Epiregulin (EPR) and amphiregulin (AR) inhibited cell death in Neuro2a cells.

<p>The cells were pretreated with EPR (0.01, 0.03, 0.1, and 1 nM) and AR (0.001, 0.01, 0.03, 0.1, and 1 nM) for 1 h and subsequently treated according to the indicated conditions. (A) EPR reduced the release of lactate dehydrogenase (LDH) at 48 h after Tm (1 μg/mL) treatment. (B) AR reduced the release of LDH at 48 h after Tm (1 μg/mL) treatment. The LDH released into the medium was expressed as a percentage of the control value. The data represent the average of 4 independent experiments for each sample. *p < 0.05.</p

Stress-Induced Neuroprotective Effects of Epiregulin and Amphiregulin

<div><p>Members of the epidermal growth factor family play important roles in the regulation of cell growth, proliferation, and survival. However, the specific roles of each epidermal growth factor family member with respect to brain injury are not well understood. Gene chip assay screens have revealed drastic increases in the expression of the epidermal growth factor family members amphiregulin and epiregulin following lipopolysaccharide stimulation, which activates an immune response. Both immune activity and endoplasmic reticulum stress are activated during cerebral ischemia. We found that the expression levels of amphiregulin and epiregulin were significantly increased under conditions of cerebral ischemia. Because endoplasmic reticulum stress increased the expression of amphiregulin and epiregulin in glial cells, endoplasmic reticulum stress may be a key mediatory factor of pathophysiological activity. Recombinant epiregulin and amphiregulin proteins effectively inhibited endoplasmic reticulum stress and the subsequent induction of neuronal cell death. Therefore, the upregulation of the epidermal growth factor family members epiregulin and amphiregulin may play a critical role in preventing endoplasmic reticulum stress-induced cell death, thus providing a potential therapy for brain injury.</p></div

Epiregulin (EPR) and amphiregulin (AR) inhibited endoplasmic reticulum stress in Neuro2a cells.

<p>Neuro2a cells were pre-incubated with (A) EPR or (B) AR for 1 h and then treated with tunicamycin (Tm) for 24 h. GRP78 and CHOP expression was detected via western blotting. Protein quantification is expressed as the mean ± standard error of 3 independent experiments. *p < 0.05 compared with the Tm-treated group.</p

Epiregulin (EPR) and amphiregulin (AR) inhibited cell death in Neuro2a cells.

<p>The cells were pretreated with EPR (0.01, 0.03, 0.1, and 1 nM) and AR (0.001, 0.01, 0.03, 0.1, and 1 nM) for 1 h and subsequently treated according to the indicated conditions. (A) EPR reduced the release of lactate dehydrogenase (LDH) at 48 h after Tm (1 μg/mL) treatment. (B) AR reduced the release of LDH at 48 h after Tm (1 μg/mL) treatment. The LDH released into the medium was expressed as a percentage of the control value. The data represent the average of 4 independent experiments for each sample. *p < 0.05.</p

Fold changes in EGF family member expression following LPS stimulation.

<p>Fold changes in EGF family member expression following LPS stimulation.</p

Fold changes in pro-inflammatory cytokines expression following LPS stimulation.

<p>Fold changes in pro-inflammatory cytokines expression following LPS stimulation.</p

Induction of epiregulin (EPR) and amphiregulin (AR) mRNA expression in primary glial cells following lipopolysaccharide (LPS) treatment (time course and dose course).

<p>(A) Total RNA was isolated from cells exposed to LPS (1 μg/ml) for the indicated time periods and subjected to RT-PCR. (B) Total RNA was isolated from cells exposed to LPS (4 h) at the indicated concentrations and subjected to RT-PCR. Data are presented as the means ± standard errors from 3 separate experiments. *p < 0.05 compared with the control.</p

Induction of epiregulin (EPR) and amphiregulin (AR) mRNA expression in primary glial cells following Tm treatment.

<p>Total RNA was isolated from cells exposed to Tm (3 μg/ml) for the indicated periods and subjected to RT-PCR. Data are presented as the means ± standard errors from 3 separate experiments. *p < 0.05 compared with the control.</p

Induction of epiregulin (EPR) and amphiregulin (AR) mRNA expression in primary glial cells under hypoxic conditions.

<p>Total RNA was isolated from cells exposed to hypoxic conditions for the indicated time periods and subjected to RT-PCR. Data are presented as the means ± standard errors from 3 separate experiments. *p < 0.05 compared with the control.</p

The CK2 inhibitor did not affect the eIF2α-CHOP arm of the ER stress-induced UPR.

<p>(A) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 4 h. Western blotting was then performed. TBB did not affect ER stress-induced eIF2α phosphorylation. Densitometric analysis of the normalization data of the phospho-eIF2α and eIF2α intensities were done. n = 3 per group. (B) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 6 h. A RT-PCR analysis was then performed. n = 3/group TBB did not affect ER stress-induced CHOP mRNA expression. (C) Primary cultured glial cells were pre-treated with 4,5,6,7-tetrabromobenzotriazole (TBB: 5 µM) for 3 h and treated with tunicamycin (Tm: 0.01 µg/mL) or thapsigargin (Tg: 0.01 µM) for 18 h. Western blotting was then performed. n = 4/group TBB did not affect ER stress-induced CHOP protein production. Results are expressed as the means ± S.E.</p