A functional genetic screen identifies TFE3 as a gene that confers resistance to the anti-proliferative effects of the retinoblastoma protein and transforming growth factor-beta.
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The helix-loop-helix transcription factor TFE3 has been suggested to play a role in the control of cell growth by acting as a binding partner of transcriptional regulators such as E2F3, SMAD3, and LEF-1. Furthermore, translocations/TFE3 fusions have been directly implicated in tumorigenesis. Surprisingly, however, a direct functional role for TFE3 in the regulation of proliferation has not been reported. By screening retroviral cDNA expression libraries to identify cDNAs that confer resistance to a pRB-induced proliferation arrest, we have found that TFE3 overrides a growth arrest in Rat1 cells induced by pRB and its upstream regulator p16(INK4A). In addition, TFE3 expression blocks the anti-mitogenic effects of TGF-beta in rodent and human cells. We provide data supporting a role for endogenous TFE3 in the direct regulation of CYCLIN E expression in an E2F3-dependent manner. These observations establish TFE3 as a functional regulator of proliferation and offer a potential mechanism for its involvement in cancer. (+info)
Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.
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E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes. (+info)
Control of the p53-p21CIP1 Axis by E2f1, E2f2, and E2f3 is essential for G1/S progression and cellular transformation.
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The E2F family of transcription factors is believed to have an essential role in the control of cellular proliferation by regulating the transcription of genes involved in cell cycle progression. Previous work has demonstrated that the targeted inactivation of E2f1, E2f2, and E2f3 results in elevated p21(CIP1) protein levels, loss of E2F target gene expression, and cell cycle arrest at G1/S and G2/M, suggesting a strict requirement for these E2Fs in the control of normal cellular proliferation. We now demonstrate that E2f1, E2f2, and E2f3 are also required for oncogene-mediated transformation of mouse embryonic fibroblasts. Analysis of synchronized populations of mouse embryonic fibroblasts revealed that the inactivation of p21(CIP1) restores the ability of E2f1-3-deficient cells to enter and transit through G1/S (but not G2/M). In contrast, loss of p53 restored the ability of these cells to progress through both G1/S and mitosis, leading to their continued proliferation. The inactivation of p53 (but not p21(CIP1)) rendered E2f1-3-deficient cells sensitive to transformation and tumorigenesis. These results suggest that the negative regulation of the p53-p21(CIP1) axis by the E2F1-3 factors is critical for cell cycle progression and cellular transformation. (+info)
Making omelets without breaking eggs: E2F-mediated induction of cardiomyoycte cell proliferation without stimulation of apoptosis.
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The fundamental role of E2F transcription factors in the regulation of proliferation is well established. According to a widely accepted model, E2F1, E2F2, and E2F3 are classified as "activating" E2Fs since they induce proliferation of quiescent cells whereas E2F4 and E2F5 do not have the power to incite cell cycle progression but are related to differentiation processes and were therefore considered to be "repressive". In addition, it has been postulated that "activating" E2Fs induce apoptosis in a wide variety of cell types depending on their expression level. However, we demonstrated recently that this 'threshold model' does not hold true for cardiomyocytes. In a series of experiments in which we overexpressed individual E2Fs we found that directed expression of E2F2, unlike E2F1, E2F3 and E2F5, did not induce apoptosis but even suppressed expression of several pro-apoptotic genes in primary cardiomyocytes. Furthermore, we established that not only E2F1, E2F2, and E2F3 but also E2F4 was able to induce S-phase entry of primary cardiomyocytes. Our results suggest that it is possible to utilize the proliferation-inducing properties of the E2Fs in cardiomyocytes without activation of potentially harmful pro-apoptotic traits. This finding might open a new access to stimulate regeneration in postmitotic tissues such as the heart. (+info)
E2f1, E2f2, and E2f3 control E2F target expression and cellular proliferation via a p53-dependent negative feedback loop.
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E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21(CIP1). Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21(CIP1) and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation. (+info)
Selective requirements for E2f3 in the development and tumorigenicity of Rb-deficient chimeric tissues.
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The tumor suppressor function of the retinoblastoma protein pRB is largely dependent upon its capacity to inhibit the E2F transcription factors and thereby cell proliferation. Attempts to study the interplay between pRB and the E2Fs have been hampered by the prenatal death of Rb; E2f nullizygous mice. In this study, we isolated Rb; E2f3 mutant embryonic stem cells and generated Rb(-/-); E2f3(-/-) chimeric mice, thus bypassing the lethality of the Rb(-/-); E2f3(-/-) germ line mutant mice. We show that loss of E2F3 has opposing effects on two of the known developmental defects arising in Rb(-/-) chimeras; it suppresses the formation of cataracts while aggravating the retinal dysplasia. This model system also allows us to assess how E2f3 status influences tumor formation in Rb(-/-) tissues. We find that E2f3 is dispensable for the development of pRB-deficient pituitary and thyroid tumors. In contrast, E2f3 inactivation completely suppresses the pulmonary neuroendocrine hyperplasia arising in Rb(-/-) chimeric mice. This hyperproliferative state is thought to represent the preneoplastic lesion of small-cell lung carcinoma. Therefore, our observation highlights a potential role for E2F3 in the early stages of this tumor type. (+info)
Rb is critical in a mammalian tissue stem cell population.
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The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). Here we show that the specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb resulted in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppressed Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restored placental development and extended the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb(-/-) embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. We conclude that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population. (+info)
Proteomic and transcriptomic analyses of retinal pigment epithelial cells exposed to REF-1/TFPI-2.
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PURPOSE: The authors previously reported a growth-promoting factor, REF-1/TFPI-2, that is specific to retinal pigment epithelial (RPE) cells. The purpose of this study was to determine the genes and proteins of human RPE cells that are altered by exposure to TFPI-2. METHODS: Human primary RPE cells were cultured with or without TFPI-2. Cell extracts and isolated RNA were subjected to proteomic and transcriptomic analyses, respectively. Proteins were separated by two-dimensional gel electrophoresis followed by gel staining and ion spray tandem mass spectrometry analyses. Transcriptomic analysis was performed using a DNA microarray to detect 27,868 gene expressions. RESULTS: Proteomic analysis revealed c-Myc binding proteins and ribosomal proteins L11 preferentially induced by TFPI-2 in human RPE cells. Transcriptomic analysis detected 10,773 of 33,096 probes in the TFPI-2 treated samples, whereas only 2186 probes were detected in the nontreated samples. Among the genes up-regulated by TFPI-2 at the protein level were c-myc, Mdm2, transcription factor E2F3, retinoblastoma binding protein, and the p21 gene, which is associated with the c-myc binding protein and ribosomal protein L11. CONCLUSIONS: The mechanisms by which TFPI-2 promotes the proliferation of RPE cells may be associated with augmented c-myc synthesis and the activation of E2F in the retinoblastoma protein (Rb)/E2F pathway at the G1 phase of the RPE cells. Activation of ribosomal protein L11 and the Mdm2 complex of the p53 pathway may be counterbalanced by the hyperproliferative conditions. (+info)