Search

Abstract Background Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells. Results A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1. Conclusions Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.

Our previous studies have found that expression of p27 in oligodendrocytes enhances myelin basic protein (MBP) gene expression through a mechanism that involves the transcription factor Sp1. In this study we show that this activation only requires the N-terminal 45 amino acids of p27 containing a functional cyclin-binding motif. In an effort to identify other cofactors that are involved in the p27-mediated activation of MBP gene expression, a yeast two-hybrid assay was performed using an N-terminal truncated p27 and a mouse embryo cDNA library. Galectin-4 was found to interact with p27 in the yeast two-hybrid assay. This novel interaction was also confirmed using a glutathione-S-transferase interaction assay and immunoprecipitation assays. Expression of galectin-4 in primary oligodendrocytes was confirmed by western blot. Additionally, the MBP promoter could be activated by expression of galectin-4 in CG4 oligodendrocytes, similar to the effects of increased p27 levels. We also show that Sp1 and galectin-4 interact in cells, while a complex of all three proteins could not be found. We conclude that galectin-4 is involved in the p27-mediated activation of the MBP gene, possibly through modulation of the glycosylation status of the transcription factor Sp1. [ABSTRACT FROM AUTHOR]

p27[sup Kip1] levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27[sup Kip1] levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27[sup Kip1] enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27[sup Kip1]. Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27[sup Kip1] can activate gene transcription by using the MBP gene in oligodendrocytes as a model. We show that the enhancement of MBP promoter activity by p27[sup Kip1] is mediated by a proximal region of the MBP promoter that contains a conserved GC box binding sequence. This sequence binds transcription factors Sp1 and Sp3. Increased expression of p27[sup Kip1] increases the level of Sp1 promoter binding to the GC box but does not change the level of Sp3 binding. The binding of Sp1 to this element activates the MBP promoter, p27[sup Kip1] leads to increased Sp1 binding through a decrease in Sp1 protein turnover. Enhancement of MBP promoter activity by an increase in the level of p27[sup Kip1] involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1. [ABSTRACT FROM AUTHOR]

Background: Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells. Results: A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5′-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1. Conclusions: Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1 reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5′- UTR of the p27Kip1 mRNA. [ABSTRACT FROM AUTHOR]

Background: The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important to identify the transcription initiation site within the p27Kip1 gene, thereby defining the promoter region of the gene and the 5'-untranslated region of the p27Kip1 mRNA. Although several previous studies have attempted to map p27Kip1 transcription start sites, the results vary widely for both the mouse and human genes. In addition, even though the mouse and human p27Kip1 gene sequences are very highly conserved, the reported start sites are notably different. Results: In this report, using a method that identifies capped ends of mRNA molecules together with RNase protection assays, we demonstrate that p27Kip1 transcription is initiated predominantly from a single site which is conserved in the human and mouse genes. Initiation at this site produces a 5'-untranslated region of 472 nucleotides in the human p27Kip1 mRNA and 502 nucleotides in the mouse p27Kip1 mRNA. In addition, several minor transcription start sites were identified for both the mouse and human genes. Conclusions: These results demonstrate that the major transcription initiation sites in the mouse and human p27Kip1 genes are conserved and that the 5'-UTR of the p27Kip1 mRNA is much longer than generally believed. It will be important to consider these findings when designing experiments to identify elements that are involved in regulating the cellular levels of p27Kip1. [ABSTRACT FROM AUTHOR]

p27Kip1 plays an important role in cell cycle progression by negatively regulating the activity of cyclin-Cdk complexes. To understand how p27Kip1 functions, the level and subcellular location of p27Kip1 in Swiss/3T3 cells following serum stimulation of quiescent cells was examined. Surprisingly, p27Kip1 was observed exclusively in the cytosol throughout G1 and into early S phase. However, as expected, p27Kip1 in the cytosolic fraction was greatly reduced following serum stimulation and reached very low levels by late G1. The decline in the level of p27Kip1 corresponded in time to an increase in the nuclear level of both Cdk2 and cyclin E. In quiescent 3T3 cells Cdk2 was inactive and co-precipitated with p27Kip1. After serum stimulation, both nuclear and cytosolic Cdk2 was activated and this corresponded to the decline in p27Kip1. Overexpression of p27Kip1 allowed accumulation of the inhibitor in the nucleus but inhibited entry of Cdk2 into the nucleus following serum stimulation. The subcellular localization of p27Kip1 was also examined in a variety of other mammalian cells. In all the cell lines examined the preponderance of p27Kip1 was found in the cytosolic fraction. However, a substantial level of nuclear p27Kip1 was observed for several cell lines. In a primary mixed glial cell culture p27Kip1 was localized to the nucleus. The results suggest that cytosolic p27Kip1 has a functional role in regulating cell cycle progression, possibly through inhibiting transport of cyclin E-Cdk 2 complexes into the nucleus. [ABSTRACT FROM AUTHOR]

The homeodomain-containing protein Nkx2.2 is critical for the development of oligodendrocyte lineage cells, but the target genes of Nkx2.2 regulation have not been identified. In the present study, we found that the myelin basic protein gene is one of the genes that is regulated by Nkx2.2. Expression of Nkx2.2 represses the expression of myelin basic protein in oligodendrocyte progenitors. Two regulatory elements in the myelin basic protein promoter were identified and found to interact with Nkx2.2 in vitro. Despite their sequence divergence, both sites were involved in the Nkx2.2-mediated repression of the myelin basic protein promoter. Binding of Nkx2.2 also blocked and disrupted the binding of the transcriptional activator Purα to the myelin basic protein promoter. Additionally Nkx2.2 recruited a histone deacetylase 1-mSin3A complex to the myelin basic protein promoter. We also found that the transcription factor Sp1 was able to compete off the binding of Nkx2.2 to its consensus binding site in vitro and reversed the repressive effect of Nkx2.2 in vivo. Our data revealed a novel role for Nkx2.2 in preventing the precocious expression of myelin basic protein in immature oligodendrocytes. Based on this study and our previous reports, a model for myelin basic protein gene control is proposed. [ABSTRACT FROM AUTHOR]

p27(Kip1) levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27(Kip1) levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27(Kip1) enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27(Kip1). Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27(Kip1) can activate gene transcription by using the MBP gene in oligodendrocytes as a model. We show that the enhancement of MBP promoter activity by p27(Kip1) is mediated by a proximal region of the MBP promoter that contains a conserved GC box binding sequence. This sequence binds transcription factors Sp1 and Sp3. Increased expression of p27(Kip1) increases the level of Sp1 promoter binding to the GC box but does not change the level of Sp3 binding. The binding of Sp1 to this element activates the MBP promoter. p27(Kip1) leads to increased Sp1 binding through a decrease in Sp1 protein turnover. Enhancement of MBP promoter activity by an increase in the level of p27(Kip1) involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells. RESULTS: A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1. CONCLUSIONS: Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.

The process of oligodendrocyte differentiation is a complex event that requires cell cycle withdrawal, followed by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. Because growth arrest precedes differentiation, we sought to investigate the role of cell cycle molecules in the activation of myelin gene promoters. We hypothesized that the cell cycle inhibitor p27(Kip1), which is primarily responsible for arresting proliferating oligodendrocyte progenitors, may be involved in the transcriptional regulation of myelin genes. In agreement with this hypothesis, overexpression of p27(Kip1) in the CG4 cell line, but not in 3T3 fibroblasts, enhances the expression of luciferase driven by the myelin basic protein (MBP) promoter. Interestingly, this effect is specific for p27(Kip1); overexpression of other cell cycle inhibitors had no effect. Additionally, this effect is independent of halting the cell cycle; treatment with the cell cycle blocker roscovitine did not affect MBP promoter usage. We conclude that p27(Kip1) contributes to oligodendrocyte differentiation by regulating transcription of the MBP gene., (Copyright 2002 Wiley-Liss, Inc.)