Our findings demonstrate for the first time a critical role for Nrf2-dependent GSH-induced signaling in lung epithelial cell proliferation and cellular protection against oxidative stress. Interestingly, we found that, despite constant production of ROS, Nrf2-deficient alveolar epithelial cells were viable and expressed normal differentiation characteristics that resembled those of their wild-type counterparts (). These results suggest that Nrf2-dependent GSH-mediated signaling, although essential for type II cell proliferation, may not be critical for maintaining the differentiation of the cells. Although previous studies using GSH-depleting chemical agents have demonstrated a role for intracellular redox status in various biological processes (15
), our study using freshly isolated primary cultures provides for the first time both genetic and pharmacologic evidence for a critical requirement for Nrf2-dependent GSH-induced signaling in lung type II alveolar cell proliferation.
Of particular interest is our observation that supplementation of Nrf2−/−
cells with NAC, a precursor of GSH, drastically lowered ROS levels but failed to induce cell proliferation to the extent seen in wild-type cells and Nrf2−/−
cells supplemented with GSH. Several studies, including ours, have shown that NAC attenuates or provides protection against prooxidant stimuli both in vitro
and in vivo
). The inability of NAC to rescue the growth arrest of Nrf2−/−
cells is not surprising, however, because these cells express diminished levels of the γ-glutamyl cysteinyl ligase (GCL) components, GCLC and GCLM (), that are critical for de novo
GSH biosynthesis and provide protection against prooxidants (21
). Thus, it is likely that Nrf2−/−
cells may only inefficiently convert NAC to GSH, a process that would otherwise cause the intracellular GSH to rise to levels that are adequate for cell proliferation (16
). Thus, it appears that in addition to its drastically reducing the high levels of ROS, signaling induced by GSH is critically required for normal proliferation of lung epithelial cells. Although the sources contributing to high levels of ROS in Nrf2−/−
cells remains to be investigated, it is likely that diminished levels of GSH may lead to an inefficient cellular detoxification of endogenous ROS levels generated by mitochondria constitutively in Nrf2−/−
cells. In addition, it is possible that Nrf2 deficiency may result in dysregulation of ROS-generating enzymes, such as Nox/Duox, Rac, or RAS, thereby contributing to elevated levels of ROS.
Unlike primary Nrf2−/−
type II epithelial cells, immortalized embryonic fibroblast cells (MEFs) lacking the Nrf2−/−
gene, established according to an NIH3T3 protocol, grow normally and do not display elevated levels of ROS or redox imbalance when compared with Nrf2+/+
MEFs. In fact, Nrf2−/−
MEFs proliferate more rapidly than do their isogenic wild-type counterparts (unpublished data). However, Nrf2−/−
MEFs are more susceptible than Nrf2+/+
cells to various stressors. There are several possible reasons for this discrepancy. One possibility is that during immortalization, the MEFs that survive selection may have undergone genetic changes that could drive cell proliferation in the absence of Nrf2. This type of discordant behavior has been reported for several genes. For example, primary MEFs lacking the Jun-D/AP1 transcription factor proliferate poorly and display senescent characteristics (23
); in contrast, established MEFs lacking Jun-D showed an increased rate of cell proliferation when compared with isogenic wild-type cells. Another likely possibility to explain our results is that the effects of Nrf2 on cell proliferation are context-dependent. However, we can probably rule out this possibility because we found that primary lung fibroblasts lacking Nrf2 also proliferate poorly and display elevated levels of ROS (data not shown).
The decreased rate of cell proliferation that we observed for wild-type cells was clearly not caused by apoptosis of Nrf2−/−
cells, since we observed no cellular or nuclear staining of Nrf2−/−
cells with trypan blue (). Consistent with this result, we found no significant differences in the activation of caspase 3, the pro-apoptotic factor, in wild-type and Nrf2−/−
cells (). Although somewhat surprising, the lack of apoptosis or activation of caspase-3 that we observed in Nrf2−/−
type II cells, despite elevated levels of ROS, suggests that high levels of ROS per se
do not initiate death-activating signals in type II cells, at least under our experimental conditions. Further studies are needed to determine whether this response pattern is an intrinsic property of type II cells, which are constantly exposed to an oxidant environment in alveolar space (24
), or whether they require additional cues, such as activation of Fas or TNF-α–activated signals. Consistent with this possible need for additional cues, exposure to H2
induced greater levels of death in Nrf2−/−
type II cells than in wild-type cells (). Moreover, we have previously reported that Nrf2 deficiency enhances the sensitivity of T-lymphocytes to Fas-mediated apoptosis (25
Although the exact mechanisms by which GSH controls the proliferation of lung type II cells remain to be investigated, recent studies have shown that an oxidized intracellular environment causes protein glutathionylation, while reducing conditions favor protein deglutathionylation (19
). Both protein glutathionylation and deglutathionylation affect the function of various proteins, including transcription factors such as NF-κB and c-Jun (see
review elsewhere; 19). We believe that decreased GSH levels in Nrf2−/−
cells may cause dysregulation of signal transduction pathways and effector transcription factor activation, which are required for gene expression. Delineating the glutathionylation and deglutathionylation mechanisms regulated by Nrf2-GSH–induced signaling may provide further insight into the roles of redox signaling in type II cell proliferation and differentiation during lung injury and repair. Preliminary expression profiling analysis has revealed that Nrf2, acting via GSH, regulates the expression of several antioxidant enzymes and genes involved in cell proliferation, including receptors, growth factors, kinases, and transcription regulators (data not shown).
In summary, the present study using freshly isolated primary cultures has provided genetic and pharmacologic evidence that Nrf2-dependent GSH-induced signaling plays a key role in lung type II cell proliferation and cellular protection against oxidant-induced death. Our findings suggest that in addition to quenching high levels of ROS, signaling induced by GSH is critically required for proper cell proliferation but may not be essential for maintaining differentiation. Since redox imbalance has been implicated in the pathogenesis of many acute and chronic lung diseases, this cell culture model (system) may be useful in further elucidating the precise mechanisms by which redox signaling regulates type II cell proliferation and cellular protection against oxidants during lung injury and repair.