Estrogen receptor agonists and estrogen attenuate TNF-α-induced apoptosis in VSC4.1 motoneurons

Abstract

Tumor necrosis factor-alpha (TNF-α) may cause apoptosis and inflammation in amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI). Recent studies suggest that estrogen (EST) provides neuroprotection against SCI. We tested whether 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (EST receptor alpha (ERα) agonist), 2,3-bis (4-hydroxyphenyl) propionitrile (DPN) (EST receptor beta (ERβ) agonist), or EST itself would prevent apoptosis in VSC4.1 motoneurons following exposure to TNF-α. Cells were exposed to TNF-α and 15 min later treated with PPT, DPN, or EST. Posttreatment with 50 nM PPT, 50 nM DPN, or 150 nM EST prevented cell death in VSC4.1 motoneurons. Treatment of VSC4.1 motoneurons with PPT, DPN, or EST induced overexpression of ERα, ERβ, or both, which contributed to neuroprotection by upregulating expression of anti-apoptotic proteins (p-AKT, p-CREB, Bcl-2, and p-Src). Our analyses also revealed that EST agonists and EST increased phosphorylation of extracellular signal-regulated kinase (ERK). The L-type Ca(2+) channel inhibitor, nifedipine (10 μM), partially inhibited EST agonist and EST-induced increase in phosphorylated ERK expression. The mitogen-activated protein kinase inhibitor, PD98059 (5 μM), partially prevented ER agonists and EST from providing neuroprotection to TNF-α toxicity. Presence of the nuclear ER antagonist, ICI 182 780 (10 μM), blocked the neuroprotection provided by all three ER agonists tested. Taken together, our data indicate that both ERα and ERβ contribute to PPT, DPN, or EST-mediated neuroprotection with similar signaling profiles. Our data strongly imply that PPT, DPN, or EST can be used as effective neuroprotective agents to attenuate motoneuron death in ALS and SCI.

Figure 1

Posttreatment with PPT, DPN, or EST prevented TNF-α-induced apoptosis in VSC4.1 motoneurons. Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). (A) Wright staining showing representative cells from each treatment group. Arrows indicate apoptotic cells. (B) ApopTag assay showing representative cells from each treatment group. (C) Bar graphs indicating the percentage of apoptotic cells counted from each group (based on Wright staining). **P<0·01 compared to control; ^#P<0·05 compared to TNF-α; ^##P<0·01 compared to TNF-α. Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0338.

Figure 2

RT-PCR and western blotting for expression of estrogen receptor beta (ERβ) and estrogen receptor alpha (ERα). Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). Representative pictures to show levels of ERβ, ERα, and GAPDH at mRNA (RT-PCR) (A) and protein levels (western blotting) (B). (C) Bar graphs indicating the changes in expression of ERβ and ERα over CON. *P<0·05 compared to control.

Figure 3

Determination of ERK phosphorylation, intracellular free [Ca²⁺], and involvement of L-type Ca²⁺ channels in VSC4.1 cells. Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). (A) Posttreatment with PPT, DPN, or EST rapidly increased ERK phosphorylation. Western blot analysis to show levels of phosphorylation of p44/42 ERK1/2 at 0·5 and 24 h. (B) Determination of intracellular free [Ca²⁺] at 0·5 and 24 h. Western blot analysis to show levels of phosphorylation of p44/42 ERK1/2 after posttreatment with nifedipine (C) and PD98059 (D). (E) Treatment with nifedipine and PD98059 increased cell viability. Trypan blue dye exclusion assay was used to assess cell viability. **P<0·01 compared to control.

Figure 4

Determination of survival proteins. Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). (A) Western blotting to show levels of p-AKT, p-CREB, p-Bad, Bcl-2, p-Src, and β-actin. (B) Densitometric analysis showing percent change in OD of the p-AKT, p-CREB, p-Bad, Bcl-2, and p-Src bands. *P<0·05 compared to control.

Figure 5

Examination of components involved in extrinsic and intrinsic apoptotic pathways. Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). (A) Western blotting to show levels of caspase-8, β-actin, tBid, and COX4. (B) Densitometric analysis showing percent change in OD of the caspase-8 and tBid bands and determination of caspase-8 activity (colorimetrically). (C) Alteration in Bax and Bcl-2 expression at mRNA and protein levels. Representative pictures to show Bax, Bcl-2, and GAPDH at mRNA (RT-PCR) and protein (western blotting) levels after different treatments. (D) Densitometric analysis showing the Bax:Bcl-2 ratio. (E) Western blotting to show levels of cytochrome c, COX4, caspase-9, and β-actin. (F) Densitometric analysis showing percent change in OD of the mitochondrial and cytosolic 15 kDa cytochrome c and 39 kDa active caspase-9, and determination of caspase-9 activity (colorimetrically). **P<0·01 compared to control; ^##P<0·01 compared to TNF-α.

Figure 6

Examination of downstream events of apoptosis in VSC4.1 cells. Treatment groups: control (CON); 50 nM PPT (24 h); 50 nM DPN (24 h); 150 nM EST (24 h); 10 μM ICI (24 h); 50 ng/ml TNF-α (24 h); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+PPT (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+DPN (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure); 50 ng/ml TNF-α (24 h)+EST (treatment at 15 min post TNF-α exposure)+ICI (treatment at 20 min post TNF-α exposure). (A) Western blotting to show levels of m-calpain, calpastatin, active caspase-3, ICAD (cytosolic), CAD (nuclear), and β-actin. (B) Densitometric analysis showing the m-calpain:calpastatin ratio and the ICAD (cytosolic):CAD (nuclear) ratio. (C) Determination of caspase-3 activation by western blotting and total caspase-3 activity by colorimetric assay. **P<0·01 compared to control; ^##P<0·01 compared to TNF-α.