Nupr1/Chop signal axis is involved in mitochondrion-related endothelial cell apoptosis induced by methamphetamine

Abstract

Methamphetamine (METH) abuse has been a serious global public health problem for decades. Previous studies have shown that METH causes detrimental effects on the nervous and cardiovascular systems. METH-induced cardiovascular toxicity has been, in part, attributed to its destructive effect on vascular endothelial cells. However, the underlying mechanism of METH-caused endothelium disruption has not been investigated systematically. In this study, we identified a novel pathway involved in endothelial cell apoptosis induced by METH. We demonstrated that exposure to METH caused mitochondrial apoptosis in human umbilical vein endothelial cells and rat cardiac microvascular endothelial cells in vitro as well as in rat cardiac endothelial cells in vivo. We found that METH mediated endothelial cell apoptosis through Nupr1-Chop/P53-PUMA/Beclin1 signaling pathway. Specifically, METH exposure increased the expression of Nupr1, Chop, P53 and PUMA. Elevated p53 expression raised up PUMA expression, which initiated mitochondrial apoptosis by downregulating antiapoptotic Bcl-2, followed by upregulation of proapoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. Interestingly, increased Beclin1, upregulated by Chop, formed a ternary complex with Bcl-2, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax was also significantly decreased, which led to translocation of cyto c and initiated more drastic apoptosis. These findings were supported by data showing METH-induced apoptosis was significantly inhibited by silencing Nupr1, Chop or P53, or by PUMA or Beclin1 knockdown. Based on the present data, a novel mechanistic model of METH-induced endothelial cell toxicity is proposed. Collectively, these results highlight that the Nupr1-Chop/P53-PUMA/Beclin1 pathway is essential for mitochondrion-related METH-induced endothelial cell apoptosis and may be a potential therapeutic target for METH-caused cardiovascular toxicity. Future studies using knockout animal models are warranted to substantiate the present findings.

Figure 1

Nupr1 expression is upregulated in endothelial cells after METH exposure in vitro and in vivo. (a) HUVECs were exposed to 1.25 mM METH for indicated times (0, 12, 24, 36 and 48 h). (c) CMECs were exposed to METH (0.5 mM) for 24 h. The protein expression of Nupr1, cleaved-PARP and cleaved-caspase-3 was determined with western blot (a and c) and quantitative analyses (b and d). Fold induction relative to cells treated with vehicle is shown. β-Actin was used as a loading control. *P<0.05 versus vehicle-treated cells. Data were analyzed with one-way ANOVA followed by LSD post hoc comparisons. Data represent mean±S.D. (n=3 replicates). (e and f) Male SD rats (n=5/group) were injected i.p. with saline or METH (15 mg/kg × 8 injections, at 12 h interval). The heart tissues were harvested at 24 h after the last dosing. Immunolabeling and confocal imaging analysis (e) showed elevated Nupr1 expression in the heart microvascular endothelial cells of METH-exposed rats compared with controls (Ctrl). TUNEL staining and confocal imaging analysis (f) was used to evaluate the endothelial cell apoptosis. Apoptotic cells were stained with TUNEL (Red). Nuclei were counterstained with DAPI (blue) and BF represents bright field

Figure 2

Nupr1 is necessary for METH-induced apoptosis in HUVECs and CMECs. (a) Synthetic Nupr1 siRNAs effectively suppressed endogenous Nupr1 expression on HUVECs. HUVECs were transfected with siRNAs targeting Nupr1 or control siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. (c) Lentivrus-mediated Nupr1 shRNAs effectively suppressed endogenous Nupr1 expression in CMECs. CMECs were incubated with viral supernatants for 24 h followed by METH (0.5 mM) treatment for another 24 h. Western blot (a and c) and quantitative analyses were performed to evaluate the efficiency of Nupr1 knockdown (b and d), and the expression of apoptosis-related proteins (cleaved-PARP (Cl-PARP) and cleaved-caspase-3 (Cl-casp3)) after knocking down Nupr1 expression in HUVECs (b) and CMECs (d). Effects of suppressing Nupr1 expression on the apoptosis caused by 1.25 mM METH in HUVECs and by 0.5 mM METH in CMECs were assessed with TUNEL (e: green for HUVECs; g: red for CMECs) and flow cytometry ((i) HUVECs and (k) CMECs). (f and h) Quantitative analysis of the percentage of apoptotic cells using a standard cell counting method with the TUNEL assay. Apoptotic cells were stained with TUNEL (green for HUVECs (f) and red for CMECs (h)). Nuclei were counterstained with DAPI (blue). (j and l) Quantitative analysis of the effects of knocking down Nupr1 on apoptosis induced by METH using flow cytometry. Representative calculations from three independent replicates are shown. The percentage of apoptotic cells is presented as mean± S.D. (n=3, *P<0.01 versus the saline vehicle-treated control group; **P<0.01 versus the scrambled+METH group; one-way ANOVA)

Figure 3

Nupr1 mediates METH-induced apoptosis through the classical mitochondrial apoptotic signaling pathways. (a and e) HUVECs were transfected with siRNAs targeting Nupr1 or control siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. (i and m) CMECs were incubated with LV-shRNA or LV-GFP control viral supernatants for 24 h followed by METH (0.5 mM) treatment for 24 h. The protein levels of Bax (b and j), Bcl-2 (c and k), cytosolic cyto c (cyto cyt-c; f and n), and mitochondrial cyto c (mit cyt-c; g and o) were measured using western blot analyses. The Bax/Bcl-2 (d and l) and cyto cyt-c/mit cyt-c (h and p) ratios were calculated. Data are presented as mean ±S.D. (n=3). *P<0.01 versus the saline vehicle-treated ctrl group, **P<0.01 versus the scrambled+METH group (one-way ANOVA)

Figure 4

Chop is involved in Nupr1-mediated apoptosis in METH-exposed endothelial cells. (a) HUVECs were exposed to 1.25 mM METH for indicated time (0, 12, 24, 36 and 48 h). (c) CMECs were exposed to METH (0.5 mM) for 24 h. (e) HUVECs were transfected with siRNAs targeting Nupr1 or ctrl siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. (g) CMECs were incubated with LV-shNupr1 or LV-GFP control viral supernatants for 24 h followed by METH (0.5 mM) treatment for 24 h. (i) HUVECs were transfected with siRNAs targeting Chop or ctrl siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. Western blot (a, c, e, g, i) and quantitative analyses (b, d, f, h and j) were performed to evaluate the expression of Chop, Nupr1, cleaved-PARP (Cl-PARP) and cleaved-caspase-3 (Cl-casp3). Effects of suppressing Chop expression on 1.25 mM METH-treated HUVECs were assessed by flow cytometry (k and l) and TUNEL (m and n). Apoptotic cells were stained with TUNEL (green). Nuclei were counterstained with DAPI (blue). (l) Quantitative analysis of the effects of Chop knockdown on apoptosis induced by METH using flow cytometry. (n) Quantitative analysis of the percentage of apoptotic cells using a standard cell counting method with the TUNEL assay. Representative calculations from three independent experiments are shown. The rate of apoptosis is presented as mean±S.D. (n=3, *P<0.01 versus the saline vehicle-treated ctrl group, **P<0.01 versus the scrambled+METH group, one-way ANOVA)

Figure 5

P53 is involved in Nupr1–Chop axis-mediated mitochondrial apoptotic signaling pathways caused by METH in endothelial cells. (a) HUVECs were exposed to METH (1.25 mM) for 24 h. (c, e and g) HUVECs were transfected with siRNAs targeting Nupr1 (c), Chop (e), P53 (g) or ctrl siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. Western blot (a, c, e, g) and quantitative analyses (b, d, f and h–j) were performed to evaluate the expression of P53, Beclin1, Bax, Bcl-2, Chop, Nupr1, cleaved-PARP (Cl-PARP) and cleaved-caspase-3 (Cl-casp3). The protein levels of cytosolic cyto c and mitochondrial cyto c (l–n) were measured using western blot analyses. The Bax/Bcl-2 (k) and cyto cyt-c/mit cyt-c (o) ratios were calculated. Data are presented as mean ±S.D. (n=3). *P<0.01 versus the saline vehicle-treated ctrl group, **P<0.01 versus the scrambled+METH group (one-way ANOVA)

Figure 6

PUMA is the initiator in the Nupr1/Chop axis-activated classical mitochondria apoptotic signaling pathways. HUVECs were transfected with siRNAs targeting Nupr1 (a), Chop (c), PUMA (e) or ctrl siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. Western blot (a, c, e, j) and quantitative analyses (b, d, f, g–i, k–m) were performed to evaluate the expression of PUMA, Bax, Bcl-2, cleaved-PARP (Cl-PARP), cleaved-caspase-3 (Cl-casp3), the ratio of Bax/Bcl-2, cytosolic cyto c (cyt cyt-c), mitochondrial cyto c (Mit cyt-c) and the Cyt cyt-c/Mit cyt-c ratio. (n) Mitochondrial membrane potential (MMP) was analyzed with fluorescent probe JC-1 assay. (o) Quantitative results of JC-1 assay. Data are presented as mean±S.D. (n=3). *P<0.05 versus the saline vehicle-treated control group and **P<0.05 versus the scrambled+METH group (one-way ANOVA)

Figure 7

Beclin1 promotes apoptosis through the Nupr1/Chop axis-activated classical mitochondria apoptotic signaling pathways. HUVECs were transfected with siRNAs targeting Nupr1 (a), Chop (c), Beclin1 (e), or control siRNA for 48 h followed by METH (1.25 mM) treatment for 24 h. Western blot (a, c, e and j) and quantitative analyses (b, d, f and g–i, k–m) were performed to evaluate the expression of Beclin1, cleaved-PARP (Cl-PARP), cleaved-caspase-3 (Cl-casp3), Bax, Bcl-2, Bax/Bcl-2 ratio, cytosolic cyto c (Cyt cyt-c), mitochondrial cyto c (Mit cyt-c) and Cyt cyt-c/Mit cyt-c ratio. (n) Mitochondrial membrane potential (MMP) was analyzed with fluorescent probe JC-1 assay. (o) Quantitative results of JC-1 assay. Data are presented as mean±S.D. (n=3). *P<0.05 versus the saline vehicle-treated ctrl group and **P<0.05 versus the scrambled+METH group (one-way ANOVA)

Figure 8

Beclin1 promotes apoptosis by physically interacting with Bcl-2. Effects of suppressing Beclin1 expression on the apoptosis caused by 1.25 mM METH in HUVECs were assessed with flow cytometry (a) and TUNEL (c: green for HUVECs). (b) Quantitative analysis of the effects of knocking down Beclin1 on apoptosis induced by METH using flow cytometry. (d) Quantitative analysis of the percentage of apoptotic cells using a standard cell counting method with the TUNEL assay. Nuclei were counterstained with DAPI (blue). (e) Endogenous Bcl-2 co-immunoprecipitated with Beclin1. Cell lysates from METH-treated HUVECs were immunoprecipitated with Beclin1 antibody or normal IgG and blotted with Bcl-2 antibody. (f) Endogenous Beclin1 co-immunoprecipitated with Bcl-2. Cell lysates from METH-treated CMECs were immunoprecipitated with Bcl-2 antibody or normal IgG and blotted with Beclin1 antibody. Representative calculations from three independent replicates are shown. The percentage of apoptotic cells is presented as mean±S.D. (n=3, *P<0.01 versus the saline vehicle-treated control group; **P<0.01 versus the scrambled+METH group; one-way ANOVA)

Figure 9

A schematic depicting the role of Nupr1–Chop/P53–PUMA/Beclin1 signaling pathway in METH-induced endothelial cell apoptosis. Briefly, Nupr1 expression is increased following METH treatment. Increased Nupr1 upregulates the expression of Chop. As a transcription factor, Chop upregulates the expression of P53, and P53 raises up PUMA expression, which mediates the mitochondrial apoptosis pathway by changing the ratio of Bax/Bcl-2. Meanwhile, Chop also increases the expression of Beclin1, which forms th eBcl-2/Beclin1 complex, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax is also significantly decreased. The increased ratio of Bax/Bcl-2 results in translocation of cyto c, an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent apoptotic pathways