RESUMEN
Although the hepatomitogenic activity of T3 is well established, the wide range of harmful effects exerted by this hormone precludes its use in regenerative therapy. The aim of this study was to investigate whether an agonist of TRß, KB2115 (Eprotirome), could exert a mitogenic effect in the liver, without most of the adverse T3/TRα-dependent side effects. F-344 rats treated with KB2115 for 1 week displayed a massive increase in bromodeoxyuridine incorporation (from 20% to 40% vs. 5% of controls), which was associated with increased mitotic activity in the absence of significant signs of liver toxicity. Noteworthy, while cardiac hypertrophy typical of T3 was not observed, beneficial effects, such as lowering blood cholesterol levels, were associated to KB2115 administration. Following a single dose of KB2115, hepatocyte proliferation was evident as early as 18 h, demonstrating its direct mitogenic effect. No increase in serum transaminase levels or apoptosis was observed prior to or concomitantly with the S phase. While KB2115-induced mitogenesis was not associated to enhance expression of c-fos, c-jun, and c-myc, cyclin D1 levels rapidly increased. In conclusion, KB2115 induces hepatocyte proliferation without overt toxicity. Hence, this agent may be useful for regenerative therapies in liver transplantation or other surgical settings.
Asunto(s)
Anilidas/química , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Alimentación Animal , Animales , Apoptosis , Peso Corporal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Corazón/efectos de los fármacos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Receptores de Hormona Tiroidea/agonistas , Medicina Regenerativa , Factores de Tiempo , Transaminasas/sangre , TriyodotironinaRESUMEN
UNLABELLED: Small noncoding RNAs comprise a growing family of molecules that regulate key cellular processes, including messenger RNA (mRNA) degradation, translational repression, and transcriptional gene silencing. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) represent a class of small RNAs initially identified in the germline of a variety of species, where they contribute to maintenance of genome stability, and recently found expressed also in stem and somatic cells, where their role and responsiveness to physiopathological signals remain elusive. Here, we investigated piRNA expression in rat liver and its response to the stimuli exerted by regenerative proliferation of this organ. Quantitative polymerase chain reaction analysis identify in the liver the RNAs encoding PIWIL2/HILI, PIWIL4/HIWI2, and other components of the piRNA biogenesis pathways, suggesting that this is indeed functional. RNA sequencing before, during, and after the wave of cell proliferation that follows partial hepatectomy (PH) identified â¼1,400 mammalian germline piRNAs expressed in rat liver, including 72 showing timed changes in expression 24-48 hours post-PH, a timing that corresponds to cell transition through the S phase, returning to basal levels by 168 hours, when organ regeneration is completed and hepatocytes reach quiescence. CONCLUSION: The piRNA pathway is active in somatic cells of the liver and is subject to regulation during the pathophysiological process of organ regeneration, when these molecules are available to exert their regulatory functions on the cell genome and transcriptome, as demonstrated by the identification of several liver mRNAs representing candidate targets of these regulatory RNAs.
Asunto(s)
Regulación de la Expresión Génica , Regeneración Hepática/genética , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Animales , Proliferación Celular , Regulación hacia Abajo/genética , Hepatectomía , Masculino , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas F344 , Análisis de Secuencia de ARNRESUMEN
UNLABELLED: Thyroid hormone (T3), like many other ligands of the steroid/thyroid hormone nuclear receptor superfamily, is a strong inducer of liver cell proliferation in rats and mice. However, the molecular basis of its mitogenic activity, which is currently unknown, must be elucidated if its use in hepatic regenerative medicine is to be considered. F-344 rats or C57BL/6 mice were fed a diet containing T3 for 2-7 days. In rats, administration of T3 led to an increased cytoplasmic stabilization and nuclear translocation of ß-catenin in pericentral hepatocytes with a concomitant increase in cyclin-D1 expression. T3 administration to wild-type (WT) mice resulted in increased hepatocyte proliferation; however, no mitogenic response in hepatocytes to T3 was evident in the hepatocyte-specific ß-catenin knockout mice (KO). In fact, T3 induced ß-catenin-TCF4 reporter activity both in vitro and in vivo. Livers from T3-treated mice demonstrated no changes in Ctnnb1 expression, activity of glycogen synthase kinase-3ß, known to phosphorylate and eventually promote ß-catenin degradation, or E-cadherin-ß-catenin association. However, T3 treatment increased ß-catenin phosphorylation at Ser675, an event downstream of protein kinase A (PKA). Administration of PKA inhibitor during T3 treatment of mice and rats as well as in cell culture abrogated Ser675-ß-catenin and simultaneously decreased cyclin-D1 expression to block hepatocyte proliferation. CONCLUSION: We have identified T3-induced hepatocyte mitogenic response to be mediated by PKA-dependent ß-catenin activation. Thus, T3 may be of therapeutic relevance to stimulate ß-catenin signaling to in turn induce regeneration in selected cases of hepatic insufficiency.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hepatocitos/fisiología , Regeneración Hepática/fisiología , Triyodotironina/fisiología , beta Catenina/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Insuficiencia Hepática/tratamiento farmacológico , Insuficiencia Hepática/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Regeneración Hepática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Endogámicas F344 , Transducción de Señal/fisiología , Triyodotironina/uso terapéutico , beta Catenina/fisiologíaRESUMEN
The growth arrest and DNA damage-inducible 45 (Gadd45) proteins act in many cellular processes. In the liver, Gadd45b (encoding Gadd45ß) is the gene most strongly induced early during both compensatory regeneration and drug-induced hyperplasia. The latter response is associated with the dramatic and rapid hepatocyte growth that follows administration of the xenobiotic TCPOBOP (1,4-bis[2-(3,5)-dichoropyridyloxy] benzene), a ligand of the nuclear receptor constitutive androstane receptor (CAR). Here, we have shown that Gadd45b-/- mice have intact proliferative responses following administration of a single dose of TCPOBOP, but marked growth delays. Moreover, early transcriptional stimulation of CAR target genes was weaker in Gadd45b-/- mice than in wild-type animals, and more genes were downregulated. Gadd45ß was then found to have a direct role in transcription by physically binding to CAR, and TCPOBOP treatment caused both proteins to localize to a regulatory element for the CAR target gene cytochrome P450 2b10 (Cyp2b10). Further analysis defined separate Gadd45ß domains that mediated binding to CAR and transcriptional activation. Although baseline hepatic expression of Gadd45b was broadly comparable to that of other coactivators, its 140-fold stimulation by TCPOBOP was striking and unique. The induction of Gadd45ß is therefore a response that facilitates increased transcription, allowing rapid expansion of liver mass for protection against xenobiotic insults.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Animales , Antígenos de Diferenciación/química , Antígenos de Diferenciación/genética , Receptor de Androstano Constitutivo , Femenino , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Regeneración Hepática/efectos de los fármacos , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Terciaria de Proteína , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Activación TranscripcionalRESUMEN
High doses of T3 are mitogenic in liver, causing hyperplasia that has numerous differences from the compensatory regeneration induced by partial hepatectomy (PH). T3 binds to the thyroid hormone receptor (TR), which directly regulates transcription, while PH acts indirectly through signal transduction pathways. We therefore carried out a proteomic analysis to compare early effects of the two treatments. Transcriptome analysis by DNA microarray also confirmed the observed proteomic changes, demonstrating that they were caused by transcriptional regulation. Among the differentially expressed proteins, many are directly or indirectly involved in energy metabolism and response to oxidative stress. Several enzymes of lipid metabolism (e.g., Acaa2, Acads, Hadh, and Echs1) were differentially regulated by T3. In addition, altered expression levels of several mitochondrial proteins (e.g., Hspa9, Atp5b, Cps1, Glud1, Aldh2, Ak2, Acads) demonstrated the known increase of mitochondrial biogenesis mediated by T3. The present results provide insights in changes in metabolic balance occurring following T3-stimulation and define a basis for dissecting the molecular pathways of hepatocyte hyperplasia.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Proteómica/métodos , Transducción de Señal , Animales , Electroforesis en Gel Bidimensional , Hepatectomía , Masculino , Redes y Vías Metabólicas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Ratas , Ratas Wistar , Receptores de Hormona Tiroidea/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transcripción Genética/efectos de los fármacos , Triyodotironina/farmacologíaRESUMEN
UNLABELLED: The Hippo kinase cascade, a growth-suppressive pathway that ultimately antagonizes the transcriptional coactivator Yes-associated protein (YAP), has been shown in transgenic animals to orchestrate organ size regulation. The purpose of this study was to determine whether in non-genetically modified mice (1) the Hippo pathway is involved in the regulation of adaptive liver enlargement caused by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor and (2) a dysregulation of this pathway occurs during the development of chemically induced hepatocellular carcinoma (HCC). We show that liver enlargement caused by TCPOBOP was associated with an increase of YAP protein levels that paralleled the increase in 2-bromodeoxyuridine incorporation. Interestingly, when a second dose of TCPOBOP was given to mice with enlarged livers, no further increases in liver mass or YAP protein levels were observed, suggesting that the Hippo pathway prevents further growth of the hyperplastic liver. Viral-mediated exogenous expression of active YAP in mouse livers was able to partially overcome the block of hepatocyte proliferation. We also show that HCCs developed in mice given diethylnitrosamine and then subjected to repeated treatments with TCPOBOP had increased levels of YAP that were associated with down-regulation of microRNA 375, which is known to control YAP expression, and with enhanced levels of alpha-fetoprotein and connective tissue growth factor, two target genes of YAP. CONCLUSION: These results suggest that the Hippo pathway regulates adaptive liver enlargement and is probably inactivated in initiated cells that escape the suppressive constrain exerted on the surrounding normal tissue, thus allowing clonal expansion to HCC.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Carcinoma Hepatocelular/fisiopatología , Hepatomegalia/fisiopatología , Neoplasias Hepáticas/fisiopatología , Hígado/patología , Fosfoproteínas/fisiología , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Dietilnitrosamina/efectos adversos , Modelos Animales de Enfermedad , Femenino , Hepatomegalia/inducido químicamente , Hepatomegalia/patología , Hiperplasia , Péptidos y Proteínas de Señalización Intracelular/fisiología , Hígado/efectos de los fármacos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Piridinas/efectos adversos , Piridinas/farmacología , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND & AIMS: Mice lacking c-jun in the liver display impaired regeneration after partial hepatectomy (PH), and were reported to be more resistant to chemically-induced hepatocellular carcinoma (HCC). We investigated the role of c-jun in normal and preneoplastic hepatocyte proliferation induced by ligands of nuclear receptors, which cause liver hyperplasia in the absence of cell loss/death. METHODS: The effect of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) on hepatocyte proliferation was determined in c-jun conditional knockout (c-jun(Δli)) or in mouse liver where c-jun has been silenced. To study the role of c-jun in HCC development, c-jun(Δli) and WT mice were given diethylnitrosamine (DENA) followed by repeated injections of TCPOBOP. RESULTS: Hepatocyte proliferation induced by TCPOBOP was associated with a stronger proliferative response and earlier S phase entry in c-jun(Δli) mice, compared to WT animals. Moreover, silencing of c-jun in the liver of CD-1 mice caused increased hepatocyte proliferation. A stronger hepatocyte proliferative response of c-jun(Δli) mice was observed also following treatment with a ligand of thyroid hormone receptor. Finally, loss of c-jun did not inhibit the development of HCC induced by DENA and promoted by TCPOBOP. CONCLUSIONS: (i) c-jun may, under certain conditions, negatively regulate proliferation of normal hepatocytes, (ii) c-jun is not an absolute requirement for DENA/TCPOBOP-induced HCC formation, suggesting that the therapeutic potential of c-jun/JNK inhibition in liver tumors might be impaired by an increased stimulation of cell growth due to blockade of the c-jun pathway.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Genes jun , Neoplasias Hepáticas Experimentales/genética , Piridinas/farmacología , Triyodotironina/farmacología , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Transformación Celular Neoplásica/efectos de los fármacos , Receptor de Androstano Constitutivo , Dietilnitrosamina , Femenino , Expresión Génica , Silenciador del Gen , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
BACKGROUND & AIMS: Thyroid hormones elicit many cellular and metabolic effects in various organs. Most of these actions, including mitogenesis, are mediated by the thyroid hormone 3,5,3'-triiodo-l-thyronine (T3) nuclear receptors (TRs). They are transcription factors, expressed as different isoforms encoded by the TRalpha and TRbeta genes. Here, experiments were performed to determine whether (i) T3-induces hepatocyte proliferation in mouse liver and pancreas, and, (ii) which TR isoform, is responsible for its mitogenic effect. METHODS: Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation after T3 or the TRbeta agonist GC-1 in liver and pancreas of CD-1, C57BL, or TRalpha(0/0) mice. Cell cycle-associated proteins were measured by Western blot. RESULTS: T3 added to the diet at a concentration of 4 mg/kg caused a striking increase in BrdU incorporation in mouse hepatocytes. Increased BrdU incorporation was associated with enhanced protein levels of cyclin D1 and PCNA and decreased levels of p27. Treatment with GC-1, a selective agonist of the TRbeta isoform, also induced a strong mitogenic response of mouse hepatocytes and pancreatic acinar cells which was similar to that elicited by T3. Finally, treatment with T3 of mice TRalpha(0/0) induced a proliferative response in the liver and pancreas, similar to that of their wild type counterpart. CONCLUSIONS: These results demonstrate that T3 is a powerful inducer of cell proliferation in mouse liver and suggest that the beta-isoform is responsible for the hepatomitogenic activity of T3. The same isoform seems to also mediate the proliferation of mouse pancreatic acinar cells.
Asunto(s)
Hepatocitos/metabolismo , Páncreas/metabolismo , Receptores beta de Hormona Tiroidea/metabolismo , Triyodotironina/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Páncreas/citología , Páncreas/efectos de los fármacos , Isoformas de Proteínas/metabolismoRESUMEN
UNLABELLED: Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S-transferase (GSTP)(+) focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19(+). Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19(+) lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19(+) early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19-associated gene expression signature accurately predicted patient survival (P < 0.009) and tumor recurrence (P < 0.006). CONCLUSION: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19-associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.
Asunto(s)
Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Células Madre/patología , Animales , Carcinoma Hepatocelular/patología , Glutatión Transferasa/análisis , Inmunohistoquímica , Queratina-19/análisis , Queratina-19/genética , Neoplasias Hepáticas/patología , Masculino , Proteína Quinasa 14 Activada por Mitógenos/análisis , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Modelos de Riesgos Proporcionales , Ratas , Ratas Endogámicas F344RESUMEN
UNLABELLED: Triiodothyronine (T3), through interaction with its intracellular thyroid hormone receptors (TRs), influences various physiological functions, including metabolism, development, and growth. We investigated the effect of T3 and the selective TR-beta agonist GC-1 in two models of hepatocarcinogenesis. Preneoplastic lesions were induced in F-344 rats via a single dose of diethylnitrosamine, followed by a choline-deficient (CD) diet for 10 weeks. Rat subgroups were then fed the CD diet or a CD diet containing either 4 mg/kg T3 or 5 mg/kg GC-1 for another week. Rats fed a CD diet alone showed a large number (65/cm(2)) of preneoplastic lesions positive for the placental form of glutathione S-transferase (GSTP). Coadministration of T3 for the last week caused an almost complete disappearance of the foci (3/cm(2)). A reduction of GSTP-positive foci was also observed in rats fed a CD + GC-1 diet (28/cm(2) versus 75/cm(2) of rats fed a CD diet alone) in the absence of significant differences in labeling or apoptotic index of preneoplastic hepatocytes between the two groups. An antitumoral effect of GC-1 was also observed with the resistant hepatocyte model of hepatocarcinogenesis. Nodule regression was associated with a return to a fully differentiated phenotype, indicated by the loss of the fetal markers GSTP and gamma glutamyl transpeptidase, and reacquisition of the activity of glucose 6-phosphatase and adenosine triphosphatase, two enzymes expressed in normal hepatocytes. CONCLUSION: Our results indicate that activated TRs negatively influence the carcinogenic process through induction of a differentiation program of preneoplastic hepatocytes. The results also suggest that TRs could be a meaningful target in liver cancer therapy.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Lesiones Precancerosas/tratamiento farmacológico , Triyodotironina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Deficiencia de Colina/complicaciones , Dietilnitrosamina , Glutatión Transferasa/metabolismo , Hepatocitos/metabolismo , Masculino , Lesiones Precancerosas/etiología , Ratas , Ratas Endogámicas F344 , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/análogos & derivados , Triyodotironina/farmacologíaRESUMEN
Nonalcoholic fatty liver disease is the most common noninfectious liver disease in clinical practice, and there is an increasing need for new therapeutic approaches for the treatment of this liver disease. Here, we examined the effect of the thyroid hormone triiodothyronine (T3) and the agonist of the thyroid hormone receptor beta isoform (TRbeta), GC-1, on fatty liver and steatohepatitis induced in rodents by a choline-methionine deficient (CMD) diet. Male Fischer 344 rats fed a CMD diet for 1 wk developed a marked fatty liver and mild hepatitis. Concurrent administration of T3 resulted in a complete prevention of the fatty change associated with increased fatty acid mitochondrial and peroxisomal beta-oxidation. To investigate whether T3 could also reverse fully established fatty liver, rats were fed a CMD diet for 10 wk and then cofed T3 for 1 wk. Coadministration of T3 resulted in a complete regression of liver steatosis associated with a decrease of lipid peroxidation, cyclooxygenase-2 expression, and activation of phospho-STAT3 and phospho-SAPK/JNK. Finally, additional experiments showed that GC-1, which has no significant side effects on heart rate, prevented and reverted CMD-induced fat accumulation, and ameliorated steatohepatitis. These results indicate that TR agonists have the potential to inhibit or reverse hepatic steatosis induced by a nutritional model.
Asunto(s)
Acetatos/farmacología , Hígado Graso/tratamiento farmacológico , Fenoles/farmacología , Receptores beta de Hormona Tiroidea/agonistas , Triyodotironina/farmacología , Animales , Secuencia de Bases , Cartilla de ADN/genética , Dieta , Modelos Animales de Enfermedad , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/prevención & control , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratas , Ratas Endogámicas F344 , Triglicéridos/metabolismoRESUMEN
Although liver regeneration occurring after partial hepatectomy (PH) is greatly reduced in aged mice, liver hyperplasia induced by xenobiotic mitogens was found to be age independent. Here, we investigated the potential utility of mitogens in stimulating liver regeneration in old mice subjected to two-third PH. Although virtually no hepatocytes entered S phase 48 h after PH, pretreatment (2 h prior to surgery) with 1,4-bis(2-(3,5-dichloropyridyloxy)benzene (TCPOBOP), a ligand of constitutive androstane receptor, induced an increase of bromodeoxyuridine incorporation and enhanced the expression of cyclin D1, cyclin A and proliferating cell nuclear antigen . Next, we investigated the potential utility of mitogens in the context of donor conditioning prior to living-related transplantation. Three days after TCPOBOP administration to intact young mice, an almost doubling of the liver mass and DNA content occurred; the regenerative response to two-third resection of the TCPOBOP-induced hyperplastic liver was similar to that of mice subjected to PH alone, suggesting that an increased liver mass at the time of surgery does not inhibit the regenerative capacity. The present results suggest that mitogen-induced hyperplasia is a promising tool in conditions characterized by reduced regenerative capacity, such as in the elderly, or when a rapid increase of liver mass is required, such as in living-related transplantation.
Asunto(s)
Regeneración Hepática/efectos de los fármacos , Trasplante de Hígado , Mitógenos/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Edad , Animales , Femenino , Hepatectomía , Hiperplasia/patología , Hígado/patología , Donadores Vivos , Ratones , Tamaño de los Órganos/efectos de los fármacosRESUMEN
Thyroid hormones regulate cell growth, cell differentiation, and metabolic functions via interaction with the thyroid hormone nuclear receptors (TRs). Recently, a small class of halogen-free high-affinity thyroid hormone agonists has been developed that are highly selective for the TRbeta subtype. Because of the selective hyperthyroidism generated by one of these agonists, GC-1, this compound has the potential to be developed as a new therapeutic agent for the treatment of a variety of metabolic disturbances, including lipid disorders and obesity; thus, it becomes important to determine whether GC-1 has other unknown effects on potential target organs. The purpose of this study was to investigate the effect of GC-1 on cell proliferation in rat liver and pancreas. Rats treated with GC-1 (50 or 100 mug/100 g body weight) were killed at different time points. Hepatic and pancreatic cell proliferation was monitored by immunohistochemical determination of bromodeoxyuridine incorporation. The expression of cell cycle-related genes was analyzed by Northern and Western analysis. The results show that GC-1 strongly stimulates rat hepatocyte proliferation in the absence of tissue injury. Although GC-1-induced hepatocyte proliferation was associated with a rapid increase in cyclin D1 mRNA levels, no change in the expression of c-jun and c-fos was observed. GC-1 also induced massive pancreatic cell proliferation. The results indicate that the TRbeta-selective agonist GC-1 is a strong mitogen for hepatocytes and pancreatic acinar cells. Furthermore, they suggest that the TRbeta receptor is the mediator for the mitogenic activity of thyroid hormone and other thyromimetics.
Asunto(s)
Acetatos/farmacología , Hígado/patología , Páncreas/patología , Fenoles/farmacología , Receptores beta de Hormona Tiroidea/agonistas , Animales , Ciclo Celular , Proliferación Celular , Hepatocitos/metabolismo , Lípidos/química , Hígado/metabolismo , Masculino , Páncreas/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Wistar , Hormonas Tiroideas/metabolismoRESUMEN
Although mammalian cardiomyocytes lose their proliferative capacity after birth, there is evidence that postmitotic cardiomyocytes can proliferate provided that cyclin D1 accumulates in the nucleus. Here we show by Northern blot, Western analysis, and immunohistochemistry that 3,5,3'-triiodothyronine (T3) treatment of adult rats caused an increase of cyclin D1 mRNA and protein levels. The increased cyclin D1 protein content was associated with its translocation into the nucleus of cardiomyocytes. These changes were accompanied by the re-entry of cardiomyocytes into the cell cycle, as demonstrated by increased levels of cyclin A, PCNA, and incorporation of bromodeoxyuridine into DNA (labeling index was 30.2% in T3-treated rats vs. 2.2% in controls). Entry into the S phase was associated with an increased mitotic activity as demonstrated by positivity of cardiomyocyte nuclei to antibodies anti-phosphohistone-3, a specific marker of the mitotic phase (mitotic index was 3.01/1000 cardiomyocte nuclei in hyperthyroid rats vs. 0.04 in controls). No biochemical or histological signs of tissue damage were observed in the heart of T3-treated rats. These results demonstrated that T3 treatment is associated with a re-entry of cardiomyocytes into the cell cycle and so may be important for the development of future therapeutic strategies aimed at inducing proliferation of cardiomyocytes.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Replicación del ADN/efectos de los fármacos , ADN/biosíntesis , Miocitos Cardíacos/efectos de los fármacos , Triyodotironina/farmacología , Animales , Bromodesoxiuridina/metabolismo , Cardiomegalia/inducido químicamente , Ciclo Celular/efectos de los fármacos , Creatina Quinasa/sangre , Creatinina/sangre , L-Lactato Deshidrogenasa/sangre , Masculino , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
We previously observed that Gadd45/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate-early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45 is known to be stimulated by nuclear factor (NF) B, which is activated by tumor necrosis factor alpha (TNF) in the early response to partial hepatectomy. We therefore investigated whether TNF and NFB also stimulated Gadd45 as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild-type, TNFR1-/-, and TNFR1-/-TNFR2-/-. The results showed that TCPOBOP did not activate NFB in any of the mice, but a strong induction of Gadd45 messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NFB did not impair CAR-mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1-/- and TNFR1-/-TNFR2-/- mice. Further studies evaluated TCPOBOP-induced gene expression in CAR-/- mice, by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45, were almost completely abolished--hence all were mediated via CAR activation. In conclusion, in the liver, Gadd45 can be induced by a distinctive pathway that requires CAR and is independent of TNF-NFB. The greater induction of proliferation in TNFR-null mice suggests negative cross-talk between the CAR and TNF-NFB controls that regulate proliferation.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Hígado/metabolismo , Hígado/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Receptor de Androstano Constitutivo , Femenino , Hiperplasia , Ratones , Ratones Endogámicos , Ratones Noqueados , FN-kappa B/metabolismo , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Factores de Transcripción/deficienciaRESUMEN
It has been shown that the magnitude of DNA synthesis and the time at which maximal DNA synthesis occurs after two-thirds partial hepatectomy (PH) is greatly reduced in the liver of aged rodents compared to young animals. This reduction could represent an intrinsic defect in proliferation or a more specialized change in the response to PH. We therefore evaluated the proliferative capacity of hepatocytes in aged animals, following treatment with primary liver mitogens. We show that treatment of 12-month-old CD-1 mice with the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) caused an increase in hepatocyte proliferation similar to that seen in young (8-week-old) mice. The labeling index was 82% in the livers of aged mice versus 76% in young animals. Histological observation demonstrated that the number of hepatocytes entering mitoses was similar in both groups; the mitotic indices were 2.5 per thousand and 2.7 per thousand, respectively. Additional experiments showed that the timing of DNA synthesis and M phase were nearly identical in both aged and young mice. Stimulation of hepatocyte DNA synthesis was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, pRb, and p107); all were comparable in aged mice and young mice. TCPOBOP treatment also increased expression of the Forkhead Box transcription factor m1b (Foxm1b) to a similar degree in both groups. In conclusion, hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. These findings suggest that therapeutic use of mitogens would alleviate the reduction in hepatocyte proliferation observed in the elderly.
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Envejecimiento/fisiología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Mitógenos/farmacología , Piridinas/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Femenino , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Hepatocitos/fisiología , Regeneración Hepática/fisiología , Ratones , Ratones Endogámicos , ARN Mensajero/análisis , Factores de Transcripción/genéticaRESUMEN
Although suppression of apoptosis has been implicated as a mechanism for the hepatocarcinogenicity of peroxisome proliferators (PPs), they can also induce cell death in rat AH130 and human HepG2 hepatoma cells. To study how PPs induce cell death and to characterize the molecular events involved, we administered the hypolipidemic BR931, a peroxisome proliferator, to rat hepatoma FaO cells. Treatment with increasing concentrations of BR931 (0.015 to 0.6 mM) reduced cell viability in a dose- and time-dependent manner, associated with DNA fragmentation and morphological changes characteristic of apoptosis. BR931 also caused phosphorylation of p53 within 3 hours, translocation of the pro-apoptotic Bax protein to mitochondria, release of cytochrome-c into the cytosol, and activation of caspase-9 and -3. These results indicated that BR931 activated the intrinsic caspase cascade. Pretreatment with three different antioxidants, N-acetylcysteine, Vitamin C and Trolox, reduced apoptosis, suggesting that reactive oxygen species (ROS) plays a role in BR931-induced apoptosis. In support of this hypothesis, BR931 produced increased levels of 8-hydroxy-deoxy-guanosine, a marker of DNA oxidative damage. Antioxidants prevented the p53 phosphorylation, up-regulation of Bax and BR931-induced apoptosis. These results suggest that BR931 can increase generation of ROS, leading to DNA damage and p53 phosphorylation, which, in turn, induces the activation of Bax, release of cytochrome-c from mitochondria and activation of caspases, culminating in cell death.
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Apoptosis/fisiología , Genes p53/fisiología , Proliferadores de Peroxisomas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Pirimidinas/farmacología , Acetilcisteína/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/genética , Ácido Ascórbico/farmacología , Western Blotting , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Citocromos c/fisiología , Citosol/metabolismo , ADN/química , Fragmentación del ADN/fisiología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Humanos , Etiquetado Corte-Fin in Situ , Neoplasias Hepáticas Experimentales/patología , Mitocondrias/metabolismo , Fosforilación , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Ratas , Factores de Tiempo , Proteína X Asociada a bcl-2RESUMEN
Partial hepatectomy (PH) and some tumor-promoting agents stimulate hepatocyte cell proliferation, but each treatment acts through distinct transcription factors. We compared mouse immediate-early gene expression changes after PH with those induced by 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene (TCPOBOP), a tumor-promoting liver mitogen. PH activates nuclear factor kappa B (NF-kappa B) and Stat3, whereas TCPOBOP is a ligand for the nuclear receptor, constitutive androstane receptor (CAR). RNA from 1 and 3 hours after each treatment was hybridized to a 9,000 complementary DNA (cDNA) microarray. Of about 6,000 messenger RNAs that had detectable expression, 127 showed reproducible up-regulation or down-regulation at a significant level. The TCPOBOP response was more discrete than the PH response; they amounted to 1% and 1.9% of positive hybridizations, respectively. Twenty-three genes were regulated only by TCPOBOP, 57 only by PH, and 59 by both treatments. More detailed analysis defined 16 clusters with common patterns of expression. These patterns and quantification of hybridization levels on the array were confirmed by Northern blots. TCPOBOP selectively activated expression of a number of detoxification enzymes. In conclusion, the genes that were regulated by both treatments suggest down-regulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components.
Asunto(s)
Genes Inmediatos-Precoces/fisiología , Regeneración Hepática/genética , Hígado/patología , Hígado/fisiología , Animales , División Celular/efectos de los fármacos , División Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Hepatectomía , Hiperplasia , Hígado/cirugía , Ratones , Ratones Endogámicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Piridinas/farmacologíaRESUMEN
The nuclear receptor Constitutive Androstane Receptor (CAR) binds DNA as a heterodimer with the retinoic-X receptor and activates gene transcription. Previously, in vitro studies have shown that the testosterone metabolites, androstenol and androstenol, inhibit the constitutive transcriptional activity of CAR, suggesting that differences might exist in the response to CAR-mediated gene activation between different sexes. In this study, we have analyzed the response of female and male CD-1 mice to stimulation of hepatocyte proliferation caused by the CAR ligand TCPOBOP. Results showed that the labelling index of female hepatocytes at 24, 30 and 36 h after treatment was much higher than that found in males. The higher proliferative activity of female hepatocytes was associated with increased hepatic levels of cyclin D1, cyclin A, E2F and enhanced phosphorylation of pRb and p107. The increased mitogenic response of females was associated with higher mRNA levels of CYP2B10, a known target of CAR. Administration of androstenol to TCPOBOP-treated mice caused a reduction of labelling index, which was accompanied by a decrease of CYP2B10 and CAR mRNA levels. In conclusion, the results show that, in addition to microsomal detoxification, another biological response elicited by the CAR ligand TCPOBOP, namely, hepatocyte proliferation, occurs at higher levels in female than male mice, suggesting that CAR transcriptional activity in males is partially counteracted by physiological higher levels of testosterone metabolites such as androstenol and androstenol.
Asunto(s)
Hepatocitos/efectos de los fármacos , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Androstanoles/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/genética , División Celular/efectos de los fármacos , Receptor de Androstano Constitutivo , Ciclina D1/genética , Familia 2 del Citocromo P450 , Femenino , Hepatocitos/fisiología , Masculino , Ratones , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/genética , Caracteres Sexuales , Esteroide Hidroxilasas/genética , Factores de Transcripción/genéticaRESUMEN
Previous work has shown that treatment with thyroid hormone (T3) decreased the incidence of rat hepatocellular carcinoma (HCC). The present study was designed to determine whether the inhibitory effect of T3 on HCC development was limited to early steps of the carcinogenetic process or, whether a similar effect could also be exerted by starting T3 treatment at later stages. Hepatic nodules were induced in Fischer rats by a single dose of DENA, followed by a 2-week exposure of the animals to 2-AAF and partial hepatectomy. Rats were then divided into 3 groups: group 1 was maintained on basal diet: group 2 was fed a diet containing 4 mg/kg T3 for a week, every month/7 months, starting 9 weeks after DENA administration: group 3 was exposed to cycles of T3 starting 8 months after initiation. Results demonstrate that inhibition of HCC development was essentially similar in rats exposed to T3 starting either 9 weeks or 8 months after initiation (50% inhibition compared to control rats). We have previously shown that T3-induced nodule regression and HCC inhibition occurred in spite of its mitogenic effect. Therefore, we next wished to determine whether a similar antitumoral effect could be exerted by other liver mitogens, such as peroxisome proliferators. Rats exposed to the initiation-promotion protocol described previously, were subjected to 11 cycles of a T3 or a ciprofibrate-supplemented diet, each cycle consisting of 7 days/month: the incidence of HCC and lung metastases was determined 13.5 months after initiation. Results showed that although treatment with T3 strongly inhibited HCC development (only 31% of T3+ rats showed HCC vs 91% of controls), rats given ciprofibrate developed the same number of HCC as T3-untreated rats. In conclusion, the results of this study showed that the anticarcinogenic effect of T3 is maintained also when treatment begins late in the process, and its antitumoral property appears to be specific and may not be shared by other liver mitogens.