Abstract
The steroid hormone estrogen has wide spread physiological effects including neuroprotective actions within the brain. The mechanisms underlying such neuronal actions are unknown but may involve an estrogenic modulation of the nerve growth factor receptor TrkA and its downstream extracellular signal-regulated kinase (ERK 1/2) pathway. Previous single molecule tracking data from our laboratory supports this proposal with estrogen increasing the periods of TrkA immobility on PC12 cells. This is important because such immobile periods are believed to be associated with enhanced intracellular signalling. The aims of this study were firstly to determine the estrogen receptor profile of PC12 cells and secondly, to determine the effect of estrogen receptor activation on both acute and chronic TrkA signalling. PC12 cells were acutely or chronically exposed to a range of 17β−estradiol (E2) concentrations (0.01-100nM) and TrkA and ERK1/2 activation (phosphorylation) monitored by Western blot. While NGF-induced TrkA phosphorylation was enhanced in a concentration-dependent manner by acute E2 treatment, the level of phosphorylated ERK1/2 decreased in a concentration-dependent manner. Although E2 treatment had no effect on its own, it altered the NGF response. Estrogen receptor expression in these cells was investigated using RT-PCR and confirmed the presence of both ERβ and GPR30 but not that of ERα. NGF is well recognized to promote PC12 cell differentiation with the resultant cessation of cell division and formation of neurite outgrowth. Chronic exposure to 1nM E2 caused PC12 cells to differentiate to a similar extent to a low concentration of NGF (30nM). Combined exposure of 1nM E2 and 30nM NGF had an additive effect on differentiation. Conversely, the more extensive proliferation produced by a high NGF concentration (300nM) was inhibited by E2. Thus the estrogenic actions on TrkA signalling are complex but may be accounted for by a model in which estrogens enhance TrkA phosphorylation by inhibiting an ERK1/2 mediated feedback which would otherwise dephosphorylate the receptor. Estrogenic inhibition of ERK1/2 may also account for the suppression of NGF-mediated cell differentiation, and suggests that this is the major differentiation pathway operating at high levels of TrkA receptor stimulation. The ability of E2 alone to promote differentiation indicates the presence of an alternate ERK1/2 independent differentiation pathway, which may also be utilized by low concentrations of NGF. In summary these data support the proposal that estrogens may modulate TrkA signalling. The importance of such an action, participating in their neuroprotective effects, remains to be determined but such an action is consistent with their observed ability to support the differentiation of PC12 cells.