Inhibition of x-box binding protein 1 reduces tunicamycin-induced apoptosis in aged murine macrophages

Abstract

Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging-associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5-2 months) and aged (16-18 months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol-requiring enzyme-1 (p-IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x-box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p-IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age-dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging-associated ER stress-induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging-associated diseases in general.

Keywords: aging; apoptosis; endoplasmic reticulum stress; macrophages.

Figure 1. Aged macrophages are more susceptible to TM-induced apoptosis than young macrophages

Aged (16–18 months) and young (1.5–2 months) peritoneal macrophages were cultured with different concentrations of TM for 4 h, then in TM-free medium for another 16 h. Apoptosis was measured by Annexin V positive staining by florescent microscopy. Apoptotic cells, Annexin V positive staining (green); nuclei, DAPI staining (blue). Representative images are shown in (A) and quantification of apoptotic cells is shown in (B). Total and cleaved caspase 3 was measured by Western blot, and representative images are shown in (C). Experiments were repeated 3 times. For each experiment, 3 mice / group were used as a source of cells. * p <0.05, ** p <0.01 (Student’s t-test). Error bars = standard error of mean (SEM). TM0 = 0μg/ml TM; TM2.5 = 2.5μg/ml TM; TM5 = 5μg/ml TM; TM10 = 10μg/ml TM10. Y: young; A: aged.

Figure 2. Aged macrophages exhibit lower p-IRE1α and higher BiP protein levels than young macrophages after tunicamycin treatment

Aged (16–18 months) and young (1.5–2 months) macrophages were treated with 5μg/ml TM for 4 h, then switched to TM-free medium. Cell lysates were obtained 16 h later. IRE1α, p-IRE1α, BiP and CHOP levels along with that of the loading control (β-actin) were determined by Western blot. Representative blots are shown in (A) and quantification based on densitometry relative to loading control is shown in (B). Experiments were repeated 3 times. For each experiment, 4 mice/group were used as a source of cells. Each lane in the Western blot represents a biological replicate. * p <0.05 (Student’s t-test). Error bars = SEM. TM0 = 0μg/ml TM; TM5 = 5μg/ml TM. Y; young; A; aged.

Figure 3. Knocking down XBP1 mRNA enhances p-IRE1α protein levels in young macrophages

Young macrophages were treated with either scrambled control (denoted as si-ct or si-control) or small interference RNA to XBP1 (denoted as si-XBP1) for 48 h, and then treated with 5μg/ml TM for 4 h followed by TM-free medium for another 16 h. Total RNA and cell lysates were collected for further analysis. TM0 = 0μg/ml TM; TM5 = 5μg/ml TM. Y; young; A; aged. (A) Gene expression of XBP1 and its direct downstream effectors, ERdj4 and p58, were measured by real time RT-PCR. Error bars = SEM (B) As per A but XBP1 protein levels were determined by Western blot analysis. (C–D) As per A but p-IREα and XBP1 protein levels were determined by Western blot analysis. Quantification of densitometry relative to loading control is shown in (D). * p <0.05 (Student’s t-test). Error bars = SEM For A–D, 3 mice/group/experiment were used as a source of cells. Experiments were repeated independently 3 times.

Figure 4. XBP1 gene silencing reduces apoptosis in aged but not in young macrophages during treatment with TM

Peritoneal macrophages were obtained from aged (16–18 months) and young (1.5–2 months) mice. Cells were treated with either scrambled control (denoted as si-control) or small interference RNA to XBP1 (denoted as si-XBP1) for 48 h, and then treated with 5μg/ml TM for 4 h followed by TM-free medium for another 16 h. The degree of apoptosis was assessed by Annexin V staining (green), and nuclei are shown by DAPI staining (blue) through florescence microscopy. Representative images are shown in (A) and quantification of apoptotic cells is shown in (B). Experiments were repeated 3 times. For each experiment, 3 mice / group were used as a source of cells. * p <0.05 (Bonferroni’s post-hoc test). Error bars = SEM. TM0 = 0μg/ml TM; TM5 = 5μg/ml TM. Y; young; A; aged. (C–G) Cell lysates from (A–B) above were obtained and p-IRE1α protein levels in young and aged macrophages were determined by Western blot analysis. Representative blots (C) and quantification based on densitometry relative to loading control (D) are shown. Fold-increases of p-IRE1α in si-XBP1 group compared to si-control groups are shown in (E). Experiments were repeated 3 times. (F) Peritoneal macrophages were obtained from aged and young mice. Cells were treated with either si-control or si-XBP1 for 48 h, and then treated with 5μg/ml TM for 4 h followed by TM-free medium for another 44 h For each experiment, 3 mice / group were used as a source of cells. * p <0.05, ***p <0.001 (Student’s t-test). Error bars = standard error of mean (SEM)

Figure 5. Reduction of apoptosis by si-XBP1 in aged macrophages undergoing ER stress is IRE1α dependent

Peritoneal macrophages were obtained from aged (16–18 months) and young (1.5–2 months) mice. Cells were treated with either: siRNA to XBP1 (si-XBP1); siRNA to IRE1α (siIRE1α); si-XBP1 plus si-IRE1α; or scrambled control sequences for 48 h, and then treated with 5μg/ml TM for 4 h followed by TM-free medium for another 16 h. The degree of apoptosis was assessed by Annexin V staining (green), and nuclei are shown by DAPI staining (blue) through florescence microscopy. Representative images are shown in (A) and quantification of apoptotic cells is shown in (B). Experiments were repeated 3 times. For each experiment, 3 mice/group were used as a source of cells. * p <0.05 (Student’s t test). Error bars = SEM. TM0 = 0μg/ml TM; TM5 = 5μg/ml TM. Y; young; A; aged.