Ovarian steroids reduce apoptosis induced by trophic insufficiency in nerve growth factor-differentiated PC12 cells and axotomized rat facial motoneurons.

Previous studies have demonstrated that ovarian steroids exert neuroprotective effects in a variety of in vitro and in vivo systems. The mechanisms underlying these effects remain poorly understood. In the present study, the neuroprotective effects of estradiol (E(2)) and progesterone (P) were examined in two models of apoptosis induced by growth factor insufficiency: partially nerve growth factor (NGF)-differentiated PC12 cells, after serum and NGF withdrawal; and axotomized immature rat facial motor motoneurons. E(2) and P both increased the survival of trophically withdrawn NGF-differentiated PC12 cells, at physiologically relevant concentrations. However, neither steroid had a significant effect on the survival of PC12 cells that had not been NGF treated. Exposure to NGF had no effect on the expression of estrogen receptor (ER)beta, but markedly increased the levels of ERalpha and altered the expression of the progesterone receptor (PR) from predominantly PR-B in NGF naive cells, to predominantly PR-A after NGF. The survival promoting effects of E(2) and P were blocked by the specific steroid receptor antagonists Faslodex (ICI 182780) and onapristone (ZK98299), respectively. Inhibitors of RNA (actinomycin D) or protein (cycloheximide) synthesis also abrogated the protective effects of both steroids. In immature rats, E(2) and P both significantly increased the numbers of surviving facial motor neurons at 21 days after axotomy. These data demonstrate significant protective effects of E(2) and P in two well-characterized models of apoptosis induced by trophic withdrawal and suggest that, at least in PC12 cells, the effects of the steroids are mediated via interaction with nuclear steroid receptor systems. The lack of steroid responsiveness in NGF-naive PC12 cells despite the presence of abundant ERbeta and PR-B are consistent with the view that ERalpha and PR-A may be particularly important as mediators of the neuroprotective effects of their corresponding hormonal ligands.

Estradiol (E2) elicits SRC phosphorylation in the mouse neocortex: the initial event in E2 activation of the MAPK cascade?

In neocortical explants, E2 activates various signaling components of the MAPK cascade, including B-Raf and MAPK kinase-dependent ERK, suggesting a possible role in the differentiative actions of E2 in the brain. To further characterize the signaling pathways activated by E2, we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of both the MAPK cascade and neuronal differentiation, may play a role in E2 signaling. The present studies show for the first time in the brain that E2 elicits phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534), and that this phosphorylation occurs despite disruption of ER alpha (in ER knockout mice). PP2, a Src family kinase inhibitor, suppressed not only E2-induced phosphorylation of c-Src, but ERK phosphorylation as well, suggesting that c-Src may be an upstream regulator of E2 signaling. E2-induced phosphorylation of c-Src is associated with increased tyrosine phosphorylation of Shc, increased association of Shc with Grb2, and induction of Ras, but not Rap1, activation. Together, these data provide evidence that E2 activates a novel c-Src-dependent signal transduction pathway in the developing brain.