Precursor Cell Physiology: Regulation of Development, Signaling and Disease by Small Changes in Intracellular Redox Status
The regulation of cell function at the level of genes and proteins is the focus of large numbers of laboratories around the world, with far fewer research teams interested in the question of how vital cell functions are regulated by changes in cell physiology. When we discovered, in 1994, that 15-20% changes in the oxidative status of cells could cause >1000% changes in cell survival (Mayer and Noble, 1994), we initiated a research path that has led to our participation in creating the field of precursor cell physiology. These research efforts have in turn led to the discovery of multiple new principles related to the regulation of normal development, and even to the discovery of novel regulatory pathways involved in functions ranging from control of normal cell function to the response of progenitor cells to environmentally relevant levels of chemically diverse toxicants.
One of the central components of our research program has been to understand control of precursor cell function at the physiological level, as a necessary complement of analyses at the genetic and proteomic levels. Physiological regulation of cell function is central to the understanding of both development and pathology. At this stage in development of the field of stem cell medicine, however, the role of physiology in precursor cell function has only been addressed by a relatively small number of laboratories.
We first became interested in physiological control of cell function as a result of our discoveries that small changes in intracellular redox state could effectively turn on or off the effects of potent signaling agents. Our initial studies (Mayer and Noble, 1994) on redox modulation demonstrated striking enhancements in the sensitivity of cells to promoters of cell survival caused by small increases (<15%) in levels of glutathione (the most prevalent reduced thiol within cells, and which is crucial for maintaining a reduced intracellular environment). For example, the response of oligodendrocytes and spinal ganglion neurons to suboptimal amounts of such survival agents as ciliary neurotrophic factor (CNTF), insulin-like growth factor-I (IGF-I) and nerve growth factor (NGF) was increased up to 1300% by co-exposure to <1 mM N-acetyl-L-cysteine (NAC, a cysteine pro-drug that is readily taken up by cells; as cysteine is the rate-limiting precursor for glutathione biosynthesis, exposure to 1mM NAC leads to small but significant increases in glutathione levels. Moreover, we found that as little as a 15% increase in glutathione content was sufficient to block cell death induced by TNF-a or glutamate, as well as to dramatically enhance response to protein survival factors. While these studies were some of the first to identify the ability of anti-oxidants to protect against a variety of inducers of cell death, it was the ability of small changes in redox state to cause disproportionately large effects on biological outcome that has been the subject of our continued experimental attention.
Our attempts to understand cell-intrinsic contributions to progenitor function led to the discovery that the probability of self-renewal was regulated by similarly small differences in intracellular redox state, a finding that has turned out to be a critical clue leading to our current understanding of the integration of progenitor cell biology, signaling pathway function and redox physiology. When we examined the effects of redox state in progenitor cell function (Smith et al., 2000), we found that intracellular redox state is a critical regulator of the balance between self-renewal and differentiation. In order to study this problem in progenitor cells, we first developed means of purifying cells on the basis of their redox state, and found that progenitor cells that were more reduced when isolated from the animal exhibited greater self-renewal when grown in basal division conditions, while those that were more oxidized exhibited more differentiation. The more oxidized cells were not committed to differentiate, however, as supplementation of their medium with N-acetyl-L-cysteine (NAC, a cysteine pro-drug that is readily taken up by cells, leading to higher levels of intracellular glutathione) promoted their self-renewal. These results suggested that redox state was important in controlling the probability of differentiation, and also demonstrated that the degree of redox change needed to affect these processes was not large.
We next found that the organism appears to use developmental, and cell-intrinsic, regulation of redox status as a means of regulating self-renewal probabilities (Power et al., 2002), providing a further linkage between redox physiology and the balance between self-renewal and differentiation. O-2A progenitor cells isolated from regions of the CNS in which myelination occurs early in development were more oxidized and exhibited a greater probability of differentiation than progenitor cells isolated from CNS regions (e.g, cortex) in which new cells are continuously generated until relatively late in development. Cortex-derived cells grown in basal division conditions continued to divide extensively long after optic nerve-derived cells had all differentiated. These differences in redox state were cell-intrinsic, such that cortical progenitor cells maintained their behavior even after several weeks of in vitro culture. Moreover, cortical cells are more resistant to inducers of differentiation or cell death.
Perhaps most surprising of all of our findings was the discovery that the ability of cell-extrinsic signaling molecules to alter redox state in specific directions was a necessary component of the mechanism of their action, thus linking together our studies on development and physiology with the analysis of more widely studied regulators of the balance between self-renewal and differentiation. Signaling molecules that enhanced self-renewal (i.e, FGF-2 and neurotrophin-3 (NT-3)) made O-2A/OPCs more reduced, while those that enhanced differentiation (i.e,. thyroid hormone (TH) and bone morphogenetic protein-4 (BMP-4)) made cells more oxidized. These redox alterations were seen well before differentiation was observable. Critically, pharmacological prevention of the redox changes associated with exposure to these signaling molecules blocked their ability to promote self-renewal (for NT-3) or promote differentiation (for TH). These studies thus revealed the remarkable finding that although these well-studied signaling molecules have a variety of other signaling functions, their ability to modify redox status is essential in order for them to exert their effects on division and differentiation.
Modulation of O-2A progenitor cell and oligodendrocyte function by redox state. Cells that are relatively more reduced will be more responsive to agents that promote cell division (1) and/or cell survival (2), and relatively less responsive to agents that promote differentiation and/or cell death. In contrast, cells that are relatively more oxidized will be less responsive to agents that promote cell division and/or cell survival, and relatively more responsive to agents that promote differentiation (3) and/or cell death (4).
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
Mark D. Noble
University of Rochester
601 Elmwood Ave.
Rochester, NY 14642
Office: MRB 2-9625