Two molecularly distinct intracellular pathways to oligodendrocyte differentiation: role of a p53 family protein

Both thyroid hormone (TH) and retinoic acid (RA) induce purified rat oligodendrocyte precursor cells in culture to stop division and differentiate. We show that these responses are blocked by the expression of a dominant-negative form of p53. Moreover, both TH and RA cause a transient, immediate early increase in the same 8 out of 13 mRNAs encoding intracellular cell cycle regulators and gene regulatory proteins, but only if protein synthesis is inhibited. Platelet-derived growth factor (PDGF) withdrawal also induces these cells to differentiate, but we show that the intracellular mechanisms involved are different from those involved in the hormone responses: the changes in cell cycle regulators differ, and the differentiation induced by PDGF withdrawal (or that which occurs spontaneously in the presence of PDGF) is not blocked by the dominant-negative p53. These results suggest that TH and RA activate the same intracellular pathway leading to oligodendrocyte differentiation, and that this pathway depends on a p53 family protein. Differentiation that occurs independently of TH and RA apparently involves a different pathway. It is likely that both pathways operate in vivo.     

Roles for p53 and p73 during oligodendrocyte development

Oligodendrocytes make myelin in the vertebrate central nervous system (CNS). They develop from oligodendrocyte precursor cells (OPCs), most of which divide a limited number of times before they stop and differentiate. OPCs can be purified from the developing rat optic nerve and stimulated to proliferate in serum-free culture by PDGF. They can be induced to differentiate in vitro by either thyroid hormone (TH) or PDGF withdrawal. It was shown previously that a dominant-negative form of p53 could inhibit OPC differentiation induced by TH but not by PDGF withdrawal, suggesting that the p53 family of proteins might play a part in TH-induced differentiation. As the dominant-negative p53 used inhibited all three known p53 family members - p53, p63 and p73 - it was uncertain which family members are important for this process. Here, we provide evidence that both p53 and p73, but not p63, are involved in TH-induced OPC differentiation and that p73 also plays a crucial part in PDGF-withdrawal-induced differentiation. This is the first evidence for a role of p73 in the differentiation of a normal mammalian cell.     

A cell-intrinsic timer that operates during oligodendrocyte development

Multicellular organisms develop on a predictable schedule that depends on both cell-intrinsic timers and sequential cell-cell interactions mediated by extracellular signals. The interplay between intracellular timers and extracellular signals is well illustrated by the development of oligodendrocytes, the cells that make the myelin in the vertebrate central nervous system. An intrinsic timing mechanism operates in each oligodendrocyte precursor cell to limit the length of time the cell divides before terminally differentiating. This mechanism consists of two components, a timing component, which depends on the mitogen platelet-derived growth factor (PDGF) and measures elapsed time, and an effector component, which depends on thyroid hormone and stops cell division and initiates differentiation at the appropriate time. The cell-cycle inhibitor p27/Kip1 accumulates in the precursor cells as they proliferate and is part of both components of the timer. It seems likely that similar timing mechanisms operate in other cell lineages. BioEssays 22:64-71, 2000.     

The role of cadherin,beta-catenin,and AP-1 in retinoid-regulated carcinoma cell differentiation and proliferation

Vitamin A derivatives (retinoids) are potent regulators of cell proliferation and differentiation. Retinoids inhibit the function of the oncogenic AP-1 and beta-catenin/TCF pathways and also stabilize components of the adherens junction, a tumor suppressor complex. When treated with retinoic acid (RA), the breast cancer cell line, SKBR3, undergoes differentiation and reduction in cell proliferation. The present work demonstrates that in SKBR3 cells, which exhibit high AP-1 activity, RA-regulation of cadherin expression and function, but not changes in AP-1 (or beta-catenin/TCF) signaling, is responsible for the epithelial differentiation. However, cadherin function and recruitment of beta-catenin to the membrane is not required for RA to regulate DNA synthesis in these cells. RA also reduces the activity of an AP-1 and TCF-sensitive cyclin D1 reporter in SKBR3 cells in a manner that is independent of the TCF site. In contrast, in SW480 cells, which have high levels of beta-catenin/TCF signaling, the activity and retinoid responsiveness of the cyclin D1 promoter was markedly inhibited by mutation of the TCF site. These data indicate that the remarkably broad effects of RA on the growth and differentiation of many different epithelial cancers may well be explained by the ability of RA to differentially regulate the activity of RAR/RXR, AP-1, and beta-catenin/TCF pathways.     

Role of thyroid hormone receptors in timing oligodendrocyte differentiation.

The timing of oligodendrocyte differentiation is thought to depend on both intracellular mechanisms and extracellular signals. Thyroid hormone (TH) helps control this timing both in vitro and in vivo, but it is still uncertain how it does so. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Previous studies suggested that TRbeta receptors may mediate the effect of TH on oligodendrocyte precursor cells (OPCs). Consistent with this possibility, we show here that overexpression of TRbeta1 promotes precocious oligodendrocyte differentiation, whereas expression of two dominant-negative forms of TRbeta1 greatly delays differentiation. Surprisingly, however, we find that postnatal TRbeta-/- mice have a normal number of oligodendrocytes in their optic nerves and that TRbeta-/- OPCs stop dividing and differentiate normally in response to TH in vitro. Moreover, we find that OPCs do not express TRbeta1 or TRbeta2 mRNAs, whereas they do express TRalpha1 and TRalpha2 mRNAs. These findings suggest that alpha receptors mediate the effect of TH on the timing of oligodendrocyte differentiation. We also show that TRalpha2 mRNA, which encodes a dominant-negative form of TRalpha, decreases as OPCs proliferate in vitro and in vivo. This decrease may help control when oligodendrocyte precursors differentiate.     

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin‐dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus‐expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur. J. Cell. Biochem. 76:270–279, 1999. © 1999 Wiley‐Liss, Inc.     

Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation.

In many tissues, progenitor cells permanently withdraw from the cell cycle prior to commitment towards a differentiated phenotype. In the oligodendrocyte lineage a counting mechanism has been proposed, linking the number of cell divisions to growth arrest and differentiation. A direct prediction of this model is that an increase in the number of cell divisions would result in a delayed onset of differentiation. Since the cell cycle inhibitor p27Kip1 is an essential component of the machinery leading to oligodendrocyte progenitor growth arrest, we examined the temporal relationship between cell cycle withdrawal and expression of late differentiation markers in vivo, in mice carrying a targeted deletion in the p27Kip1 gene. Using bromodeoxyuridine to label proliferating cells, quaking (QKI) to identify embryonic glial progenitors, NG2 to identify neonatal oligodendrocyte progenitors, and myelin basic protein to label differentiated oligodendrocytes, we found an increased number of proliferating QKI- and NG2-positive cells in germinal zones of p27Kip1(-/-) mice at the peak of gliogenesis. However, no delay was observed in these mice in the appearance of the late differentiation marker myelin basic protein in the developing corpus callosum and cerebellum. Significantly, a decrease in cyclin E levels was observed in the brain of p27Kip1 null mice coincident with oligodendrocyte growth arrest. We conclude that two distinct modalities of growth arrest occur in the oligodendrocyte lineage: a p27Kip1-dependent mechanism of growth arrest affecting proliferation in early phases of gliogenesis, and a p27Kip1-independent event leading to withdrawal from the cell cycle and differentiation.     

Normal timing of oligodendrocyte development depends on thyroid hormone receptor alpha 1 (TRalpha1)

The timing of oligodendrocyte development is regulated by thyroid hormone (TH) in vitro and in vivo, but it is still uncertain which TH receptors mediate this regulation. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Here, we provide direct evidence for the involvement of the TRalpha1 receptor isoform in vivo, by showing that the number of oligodendrocytes in the postnatal day 7 (P7) and P14 optic nerve of TRalpha1-/- mice is decreased compared with normal. We demonstrate that TRalpha1 mediates the normal differentiation-promoting effect of TH on oligodendrocyte precursor cells (OPCs): unlike wild-type OPCs, postnatal TRalpha1-/- OPCs fail to stop dividing and differentiate in response to TH in culture. We also show that overexpression of TRalpha1 accelerates oligodendrocyte differentiation in culture, suggesting that the level of TRalpha1 expression is normally limiting for TH-dependent OPC differentiation. Finally, we provide evidence that the inhibitory isoforms of TRalpha are unlikely to play a part in the timing of OPC differentiation.     

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin-dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus-expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur.     

TGFβ and Retinoic Acid Intersect in Immune-Regulation

Transforming growth factor (TGFβ) prevents TH1 and TH2 differentiation and converts naïve CD4 cells into Foxp3-expressing T regulatory (Treg) cell1, 2. In sharp contrast, in the presence of pro-inflammatory cytokines, including IL-6, TGFβ not only inhibits Foxp3 expression but also promotes the differentiation of pro-inflammatory IL17-producing CD4 effector T (TH17) cells3-5. This reciprocal TGFβ-dependent differentiation imposes a critical dilemma between pro- and anti-inflammatory immunity and suggests that a sensitive regulatory mechanism must exist to control TGFβ-driven TH17 effector and Treg differentiation. A vitamin A metabolite, retinoic acid (RA), was recently identified as a key modulator of TGFβ-driven immune deviation capable of suppressing TH17 differentiation while promoting Foxp3+Treg generation 6-10.     

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27^Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 431–440, 1998     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

PDGF and intracellular signaling in the timing of oligodendrocyte differentiation

In the rat optic nerve, bipotential O-2A progenitor cells give rise to oligodendrocytes and type 2 astrocytes on a precise schedule. Previous studies suggest that PDGF plays an important part in timing oligodendrocyte development by stimulating O-2A progenitor cells to proliferate until they become mitotically unresponsive to PDGF, stop dividing, and differentiate automatically into oligodendrocytes. Since the loss of mitotic responsiveness to PDGF has been shown not to be due to a loss of PDGF receptors, we have now examined the possibility that the unresponsiveness results from an uncoupling of these receptors from early intracellular signaling pathways. We show that (a) although PDGF does not stimulate newly formed oligodendrocytes to synthesize DNA, it induces an increase in cytosolic Ca2+ in these cells; (b) a combination of a Ca2+ ionophore plus a phorbol ester mimics the effect of PDGF, both in stimulating O-2A progenitor cell division and in reconstituting the normal timing of oligodendrocyte differentiation in culture; and (c) the same combination of drugs does not stimulate newly formed oligodendrocytes to proliferate, even in the presence of PDGF or dibutyryl cAMP. The most parsimonious explanation for these results is that O-2A progenitor cells become mitotically unresponsive to PDGF because the intracellular signaling pathways from the PDGF receptor to the nucleus are blocked downstream from the receptor and some of the early events that are triggered by receptor activation.