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.

Persistent orofacial muscle pain

“…When NGF was systemically administered to healthy human subjects muscle pain particularly in the craniofacial region was observed but was more pronounced in females than in males (Petty et al , 1994). Interactions between NGF and oestrogen have been shown (Gollapudi and Oblinger, 1999) but the mechanisms involved in POMP are unclear.…”

Mechanisms of gender-linked ischemic brain injury

“…Other proposed mechanisms of protection by E2 include the suppression of excitotoxicity (Singer et al, 1996; Smith et al, 1987; Weaver et al, 1997), apoptosis (Alkayed et al, 2000; Alkayed et al, 2001), β amyloid toxicity (Goodman et al, 1996; Shi et al, 1998), edema formation (Roof et al, 1993; Roof et al, 1994), antioxidant production (Ayres et al, 1998; Bruce-Keller et al, 2000; Culmsee et al, 1999; Sawada et al, 1998) and regulation of growth factors (Gollapudi & Oblinger, 1999; Toran-Allerand, 1996). Finally, E2 is anti-inflammatory (Hunt et al, 1997; Ray et al, 1997; Salem et al, 2000; St Clair, 1997; Vegeto et al, 2001) in cerebral ischemia, reducing the number of active microglia (Lei et al, 2003), decreasing intravascular leukocyte adhesion and migration into brain (Santizo et al, 2000), and suppressing endothelial expression of adhesion molecules (Mori et al, 2004; Nathan et al, 1999).…”

“…This common pathway may explain the ability of E2 and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation. The interaction of E2 with growth factors may also prevent cell death by preventing initiation of apoptosis (Gollapudi & Oblinger, 1999). However, as mentioned above, E2 can also prevent neuronal cell death by directly regulating expression of effectors of cell death, such as members of the bcl-2 family of cell death-associated proteins (Alkayed et al, 2001b).…”

Estrogen‐mediated neuroprotection against β‐amyloid toxicity requires expression of estrogen receptor α or β and activation of the MAPK pathway

“…Studies in various systems have shown that estrogen protects neurons from insults including β‐amyloid peptide (Green et al . 1996), serum‐deprivation (Gollapudi and Oblinger 1999a), excitotoxicity (Singer et al . 1998), and oxidative stress (Moosmann and Behl 1999).…”