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The Redox/Fyn/c-Cbl Pathway: Environmental Toxicants and Other Pro-oxidative Stimuli Disrupt Progenitor Cell Function by Enhancing Degradation of Specific Receptor Tyrosine Kinases

Project Overview

One of the areas in which we recently have become actively interested is that of toxicology. Integration of the fields of precursor cell biology and toxicology represents a critical, but under-explored, opportunity for stem cell biology to make substantive advances relevant to understanding how toxicant exposure contributes to abnormal development and tissue function. The World Health Organization estimates that environmental factors may be responsible for 30-40% of the burden of childhood disease. While some environmental insults represent nutritional deficiencies, many others represent exposure to environmental toxicants. Insults that occur during periods of maximal stem and progenitor cell function (e.g., during gestation) are particularly disruptive to normal development, suggesting that disruption of precursor cell function may be an important means by which such insults exert their adverse effects.

Our studies thus far have led to the discovery of a novel regulatory pathway that converts small increases in oxidative status into enhanced degradation of receptors on the cell surface that are essential for promoting precursor cell division and survival. This pathway, called the redox/Fyn/c-Cbl pathway, provides one of the few general mechanistic principles that applies to understanding the action of environmentally relevant levels of chemically diverse toxicants, and the only such principle thus far identified that integrates precursor cell biology, toxicology, redox biology and signaling pathway analysis in a predictive framework of broad potential relevance to the understanding of toxicant-mediated disruption of precursor cell function. Our studies suggest new strategies for addressing the challenge offered by the fact we lack even the most basic toxicological information on the great majority of the many tens of thousands of chemicals that are produced – which means these substances can be released without adequate regulation. Moreover, it seems increasingly likely that the redox/Fyn/c-Cbl pathway is also central to understanding how the signaling molecules of normal development regulate the balance between division, differentiation and survival in precursor cell populations.

One of the greatest obstacles to analysis of environmental toxicants on development is the need to identify mechanistic principles responsible for the effects of exposure to environmentally relevant levels of chemically diverse substances, a challenge that offers an intriguing point of integration of precursor cell biology with toxicology. Unless such general principles are identified, effective analysis of estimated 80,000 – 150,000 registered chemicals for which no toxicological information exists (and which therefore are not subject to regulatory control) is difficult to envisage. Despite the importance of addressing this challenge, however, relatively few mechanisms have been identified that apply to analysis of low levels of exposure to chemically diverse toxicants and candidate toxicants.

The likely effects of toxicants on precursor cell function, and the difficult challenges posed by the need to identify general mechanisms by which these act were already sufficient to attract our attention – but we were also attracted by observations that O-2A progenitor cells are the targets of multiple environmental toxicants and that many of these substances make cells more oxidized. For example, O-2A progenitor cells are targets of such chemically diverse substances as lead, ethanol, and triethyltin and multiple other substances (including, from our own studies, multiple chemotherapeutic agents). The sensitivity of O-2A progenitor cells to such agents was of particular interest in that one of the few effects of toxicant exposure that appears to be common to multiple chemically diverse substances is the ability of these agents to cause cells to become more oxidized. The range of toxicants reported to induce oxidative status is very broad, and includes metal toxicants such as methylmercury, lead, trimethyltin, cadmium and arsenic. Ethanol exposure also is associated with oxidative stress, as is exposure to a diverse assortment of agricultural chemicals, including herbicides (e.g., paraquat, pyrethroids, and organophosphate and carbamate inhibitors of cholinesterase. Thus, the ability to cause cells to become more oxidized is shared by many toxicants, regardless of their chemical structure. The regulation of multiple functions of O-2A progenitor cells and other cell types by small changes in redox balance (Mayer and Noble, 1994; Power et al., 2002; Smith et al., 2000) suggested that this property of chemically diverse toxicants might be of significant biological interest.

After discovering that low and environmentally relevant levels of exposure to methylmercury (MeHg) caused O-2A progenitor death and, at still lower exposure levels, suppression of division, we proceeded to dissect the effects of MeHg on PDGF-mediated signaling. While it has been widely believed that changes in oxidative status were global regulators of cell signaling, we found that effects of MeHg (and of every other pro-oxidant we have examined) are remarkably pathway specific. We also found that chemically diverse toxicants (e.g., lead, paraquat) had effects identical to those of MeHg.

Our analyses of the mechanisms by which environmentally relevant levels of chemically diverse toxicants caused similar changes in O-2A progenitor cell function have revealed a new regulatory pathway, the redox/Fyn/c-Cbl pathway (Li et al., 2007), that links small changes in redox status with pathway-specific degradation of receptor tyrosine kinases (RTKs), thus suppressing all signaling downstream of the degraded RTKs. In this pathway, small increases in oxidative status cause activation of Fyn kinase One of Fyn’s targets is c-Cbl, a ubiquitin ligase that is an important negative regulator of RTK signaling. When c-Cbl is activated, its targets are ubiquitylated and targeted for degradation. RTKs that are targets of c-Cbl include the PDGF receptor-a (PDGFRa), the epidermal growth factor receptor (EGFR) and the c-Met receptor for hepatocyte growth factor all of which are expressed by O-2A progenitor cells. Activation of Fyn and c-Cbl associated with reductions in signaling (e.g., Erk1/2, Akt) and in nuclear transcription (e.g., serum response element (SRE) and NF-kB) downstream of PDGFRa (or other receptors that are c-Cbl targets). In contrast, signaling mediated by receptors that are not c-Cbl targets (e.g, the TrkC receptor for NT-3) is not affected, nor are levels of these receptors altered by activation of Fyn and c-Cbl.

Diagrammatic summary of the redox/Fyn/c-Cbl pathway. Changes in redox state cause activation of Fyn kinase, which in turn causes activation of c-Cbl, an E3 ubiquitin ligase. c-Cbl has as one of its functions the negative regulation of receptor tyrosine kinase (RTK) signaling from specific RTKS, with PDGFRa being an RTK of particular relevance to the division and survival of O-2A progenitor cells. c-Cbl activation causes enhanced degradation of PDGFRa, leading to reductions in all downstream signaling from this receptor, with consequent reductions in cell survival or cell division (depending upon the extent of loss of PDGFRa).

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The general importance of the signaling pathways regulated by Fyn and c-Cbl suggests that the ability of chemically diverse toxicants to converge on this pathway may be of very broad relevance to the understanding of toxicant action. The c-Cbl target PDGFRa plays critical roles in the proliferation and survival of O-2A progenitor cells with disruption of PDGF function in vivo being associated with reductions in O-2A progenitor and oligodendrocyte numbers in vivo, but this receptor also plays important roles in development of neurons and in other tissues. The EGFR is thought to play roles in such a diverse range of processes as cortical neurogenesis, maintenance of the subventricular zone and astrocyte development, as well as playing multiple important roles in non-CNS tissues. Hepatocyte growth factor and c-Met are involved in regulating development of cortical pyramidal dendrites, neural induction, motoneuron survival and pathfinding, sympathetic neuroblast survival and hippocampal neuron neurite outgrowth, as well as having extensive effects on development of kidney, lung, breast and other tissues. Indeed, the wide range of targets of c-Cbl offers a rich fabric of potentially critical regulatory molecules that would be effected by changes in activity of this protein, with the importance of particular proteins being dependent on the cell type and developmental stage under consideration. In addition, Fyn regulation of the Rho/ROCK signaling pathway could be of relevance in understanding toxicant-mediated effects on cell migration, neurite outgrowth and development of dendritic morphology. Thus, the ability of exposure to low levels of toxicants with pro-oxidant activity to activate Fyn may also be relevant to understanding toxicant-mediated alterations in cytoskeletal function. As Fyn, c-Cbl, PDGFRa and other c-Cbl targets are expressed in multiple tissues of the body, our observations may additionally help explain the effects of toxicant exposure in non-CNS tissues.

The redox/Fyn/c-Cbl pathway is activated in O-2A progenitor cells by levels of MeHg that are frequently encountered in the environment. For example, exposure of O-2A progenitor cells to 20 nM MeHg was sufficient these cells more oxidized and to cause a ~25% drop in the percentage of O-2A/OPCs incorporating BrdU in response to stimulation with PDGF. When examined at the clonal level, MeHg exposure was associated with a reduction in the number of large clones and an increase in the number of small clones, as seen for other pro-oxidant stimuli. These effects of MeHg were seen at exposure levels as low as 20 nM, less than the >5.8 mg/L (i.e., parts per billion) of MeHg found in cord blood specimens of as many as 600,000 infants in the United States each year and < 0.3% of the exposure levels previously found to induce oxidative changes in astrocytes. As the human brain is thought to concentrate MeHg 5-7 fold over levels found in the bloodstream, the levels of sensitivity that we have discovered are of profound concern. Moreover, we found that developmental exposure to MeHg disrupted progenitor cell division and caused reductions in levels of both EGFR and PDGFRα in the cerebellum and hippocampus, even at exposure levels as low as 100 ppb MeHg (i.e, 1/10 of levels previously considered to be subthreshold for detecting effects).

By combining our analyses of progenitor cell biology with toxicological investigations, we have revealed one of the few general mechanistic principles that applies to understanding the action of environmentally relevant levels of chemically diverse toxicants, and the only such principle thus far identified that integrates precursor cell biology, toxicology, redox biology and signaling pathway analysis in a predictive framework of broad potential relevance to the understanding of toxicant-mediated disruption of precursor cell function. Our discovery of the redox/Fyn/c-Cbl pathway provides a new general principle and mechanistic target that may apply to the understanding of the action of a large number of chemically diverse toxicants. As the outcomes we have identified occur at quite low toxicant exposure levels, they may provide a particularly useful unifying principle for the analysis of toxicant effects.

The principles revealed by our studies of precursor cell physiology and toxicology are particularly broad in their potential applicability, and extend beyond the boundaries of toxicology back to normal development. These studies, combined with our previous analysis of the central importance of intracellular redox state in modulating progenitor cell function lead to the prediction that any toxicant with pro-oxidant activity will exhibit these effects. While toxicants of differing chemical structures also will have additional activites, the convergence of small increases in oxidative status on regulation on the Fyn/c-Cbl pathway provides a specific means by which exposure to low levels of a wide range of chemically diverse toxicants might have similar classes of effects on development. Our findings also provide a strategy for rapid identification of such effects by any of the estimated 80,000 to 150,000 chemicals for which toxicological information is limited or non-existent, thus enabling a preliminary identification of compounds that would need to be examined in vivo. The sensitivity of O-2A/OPCs to environmentally relevant levels of MeHg and Pb provides a great advantage over established cell lines and such other neural cells as astrocytes, for which these low exposure levels may have little effect, and the importance of understanding the effects of toxicants on progenitor cell function provides a direct link between our studies and the broad field of developmental toxicology. In addition, the ability of NAC to protect progenitor cells against the adverse effects of chemically diverse toxicants raises the possibility that this benign therapeutic agent may be of benefit in protecting children known to be at increased risk from the effects of toxicant exposure during critical developmental periods.

Moving outside of the field of toxicology, ongoing studies demonstrate that the redox/Fyn/c-Cbl pathway is also a convergence point for biological agents that control the balance between self-renewal and differentiation, including such factors as FGF-2, NT-3, TH and BMP-4. This pathway is also active in cell types other than O-2A progenitor cells. Thus, these studies provide an example of how our interests in precursor cell physiology have led us to new understandings of both development and pathology. What is particularly exciting is that this part of the fields of stem cell biology and stem cell medicine is in its very earliest stages, and there are certain to be many more equally powerful integrative discoveries to be made.

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Li, Z., Dong, T., Pröschel, C., and Noble, M. (2007). Chemically diverse toxicants converge on fyn and c-Cbl to disrupt precursor cell function. PLoS Biol. 5, e35 (doi:10.1371/journal.pbio.0050035). link

Mayer, M., and Noble, M. (1994). N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro. Proc. Natl. Acad. Sci. U.S.A. 91, 7496-7500. link

Power, J., Mayer-Proschel, M., Smith, J., and Noble, M. (2002). Oligodendrocyte precursor cells from different brain regions express divergent properties consistent with the differing time courses of myelination in these regions. Dev. Biol. 245, 362-375. link

Smith, J., Ladi, E., Mayer-Pröschel, M., and Noble, M. (2000). Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. Proc. Natl. Acad. Sci. U.S.A. 97, 10032-10037. link

Contact

Mark D. Noble
University of Rochester
Box 633
601 Elmwood Ave.
Rochester, NY 14642
Office: MRB 2-9625
+1-585-273-1448 mark_noble@urmc.
rochester.edu