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Endocrinology, doi:10.1210/en.2003-0803
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Endocrinology Vol. 145, No. 1 95-103
Copyright © 2004 by The Endocrine Society

Regulation of Cytokine-Induced Neuron Death by Ovarian Hormones: Involvement of Antiapoptotic Protein Expression and c-JUN N-Terminal Kinase-Mediated Proapoptotic Signaling

Carol Lee Koski, Sorana Hila and Gloria E. Hoffman

Departments of Neurology, and Anatomy and Neurobiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Carol Lee Koski, M.D., N4W46, Neurology, UMMS, 22 South Greene Street, Baltimore, Maryland 21201. E-mail: ckoski{at}umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mechanisms underlying the divergent effects of ovarian hormones on neuron death induced by TNF{alpha} were investigated in differentiated PC12 cells (dPC12). dPC12 cells were exposed to 17ß-estradiol (E, 1.0 nM), progesterone (P, 100 nM), or a combination of both hormones for 0–72 h before treatment with TNF{alpha} (0–150 ng) to induce cell death. Cells undergoing apoptosis were identified by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay and fluorescence-activated cell sorting after 18 h. Cell death induced by TNF{alpha} was decreased 89% after E treatment and increased 2-fold after P treatment compared with cells treated with TNF{alpha} alone. Treatment with E for 24 h before TNF{alpha} exposure was required for maximum neuroprotection, whereas P-enhanced death was maximal after a 30-min P treatment. TNF{alpha} induced a 3-fold increased activity of c-JUN-N-terminal kinase (JNK) 1 in d PC12 cells within 20 min that could be increased 5- to 8-fold by P together with TNF{alpha}. A peptide inhibitor of JNK1 abrogated P enhancement of TNF{alpha}-mediated dPC12 death but had only a minimal effect on cell death by TNF{alpha} alone. Inhibition of caspase-8 activation reduced death induced by TNF{alpha} alone but was much less effective for P+TNF. P alone did not activate caspase-8. E increased estrogen receptor {alpha} (ER{alpha}) and Bcl-xL expression and all but abolished TNF{alpha} receptor 1 (TNFR1) expression. P decreased ER{alpha} and Bcl-xL expression and doubled TNFR1 expression. These data suggest that P regulates apoptosis or survival through augmentation of JNK signaling and altered TNFR1 expression, whereas E mainly affects the expression of BCL-xL, TNFR1, and ER{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGRAMMED CELL DEATH or apoptosis of neurons is a normal physiologic process that regulates cell numbers during development but is also a consequence of disease. Exposure of neurons to stressful stimuli including growth factor withdrawal, ionizing radiation, hypoxia, lipid second messengers such as ceramide, or activation of death domain receptors by exogenous inflammatory mediators including Fas-ligand and TNF{alpha} result in enzymatic and genomic consequences leading to apoptosis. Altered levels of TNF{alpha} reported in seizures and other excitotoxic insults (1, 2, 3, 4, 5, 6, 7), ischemia (8, 9, 10, 11, 12, 13, 14), traumatic injury (11, 15), tissue responses to transplantation (16), hemorrhage (17), infection (18, 19, 20, 21, 22), and inflammation (23) led to the hypothesis that TNF{alpha} contributes to neuronal death in multiple diseases such as Parkinson’s disease, Alzheimer’s disease, cerebral palsy, and multiple sclerosis (24, 25, 26, 27, 28).

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) with exacerbations of inflammation that are initially intermittent. Although demyelination is the primary CNS damage in MS, axonal injury associated with inflammation also determines the severity of disease and the level of disability (29, 30, 31). Neuronal loss subsequent to axonal injury is proposed in MS (32), and a significant increase in apoptotic neurons was recently demonstrated in demyelinated cortex, compared with myelinated cortex of MS brains (33). This is further corroborated by the ability of spinal fluid of MS patients to damage axons and induce apoptosis in vitro (34, 35).

In MS and its experimental model, experimental autoimmune encephalomyelitis (EAE), a role for hormones is suggested by gender susceptibility (36, 37), disease amelioration during pregnancy when ovarian hormones are elevated, and disease exacerbation post partum when hormones drop precipitously (36, 37, 38, 39, 40). Estrogen treatment of EAE animals affects the immune response by decreasing interferon {gamma} and TNF{alpha}, reducing the mononuclear infiltration in the CNS, and limiting the development of clinical signs (41, 42, 43, 44, 45, 46). Earlier studies in our laboratories examining neuronal cell death in EAE revealed mild neuronal losses in the superficial dorsal horn of the spinal cord in ovariectomized rats, which was greatly attenuated by estrogen replacement and greatly exacerbated by replacement with progesterone (P) (47). This finding challenged the prevailing concepts that physiologic doses of P beneficially supplement the effect of 17ß-estradiol (E) on immune induction (48, 49, 50, 51, 52, 53) and that large nonphysiologic doses are needed for steroids to confer neuroprotection after CNS injury (54).

Ovarian hormones, estrogen, and P regulate the functions of cells specific to reproductive system as well as other cells derived from a diverse origins. Estrogen and P affect target cells by inducing gene expression through binding to nuclear receptors that can act as transcription factors, acting at the cell membrane to alter signal transduction pathways, and serving as antioxidants (55, 56, 57, 58, 59). Each of these processes is implicated in steroid-induced neuroprotection, yet study of cytokine-induced cell death is largely unexplored. In the current study, we explored the possible mechanisms by which estrogen reduced and P increased neuronal cell death in an in vitro model of cytokine-mediated injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PC12 preparation and culture
PC12 cells (5 x 105, American Type Tissue Collection, Manassas, VA) were plated onto rat tail collagen type I (Sigma-Aldrich Co., St. Louis, MO)-coated dishes in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated horse serum (HS) (Life Technologies, Inc.), 5% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Inc.), and 1% of an antibiotic-antimycotic (penicillin-streptomycin-amphotericin) solution containing 10,000 U penicillin, 10 mg streptomycin, and 25 µg amphotericin per milliliter (Sigma-Aldrich), 5% CO2. Cells were incubated for 24 h at 37 C before treatment with nerve growth factor (NGF) 2.5S prepared from mouse submaxillary glands (Roche Molecular Biochemicals, Indianapolis, IN) at a final concentration of 50 ng/ml for 8–11 d in RPMI 1640 medium supplemented with 2% HS and 1% FBS. Cells became adherent and developed extensive neurite outgrowth (Fig. 1Go). All sera used throughout these studies were charcoal stripped to remove endogenous steroids.



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  		FIG. 1. NGF-differentiated PC12 cells. PC12 cells were plated onto rat tail collagen-coated dishes and treated with NGF, 50 ng/ml, for 8–11 d in RPMI 1640 medium with charcoal-treated 2% HS and 1% FBS. NGF induced phenotypic changes characterized by adherence and neurite outgrowth.

 
Hormones and cytokines
After differentiation, cultures were treated for 0–72 h with one of the following: E (10-9 M) (Sigma-Aldrich), P (10-7 M) (Sigma-Aldrich), E together with P, or medium alone. In all time course experiments, steroid-containing medium was removed before the addition of fresh medium with TNF{alpha} (Calbiochem, San Diego, CA). NGF and serum factors were maintained throughout all cell manipulations. Apoptosis after 18 h incubation was assessed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. For experiments measuring activation products of signal transduction, P was added together with TNF{alpha}.

TUNEL staining and quantification
Adherent PC12 cells (106) were detached from culture dishes with 0.02% EDTA and washed twice in PBS before being fixed 1 h in 4% paraformaldehyde, 4 C, and permeabilized with 0.1% Triton X-100 in 1% sodium citrate for 5 min, 4 C. Apoptosis was determined by detecting DNA strand breaks with a TUNEL assay, using the in situ death detection kit (Roche Molecular Biochemicals). Ninety-seven percent of cells initially plated were recovered for analysis. The percentage of TUNEL-positive cells was determined with fluorescence-activated cell sorter (FACS) analysis, using the Consort-40 software (Becton Dickinson, Lincoln Park, NJ). Cell populations were gated to a negative control in which neuronal cells were treated with labeling solution alone containing fluorescein-labeled deoxyuridine 5-triphosphate but no terminal deoxynucleotidyl transferase.

c-JUN N-terminal kinase 1 (JNK1) activity assay
PC12 cells were washed twice in ice-cold PBS and harvested in a stress-activated protein kinase lysis buffer containing 20 mM HEPES-KOH (pH 7.4), 2 mM EDTA, 50 mM ß-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol (DTT), 1 mM sodium orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml aprotinin, and 0.5 µg/ml leupeptin. One hundred micrograms of protein of each lysate was immunoprecipitated overnight at 4 C with 10 µl each of anti-JNK1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A/G-agarose. After washing with lysis buffer, JNK1 activity was determined by incubation for 0–40 min at 30 C with 1 µg GST-c-jun (1–169) (Upstate Biotechnology, Lake Placid, NY) in a reaction buffer [20 mM 3[N-morholino]propanesulfonic acid (pH 7.2), 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM DTT, 1 mM sodium orthovanadate] containing [{gamma}-32P]ATP, 1 µCi per sample. Reactions were terminated by the addition of 65 µl of Laemmli sample buffer, and samples were analyzed on 12% SDS-PAGE, transferred to nitrocellulose, and labeled c-Jun bands detected with autoradiography. Autoradiograms were scanned and the band density determined with Scion Image (Scion Corp., Frederick, MD) analysis system.

Caspase-8 activation
Caspase-8 cleavage was studied in dPC12 cells lysed in SDS sample buffer: 62.5 mM Tris-HCl (pH 6.8 at 25 C), 2% (wt/vol) SDS, 10% glycerol, 50 mM DTT, and 0.01% (wt/vol) bromophenol blue, sonicated for 5–10 sec, heated 5 min at 95–100 C, and cooled on ice. Equal quantities of protein were separated on 12% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with rabbit polyclonal anticaspase-8 (FLICE) Ab-4 (Lab Vision Corp., Fremont, CA), which detects the full-length protein and the processed forms (42/44, 25, and 14 molecular weight) of caspase-8. Bands were detected with an enhanced chemiluminescence system (Amersham, Piscataway, NJ).

Pharmacological inhibition of signal transduction
Where appropriate, cultures of PC12 cells were preincubated for 30 min with inhibitors including the L-stereoisomer of a peptide inhibitor of JNK (L-JNKI1, 20 µM, Allexis Biochemicals, San Diego, CA) that competitively blocks JNK and c-JUN interaction and/or 50 µM caspase-8 inhibitor II (z-IEDT-FMK) (Calbiochem) before stimulation with hormones and/or TNF{alpha}. To rule out the possible effects of P on phosphatidyl inositol-3 kinase (PI3 kinase) and mitogen-activated protein kinase kinase (MEK) signaling pathways, which modulate cell apoptosis, similarly prepared cultures were preincubated with LY294002 (10 µM, a PI3 kinase inhibitor, Biomol Research Laboratories, Plymouth Meeting, PA) or PD 098,059 (25 µM, a MEK inhibitor, Upstate Biotechnology). The concentrations used were established with dose-response curves to minimize nonspecific toxicity.

FACS analysis of estrogen receptor (ER){alpha}, TNF receptor, and BCL-xL expression
dPC 12 cells (5 x 105) were suspended, fixed, and permeabilized as described earlier and incubated with a 1:5000 dilution of a rabbit anti-ER{alpha} (Upstate Biotechnology) or a 1:1000 dilution of rabbit anti-BCL-xL polyclonal IgG (Santa Cruz Biotechnology) for 30 min at 4 C. Binding was detected with a fluorescein isothiocyanate-conjugated goat antirabbit IgG (Sigma 1:10,000 dilution) and FACS analysis using Cell Quest software. TNF receptor (TNFR) 1 expression was assayed on unfixed cells with an optimal dilution of a goat polyclonal TNFR1 antibody (Santa Cruz Biotechnology) and binding was detected with a fluorescein isothiocyanate-conjugated mouse antigoat IgG (Sigma). Cells were fixed in 4% paraformaldehyde before FACS analysis. All samples were gated based on light scatter distribution to exclude dead and fragmented cells. The specific binding is expressed as specific mean fluorescence intensity defined as [mean fluorescent intensity of specific polyclonal IgG] - [mean fluorescent intensity of a nonspecific polyclonal IgG].

Western blot analysis
dPC12 cells (106) were washed twice with PBS (pH 7.4) and lysed in a buffer containing 0.15 M NaCl, 30 mM Tris-HCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 2 mM MgCl2, 1 mM sodium orthovanadate, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 0.5 mM phenylmethyl-sulfonylfluoride. The lysates were sonicated four times for 10 sec on ice, centrifuged 15 min at 12,000 x g, and the protein concentrations determined using the BCA protein assay (Pierce, Rockford, IL).

ER{alpha}.
Twenty micrograms of protein from cell extracts were loaded in each lane of a 10% SDS-PAGE gel. Membranes were blocked with 5% nonfat dry milk in 10 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20 for 24 h at 4 C before incubation for 1 h with 1:3000 rabbit anti-ER{alpha} (Upstate Biotechnology, antibody no. C1355). Antirabbit IgG secondary antibody, horseradish peroxidase conjugated 1:20,000 (Upstate Biotechnology) was used as the secondary antibody. The blots were developed with an enhanced chemiluminescence system (Amersham).

BCL-xL.
One hundred fifty micrograms of protein were immunoprecipitated overnight at 4 C with a rabbit antirat BCL-xL polyclonal antibody (Santa Cruz Biotechnology) in the presence of protein A/G agarose beads. The beads were washed twice with lysis buffer and the samples eluted by boiling in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% wt/vol SDS; 10% glycerol; 50 mM DTT; 0.1% wt/vol bromophenol blue). Lysates were separated on reducing 12% SDS-PAGE gels and blotted onto nitrocellulose. Membranes were blocked as above and BCL-xL detected with 1:1000 rabbit antirat BCL-xL, and 1:10,000 horseradish peroxidase-conjugated goat antirabbit antibody (Santa Cruz Biotechnology). Binding was determined with chemiluminescence. Antibody specificity was confirmed with a blocking peptide for BCL-xL (Santa Cruz Biotechnology). In each case, the density of protein bands was quantified using Scion Image analysis system.

Statistical analysis
Each experiment was repeated at least three times, and triplicate samples were counted for each condition. Statistical analysis was done using ANOVA followed by a two-tailed t test, with P < 0.01 considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovarian hormones affect cytokine-mediated cell death
dPC12 cell death induced by TNF{alpha}.
Treatment of PC12 cells with NFG for 8–11 d resulted in cell adherence and extensive neurite outgrowth (Fig. 1Go). Exposure to TNF{alpha} caused rounding up, detachment, and subsequent death of cells, as determined by a TUNEL assay (Fig. 2Go). Treatment with TNF{alpha}, 18 h, induced dPC12 cell death in a dose-dependent manner. TUNEL-positive cells reached 30 ± 4.25%, 37 ± 5.62%, and 65 ± 10.3%, with 50, 100, and 150 ng/ml TNF{alpha}, respectively (Fig. 2Go). Background cell death without TNF{alpha} was 8.1 ± 1.3%. With 50 ng/ml TNF{alpha}, TUNEL-positive cells increased to 54 ± 5.0% at 24 h from 30 ± 4.25% at 18 h (data not shown). In all subsequent experiments to study the effects of hormones, cells were exposed to TNF{alpha} for 18 h.



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  		FIG. 2. Ovarian hormones affected PC12 cytokine-mediated death. Treatment of differentiated PC12 cells with TNF{alpha} for 18 h induced a dose-dependent apoptosis as determined with a TUNEL assay and FACS analysis ({diamondsuit}). Pretreatment of cells with estradiol, 1 nM ({square}), for 72 h decreased cell death, whereas P, 100 nM ({blacktriangleup}), increased specific cell death 2-fold. Results are the mean of three experiments ± SE of the mean.

 
Consequences of hormone treatment on cell survival.
dPC12 cells were pretreated with 10-9 M E for 72 h and then incubated for 18 h with TNF{alpha} (1–150 ng/ml) (Fig. 2Go). E dramatically reduced TNF{alpha}-induced cell death, by 89% for 50 ng/ml TNF{alpha} (from 22.4 ± 4.25% to 2 ± 0.71%, P < 0.001), 69% for 100 ng/ml TNF{alpha} (from 29 ± 5.62% to 9 ± 4.1%, P < 0.001), and 32% for 150 ng/ml TNF{alpha} (from 57 ± 10.2% to 39 ± 5.35% TUNEL-positive cells, P < 0.01). In sharp contrast, pretreatment of dPC12 cells for 72 h with 10-7 M P significantly enhanced TNF{alpha}-induced cell death. The increase was almost 2-fold with 50 ng/ml TNF{alpha} (from 22.4 ± 4.25% to 42 ± 6.25%, P < 0.001) and 1.7-fold with 100 ng/ml TNF{alpha} (from 29 ± 5.62% to 49 ± 7.62%, P < 0.001). P had little effect on the cell death induced by 150 ng/ml TNF{alpha} or on the background cell death.

Time course of affect of ovarian hormones.
Both early membrane-associated signaling events and delayed cell responses requiring gene transcription and protein synthesis have been implicated in ovarian hormone-mediated target cell modulation. We have therefore investigated the time required for ovarian hormones to affect the cytokine-mediated PC12 cell death. dPC12 cells were stimulated with P (10-7 M) for from 5 min to 360 min (6 h); the steroid was then removed and fresh media applied before treating the cells with 50 ng/ml TNF{alpha} for 18 h (Fig. 3AGo). In the absence of TNF{alpha}, P failed to affect the proportion of TUNEL-positive cells. However, TNF{alpha}-induced apoptotic cell death was significantly increased by P, even with only a brief exposure. A 30-min exposure to P increased the TNF-induced cell death to 55.6 ± 8.14% from 38.33 ± 3.05%, which was not significantly increased further by a 6-h exposure (61.33 ± 7.5%). To determine the time required for E to mediate rescue, dPC12 cells were pretreated with E (10-9 M) from 3 to 72 h and then exposed to 50 ng/ml TNF{alpha} (Fig. 3BGo). A significant reduction of cell death was first detected after an 18-h preincubation of E (from 62.5 ± 4.08% to 40.3 ± 4.24%, P < 0.001). This protective effect was maximal after 24 h preincubation and resulted in survival of 90 ± 1.41% dPC12 cells (Fig. 3BGo). These data suggested that P facilitates the TNF{alpha}-induced apoptosis in dPC12 cells through early postbinding signaling mechanisms, and E protected these cells from TNF-induced death by different mechanisms that may involve gene activation and protein synthesis.



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  		FIG. 3. Time course of hormone effect on PC12 survival. A, Pretreatment of PC12 cells with P, 100 nM, for 30 min before exposure to TNF{alpha}, 50 ng/ml ({blacksquare}), significantly increased cell death, compared with that with TNF{alpha} (50 ng/ml) alone ({triangleup}). P alone was not cytotoxic ({square}). B, A 24-h preincubation with 1 nM E ({diamondsuit}) before the addition of TNF{alpha} (50 ng/ml) was required to achieve maximal neuroprotection and prevent significant cell death caused by TNF{alpha} alone ({blacksquare}). Death was determined with a TUNEL assay and FACS analysis. Data represent the mean of three independent experiments ± SEM.

 
P enhanced JNK activity in the presence of TNF
Activation of the JNK pathway is a common response to stress stimuli, including TNF{alpha}. Because L-JNKI 1, a specific inhibitor for JNK, abolished the apoptotic effects of TNF{alpha} in the presence of P, we determined specific JNK1 activity in dPC12. As shown in Fig. 4Go, stimulation with TNF{alpha} alone induced an early 3.4 ± 0.79-fold increase in JNK activity at 20 min, and this activity was sustained at 40 min (3.8 ± 0.66-old). The addition of P to TNF{alpha} induced much higher JNK1: 5.26 ± 0.81-fold increase at 20 min and 7.5 ± 0.9-fold increase at 40 min. P alone had only slightly higher JNK1 activity, 1.4 ± 0.37-fold at 20 min and 1.6 ± 0.32-fold at 40 min that was not associated with increased apoptosis.



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  		FIG. 4. JNK activity in stimulated PC12 cells. PC12 cells were incubated with P, TNF{alpha}, 50 ng/ml (TNF), P+TNF, or media alone (CTL) for up to 40 min. The 100 µg cell lysate protein, immunoprecipitated with anti-JNK1 antibody and protein A/G agarose beads, was assayed for its ability to phosphorylate a GST-c-JUN substrate. Reaction mixtures were separated on 12% SDS-PAGE and Western blot. Labeled c-JUN was detected by SDS-PAGE and autoradiography (A). The phosphorylated c-JUN bands were quantitated with Scion software (B). P in the presence of TNF{alpha} stimulated increased JNK activity within 40 min 2-fold over that seen with TNF{alpha} alone. Data represent the mean of three independent experiments ± SEM.

 
L-JNKI-1 effected P-mediated neuronal cell death
Apoptosis of dPC12 cells induced by 50 ng/ml TNF{alpha} with or without P (10-7 M) was evaluated using signaling pathway inhibitors: LY294002 (10 µM), PD098, 059 (25 µM), and L-JNKI1 (20 µM), respectively, inhibiting PI3 kinase, MEK1, and JNK1 (Fig. 5Go). Neither P nor any of the inhibitors at concentrations used induced additional apoptosis in the absence of TNF{alpha} over the background cell death. LY294002 and PD098,059 did not affect the TNF{alpha}-induced cell death, thus excluding the involvement of PI3 kinase- or MEK1-dependent pathway for TNF{alpha}. Inhibition of JNK activation by a peptide inhibitor, L-JNKI 1, showed a small but significant effect on reducing apoptosis induced by TNF{alpha} alone (29.3 ± 1.88% from 40.5 ± 2.6% cell death) (Fig. 5Go). In cells exposed to P and TNF{alpha}, inhibition of JNK1 activity produced a significant effect, decreasing apoptosis from 59.2 ± 7.2% to 28.3 ± 4.13% (P < 0.001) (Fig. 5Go). These data indicated that JNK1 activity plays a limited role in TNF{alpha}-induced apoptosis, which P is able to significantly increase.



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  		FIG. 5. L-JNKI-1 affected P-mediated cell death. Preincubation for 30 min with the specific peptide inhibitor of JNK (JNKI-1; black bars) abrogated the ability of a 30-min incubation with P to enhance the TNF{alpha}-mediated PC12 cell death (**). JNKI-1 also had a small but significant effect on cell death produced by TNF{alpha} in the absence of P (*). Other inhibitors, 25 µM PD 098,059 (PD) and 10 µM LY294002 (LY), had no significant effect on cell death with either TNF{alpha} or TNF{alpha} + P. Data represent the mean of three independent experiments ± SEM.

 
Caspase-8 activation in TNF{alpha}-induced apoptosis in dPC-12
Recruitment and activation of caspase-8 are involved in apoptosis induced by TNF receptor (TNFR){alpha}-mediated activation of TNFR1-associated death domain protein, and the Fas-associated death domain protein (FADD) (60, 61, 62, 63). To determine the involvement of caspase-8 in dPC12 cell death in our system, we used z-IEDT-FMK, a caspase-8 inhibitor II. Pretreatment of dPC12 with 50 µM z-IEDT-FMK reduced the level of apoptosis induced by 50 ng/ml TNF{alpha} to 24.0 ± 4.72% from 45 ± 3.60% (P < 0.001). z-IEDT-FMK reduced cell death from 62.6 ± 3.44% to 40.9 ± 5.4%, when P-treated cells were exposed to TNF{alpha} (Fig. 6Go). Pretreatment of dPC12 with inhibitors of JNK1 together with a caspase-8 inhibitor before P/TNF{alpha} treatment reduced cell death to the background level. JNKI by itself was able to only modestly reduce dPC12 cell death mediated by TNF{alpha} by 15%. These findings suggested that both caspase-8 and JNK are involved in TNF{alpha}-induced PC12 apoptosis and that progesterone may modulate this process through increased activation of JNK pathways.



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  		FIG. 6. Caspase-8 and JNK mediate cell death induced by TNF{alpha} and P. Pretreatment with the inhibitor of caspase 8, z-IEDT-FMK (gray bars), limited TNF{alpha} cell death by 50% but only limited that by P+TNF by 25%. Addition of the JNK inhibitor, JNKI-1 (black bars), reduced cell death to background levels (8–10%). Neither of the inhibitors in the absence of P or TNF caused significant cell death. Cell death was determined with a TUNEL assay and FACS analysis. Each bar represents the mean of three individual experiments ± SEM.

 
Activation of caspase-8 by TNF{alpha} was demonstrated by immunoblotting for cleaved caspase-8. Activation of caspase-8, a 53/51 molecular weight inactive proenzyme, involves cleavage to a 42/44 and 25 molecular weight fragments and further proteolysis to generate a 14 molecular weight peptide. The active caspase-8 subunit was detected 5 h after TNF{alpha} treatment with or without exposure of cells to P (Fig. 7Go). This TNF{alpha}-dependent cleavage of caspase-8 was completely inhibited by z-IEDT-FMK.



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  		FIG. 7. Caspase-8 activation. PC12 cells were treated with P, P + TNF{alpha}, 50 ng/ml (TNF), or TNF alone for 2.5 and 5 h. Proteins of individual cell lysates, separated on 12% SDS-PAGE and blotted onto nitrocellulose, were immunoblotted with polyclonal antibody to caspase-8. Activation was detected by the presence of cleavage fragments detected with chemiluminescence. The representative blot above showed increased expression of a 14-kDa fragment at 5 h after treatment with TNF and TNF+P but not with P alone.

 
Effect of hormones on ER{alpha}, BCl-xL, and TNFR 1
Because E protected dPC12 cells from TNF{alpha}-induced apoptosis (Fig. 2Go), we investigated whether E modulated TNFR1 and/or ER{alpha} expression. We also examined the regulation of anti-apoptotic protein Bcl-xL by E.

Treatment with E (10-9 M) for 72 h increased ER{alpha} expressing cells to 70.25 ± 3.18% from 29.5 ± 2.12% in dPC12 cells unstimulated with steroids (Fig. 8AGo). On the other hand, treatment with P (10-7 M) for 72 h dramatically reduced the number of ER{alpha}-positive cells to 11.5 ± 0.7%, whereas combined treatment of P and estrogen failed to affect the ER{alpha} expression. Western blot analysis of ER{alpha} protein in cell treatment with E, P, or both, as described in Materials and Methods, is shown in Fig. 8BGo. Densitometric band analysis confirmed the expression of ER{alpha} protein affected by E and P by FACS analysis (Fig. 8AGo).



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  		FIG. 8. Ovarian hormones induce ER{alpha}, TNFR1, and Bcl-xL. A, Estrogen (E) treatment for 72 h increased ER{alpha} expression on PC12 cells greater than 2-fold, whereas that with P was reduced to 29% of control values. Expression was detected with FACS analysis. ER{alpha} on cells treated with both E+P was similar to untreated cells. Results were confirmed on Western blot (B). E also increased expression of BCL-xL 3.5-fold, which was reduced to control levels on coincubation with P (B and D) as analyzed by Western blot and FACS. Cellular expression of TNFR1 was reduced by E and enhanced 2-fold by P (C). Each of the bars represents the mean of at least three individual experiments ± SEM.

 
Expression of TNFR1 by FACS analysis after treatment with ovarian hormones and then TNF{alpha} is shown in Fig. 8CGo. Pretreatment with P for 72 h increased the mean specific fluorescence of TNFR1 almost 2-fold (from 18 ± 2.85 in unstimulated cells to 33 ± 3.29). Costimulation with TNF{alpha} for 18 h further increased the value (47 ± 4.58). In contrast, E abrogated TNFR1 expression (2.7 ± 0.52), and this was not significantly increased by a further exposure to TNF{alpha} (6 ± 0.26).

To examine whether apoptosis is also modulated by E and P through regulating the antiapoptotic BCL-xL protein, we performed Western blotting and FACS analysis to measure the protein (Fig. 8Go, B and D). Unstimulated dPC12 cells expressed low levels of BCL-xL (specific mean fluorescence 15.1 ± 2.9), and this was further decreased to 7.7 ± 2.6 with exposure to P. Estrogen increased BCL-xL protein expression 3.55 ± 0.34-fold (P < 0.01), which was only minimally affected by TNF{alpha} (50 ng/ml) (3.05 ± 0.26-fold). We confirmed these results by densitometry of Western blots (Fig. 8BGo). Costimulation with E and P induced a moderate 1.9-fold increase of Bcl-xL expression. These data suggested that the neuroprotective effect of E reflects a delayed and complex mechanism reflecting up-regulation of ER{alpha} expression, down-regulation of TNFR 1, and an increase in Bcl-xL protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we explored the role of ovarian hormones, E and P, in neuron survival using an in vitro model of TNF{alpha}-mediated apoptotic death of differentiated PC12 cells. As previously described, PC12 cells differentiated in vitro in the presence of NGF showed extensive neurite outgrowth (64, 65) and became susceptible to TNF{alpha}-induced apoptotic death in a dose- and time-dependent manner. At physiologic concentrations of E and P, the dPC12 cell death induced by TNF{alpha} was markedly reduced by E but significantly increased by P. These findings are consistent with our observations made using the rat EAE model in which disease activity and neuronal death were reduced by treatment of the animals with physiological levels of E, but the disease was exacerbated when animals were administered P also at physiological levels (47). The use of dPC12 cells enabled characterization of the duration of steroid exposure needed to produce effects on cell death.

In the present study, significant protection of dPC12 cells by E was a delayed event, requiring 18–24 h incubation preceding TNF{alpha} treatment. E enhances neuron survival in a variety of models through specific receptor-mediated transcriptional regulation of genes encoding pro- and antiapoptotic proteins, cytokines including TNF{alpha}, and cytokine receptors (44, 45, 46, 66, 67, 68, 69, 70). After trophic factor withdrawal, E-mediated survival of dPC12 cells required ER{alpha} expression, and survival was blocked by both transcriptional inhibitors and specific ER antagonists (70). The increased dPC12 cell survival produced by E in our system is consistent with a transcriptional mechanism of a similar type because E increased the expression of ER{alpha} and the antiapoptotic BCL-xL protein, whereas it decreased TNFR1 expression. Neuroprotective changes dependent on ER{alpha} were also observed after E treatment in the penumbra of an acute rodent stroke model as well as in a study of cultured cortical neurons exposed to kainic acid or potassium cyanide/2-deoxyglucose to mimic ischemia after stroke (71, 72).

ER{alpha} and ERß are widely recognized for their activities as transcription factors. However, existence of membrane receptors for E is also suggested by experiments showing rapid activation of signaling, such as ERK1 and PI3 kinase pathways. This rapid activation of the ERK1 and PI3 kinase/Akt pathways has been linked to cell proliferation and survival (73, 74, 75). To date, however, no clearly identifiable estrogen membrane receptors have been cloned. Rescue of dPC12 cells from TNF{alpha}-induced apoptosis is less likely to be through estrogen membrane receptor activation because 24-h incubation with estrogen was required to increase significant cell survival. This same period of estrogen exposure was critically important for survival of cortical neurons to insult as seen in the studies by Wise and colleagues (71, 72). Our findings are thus more consistent with ER{alpha}-mediated transcription of genes encoding proteins involved in various aspects of antiapoptotic activity, although it is still possible that some signaling intermediate must accumulate over several hours to effect rescue through an ER{alpha}-independent mechanism.

Both E and P at high doses have antioxidant properties associated with neuroprotection (54, 66, 76). Physiologic doses of P similar to those used in this study were neuroprotective in dPC12 cells after NGF withdrawal and were mediated through a receptor-dependent mechanism (70). Antioxidant neuroprotective properties of P were also not observed in our study of cytokine-mediated injury. Rather, P further increased the TNF{alpha}-induced dPC12 cell death. Such proapoptotic activity accompanied by reduced BCL2/BCL-xL expression was reported in an in vitro model of breast tissue using micromolar levels of progestins (77, 78) but was previously unrecognized in neurons. Although we too noted a reduction in BCL-xL expression after P exposure, we also detected decreased ER{alpha} and TNFR1 expression. Three receptors for P (PRs) have been cloned. PR-A and PR-B are ligand-activated transcription factors of the steroid hormone family. Activity through a nuclear PR could explain the changes in TNFR1, BCLxl, and ER{alpha} expression associated with P. Recently a membrane receptor for P has been identified and is associated with rapid and nongenomic P effects (68, 79). The detrimental effect of P on neuronal survival induced in as little as 15–30 min could reflect modulation of a downsteam proapoptotic signaling cascade through such a membrane receptor.

TNF{alpha} activates downstream signaling molecules via its receptors, TNFR1 and TNFR2. Activation of the major receptor, TNFR1, leads to recruitment and activation of receptor-associated proteins TNFR1-associated death domain protein, FADD, and the TNFR-associated factor (TRAF) 2, TRAF2 (80). Activation of apoptotic pathway may be initiated by the binding and activation of caspase-8 to FADD. TNF{alpha}-induced apoptosis via TNFR1 also involves TRAF2-mediated activation of {rho} GTPase, MEKK1, and JNK1 pathway (75, 80, 81). This pathway is relevant to neuronal death because differentiated neurons may undergo apoptosis through the JNK pathway (32, 33, 75, 82), a pathway that can also be activated by growth factors involved in cell growth (75). These two upstream signaling pathways involving caspase-8 and JNK may be activated in PC12 cells exposed to TNF{alpha}, and they may act in synergy at the level of mitochondrial damage through caspase-8-mediated activation of BID and BAX (83, 84) and through JNK-mediated inactivation of BCL-2/BCL-xL. Activation of BID and BAX by proteolysis and phosphorylation may play a critical role in inducing mitochondrial membrane permeability by forming BAX dimers and oligomers (81). JNK can also phosphorylate BCL-2 to cause loss of antiapoptotic property by releasing proapoptotic BAX and BAK.

For TNF{alpha}-induced PC12 cell death, our data support a dependence on caspase-8 because the active cleavage fragment of caspase-8 was maximally generated by TNF{alpha} and inhibition of caspase-8 markedly reduced TNF{alpha}-induced specific cell death. Mielke and Herdegen (64) also studied the cascades associated with TNF{alpha}-mediated dPC12 cell death. They concluded that JNK activation cascade was required for induction of TNF{alpha}-mediated apoptosis but did not study caspase-8. Although our data also supported a role for JNK activation in TNF{alpha}-mediated dPC12 death, we observed an important if not dominant role for caspase-8. Similar concentrations of NGF, TNF, and differentiation protocols were used by both studies. The differences in our conclusions concerning the extent of the role that JNK activity plays in TNF{alpha}-mediated dPC12 death may reflect the maintenance of NGF in our studies throughout all manipulations including during cytokine stimulation and charcoal treatment of our medium.

With regard to P’s actions on PC12 cells exposed to TNF{alpha}, our data support effects on the TNF signaling cascade. First, P’s effects were very rapid, producing maximal increases in apoptosis after only 30 min of steroid exposure before TNF{alpha}. P by itself was unable to induce apoptosis or JNK activity in PC12 cells, but the steroid increased the level of JNK activated by TNF{alpha} more than 2-fold over that seen with TNF{alpha} alone. By 30 min the full P effect on dPC12 apoptosis mediated by TNF{alpha} was manifest and inhibition of JNK activation prevented the augmentation of apoptosis. The rapid P effect and inhibitor data tend to support a mechanism of nongenomic signal transduction, although genomic effects cannot be completely excluded because no protein or transcription inhibitors were used in these experiments. Further study is needed to assess whether the changes in JNK activity and the observed changes in gene expression are independent. It is important to note that the two mechanisms are not mutually exclusive. Rather, the rapid and delayed actions of P could act in concert.

Our data indicate that survival in this neuronal cell model system is greatly affected by both E and P and that although E promotes neuron survival, the unopposed action of P can enhance neuronal death. Although the impact of the two steroids on expression levels of receptors and pro/antiapoptotic molecules might conflict when both steroids are present, the ability of P to rapidly enhance the proapoptotic signaling cascade may not be easily opposed by E coadministration. This feature may have clinical significance and shed some light on recent trials in which the synthetic progestin, medroxyprogesterone acetate, in combination with conjugated estrogens did not limit the development of cognitive impairment in postmenopausal women but rather increased it (85, 86, 87). Our data showing the regulation of JNK, BCL-xL, TNFR1, and ER{alpha} by P and E clearly indicate that apoptosis or survival is regulated by these hormones in a pleiotropic and coordinate manner. Understanding the mechanisms by which ovarian hormones limit or contribute to CNS cytokine-mediated damage will enable the development of treatment paradigms to preserve or protect primary CNS cells during inflammatory disease.


   Footnotes
 
This work was supported by a grant from the Wadsworth Foundation (to C.L.K.).

Abbreviations: CNS, Central nervous system; DTT, dithiothreitol; EAE, experimental autoimmune encephalomyelitis; ER{alpha}, estrogen receptor {alpha}; FACS, fluorescence-activated cell sorter; FADD, Fas-associated death domain protein; FBS, fetal bovine serum; HS, horse serum; JNK, c-JUN-N-terminal kinase; L-JNKI1, L-stereoisomer of a peptide inhibitor of JNK; MEK, mitogen-activated protein kinase kinase; MS, multiple sclerosis; NGF, nerve growth factor; P, progesterone; PI3 kinase, phosphatidyl inositol-3 kinase; PR, P receptor; TNFR, TNF receptor; TRAF, TNFR-associated factor; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.

Received June 26, 2003.

Accepted for publication September 15, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
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 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals