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Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons. In the central nervous system, MBP is synthesized by differentiating oligodendrocytes. The expression of MBP in oligodendrocytes is regulated mainly at the transcriptional level. The Sp1 family of transcription factors has been shown to be important in the regulation of many genes. Binding of Sp1 to the GC box in the proximal MBP promoter has been shown to be indispensable for the activation of MBP gene expression. Previous results from our laboratory have shown that the increase in p27Kip1 that accompanies oligodendrocyte differentiation is paralleled by an increase in Sp1. We also have shown that the increase in MBP expression resulting from elevated p27Kip1 levels is mediated through Sp1 and that this effect occurs specifically in oligodendrocytes. In this study, we found that increased expression of p27Kip1 together with the nervous-system-specific transcription factor Sox10 can activate the MBP promoter even in nonoligodendrocyte cells. This indicates that Sox10 confers cell type specificity on the expression of MBP. Both Sp1 and Sox10 can enhance MBP promoter activity when expressed alone. Cotransfection of plasmids encoding Sp1 and Sox10 induces increased activation of the MBP promoter over expression of either transcription factor alone. This effect is not limited to oligodendrocyte cell lines, in that Sp1 and Sox10 can also synergistically activate the MBP promoter when expressed in NIH3T3 fibroblasts. Mutation of the Sp1 binding sites in the MBP promoter eliminates Sox10 stimulated activation, suggesting that the MBP promoter is activated, at least in part, through protein-protein interactions between Sp1 and Sox10., ((c) 2004 Wiley-Liss, Inc.)

Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27(Kip1) and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27(Kip1) and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27(Kip1) levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27(Kip1) is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.

Expression of cyclin dependent kinase (Cdk) inhibitor p27(Kip1), which blocks cell cycle progression from G(1) to S phase, can be regulated via multiple mechanisms including transcription, protein degradation, and translation. Recently, it was shown that p27(Kip1) plays an important role in the cellular response to hypoxia. However, the mechanisms involved in the hypoxia-induced regulation of p27(Kip1) expression are still not clear. In this study, we compare the expression of p27(Kip1) in two related murine hepatoma cell lines, Hepa-1 and c4. Hepa-1 produces functional aryl hydrocarbon receptor nuclear translocator (ARNT). c4 cells are derived from Hepa-1, but are ARNT deficient. Interestingly, we observed cell line-dependent effects of hypoxia on the expression of p27(Kip1). The level of p27(Kip1) protein in Hepa-1 cells is enhanced by hypoxia, but is reduced by hypoxia in c4 cells. Further investigation demonstrated that hypoxia-induced, ARNT-mediated, transactivation of the p27(Kip1) gene in Hepa-1 cells is responsible for the increase in p27(Kip1) protein. Once c4 cells were stably transfected with the wild type ARNT gene, a hypoxia-induced increase in p27(Kip1) mRNA was observed and reduction of p27(Kip1) protein caused by hypoxia was blocked. Hence, our data indicate that ARNT is involved in transcriptional upregulation of the p27(Kip1) gene under hypoxic conditions., (Copyright 2003 Wiley-Liss, Inc.)

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.

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.)