Role ofp27Kipl

It is uncertain whether the intrinsic timer in OPCs primarily controls the onset of differentiation, the cessation of proliferation, or both. It seems likely, however, that the timer at some point interacts with the cell-cycle control system that regulates progress through the cell cycle. As the cells stop dividing and differentiate in G1, it is the part of the control system that operates in G1 that is most likely to be relevant. In principle, the components of the cell-cycle control system could be components ofthe timer. The timer, for example, could depend on a decrease in one or more positive intracellular regulators, such as a cyclin or a Cdk that promotes progression through G1, an increase in one or more negative intracellular regulators, such as Cdk inhibitors that block such progression, or on both types of changes. It would be advantageous to have the timer involve changes in multiple components, as this would make it more robust: if one component were defective, the timer would still work, although inaccurately.

Several lines of evidence suggest that an increase in the Cdk inhibitor p27/ Kipl (p27) is part of the timer. First, p27 is high in all oligodendrocytes, whereas it is variable in OPCs (Durand et al 1997). Second, p27 progressively increases as precursor cells proliferate in culture in the presence of PDGF and the absence of TH, even though most of the cells do not stop dividing and differentiate in these conditions (Durand et al 1997). p27 reaches a plateau value at around the time that most of the cells would have stopped dividing and differentiated were TH present, consistent with the possibility that the progressive rise in p27 is part of the timing mechanism. Since the cells continue to divide with this high level of p27, however, it is clear that the rise in p27 is not enough on its own to stop the cell cycle. Third, p27 levels rise faster at 33 °C than at 37 °C (Gao et al 1997), which may be one reason why the timer runs faster at the lower temperature. Fourth, artificially increasing p27 levels by transfection speeds up the timer (J. Apperly, unpublished work). Fifth, many OPCs isolated from the optic nerves of p27-deficient mice go through one or two more divisions in clonal culture in the presence of PDGF and TH before they differentiate than do any OPCs from wild-type mice of the same age (Durand et al 1998).

The p27-deficient mice are larger than normal and have more cells in all organs that have been examined (Fero et al 1996, Kiyokawa et al 1996, Nakayama et al 1996), suggesting that p27 may be required in many cell lineages for normal exit from the cell cycle. Genetic studies indicate that p27-like proteins are also present in Caenorhabditis elegans (Hong et al 1998) and Drosophila (de Nooij et al 1996, Lane et al 1996). If the genes encoding these proteins are inactivated by mutation, developing cells in multiple lineages go through one or more extra divisions before withdrawing from the cell cycle (de Nooij et al 1996, Lane et al 1996). It seems that Cdk inhibitors are involved in stopping the cell cycle at the appropriate time during development in all animals. On the other hand, precursor cells still stop dividing and differentiate in the mutant mice, worms and flies, suggesting that p27 and its relatives are only part of the stopping mechanism. What are the other components of the stopping mechanism? The phenotype of mice deficient in another Cdk inhibitor, p18/Ink4c (p18), is very similar to that of p27-deficient mice, suggesting that p18 may be another component in mice (Franklin et al 1998). In mice deficient in both p27 and p18 some organs are even larger than in mice deficient in either protein alone (Franklin et al 1998), but even in these doubly-deficient mice precursor cells still stop dividing and differentiate during development, suggesting that there are additional components that stop the cell cycle in these mice.

How are the levels of p27 controlled such that they progressively increase in dividing OPCs? RT-PCR studies indicate that mRNA levels do not change appreciably as the protein levels increase, suggesting that the controls on p27 protein levels in these cells are all post-transcriptional (Y. Tokumoto, unpublished work). Transcriptional controls, however, may also play a part in the timer. This is suggested by studies of the inhibitory helix-loop-helix protein Id4, which decreases progressively as OPCs proliferate in vitro and in vivo with a time course expected if Id4 is part of the timer (Kondo & Raff 2000a). Enforced expression of ld4 stimulates OPC proliferation and inhibits OPC differentiation, suggesting that the normal progressive fall in Id4 protein may help determine when OPCs withdraw from the cell cycle and differentiate (Kondo & Raff 2000a). Id4 protein and mRNA decrease in parallel as OPCs proliferate in vitro and in vivo, suggesting that the control of Id4 expression in these cells is transcriptional (Kondo & Raff 2000a).

Thus the intrinsic timer in OPCs is complex. Some proteins increase over time and others decrease, and both transcriptional and post-transcriptional mechanisms seem to be involved.

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