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 , TransfecciónRESUMEN
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 , RatasRESUMEN
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.