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1.
J Biol Chem ; 299(12): 105407, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38152849

RESUMEN

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.


Asunto(s)
Vías Biosintéticas , Ciclina D1 , Hepatocitos , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Hepatocitos/metabolismo , Proteómica , Pirimidinas/biosíntesis , Humanos , Animales , Ratones , Línea Celular
3.
Science ; 371(6532)2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33632817

RESUMEN

The liver is organized into zones in which hepatocytes express different metabolic enzymes. The cells most responsible for liver repopulation and regeneration remain undefined, because fate mapping has only been performed on a few hepatocyte subsets. Here, 14 murine fate-mapping strains were used to systematically compare distinct subsets of hepatocytes. During homeostasis, cells from both periportal zone 1 and pericentral zone 3 contracted in number, whereas cells from midlobular zone 2 expanded in number. Cells within zone 2, which are sheltered from common injuries, also contributed to regeneration after pericentral and periportal injuries. Repopulation from zone 2 was driven by the insulin-like growth factor binding protein 2-mechanistic target of rapamycin-cyclin D1 (IGFBP2-mTOR-CCND1) axis. Therefore, different regions of the lobule exhibit differences in their contribution to hepatocyte turnover, and zone 2 is an important source of new hepatocytes during homeostasis and regeneration.


Asunto(s)
Hepatocitos/fisiología , Regeneración Hepática , Hígado/fisiología , Animales , Sistema Biliar/citología , Enfermedades de las Vías Biliares/fisiopatología , Proliferación Celular , Ciclina D1/metabolismo , Técnicas de Sustitución del Gen , Homeostasis , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Hígado/citología , Ratones , Serina-Treonina Quinasas TOR/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(29): 17177-17186, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32631996

RESUMEN

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.


Asunto(s)
Ciclo Celular/fisiología , Ciclina D1/genética , Ciclina D1/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Hígado/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Hepatocitos/metabolismo , Hepatocitos/patología , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados
5.
Hepatol Commun ; 3(3): 406-422, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30859152

RESUMEN

During normal proliferation, hepatocytes accumulate triglycerides (TGs) in lipid droplets (LDs), but the underlying mechanisms and functional significance of this steatosis are unknown. In the current study, we examined the coordinated regulation of cell cycle progression and LD accumulation. As previously shown, hepatocytes develop increased LD content after mitogen stimulation. Cyclin D1, in addition to regulating proliferation, was both necessary and sufficient to promote LD accumulation in response to mitogens. Interestingly, cyclin D1 promotes LD accumulation by inhibiting the breakdown of TGs by lipolysis through a mechanism involving decreased lipophagy, the autophagic degradation of LDs. To examine whether inhibition of lipolysis is important for cell cycle progression, we overexpressed adipose TG lipase (ATGL), a key enzyme involved in TG breakdown. As expected, ATGL reduced LD content but also markedly inhibited hepatocyte proliferation, suggesting that lipolysis regulates a previously uncharacterized cell cycle checkpoint. Consistent with this, in mitogen-stimulated cells with small interfering RNA-mediated depletion of cyclin D1 (which inhibits proliferation and stimulates lipolysis), concurrent ATGL knockdown restored progression into S phase. Following partial hepatectomy, a model of robust hepatocyte proliferation in vivo, ATGL overexpression led to decreased LD content, cell cycle inhibition, and marked liver injury, further indicating that down-regulation of lipolysis is important for normal hepatocyte proliferation. Conclusion: We suggest a new relationship between steatosis and proliferation in hepatocytes: cyclin D1 inhibits lipolysis, resulting in LD accumulation, and suppression of lipolysis is necessary for cell cycle progression.

8.
Oncotarget ; 7(30): 47674-47686, 2016 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-27351284

RESUMEN

Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation.


Asunto(s)
Ciclina D1/metabolismo , Ácidos Grasos/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , PPAR alfa/metabolismo , Animales , Línea Celular Tumoral , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidación-Reducción , Proto-Oncogenes Mas , Ratas , Ratas Sprague-Dawley , Transfección
9.
J Biol Chem ; 288(29): 21389-21398, 2013 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-23740243

RESUMEN

Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced ß-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and ß-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.


Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Regeneración Hepática , Sulfatasas/metabolismo , Vía de Señalización Wnt , Animales , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Hepatectomía , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Regeneración Hepática/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Sulfatasas/deficiencia , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt3A/farmacología , Proteína con Dedos de Zinc GLI1 , beta Catenina/metabolismo
10.
Cell Cycle ; 11(14): 2681-90, 2012 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-22751438

RESUMEN

Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.


Asunto(s)
Ciclina D1/metabolismo , Glucosa/farmacología , Factor Nuclear 4 del Hepatocito/metabolismo , Lipogénesis/efectos de los fármacos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células Cultivadas , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Proteínas Nucleares/genética , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/genética
11.
PLoS One ; 6(11): e27360, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22073316

RESUMEN

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.


Asunto(s)
Ciclo Celular , Ciclina D1/metabolismo , Vestíbulo del Laberinto/citología , Adulto , Apoptosis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Humanos
12.
J Clin Invest ; 121(7): 2821-32, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21633171

RESUMEN

Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.


Asunto(s)
Antibióticos Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Regeneración Hepática/fisiología , Hígado/efectos de los fármacos , Hígado/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Sirolimus/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Genotipo , Hepatectomía , Hepatocitos/citología , Hepatocitos/fisiología , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/citología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos , Proteínas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Serina-Treonina Quinasas TOR
13.
Hepatology ; 54(2): 609-19, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21574170

RESUMEN

UNLABELLED: The liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genomewide microRNA (miRNA) microarray studies during liver regeneration in rats after 70% partial hepatectomy (PH) at early and mid time points to more thoroughly understand their role. At 3, 12, and 18 hours post-PH ∼40% of the miRNAs tested were up-regulated. Conversely, at 24 hours post-PH, ∼70% of miRNAs were down-regulated. Furthermore, we established that the genomewide down-regulation of miRNA expression at 24 hours was also correlated with decreased expression of genes, such as Rnasen, Dgcr8, Dicer, Tarbp2, and Prkra, associated with miRNA biogenesis. To determine whether a potential negative feedback loop between miRNAs and their regulatory genes exists, 11 candidate miRNAs predicted to target the above-mentioned genes were examined and found to be up-regulated at 3 hours post-PH. Using reporter and functional assays, we determined that expression of these miRNA-processing genes could be regulated by a subset of miRNAs and that some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that overexpression of these miRNAs inhibited cell proliferation and modulated cell cycle in both Huh-7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up-regulation of miRNAs in the early phase after PH was involved in the priming and commitment to liver regeneration, whereas the subsequent genomewide down-regulation of miRNAs was required for efficient recovery of liver cell mass. CONCLUSION: Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady-state regulation of liver regeneration.


Asunto(s)
Regulación hacia Abajo , Retroalimentación Fisiológica , Estudio de Asociación del Genoma Completo , Regeneración Hepática/genética , MicroARNs/genética , Animales , Células Cultivadas , Humanos , Masculino , Ratas , Ratas Sprague-Dawley
14.
Am J Physiol Gastrointest Liver Physiol ; 298(6): G884-95, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20338923

RESUMEN

Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.


Asunto(s)
Andrógenos/biosíntesis , Ciclina D1/metabolismo , Estrógenos/biosíntesis , Hígado/metabolismo , Animales , Inhibidores de la Aromatasa/farmacología , Línea Celular Tumoral , Ciclina D1/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Letrozol , Regeneración Hepática , Masculino , Ratones , Ratones Endogámicos BALB C , Nitrilos/farmacología , Triazoles/farmacología
15.
Cell Cycle ; 8(17): 2802-9, 2009 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-19652536

RESUMEN

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2(-/-) mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication and survival. Cdk2(-/-) hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2(-/-) liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2(-/-) hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2(-/-) mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.


Asunto(s)
Ciclo Celular , Ciclina D1/metabolismo , Quinasa 2 Dependiente de la Ciclina/fisiología , Hepatocitos/enzimología , Animales , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Centrosoma/metabolismo , Ciclina A/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Regeneración Hepática , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Purinas/farmacología , Roscovitina , Factores de Tiempo , Transfección
16.
Cell Cycle ; 7(14): 2215-24, 2008 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-18635970

RESUMEN

The D-type cyclins (D1, D2 and D3) are components of the cell cycle machinery and govern progression through G(1) phase in response to extracellular signals. Although these proteins are highly homologous and conserved in evolution, they contain distinct structural motifs and are differentially regulated in various cell types. Cyclin D1 appears to play a role in many different types of cancer, whereas cyclins D2 and D3 are less frequently associated with malignancy. In this study, we transiently expressed cyclin D1, D2 or D3 in hepatocytes and analyzed transcriptional networks regulated by each. All three D-type cyclins promoted robust hepatocyte proliferation and marked liver growth, although cyclin D3 stimulated less DNA synthesis than D1 or D2. Accordingly, the three D-type cyclins similarly activated genes associated with cell division. Cyclin D1 regulated transcriptional pathways involved in the metabolism of carbohydrates, lipids, amino acids, and other substrates, whereas cyclin D2 did not regulate these pathways despite having an equivalent effect on proliferation. Comparison of transcriptional profiles following 70% partial hepatectomy and cyclin D1 transduction revealed a highly significant overlap, suggesting that cyclin D1 may regulate diverse cellular processes in the regenerating liver. In summary, these studies provide the first comparative analysis of the transcriptional networks regulated by the D-type cyclins and provide insight into novel functions of these key cell cycle proteins. Further study of the unique targets of cyclin D1 should provide further insight into its prominent role in proliferation, growth and cancer.


Asunto(s)
Ciclinas/metabolismo , Transcripción Genética , Animales , Proteínas de Ciclo Celular/genética , Proliferación Celular , Ciclina D , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina D2 , Ciclina D3 , Ciclinas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hepatectomía , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas
17.
Exp Cell Res ; 314(11-12): 2266-78, 2008 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-18570922

RESUMEN

Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBPbeta mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.


Asunto(s)
Diferenciación Celular/fisiología , Ciclinas/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético , Mioblastos/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas CELF1 , Fusión Celular , Línea Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/genética , Humanos , Ratones , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Mioblastos/citología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/genética , Serina/metabolismo , Transducción de Señal/fisiología
18.
J Biol Chem ; 282(29): 21244-52, 2007 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-17517888

RESUMEN

The control of hepatocyte growth is relevant to the processes of liver regeneration, development, metabolic homeostasis, and cancer. A key component of growth control is the protein kinase Akt, which acts downstream of mitogens and nutrients to affect protein translation and cell cycle progression. In this study, we found that transient transfection of activated Akt triggered a 3-4-fold increase in liver size within days but only minimal hepatocyte proliferation. Akt-induced liver growth was associated with marked up-regulation of cyclin E but not cyclin D1. Analysis of liver polyribosomes demonstrated that the post-transcriptional induction of cyclin E was associated with increased translational efficiency of this mRNA, suggesting that cell growth promotes expression of this protein through a translational mechanism that is distinct from the cyclin D-E2F pathway. Treatment of Akt-transfected mice with rapamycin only partially inhibited liver growth and did not prevent the induction of cyclin E protein, indicating that target of rapamycin activity is not necessary for this response. In the enlarged livers, cyclin E-Cdk2 complexes were present in high abundance but were inactive due to increased binding of p21 to these complexes. Akt transfection of p21(-/-) mice promoted liver growth, activation of Cdk2, and enhanced hepatocyte proliferation. In conclusion, growth promotes cyclin E expression through a novel translational mechanism in the liver, suggesting a new link between cell growth and the cell cycle machinery. Furthermore, p21 suppresses proliferation in the overgrown livers and may play a role in preventing cell cycle progression in response to organ size homeostatic mechanisms.


Asunto(s)
Ciclina E/biosíntesis , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Ciclo Celular , Proliferación Celular , Ciclina D1/metabolismo , Ciclina E/metabolismo , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Biológicos , Biosíntesis de Proteínas , Quinasas p21 Activadas
19.
J Biol Chem ; 282(2): 1468-78, 2007 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-17107955

RESUMEN

Growth hormone (GH), which is reduced with age, corrects the impaired proliferative capacity of livers of old animals. In this paper, we present a mechanism by which GH eliminates age-dependent negative control of proliferation and increases transcription of liver-specific genes in livers of old mice. The reduced proliferative capacities of the liver of old animals are associated with the CCAAT/enhancer-binding protein alpha (C/EBPalpha)-Brm complex, which inhibits E2F-dependent promoters. We found that a sequestration of C/EBPalpha into complexes with Brm leads to a weak interaction of C/EBPalpha with promoters of liver-specific genes, expression of which is reduced in old animals. Injection of either GH or the regulator of the amplitude of endogenous GH release, ghrelin, reduces the C/EBPalpha-Brm complex in livers of old mice, leading to a derepression of E2F targets, to increased interactions of C/EBPalpha with promoters of liver-specific genes, and to correction of their expression. GH-dependent elimination of the complex is mediated by the inhibition of cyclin D3-CDK4 activity and by elevation of a phosphatase, protein phosphatase 2A, which dephosphorylates C/EBPalpha and dissociates the complex.


Asunto(s)
Envejecimiento/fisiología , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Hormona del Crecimiento/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , División Celular/fisiología , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Regulación hacia Abajo/fisiología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Ghrelina , Hormona del Crecimiento/metabolismo , Hígado/enzimología , Ratones , Datos de Secuencia Molecular , Hormonas Peptídicas/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Regiones Promotoras Genéticas/fisiología , Proteína Fosfatasa 2 , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología
20.
J Biol Chem ; 281(43): 32806-19, 2006 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-16931514

RESUMEN

The RNA-binding protein CUGBP1 regulates translation of proteins in a variety of biological processes. In this study, we show that aging liver increases CUGBP1 translational activities by induction of a high molecular weight protein-protein complex of CUGBP1. The complex contains CUGBP1, subunits alpha, beta, and gamma of the initiation translation factor eIF2, and four proteins of the endoplasmic reticulum, eR90, CRT, eR60, and Grp78. The induction of the CUGBP1-eIF2 complex in old livers is associated with the elevation of protein levels of CUGBP1 and with the hyper-phosphorylation of CUGBP1 by a cyclin D3-cdk4 kinase, activity of which is increased with age. We have examined the role of the elevation of CUGBP1 and the role of cyclin D3-cdk4-mediated phosphorylation of CUGBP1 in the formation of the CUGBP1-eIF2 complex by using CUGBP1 transgenic mice and young animals expressing high levels of cyclin D3 after injection with cyclin D3 plasmid. These studies showed that both the increased levels of CUGBP1 and cdk4-mediated hyper-phosphorylation of CUGBP1 are involved in the age-associated induction of the CUGBP1-eIF2 complex. The CUGBP1-eIF2 complex is bound to C/EBPbeta mRNA in the liver of old animals, and this binding correlates with the increased amounts of liver-enriched activator protein and liver-enriched inhibitory protein. Consistent with these observations, the purified CUGBP1-eIF2 complex binds to the 5' region of C/EBPbeta mRNA and significantly increases translation of the three isoforms of C/EBPbeta in a cell-free translation system, in cultured cells, and in the liver. Thus, these studies demonstrated that age-mediated induction of the CUGBP1-eIF2 complex changes translation of C/EBPbeta in old livers.


Asunto(s)
Envejecimiento/fisiología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Hígado/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas CELF1 , Carcinoma Hepatocelular/patología , Línea Celular , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Transfección
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