RESUMEN
Smad3 is a well-characterized intracellular effector of the transforming growth factor beta (TGF-beta) signaling pathway and was implicated recently in the potentiation of vitamin D receptor (VDR)-mediated signaling. Given that both TGF-beta and vitamin D are important regulators of bone remodeling, it is expected that Smad3 plays an integral role in normal maintenance of bone. However, the exact mechanisms by which Smad3 functions in bone remodeling are unknown. Here, we show that mice with targeted deletion of Smad3 are osteopenic with less cortical and cancellous bone compared with wild-type littermates. Decreases in bone mineral density (BMD) in Smad3 null mice reflect the inability of osteoblasts to balance osteoclast activity, although osteoclast numbers are normal and vitamin D mediated serum calcium homeostasis is maintained. The osteopenia of Smad3 null mice is attributed to a decreased rate of bone formation associated with increased osteocyte number and apoptosis. These findings are supported by studies with isolated primary osteoblasts that show TGF-beta can no longer inhibit the differentiation of osteoblasts in the absence of Smad3; yet, TGF-beta-stimulated proliferation remains intact. Together these data support a model that a loss of Smad3 increases the osteocyte fate of the osteoblast and decreases the duration of osteoblast function by shortening lifespan, ultimately resulting in osteopenia.
Asunto(s)
Apoptosis , Enfermedades Óseas Metabólicas/metabolismo , Proteínas de Unión al ADN/fisiología , Osteoblastos/citología , Osteogénesis/fisiología , Transactivadores/fisiología , Animales , Huesos/patología , Calcio/sangre , Recuento de Células , Diferenciación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/metabolismo , Osteoclastos/citología , Osteocitos/citología , Proteína smad3 , Transactivadores/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We have previously shown that RB plays an important role in the maintenance of the epithelial phenotype. p21 is also involved in several terminal differentiation systems including keratinocytes. We report here that p21 is an RB target gene in epithelial cells, but not in fibroblasts where RB is unable to transactivate p21 transcriptional expression. In epithelial cells, when RB family factors were inactivated by SV40 T antigen (LT), p21 expression was strongly repressed, whereas its expression was not affected when the cells were transformed by a mutated LT leaving RB active but inactivating p53. Moreover, retransformation by RB of LT transformed epithelial cells totally restored p21 expression. By cotransfection experiments and using deletions and point mutations of the p21 promoter, we show that the minimal region required for the RB-mediated transcriptional activation maps to a GC-rich region located between -83 and -74. This region is shown to interact specifically with the transcription factor Sp1 and Sp3. Thus for the first time, we show a positive transcriptional relationship between RB and p21 in epithelial cells. Since p21 keeps RB in a hypophosphorylated state important for its transcriptional activity during differentiation, our results imply an auto-loop of regulation between RB and p21 that may be essential for the maintenance of the differentiation state. We propose that this transcriptional relationship might be necessary of their roles in cell cycle arrest and in several differentiation pathways.
Asunto(s)
Ciclinas/genética , Células Epiteliales/fisiología , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Animales , Ciclo Celular , Diferenciación Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Perros , Células Epiteliales/citología , Retroalimentación , Fibroblastos/fisiología , Ratones , Modelos Genéticos , Fenotipo , Mutación Puntual , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta , Eliminación de Secuencia , Factor de Transcripción Sp3 , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
p21Cip1/Waf1 cyclin-dependent kinase inhibitor (p21) is inducible by Raf and mitogen-activated protein kinase kinase (MAPKK), but the level of regulation is unknown. We show here by conditional and transient Ras-expression models that Ras induces p21. Induction of p21 in conditionally Ras-expressing cells is posttranscriptional utilizing mitogen-activated protein kinase (MAPK) pathway. Transient, high-level Ras-expression induces transcriptional activation of p21 mediated by a GC-rich region in p21 promoter -83-54 bp relative to the transcription initiation site containing binding sites for Sp1-family transcription factors. Mutation of either Sp1-binding site 2 or 4 in this region decreases the magnitude of induction of promoter activity by Ras, but only the simultaneous mutation of both sites abolishes fully the induction. Electrophoretic mobility shift assays using an oligonucleotide corresponding to Sp1-binding site 2 indicate that both Sp1 and Sp3 transcription factors bind to this region. The results demonstrate that the central cytosolic growth regulator Ras is a potent transcriptional and posttranscriptional inducer of the nuclear growth inhibitor p21.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclinas/biosíntesis , ADN/genética , Regulación de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Células 3T3 , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , ADN/metabolismo , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Eliminación de SecuenciaRESUMEN
We sought to characterize the pathway by which the multifunctional cytokine transforming growth factor-beta (TGF-beta) inhibits the proliferation of normal astrocytes, and we analyzed the alterations in the TGF-beta pathway in human glioma cell lines. Upon TGF-beta treatment, primary rat astrocytes showed a significant decrease in DNA synthesis upon thymidine incorporation with a cell cycle arrest in the G(1) phase. Western analysis of the astrocytes revealed that the expression of the cyclin-dependent kinase inhibitor (CdkI) p15(INK4B) was significantly up-regulated upon TGF-beta treatment without a change in other CdkI levels. The retinoblastoma protein (Rb) became hypophosphorylated, and Cdk2 activity decreased. Analysis of Smad3 null mouse astrocytes showed a significant loss of both TGF-beta-mediated growth inhibition and p15(INK4B) induction compared with wild-type mouse astrocytes. Infection of rat astrocytes by SMAD3 and SMAD4 adenoviruses failed to induce increased expression of p15(INK4B), implying indirect transcriptional regulation of p15(INK4B) by SMAD3. High-grade human gliomas secrete TGF-beta, yet are resistant to its growth inhibitory effects. Analysis of the effects of TGF-beta on 12 human glioma cell lines showed that TGF-beta mildly inhibited the growth of six lines, had no effect on four lines, and stimulated the growth of two lines. The majority of glioma lines had homozygous deletions of the p15(INK4B) gene, except for two lines that expressed p15(INK4B) protein, which was induced further upon TGF-beta treatment. Three lines mildly induced CdkI p21(WAF1) expression in response to TGF-beta. Most tumor lines retained other TGF-beta-mediated responses, including extracellular matrix protein and angiogenic factor secretion, which may contribute to increased malignant behavior. This suggests that the loss of p15(INK4B) may explain, in part, the selective loss of growth inhibition by TGF-beta in gliomas to form a more aggressive tumor phenotype.
Asunto(s)
Astrocitos/metabolismo , Quinasas CDC2-CDC28 , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Proteínas de Unión al ADN/metabolismo , Glioma/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proteínas Supresoras de Tumor , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Southern Blotting , Western Blotting , División Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas de Unión al ADN/fisiología , Citometría de Flujo , Fase G1/efectos de los fármacos , Glioma/patología , Humanos , Ratones , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Transducción de Señal , Proteína smad3 , Transactivadores/fisiología , Células Tumorales CultivadasRESUMEN
Addition of nerve growth factor (NGF) to PC12 cells promotes neuronal differentiation while inhibiting cell proliferation. In order to understand how NGF exerts its antimitogenic effect during differentiation, we have studied the mechanism by which this factor activates the promoter of the CDK inhibitor p21W4F1/CIP1. The minimal region of the p21 promoter required for the NGF-induction was mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. This GC-rich region was shown to interact specifically with the transcription factor Sp1 and the related protein Sp3, in either exponentially-growing or NGF-treated PC12 cells. The addition of NGF resulted in an accumulation of the transcriptional co-activator p300 in complexes associated with the NGF-responsive region. Transcriptional activity of Sp1, Sp3 and p300 was specifically induced by NGF in a Gal4-fusion assay, indicating that induction of p21 during neuronal differentiation may involve regulation of the activity of these factors by NGF. Furthermore, p300 was able to act as a co-activator for Sp1-mediated transcriptional activation in PC12 cells, suggesting that p300 and Sp1 may cooperate in activating p21 transcription during the withdrawal of neuronal precursors from the cell cycle. This hypothesis is supported by experiments showing that p300 and Sp1 form complexes in PC12 cells.
Asunto(s)
Ciclinas/genética , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína p300 Asociada a E1A , Galactosidasas/genética , Galactosidasas/metabolismo , Humanos , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células PC12/metabolismo , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción Sp3 , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Smad3 and Smad4 are sequence-specific DNA-binding factors that bind to their consensus DNA-binding sites in response to transforming growth factor beta (TGFbeta) and activate transcription. Recent evidence implicates Smad3 and Smad4 in the transcriptional activation of consensus AP-1 DNA-binding sites that do not interact with Smads directly. Here, we report that Smad3 and Smad4 can physically interact with AP-1 family members. In vitro binding studies demonstrate that both Smad3 and Smad4 bind all three Jun family members: JunB, cJun, and JunD. The Smad interacting region of JunB maps to a C-terminal 20-amino acid sequence that is partially conserved in cJun and JunD. We show that Smad3 and Smad4 also associate with an endogenous form of cJun that is rapidly phosphorylated in response to TGFbeta. Providing evidence for the importance of this interaction between Smad and Jun proteins, we demonstrate that Smad3 is required for the activation of concatamerized AP-1 sites in a reporter construct that has previously been characterized as unable to bind Smad proteins directly. Together, these data suggest that TGFbeta-mediated transcriptional activation through AP-1 sites may involve a regulated interaction between Smads and AP-1 transcription factors.
Asunto(s)
Proteínas de Unión al ADN/genética , Transactivadores/genética , Factor de Transcripción AP-1/genética , Activación Transcripcional , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Células COS , Datos de Secuencia Molecular , Transducción de Señal/genética , Proteína smad3 , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The Smads are a family of nine related proteins which function as signaling intermediates for the transforming growth factor beta (TGF-beta) superfamily of ligands. To discern the in vivo functions of one of these Smads, Smad3, we generated mice harboring a targeted disruption of this gene. Smad3 null mice, although smaller than wild-type littermates, are viable, survive to adulthood, and exhibit an early phenotype of forelimb malformation. To study the cellular functions of Smad3, we generated Smad3 null mouse embryonic fibroblasts (MEFs) and dermal fibroblasts. We demonstrate that null MEFs have lost the ability to form Smad-containing DNA binding complexes and are unable to induce transcription from the TGF-beta-responsive promoter construct, p3TP-lux. Using the primary dermal fibroblasts, we also demonstrate that Smad3 is integral for induction of endogenous plasminogen activator inhibitor 1. We subsequently demonstrate that Smad3 null MEFs are partially resistant to TGF-beta's antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-beta-mediated growth inhibition. We next examined cells in which Smad3 is most highly expressed, specifically cells of immune origin. Although no specific developmental defect was detected in the immune system of the Smad3 null mice, a functional defect was observed in the ability of TGF-beta to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-beta-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-beta and implicate Smad3 as a potential effector for TGF-beta in modulating immune system function.
Asunto(s)
Proteínas de Unión al ADN/genética , Transactivadores/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Antineoplásicos , Constitución Corporal , Fibroblastos/metabolismo , Miembro Anterior/patología , Regulación de la Expresión Génica , Sistema Inmunológico , Ratones , Ratones Mutantes , Penetrancia , Fenotipo , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Elementos de Respuesta , Transducción de Señal , Proteína smad3 , Distribución TisularRESUMEN
p53 target genes p21(Cip1/Waf1) cyclin-kinase inhibitor (p21 CKI), GADD45, bax, and cyclin G and genes affecting the redox state of the cells are implicated in p53 damage control responses. In order to attribute their functions and dependency of p53 in UV-damaged cells we undertook an analysis of UVC responses of fibroblasts derived from p53 knock-out mice. UVC radiation efficiently and rapidly inhibited DNA replication in both p53 -/- and +/+ cells. The arrest was persistent in p53 -/- fibroblasts and cells underwent apoptosis, whereas p53 +/+ cells recovered and reentered the cycle. Protein and mRNA analyses of p21 expression showed that it was induced up to sixfold with similar kinetics both in the presence and in the absence of p53. However, high doses of UV abrogated the p21 response in p53 -/- cells, whereas it was maintained in cells with normal p53. UVC radiation transcriptionally activated p21 expression as demonstrated by luciferase reporter assays using deletion constructs of the p21 promoter. The promoter assays further confirmed the independency of p53-binding sites in the activation and linked UV-responsive transcriptional regulation of p21 to two Sp1 consensus binding sites within -61 bp of the transcription initiation site. A weaker regulation was mediated by elements between -1300 to -500 bp relative to the transcription initiation site. The results suggest that in fibroblasts UVC radiation is a rapid and efficient inducer of p21 expression also in a p53-independent manner.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/genética , Inhibidores Enzimáticos , Regulación de la Expresión Génica/efectos de la radiación , Transcripción Genética/efectos de la radiación , Animales , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ratones , Ratones Noqueados , Factor de Transcripción Sp1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología , Rayos UltravioletaRESUMEN
Transcriptional regulation by transforming growth factor beta (TGF-beta) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. Here, we describe a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter, one a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each was able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements were nonadditive; disruption of either sequence resulted in complete or severe reductions in TGF-beta responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-beta-induced transcriptional activation of the c-Jun promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Conejos , Proteína smad3 , Proteína Smad4 , TATA BoxRESUMEN
Smads are intermediate effector proteins that transduce the TGF-beta signal from the plasma membrane to the nucleus, where they participate in transactivation of downstream target genes. We have shown previously that coactivators p300/CREB-binding protein are involved in TGF-beta-mediated transactivation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibility that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-terminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-beta-mediated phosphorylation of Smad3 potentiates the association between Smad3 and p300, likely because of an induced conformational change that removes the autoinhibitory interaction between the N- and C-terminal domains of Smad3. Consistent with a role for p300 in the transcription regulation of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-beta-responsive reporter constructs. The adenoviral oncoprotein E1A can partially block Smad-dependent transcriptional activation by directly competing for binding to p300. Taken together, these findings define a new role for phosphorylation of Smad3: in addition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 modulates its interaction with coactivators, leading to transcriptional regulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proteínas E1A de Adenovirus/metabolismo , Sitios de Unión , Unión Competitiva , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Expresión Génica , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Transducción de Señal , Proteína smad3 , Transactivadores/química , Transactivadores/genética , Activación TranscripcionalRESUMEN
Transforming growth factor beta (TGF-beta) causes growth arrest at the G1 phase of the cell cycle in most cell types. Both the cyclin dependent kinase inhibitor p15(INK4B) and p21(Cip1/WAF1) genes have been found to be induced by TGF-beta in human keratinocyte HaCaT cells. Analyses of the human p15 and p21 promoters have led to the identification of GC-rich sequences capable of binding to Sp1 transcription factors as necessary elements for the TGF-beta induction of both promoters. We report here that canonical Sp1 binding sites derived from the SV40 21 bp repeat could also support promoter induction by TGF-beta when placed upstream of a minimal luciferase reporter construct containing only the TATA and Inr elements. Gel retardation assays identified Sp1, Sp3 and DeltaSp3 as major factors binding to the canonical Sp1 sites in HaCaT cells and that TGF-beta treatment did not change their binding activities over a 24 h period. More importantly, GAL4-Sp1, but not GAL4-Sp3, chimeric protein supported TGF-beta mediated gene induction from a luciferase reporter construct driven by five GAL4 DNA binding sites. Our results suggest that Sp1 binding site can function as a distinct TGF-beta responsive element for TGF-beta mediated promoter expression and Sp1 per se can mediate this response.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/genética , Factor de Crecimiento Transformador beta/farmacología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/fisiología , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/genética , Humanos , Queratinocitos , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteínas Recombinantes de Fusión , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Eliminación de Secuencia , Virus 40 de los Simios/genética , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3 , Factores de Transcripción/genéticaRESUMEN
Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGF-beta)-mediated signal transduction. In response to TGF-beta, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-beta on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-beta-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-beta-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-beta. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Expresión Génica , Genes Supresores de Tumor , Humanos , Datos de Secuencia Molecular , Inhibidor 1 de Activador Plasminogénico/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Transducción de Señal , Proteína smad3 , Proteína Smad4 , Transactivadores/genética , Factor de Transcripción AP-1/metabolismo , Activación TranscripcionalRESUMEN
The adenovirus early gene product E1A is a potent stimulator of cellular proliferation, which when overexpressed can overcome the growth-inhibitory effects of the polypeptide hormone transforming growth factor beta (TGF-beta). The ability of TGF-beta to arrest cell growth in G1 correlates with the transcriptional induction of the cyclin-dependent kinase inhibitors, p15/INK4B and p21/WAF1/Cip1; an inhibition of the G1 cyclin-Cdk complexes; and a maintenance of the retinoblastoma susceptibility gene product, Rb, in a hypophosphorylated state. The ability of E1A to overcome TGF-beta-mediated growth inhibition derives, in part, from its ability to sequester Rb and Rb family members. We report here that E1A also acts upstream of Rb by blocking the TGF-beta-mediated induction of p15 and p21. Consistent with these findings, E1A expression also blocks the ability of TGF-beta to inhibit Cdk2 kinase activity, as well as its ability to hold Rb in a hypophosphorylated state. The effect of E1A on the induction of p15 and p21 is independent of E1A's Rb binding activity. The E1A-mediated decrease in p15 levels is primarily the result of a block at the level of transcriptional activation by TGF-beta. This effect is dependent on E1A's ability to bind p300, one of E1A's target proteins. Thus, the ability of E1A to affect p15 and p21 expression represents an additional possible mechanism by which E1A can circumvent the negative regulation of cell cycle progression.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas Portadoras/biosíntesis , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Ciclinas/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Proteínas Supresoras de Tumor , Proteínas Portadoras/genética , Línea Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/genética , Inhibidores Enzimáticos/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Activación TranscripcionalRESUMEN
The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors that inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and activities of the G1 cyclins and their cyclin-dependent kinase partners. The ability of TGF-beta to inhibit the activity of these kinase complexes derives in part from its regulatory effects on the cyclin-dependent kinase inhibitors, p21/WAF1/Cip1, p27Kip1, and p15. Upon treatment of cells with TGF-beta, these three inhibitors bind to and block the activities of specific cyclin-cyclin-dependent kinase complexes to cause cell cycle arrest. Little is known, however, on the mechanism through which TGF-beta activates these cyclin-dependent kinase inhibitors. In the case of p21, TGF-beta treatment leads to an increase in p21 mRNA. This increase in p21 mRNA is partly due to transcriptional activation of the p21 promoter by TGF-beta. To further define the signaling pathways through which TGF-beta induces p21, we have performed a detailed functional analysis on the p21 promoter. Through both deletion and mutation analysis of the p21 promoter, we have defined a 10-base pair sequence that is required for the activation of the p21 promoter by TGF-beta. In addition, this sequence is sufficient to drive TGF-beta-mediated transcription from a previously nonresponsive promoter. Preliminary gel shift assays demonstrate that this TGF-beta responsive element binds specifically to several proteins in vitro. Two of these proteins are the transcription factors Sp-1 and Sp-3. These studies represent the initial steps toward defining the signaling pathways involved in TGF-beta-mediated transcriptional activation of p21.
Asunto(s)
Ciclinas/genética , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta/fisiología , Secuencia de Bases , Ciclo Celular/fisiología , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Análisis Mutacional de ADN , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas/metabolismo , Eliminación de Secuencia , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors which inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of small proteins, p21 (WAF1, Cip1), p27Kip1, p16, and p15INK4B, that physically associate with cyclins, cyclin-dependent kinases, or cyclin-Cdk complexes. p21 has been previously shown to be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-beta also causes a rapid transcriptional induction of p21, suggesting that p21 can respond to both intracellular and extracellular signals for cell cycle arrest. In contrast to DNA damage, however, induction of p21 by TGF-beta is not dependent on wild-type p53. The cell line studied in these experiments, HaCaT, contains two mutant alleles of p53, which are unable to activate transcription from the p21 promoter when overexpressed. In addition, TGF-beta and p53 act through distinct elements in the p21 promoter. Taken together, these findings suggest that TGF-beta can induce p21 through a p53-independent pathway. Previous findings have implicated p27Kip1 and p15INK2B as effectors mediating the TGF-beta growth inhibitory effect. These results demonstrate that a single extracellular antiproliferative signal, TGF-beta, can act through multiple signaling pathways to elicit a growth arrest response.
Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Inhibition of cell growth by type beta transforming growth factor (TGF-beta) occurs in mid-G1 and is associated with decreased G1 cyclin-dependent kinase activity and maintenance of the retinoblastoma tumor suppressor protein Rb in an underphosphorylated, growth-suppressive state. A variety of recent experiments suggest that a functional target of Rb is the E2F transcription factor. In addition, the growth-suppressive effects of TGF-beta can be overcome by expression of viral oncogene products that dissociate E2F from Rb and Rb-related polypeptides. These results suggest the possibility that control of E2F may be a downstream event of TGF-beta action. Consistent with that possibility is the observation that E2F1 RNA levels are drastically reduced in TGF-beta-treated cells. We have also used a recombinant adenovirus containing the human E2F1 gene to overexpress the E2F1 product in mink lung epithelial cells that were growth arrested with TGF-beta. We find that overexpression of E2F1 can overcome the TGF-beta-mediated effect as measured by the activation of cellular DNA synthesis. These results suggest that a likely downstream target for the cyclin-dependent kinases, which are controlled by TGF-beta, is the activation of E2F.