Estrogen and NGF synergistically protect terminally differentiated, ERα‐transfected PC12 cells from apoptosis

Abstract

The potential cytoprotective effects of the gonadal steroid estrogen, acting via its receptor (ER) on target neurons, are of considerable interest in aging, disease, and trauma. In this study, we examined the effects of estrogen on a prototypical neuronal-like cell, namely, nerve growth factor (NGF) differentiated PC12 cells when these cells are placed into an apoptosis-inducing environment. A clonal line of PC12 cells stably transfected to express full-length rat ERalpha mRNA and protein (PCER cells), as well as control cells (vector DNA transfected: PCCON cells), were differentiated into neurite bearing cells by 14 days of NGF treatment. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry for ERalpha revealed that terminally differentiated PCER cells continue to show robust ER mRNA and protein expression after differentiation. PCCON cells do not express ERalpha either before or after NGF-induced differentiation. After differentiation, serum and NGF withdrawal from wild type PC12 cells is well known to induce cell death via apoptotic mechanisms. We challenged differentiated PCER and PCCON cells with serum-free media and assessed the effects of various treatments on cell survival and apoptosis using trypan blue staining and quantitative TUNEL staining. When differentiated PCER cells were treated with 17beta-estradiol (E2) plus NGF during a 2-day serum-free media challenge, cell survival was significantly greater, and the apoptotic index was significantly lower, than when PCER cells were treated with NGF only during the challenge. No additional improvement in cell survival, and no further reduction in apoptosis, was observed in non-ER expressing cells (PCCON) by E2 plus NGF treatment over that obtained by NGF alone. The effects of E2 on the expression of several apoptosis-related genes in this system were also examined. We found that E2 treatment of differentiated PCER cells in serum-free media increased the levels of Bcl-XL mRNA and reduced the levels of BAD mRNA relative to those in vehicle-treated PCER cells, and also relative to those in PCCON cells. Thus, estrogen's protective effects on PCER cells appear to, at least partly, involve a modulation of apoptosis-regulating genes.

Pathophysiology of Cerebral Ischemia and Brain Trauma: Similarities and Differences

“…These include cerebral blood flow (Alkayed et al, 1998a;Hurn et al, 1995;Roof and Hall, 2001), leukocyte adhesion (Santizo et al, 2000), edema formation (Roof et al, 1992;Roof et al, 1993;Roof et al, 1994), antioxidant production (Ayres et al, 1998;BruceKeller et al, 2000;Culmsee et al, 1999;Sawada et al, 1998), inflammation Ray et al, 1997;Salem et al, 2000;St Clair, 1997;Vegeto et al, 2001), excitotoxicity (Singer et al, 1996;Smith et al, 1987;Weaver et al, 1997), apoptosis (Alkayed et al, 1998b;Alkayed et al, 2001), ␤ amyloid (Goodman et al, 1996;Shi et al, 1998), and growth factor regulation (Gollapudi and Oblinger, 1999;Toran-Allerand, 1996). Hormones have the ability to target multiple pathways that are involved in injury.…”

Estrogen Regulates Bcl-w and Bim Expression: Role in Protection against β-Amyloid Peptide-Induced Neuronal Death

“…One putative mechanism of estrogen neuroprotection is regulation of the Bcl-2 family, pivotal regulators of apoptosis that include both proteins that promote cell survival (e.g., Bcl-2, Bcl-x L , and Bcl-w) and others that antagonize it (e.g., Bax, Bak, Bad, Bik, BNIP3, Bid, and Bim) (Antonsson and Martinou, 2000). Upregulation of antiapoptotic proteins, such as Bcl-2 (Garcia-Segura et al, 1998;Singer et al, 1998;Dubal et al, 1999;Nilsen and Diaz Brinton, 2003) and Bcl-x L (Patrone et al, 1999;Pike, 1999;Koski et al, 2004), or downregulation of proapoptotic Bad (Gollapudi and Oblinger, 1999) may contribute to the protective effects of estrogen. However, the relationship between estrogen and neuronal expression of Bcl-2 family members remains incompletely defined.…”

Nerve Growth Factor Withdrawal-mediated Apoptosis in Naïve and Differentiated PC12 Cells through p53/Caspase-3-dependent and -independent Pathways

“…Thus trophic factor withdrawal allows a 2-fold approach toward studying different aspects of apopto-sis: (a) serum withdrawal in naïve PC12 cells and (b) NGF withdrawal in terminally differentiated, neuronal-like PC12 cells. Trophic factor withdrawal-induced apoptosis is a relevant approach toward programmed cell death, because it mimics what happens, for example, in the dysfunctional nervous system as trophic factor production is compromised, contributing to neurodegenerative disorders such as Alzheimer's and Parkinson's disease (31)(32)(33)(34)(35)(36)(37).…”