehydrogenase (LCHAD). The LCHAD exon 15 deletion was embryonic lethal to the homozygous mice whereas heterozygous mice (HT) develop significant hepatic steatosis starting at young age (3 months old) and HCC at older age (>13 months old) without any evidence of fibrosis or cirrhosis. None of the wild-type (WT) mice developed steatosis and HCC (n = 39), whereas HT-LCHAD mice (n = 41) showed steatosis and ~20% (8/41) developed liver masses with histological features of HCC. Proteomic analysis of liver tissues from WT-mice and HT-mice with no signs of HCC was conducted. Proteins with significant changes in abundance were identified by mass spectrometry. Abundance of 24 proteins was significantly different (p < 0.01) between WT and HT-LCHAD mice. The proteins found to vary in abundance are associated with different cellular response processes ranging from intermediary metabolism of carbohydrate, protein and lipid to oxidative stress, signal transduction and the process of tumorigenesis. Protein expression pattern of the HT-LCHAD mouse liver indicates predisposition to HCC and suggests that impaired hepatic mitochondrial fatty acid oxidation plays an important role in the development and progression of HCC. To assess the implication of these studies in human disease, we demonstrated significant downregulation of HADHA transcripts in HCC patients.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/genética , Carcinoma Hepatocelular/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias Hepáticas/genética , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ácidos Grasos/metabolismo , Regulación Neoplásica de la Expresión Génica , Heterocigoto , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/metabolismo , Ratones , Mitocondrias Hepáticas/metabolismo , Subunidad alfa de la Proteína Trifuncional Mitocondrial/genética , Mutación , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Oxidación-ReducciónRESUMEN
Immune cell function can be modulated by changes in lipid metabolism. Our studies indicate that cholesterol and fatty acid synthesis increases in macrophages between 12 and 18 h after the activation of Toll-like receptors with proinflammatory stimuli and that the upregulation of lipogenesis may contribute to the resolution of inflammation. The inflammation-dependent increase in lipogenesis requires the induction of the liver X receptors, members of the nuclear receptor superfamily of transcription factors, by type I interferons in response to inflammatory signals. Instead of the well-established role for liver X receptors in stimulating cholesterol efflux, we demonstrate that liver X receptors are necessary for the proper resumption of cholesterol synthesis in response to inflammatory signals. Thus, liver X receptors function as bidirectional regulators of cholesterol homeostasis, driving efflux when cholesterol levels are high and facilitating synthesis in response to inflammatory signals. Liver X receptor activity is also required for the proper shutdown of a subset of type I interferon-stimulated genes as inflammation subsides, placing the receptors in a negative-feedback loop that may contribute to the resolution of the inflammatory response.
Asunto(s)
Colesterol/metabolismo , Inflamación/metabolismo , Lipogénesis , Receptores X del Hígado/metabolismo , Animales , Línea Celular , Células Cultivadas , Células HEK293 , Humanos , Macrófagos/metabolismo , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: Inflammatory macrophages promote the development of atherosclerosis. We have identified the adaptor protein Dab2 (disabled homolog 2) as a regulator of phenotypic polarization in macrophages. The absence of Dab2 in myeloid cells promotes an inflammatory phenotype, but the impact of myeloid Dab2 deficiency on atherosclerosis has not been shown. APPROACH AND RESULTS: To determine the role of myeloid Dab2 in atherosclerosis, Ldlr-/- mice were reconstituted with either Dab2-positive or Dab2-deficient bone marrow and fed a western diet. Consistent with our previous finding that Dab2 inhibits NFκB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling in macrophages, Ldlr-/- mice reconstituted with Dab2-deficient bone marrow had increased systemic inflammation as evidenced by increased serum IL-6 (interleukin-6) levels and increased inflammatory cytokine expression levels in liver. Serum lipid levels were significantly lower in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow, and further examination of livers from these mice revealed drastically increased inflammatory tissue damage and massive infiltration of immune cells. Surprisingly, the atherosclerotic lesion burden in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow was decreased compared with Ldlr-/- mice reconstituted with wild-type bone marrow. Further analysis of aortic root sections revealed increased macrophage content and evidence of increased apoptosis in lesions from Ldlr-/- mice reconstituted with Dab2-deficient bone marrow but no difference in collagen or α-smooth muscle actin content. CONCLUSIONS: Dab2 deficiency in myeloid cells promotes inflammation in livers and atherosclerotic plaques in a mouse model of atherosclerosis. Nevertheless, decreased serum lipids as a result of massive inflammatory liver damage may preclude an appreciable increase in atherosclerotic lesion burden in mice reconstituted with Dab2-deficient bone marrow.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Hepatitis/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica , Receptores de LDL/deficiencia , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Proteínas Reguladoras de la Apoptosis , Aterosclerosis/genética , Aterosclerosis/patología , Caspasas/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Hepatitis/genética , Hepatitis/patología , Humanos , Interleucina-6/sangre , Células Jurkat , Lípidos/sangre , Hígado/patología , Macrófagos/patología , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fenotipo , Receptores de LDL/genética , Transducción de Señal , Triglicéridos/metabolismoRESUMEN
OBJECTIVE: The ability of high-density lipoprotein (HDL) particles to accept cholesterol from peripheral cells, such as lipid-laden macrophages, and to transport cholesterol to the liver for catabolism and excretion in a process termed reverse cholesterol transport (RCT) is thought to underlie the beneficial cardiovascular effects of elevated HDL. The liver X receptors (LXRs; LXRα and LXRß) regulate RCT by controlling the efflux of cholesterol from macrophages to HDL and the excretion, catabolism, and absorption of cholesterol in the liver and intestine. Importantly, treatment with LXR agonists increases RCT and decreases atherosclerosis in animal models. Nevertheless, LXRs are expressed in multiple tissues involved in RCT, and their tissue-specific contributions to RCT are still not well defined. APPROACH AND RESULTS: Using tissue-specific LXR deletions together with in vitro and in vivo assays of cholesterol efflux and fecal cholesterol excretion, we demonstrate that macrophage LXR activity is neither necessary nor sufficient for LXR agonist-stimulated RCT. In contrast, the ability of LXR agonists primarily acting in the intestine to increase HDL mass and HDL function seems to underlie the ability of LXR agonists to stimulate RCT in vivo. CONCLUSIONS: We demonstrate that activation of LXR in macrophages makes little or no contribution to LXR agonist-stimulated RCT. Unexpectedly, our studies suggest that the ability of macrophages to efflux cholesterol to HDL in vivo is not regulated by macrophage activity but is primarily determined by the quantity and functional activity of HDL.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Transporte Biológico , Línea Celular , Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/genética , Colesterol en la Dieta/metabolismo , HDL-Colesterol/metabolismo , Heces/química , Humanos , Hidrocarburos Fluorados/farmacología , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/deficiencia , Receptores Nucleares Huérfanos/genética , Sulfonamidas/farmacología , Factores de TiempoRESUMEN
The ability of cells to precisely control gene expression in response to intracellular and extracellular signals plays an important role in both normal physiology and in pathological settings. For instance, the accumulation of excess cholesterol by macrophages initiates a genetic response mediated by the liver X receptors (LXRs)-α (NR1H3) and LXRß (NR1H2), which facilitates the transport of cholesterol out of cells to high-density lipoprotein particles. Studies using synthetic LXR agonists have also demonstrated that macrophage LXR activation simultaneously induces a second network of genes that promotes fatty acid and triglyceride synthesis that may support the detoxification of excess free cholesterol by storage in the ester form. We now show that treatment of human THP-1 macrophages with endogenous or synthetic LXR ligands stimulates both transcriptional and posttranscriptional pathways that result in the selective recruitment of the LXRα subtype to LXR-regulated promoters. Interestingly, when human or mouse macrophages are loaded with cholesterol under conditions that mimic the development of atherogenic macrophage foam cells, a selective LXR response is generated that induces genes mediating cholesterol transport but does not coordinately regulate genes involved in fatty acid synthesis. The gene-selective response to cholesterol loading occurs, even in the presence of LXRα binding to the promoter of the gene encoding the sterol regulatory element-binding protein-1c, the master transcriptional regulator of fatty acid synthesis. The ability of promoter bound LXRα to recruit RNA polymerase to the sterol regulatory element-binding protein-1c promoter, however, appears to be ligand selective.
Asunto(s)
Colesterol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Línea Celular , Semivida , Humanos , Ligandos , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Ratones , Receptores Nucleares Huérfanos/agonistas , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Elementos de Respuesta/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Liver X receptors (LXRα and LXRß) are important regulators of cholesterol and lipid metabolism, and their activation has been shown to inhibit cardiovascular disease and reduce atherosclerosis in animal models. Small molecule agonists of LXR activity are therefore of great therapeutic interest. However, the finding that such agonists also promote hepatic lipogenesis has led to the idea that hepatic LXR activity is undesirable from a therapeutic perspective. To investigate whether this might be true, we performed gene targeting to selectively delete LXRα in hepatocytes. Liver-specific deletion of LXRα in mice substantially decreased reverse cholesterol transport, cholesterol catabolism, and cholesterol excretion, revealing the essential importance of hepatic LXRα for whole body cholesterol homeostasis. Additionally, in a pro-atherogenic background, liver-specific deletion of LXRα increased atherosclerosis, uncovering an important function for hepatic LXR activity in limiting cardiovascular disease. Nevertheless, synthetic LXR agonists still elicited anti-atherogenic activity in the absence of hepatic LXRα, indicating that the ability of agonists to reduce cardiovascular disease did not require an increase in cholesterol excretion. Furthermore, when non-atherogenic mice were treated with synthetic LXR agonists, liver-specific deletion of LXRα eliminated the detrimental effect of increased plasma triglycerides, while the beneficial effect of increased plasma HDL was unaltered. In sum, these observations suggest that therapeutic strategies that bypass the liver or limit the activation of hepatic LXRs should still be beneficial for the treatment of cardiovascular disease.
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Colesterol/metabolismo , Homeostasis , Receptores Nucleares Huérfanos/metabolismo , Animales , Anticolesterolemiantes/farmacología , Anticolesterolemiantes/uso terapéutico , Aterosclerosis/sangre , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/metabolismo , Transporte Biológico , Células Cultivadas , Colesterol/sangre , Heces/química , Femenino , Técnicas de Inactivación de Genes , Hidrocarburos Fluorados/farmacología , Hidrocarburos Fluorados/uso terapéutico , Metabolismo de los Lípidos , Lipoproteínas/sangre , Lipoproteínas/metabolismo , Hígado/metabolismo , Receptores X del Hígado , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/genética , Tamaño de la Partícula , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
Oncogenic K-ras is one of the most common genetic alterations in human lung adenocarcinomas. In addition, inactivation of clusters of tumor suppressor genes is required to bring about classical characteristics of cancer including angiogenesis as a prelude to invasion and metastasis. Transforming growth factor-beta (TGF-beta) 1 is a tumor suppressor gene that is implicated in lung cancer progression. Although in vitro studies have shown that TGF-beta1 and Ras pathways cooperate during tumorigenesis, the biology of interaction of TGF-beta1 and Ras has not been studied in in vivo tumorigenesis. We hypothesized that inactivation of TGF-beta1 in addition to oncogeneic activation of K-ras would lead to early initiation and faster progression to lung adenocarcinoma and invasion and metastasis. Heterozygous (HT) TGF-beta1 mice were mated with latent activatable (LA) mutated K-ras mice to generate TGF-beta1(+/+), K-ras LA (wild-type (WT)/LA) and TGF-beta1(+/-), K-ras LA (HT/LA) mice. Both HT/LA and WT/LA mice developed spontaneous lung tumors, but HT/LA mice progressed to adenocarcinomas significantly earlier compared with WT/LA mice. In addition, WT/LA adenocarcinomas had significantly higher angiogenic activity compared with HT/LA adenocarcinomas. Thus, while oncogenic K-ras mutation and insensitivity to the growth regulatory effects of TGF-beta1 is essential for initiation and progression of mouse lung tumors to adenocarcinoma, a full gene dosage of TGF-beta1 is required for tumor-induced angiogenesis and invasive potential. This study identifies a number of genes not previously associated with lung cancer that are involved in tumor induction and progression. In addition, we provide evidence that progression to invasive angiogenic lesions requires TGF-beta1 responsiveness in addition to Ras mutation.
Asunto(s)
Adenocarcinoma/metabolismo , Genes ras/fisiología , Neoplasias Pulmonares/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/genética , Animales , Progresión de la Enfermedad , Heterocigoto , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Neovascularización Patológica , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND & AIMS: Little is known about the role of mitochondrial beta-oxidation in development of nonalcoholic fatty liver disease (NAFLD). Mitochondrial trifunctional protein (MTP) catalyzes long-chain fatty acid oxidation. Recently, we generated a mouse model for MTP deficiency and reported that homozygous (MTPa-/-) mice suffer neonatal death. In this study, we investigated effects of heterozygosity for the MTP defect on hepatic oxidative stress, insulin resistance, and development of NAFLD in mice. METHODS: We evaluated liver histopathology, serum alanine aminotransferase (ALT), glucose, fatty acids, and insulin levels in MTPa+/- and MTPa+/+ littermates. Insulin resistance was evaluated using glucose tolerance test (GTT) and insulin tolerance test (ITT). Liver tissues were used to measure triglyceride and fatty acid content, activity of superoxide dismutases (SOD) and glutathione peroxidase (GPx), glutathione (GSH), and cytochrome P-450 2E1 expression. RESULTS: Aging but not young MTPa+/- mice developed hepatic steatosis with elevated ALT, basal hyperinsulinemia, and increased insulin area under curve (AUC) on GTT compared with MTPa+/+ littermates. In response to insulin challenge, aging MTPa+/- mice had slower rate of glucose disappearance and increased glucose AUC. Significant hepatic steatosis and insulin resistance developed concomitantly in the MTPa+/- mice at 9-10 months of age. Aging MTPa+/- mice had higher antioxidant activity of total SOD and GPx, lower GSH, and increased expression of cytochrome P-450 2E1, consistent with increased hepatic oxidative stress. CONCLUSIONS: Heterozygosity for beta-oxidation defects predisposes to NAFLD and insulin resistance in aging mice. Impairment of mitochondrial beta-oxidation may play an important role in pathogenesis of NAFLD.
Asunto(s)
Hígado Graso/genética , Hígado Graso/metabolismo , Resistencia a la Insulina/genética , Complejos Multienzimáticos/genética , Animales , Ácidos Grasos/metabolismo , Hígado Graso/patología , Heterocigoto , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Proteína Trifuncional Mitocondrial , Complejos Multienzimáticos/metabolismo , Estrés Oxidativo/fisiologíaRESUMEN
Differentially expressed nucleolar TGF-beta1 target (DENTT) is a recently identified gene whose mRNA is differentially affected by TGF-beta1 in TGF-beta1-responsive human lung cancer cells and who is a new member of the TSPY/TSPY-like/SET/NAP-1 (TTSN) protein superfamily. Here, we report that mouse DENTT mRNA contains a 2031-bp open reading frame that encodes a predicted polypeptide of 677-amino acids with a relative molecular mass of 77,671 Da. The mouse and human DENTT sequences show 77% and 78% homology at the nucleotide and amino acid level, respectively. Mouse DENTT is predicted to be a nuclear protein with two nuclear localization signals (NLS), two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. Green fluorescent protein (GFP)-tagged full-length mouse DENTT transfected into COS-7 cells showed localization predominantly in the nucleolus. Reverse transcription-polymerase chain reaction amplification, Northern hybridization, and Western blot analyses showed expression of mouse DENTT mRNA and protein throughout mouse embryogenesis. Immunohistochemical staining analysis showed that DENTT is expressed in multiple tissues in a defined spatiotemporal pattern during mouse embryogenesis. The heart and primitive brain were the first organs of the embryo that showed immunoreactivity for the DENTT antibody by day 8 of development (E8). In the developing mouse brain, the choroid plexus was intensely stained for DENTT in all stages of development. The spinal cord and dorsal root ganglia were also positive for DENTT staining beginning in the 11-day-old embryo (E11), where homogeneous immunostaining was observed throughout the developing neurons. By day 16 of development (E16), only a small subset of the neuronal population in the spinal cord and dorsal root ganglia was positively stained for DENTT. DENTT immunoreactivity increased steadily with maturation as the differentiation of cartilage and osteoblasts proceeded and reached a maximum in the growth plate during endochondral ossification. DENTT expression was also detected in multiple rodent cell types in vitro, including mouse F9 embryonal carcinoma (EC) cells. Addition of retinoic acid or sodium butyrate to F9 EC cells showed a rapid decrease in expression of DENTT protein occurring by 1 hr that continued to decrease to almost undetectable levels after 24 hr. Cotransfection of full-length mouse DENTT expression plasmid with 3TPLux or COL7A1Luc Luciferase reporter plasmids into F9 EC cells significantly increased the level of 3TPLux reporter transcription while decreasing the level of COL7A1Luc reporter transcription, suggesting that DENTT may play multiple roles in modulating transcriptional responses. These findings suggest new roles for the TTSN superfamily during embryogenesis and differentiation.
Asunto(s)
Nucléolo Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/genética , Células 3T3 , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Huesos/embriología , Sistema Cardiovascular/embriología , Proteínas de Ciclo Celular , Diferenciación Celular/fisiología , Corion/química , Proteínas de Unión al ADN , Sistema Digestivo/embriología , Femenino , Genes Reporteros , Corazón/embriología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Sistema Nervioso/embriología , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Placenta/química , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
Differentially expressed nucleolar TGF-beta1 target (DENTT) is a novel member of the TSPY/TSPY-L/SET/NAP-1 (TTSN) superfamily that we have previously identified in human lung cancer cells. Here, we have investigated the expression of this protein in the adult mouse. By Western analysis, DENTT is highly expressed in the pituitary gland and moderately in the adrenals, brain, testis, and ovary. Immunohistochemical staining analysis for DENTT showed differential cytoplasmic and nuclear staining patterns in several cell types. The pituitary gland showed the highest level of immunostaining for DENTT, with strong cytoplasmic immunoreactivity in the anterior lobe, moderate levels in the posterior lobe, and a few cells showing nuclear staining in the intermediate lobe. In contrast, the intermediate lobe of the pituitary showed intense cytoplasmic staining for TGF-beta1. Nuclear and cytoplasmic staining for DENTT was present in the islets of Langerhans in the pancreas. Cytoplasmic staining for DENTT was particularly intense in the cortex of the adrenal gland, whereas the medulla showed weak nuclear staining. In the nervous system, the choroid plexus showed the highest immunoreactivity, with cortical motoneurons and Purkinje cells having relatively high levels of staining for DENTT as well. DENTT immunoreactivity was found in Leydig interstitial cells, Sertoli cells, and primary spermatocytes in the testis. In the female reproductive system, DENTT immunoreactivity was present in oocytes, thecal cells, and corpora lutea. The bronchial epithelium of the lung showed moderate levels of staining for DENTT localized to the cell nucleus. Additionally, three rodent pituitary cell lines (AtT20, GH3, and alphaT3-1, representing corticotropes, lactotropes, and gonadotropes, respectively) showed expression of DENTT. Addition of TGF-beta1 or serum to AtT20 cells increased DENTT protein production by 4 hr and, after reaching maximal levels at 2.4-fold above basal level by 8 hr, decreased, whereas no more than a 1.5-fold increase in DENTT protein occurred in GH3 or alphaT3-1 cells. Transient transfection studies showed that ectopic DENTT expression significantly increased the level of p3TP-Lux reporter transcription in AtT20 cells, but not in GH3 or alphaT3-1 cells. Interestingly, addition of TGF-beta1 had no significant effect on the ability of DENTT expression to influence p3TP-Lux reporter transcription in AtT20 cells. This report is the first detailed immunohistochemical examination of a member of the TTSN superfamily in the adult mouse. Expression of DENTT in endocrine tissues, nervous system, lung, oocytes, and thecal cells, in addition to the testis, suggests new roles for the TTSN superfamily. The differential patterns of expression of DENTT and TGF-beta1 in some tissues, including the pituitary, suggest that other factors are likely to be regulators of DENTT besides TGF-beta1.
Asunto(s)
Nucléolo Celular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Animales , Western Blotting , Proteínas de Ciclo Celular , Línea Celular , Femenino , Inmunohistoquímica , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Hipófisis/metabolismo , Distribución Tisular , TransfecciónRESUMEN
We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy.