This study represents the first demonstration that ROS can regulate the expression of MIF in CNS neurons. Furthermore, we have shown that this effect of H2O2 involves intracellular events that are specific to neurons, and the data suggest that the increase in MIF involves de novo transcription. Finally, the observation that H2O2 fails to elicit an increase in MIF in neurons cultured from SHR, in contrast to their normotensive controls, provides support for the contention that the MIF gene responds in a specific and regulated fashion to redox signaling.
In this study, H2
was selected as the ROS donor for many reasons. First, the aim was to study a ROS that is downstream of Ang II and the AT1R in neurons. It has already been established that Ang II is capable of producing intracellular H2
in many cell-types, including neurons, and that this H2
has significant physiological effects (e.g. influencing sympathetic activity in the brain) [12
]. Second, it is readily cell permeant, with exogenously-applied H2
establishing equilibrium across the cell membrane within minutes [16
]. Finally, it is relatively stable since, while it is a ROS, it is not a free-radical. While the concentration of H2
[30 μM] that was required to induce MIF expression is relatively high, the cytotoxicity, protein carbonyl, and PEG-catalase experiments support the notion that this ROS is functioning as a signaling agent in our neuronal cultures, rather than a mediator of cell death and/or oxidative stress. The necessity of a relatively high concentration of H2
is likely due to the presence of a small population of glia (astrocytes, oligodendrocytes, microglia) within the neuronal cultures. These cells all have the capacity to detoxify H2
], and so it is possible that a portion of the exogenous H2
is rapidly degraded when added to the medium of the neuronal cultures, and, consequently, the neurons are not exposed to doses high enough to create oxidative stress in the intracellular environment. Moreover, the knowledge that a bolus application of H2
can be rapidly detoxified by many of the cell types in our neuronal cultures prompted us to perform the experiments utilizing glucose oxidase, which represents a more chronic means of administering H2
, to confirm that this ROS can induce MIF expression.
The results raise important questions as to the mechanism by which H2
is inducing MIF expression in CNS neurons. It is now widely recognized that H2
, like nitric oxide, may be a readily-diffusible small molecule that acts as a signaling agent. Indeed, in prokaryotes and yeast, systems that sense and signal in response to H2
are well-characterized [11
]. In higher mammals, many of the signaling pathways affected by ROS are still under investigation. Nevertheless, it is becoming clear that several kinase pathways are modulated by ROS and the activity of many transcription factors is subject to redox regulation [18
]. For example, transcription factors such as AP-1, SP-1, CREB, and NFκB are sensitive to redox regulation [21
], and binding sites for these transcription factors have been identified in the promoter of the human MIF gene [24
]. Furthermore, SP-1 and CREB are important transcriptional regulators of the MIF gene [25
]. Experiments to determine if these transcription factors may be the mediators of redox regulation of the MIF gene in neuronal cell lines are ongoing.
This study is also significant because, as we have established in prior reports, MIF is up-regulated in neurons in response to Ang II signaling via the AT1R [7
]. MIF then serves, either directly or indirectly, as a negative regulator of the chronotropic actions of Ang II in neurons that lie along key sympathetic and neuroendocrine pathways in the brain such as the PVN [8
]. Intriguingly, a recent publication has shown that H2
produced in the PVN in response to Ang II may play a role in regulating sympathetic activity [12
]. Accordingly, it is tempting to visualize a feed-back loop such that Ang II causes H2
production in the PVN, which stimulates MIF production [9
], subsequently feeding back to decrease the sensitivity of the neuron to Ang II and, perhaps, influencing the central sympathetic and/or neuroendocrine actions of Ang II. We believe that MIF may act in this regard by scavenging ROS, as do some other proteins that contain TPOR motifs (e.g. Trx, peroxiredoxins) [5
], but the exact mechanism is still under investigation.
A further interesting point is the failure of H2
to induce MIF expression in SHR neuronal cultures. This finding provides further evidence that the effect of H2
on neuronal MIF expression is a specific signaling event, rather than a non-selective oxidative stress-mediated mechanism. Aside from this, we previously demonstrated that Ang II does not induce MIF expression in SHR neurons in culture [26
] and in PVN neurons of SHR [27
], and this lack of MIF induction may contribute to the hyper-sensitivity of these neurons to Ang II, and perhaps even the development and/or maintenance of hypertension in these animals. This idea is borne out by recent findings that viral-mediated over-expression of MIF in the PVN of young SHRs attenuates the development of hypertension [27
]. Future studies will include investigating why Ang II and H2
fail to elicit MIF expression in SHR neurons.
In conclusion, this study establishes that MIF expression in neurons can be regulated by ROS. It serves as a basis for further studies on whether H2O2 is a mediator of Ang II-induced MIF expression in normal rat neurons, and whether H2O2 signaling is “broken” in SHR neurons and, hence, leads to an inability of Ang II to induce MIF expression.