RESUMEN
Trophoblast cell differentiation is of paramount importance for successful pregnancy. Krüppel-like factor 6 (KLF6), a transcription factor with diverse roles in cell physiology and tumor biology, is required for trophoblast differentiation through the syncytial pathway. Herein, we demonstrate that extravillous trophoblast (EVT) cell migration and mesenchymal phenotype are increased upon KLF6 downregulation or the expression of a deletion mutant lacking its transcriptional regulatory domain (KΔac). Raman spectroscopy revealed molecular modifications compatible with increased differentiation in cells stably expressing the KΔac mutant. Moreover, abnormally invasive placenta showed lower KLF6 immunostaining compared with the normal placenta. Thus, impaired KLF6 expression or function stimulates EVT migration and differentiation in vitro and may contribute to the physiopathology of the abnormally invasive placenta.
Asunto(s)
Placenta , Trofoblastos , Diferenciación Celular/genética , Movimiento Celular/genética , Femenino , Regulación de la Expresión Génica , Humanos , Factor 6 Similar a Kruppel/genética , Factor 6 Similar a Kruppel/metabolismo , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
PURPOSE: To analyse the relationship between the transcriptional expression of Krüppel-like factor-6 (KLF6) and local response to treatment with radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: We determined the transcriptional expression of KLF6 in tumour biopsies obtained before treatment with radiotherapy in 83 HNSCC patients. The KLF6 expression was categorized according to the local control of the disease with a recursive partitioning analysis. RESULTS: During the follow-up period, 27 patients (32.5%) had a local recurrence of the tumour. Patients with local recurrence had significantly higher levels of KLF6 expression than patients in which radiotherapy achieved local control of the disease (P = 0.029). Five-year local recurrence-free survival for patients with a high transcriptional expression of KLF6 (n = 46) was 51.1% (95% CI 36.4-66.2%), and for patients with low expression it was 85.6% (95% CI 73.9-97.3%) (P = 0.0001). The results of a multivariate analysis showed that patients with a high KLF6 expression had a 3.8 times higher risk of local recurrence after treatment with radiotherapy (95% CI 1.4-10.5, P = 0.008). CONCLUSION: Transcriptional expression of KLF6 was significantly related to local control in HNSCC patients treated with radiotherapy. Patients with high levels of KLF6 expression had a significantly higher risk of local recurrence after treatment.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Neoplasias de Cabeza y Cuello/patología , Factor 6 Similar a Kruppel/metabolismo , Radioterapia/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Anciano , Biomarcadores de Tumor/genética , Femenino , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Factor 6 Similar a Kruppel/genética , Masculino , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Pronóstico , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Tasa de SupervivenciaRESUMEN
The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.
Asunto(s)
Hipoxia/metabolismo , Factor 6 Similar a Kruppel/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Trofoblastos/metabolismo , Línea Celular , Femenino , Regulación de la Expresión Génica , Humanos , Placentación/fisiología , Embarazo , Regulación hacia ArribaRESUMEN
Liver steatosis is characterised by lipid droplet deposition in hepatocytes that can leads to an inflammatory and fibrotic phenotype. Peroxisome proliferator-activated receptors (PPARs) play key roles in energetic homeostasis by regulating lipid metabolism in hepatic tissue. In adipose tissue PPARγ regulates the adipocyte differentiation by promoting the expression of lipid-associated genes. Within the liver PPARγ is up-regulated under steatotic conditions; however, which transcription factors participate in its expression is not completely understood. Krüppel-like transcription factors (KLFs) regulate various cellular mechanisms, such as cell proliferation and differentiation. KLFs are key components of adipogenesis by regulating the expression of PPARγ and other proteins such as the C-terminal enhancer binding protein (C/EBP). Here, we demonstrate that the transcript levels of Klf6, Klf9 and Pparγ are increased in response to a steatotic insult in vitro. Chromatin immunoprecipitation (ChIp) experiments showed that klf6 and klf9 are actively recruited to the Pparγ promoter region under these conditions. Accordingly, the loss-of-function experiments reduced cytoplasmic triglyceride accumulation. Here, we demonstrated that KLF6 and KLF9 proteins directly regulate PPARγ expression under steatotic conditions.
Asunto(s)
Hígado Graso/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , PPAR gamma/genética , Ácido Palmítico/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Hígado Graso/genética , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , PPAR gamma/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Activación Transcripcional , Triglicéridos/metabolismoRESUMEN
Ewing sarcoma is the third most common sarcoma in children and young adults. Its characteristic chromosomal rearrangement results in a chimerical EWSR1-ETS transcription factor. Secondary genetic alterations are very common. Membranous expression of CD99 is seen in almost all tumors. We report 2 unusual cytogenetic findings in a pediatric Ewing sarcoma, an insertion of the MIC2 gene encoding CD99 from Xp to 10p and a submicroscopic deletion of the well-known tumor supressor gene KLF6. The latter has not been described previously in pediatric neoplasms. Molecular pathways in tumorigenesis and genetic complexity in cancer are discussed.
Asunto(s)
Neoplasias Óseas/genética , Eliminación de Gen , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Proto-Oncogénicas/genética , Sarcoma de Ewing/genética , Antígeno 12E7 , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Niño , Humanos , Hibridación Fluorescente in Situ , Factor 6 Similar a Kruppel , MasculinoRESUMEN
Cell-cell fusion is an essential event during life. Throughout human pregnancy, the syncytiotrophoblast (STB) layer of the placenta is formed by continuous fusion of the underlying villous cytotrophoblasts, thus maintaining placental functionality. Defects in this process are associated with pathologies like pre-eclampsia and intrauterine growth restriction. Krüppel-like factor 6 (KLF6) is a transcription factor highly expressed in human and murine placenta. However, KLF6 functions in trophoblast cells remain largely unexplored. The aim of this work was to address the role of KLF6 during STB formation. KLF6 knockdown through small interfering RNA experiments hindered cell-cell fusion revealed by immunofluorescence microscopy in human primary villous cytotrophoblast as well as in the human placental-derived BeWo cell line. Furthermore, KLF6 silencing led to a decrease in the expression of the fusogenic protein Syncytin-1 and the cell cycle regulator p21 CIP1/WAF1: measured by quantitative RT-PCR and western blot assays. On the contrary, transcript levels of genes that encode for proteins involved in STB formation such as Syncytin-1, Syncytin-2, Connexin-43 and Zonula Occludens-1 increased when KLF6 was overexpressed in differentiating villous cytotrophoblasts and in non-fusing placental-derived JEG-3 cells. Interestingly, the expression of two trophoblast biochemical differentiation markers, ßhCG and PSG3, were not reduced after KLF6 silencing in differentiating trophoblast cells. Present results support the notion that KLF6 is a relevant participant in cytotrophoblast fusion.
Asunto(s)
Vellosidades Coriónicas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Trofoblastos/metabolismo , Adulto , Fusión Celular , Gonadotropina Coriónica Humana de Subunidad beta/genética , Gonadotropina Coriónica Humana de Subunidad beta/metabolismo , Vellosidades Coriónicas/crecimiento & desarrollo , Conexina 43/genética , Conexina 43/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Regulación de la Expresión Génica , Productos del Gen env/genética , Productos del Gen env/metabolismo , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Trofoblastos/citología , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
KLF6 is a member of the Krüppel-like factor family of transcription factors, with diverse roles in the regulation of cell physiology, including proliferation, signal transduction, and apoptosis. Mutations or down-regulation of KLF6 have been described in several human cancers. In this work, we found that KLF6-knockdown resulted in the formation of transformed foci and allowed the spontaneous conversion of NIH3T3 cells to a tumorigenic state. We further assessed the role of KLF6 in the context of oncogenic Ras. We showed that KLF6 was up-regulated by H-Ras(G12V) expression in a Jun N-terminal kinase (JNK)-dependent manner, correlated with enhanced klf6 promoter activity. We found that ectopic KLF6 expression induced a G1-phase cell cycle arrest, thereby decreasing the cell proliferation rate. In addition, constitutive KLF6 expression impaired H-Ras(G12V)-mediated loss of density-dependent growth inhibition and anchorage-independent growth. Moreover, growth of H-Ras(G12V)-driven tumors was reduced in mice challenged with cells stably expressing KLF6. KLF6 expression correlated with the up-regulation of p21, whereas neither p53 induction nor apoptotic cell death was detected. Further, p21 knockdown impaired KLF6-induced cell cycle arrest. These findings provide novel evidence highlighting KLF6 function in response to malignant transformation, suggesting the relevance of KLF6 in controlling cell proliferation and hindering tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica , Genes ras , Factores de Transcripción de Tipo Kruppel/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Células 3T3 NIH , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cervical cancer (CC) as other cancer types, presents molecular deregulations, such as the alterations of transcription factors. Krüppel-like factors (KLF) are a family of transcriptional regulators. They are involved in diverse cellular processes, such as proliferation, apoptosis, and angiogenesis among others. Here, we analyzed the expression of all 17 KLF members at messenger RNA (mRNA) level, and protein expression of the two most commonly altered KLF5 and KLF6 in cervical tissues. Fifty-nine cervical tissues ranging from normal tissue to CC were evaluated for KLF1-17 mRNA expression by end-point RT-PCR and KLF5 by qRT-PCR. For KLF5 and KLF6 protein analysis, a tissue microarray was constructed containing the 59 cases and subjected for immunohistochemistry assay and KLF6 IVS1-27G>A single nucleotide polymorphism by direct DNA sequencing. KLF2-16 expressions were present in normal tissue, whereas all 17 were present in Low-Grade Squamous Intraepithelial Lesion, High-Grade-SIL and CC, unrelated to presence of human papillomavirus (HPV). KLF5 mRNA expression gradually increased throughout the subgroups and overexpressed in CC (p=0.01). KLF5 and KLF6 proteins were immunodetected in all samples. For the KLF6 SNP analysis, 80% of the CC population analyzed presented GG genotype and the remaining 20% presented GA genotype (p=0.491). Our present data show that KLFs expression could not be related to HPV presence, at least at transcriptional level, and KLF5 mRNA overexpression could represent a potential molecular marker for CC; KLF6 SNP has no relation to increased risk of CC.
Asunto(s)
Adenocarcinoma/diagnóstico , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias del Cuello Uterino/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Estudios de Casos y Controles , Cuello del Útero/metabolismo , Cuello del Útero/patología , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Polimorfismo de Nucleótido Simple/genética , Pronóstico , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs) up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140), was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA). This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT) function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.
Asunto(s)
Histonas/genética , Placenta/metabolismo , Glicoproteínas beta 1 Específicas del Embarazo/genética , Regulación hacia Arriba/fisiología , Acetilación/efectos de los fármacos , Línea Celular , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Lisina/genética , Lisina/metabolismo , Placenta/efectos de los fármacos , Embarazo , Glicoproteínas beta 1 Específicas del Embarazo/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Regulación hacia Arriba/efectos de los fármacos , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
BACKGROUND: Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human ß-chorionic gonadotropin (ßhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated ßhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter. CONCLUSIONS/SIGNIFICANCE: Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as ßhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization.
Asunto(s)
Diferenciación Celular , Gonadotropina Coriónica Humana de Subunidad beta/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Placenta/metabolismo , Proteínas Gestacionales/genética , Proteínas Proto-Oncogénicas/metabolismo , Activación Transcripcional , Trofoblastos/citología , Animales , Biomarcadores/metabolismo , Línea Celular , Femenino , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Placenta/citología , Embarazo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Transcripción Genética , Trofoblastos/metabolismoRESUMEN
The mammalian Krüppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC(50)). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p<0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC(50) concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable marker for the efficiency of cell death upon cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Inhibidores de Crecimiento/farmacología , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias/genética , Estrés Oxidativo , Proteínas Proto-Oncogénicas/genética , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Genes p53 , Células Hep G2 , Humanos , Factor 6 Similar a Kruppel , Mutágenos/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
An essential role for the Krüppel-like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel-like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss-of-heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross-talk with the c-Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c-Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.
Asunto(s)
Apoptosis , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Proteínas Proto-Oncogénicas , Transducción de Señal , Animales , Apoptosis/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Genes Supresores de Tumor/fisiología , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Krüppel-like factor 6 (KLF6) is an evolutionarily conserved and ubiquitously expressed protein that belongs to the mammalian Sp1/KLF family of transcriptional regulators. Though KLF6 is a transcription factor and harbors a nuclear localization signal it is not systematically located in the nucleus but it was detected in the cytoplasm of several tissues and cell lines. Hence, it is still not fully settled whether the tumor suppressor function of KLF6 is directly associated with its ability to regulate target genes. METHODOLOGY/PRINCIPAL FINDINGS: In this study we analyzed KLF6 expression and sub-cellular distribution by immunohistochemistry in several normal and tumor tissues in a microarray format representing fifteen human organs. Results indicate that while both nuclear and cytoplasmic distribution of KLF6 is detected in normal breast tissues, breast carcinomas express KLF6 mainly detected in the cytoplasm. Expression of KLF6 was further analyzed in breast cancer tissues overexpressing ERBB2 oncoprotein, which is associated with poor disease prognosis and patient's survival. The analysis of 48 ductal carcinomas revealed a significant population expressing KLF6 predominantly in the nuclear compartment (X(2)p = 0.005; Fisher p = 0.003). Moreover, this expression pattern correlates directly with early stage and small ductal breast tumors and linked to metastatic events in lymph nodes. CONCLUSIONS/SIGNIFICANCE: Data are consistent with a preferential localization of KLF6 in the nuclear compartment of early stage and small HER2-ERBB2 overexpressing ductal breast tumor cells, also presenting lymph node metastatic events. Thus, KLF6 tumor suppressor could represent a new molecular marker candidate for tumor prognosis and/or a potential target for therapy strategies.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Núcleo Celular/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Inmunohistoquímica , Factor 6 Similar a Kruppel , Fracciones Subcelulares/metabolismoRESUMEN
The Krüppel-like transcription Factor 6 (KLF6) is regulated during cell proliferation and differentiation events like mammalian development and tissue regeneration, while its aberrant expression is associated with tumor formation. To investigate KLF6 transcriptional control, the genomic organization of human KLF6 together with its cis-regulatory region was analyzed. A high sequence homology of KLF6 regulatory regions was found in mammals, which in turn predicts a high degree of evolutionary conserved transcriptional mechanisms. A transcription start site was identified at the first nucleotide downstream of a potential initiator element. Also, the role of KLF6 regulatory regions was determined by transfection experiments. A minimal promoter region lacking a TATA-box yet containing an Initiator was identified and found to be active in all cells analyzed. In addition, two strong activating sequences were located between positions -407/-344 and -307/-207, where the latter contained Sp1 and CAAT-box sites. Furthermore, ectopic expression of Sp1 increased the transcriptional activity of the KLF6 promoter. In conclusion, our data revealed that KLF6 gene transcription is under control of a TATA-box independent initiation mechanism together with an evolutionary conserved array of positive cis-acting elements.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/genética , Transactivadores/fisiología , Animales , Secuencia de Bases , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Mamíferos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , TATA Box , Transcripción Genética , TransfecciónRESUMEN
Kruppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that all KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6's role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.
Asunto(s)
Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Animales , Células COS , División Celular/fisiología , Núcleo Celular/metabolismo , Humanos , Hidrólisis , Células Jurkat , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-jun/fisiología , Transactivadores/metabolismo , Transcripción Genética/fisiologíaRESUMEN
The human core promoter binding protein (hCPBP) has been identified as a DNA-binding protein involved in the regulation of TATA box-less genes like those encoding the pregnancy-specific glycoproteins. Structurally, hCPBP contains three zinc fingers in the C-terminal domain, which is highly conserved in a number of proteins that constitute the Krüppel-like family of transcription factors. In the present work, we report the molecular cloning of the mouse CPBP (mCPBP) and its expression pattern during development as well as in adult tissues. The mouse cDNA encodes a protein of 283 amino acids that share 94.4% of identity with the hCPBP. The highest level of mCPBP transcript was detected in placenta, and its expression was lower in total embryos and in adult tissues. We also show by in situ hybridization that during embryonic development the mCPBP gene is mainly expressed in extra-embryonic structures throughout gestation; essentially no specific expression was detected in embryonic tissues. Our data demonstrate that CPBP transcript is enriched in the trophoblastic tissue and strongly suggest that its encoded polypeptide regulates target genes involved in placental development and pregnancy maintenance.
Asunto(s)
Desarrollo Embrionario y Fetal , Expresión Génica , Placenta/metabolismo , Proteínas Proto-Oncogénicas , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Femenino , Humanos , Hibridación in Situ , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Ratones , Datos de Secuencia Molecular , Placenta/química , Embarazo , ARN Mensajero/análisis , Transactivadores/química , Dedos de ZincRESUMEN
We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.