Beclin-1-mediated Autophagy Protects Against Cadmium-activated Apoptosis via the Fas/FasL Pathway in Primary Rat Proximal Tubular Cell Culture

Abstract

The Fas/FasL signaling pathway is one of the primary apoptosis pathways, but the involvement and regulatory mechanism of this pathway by autophagy remain unclear. Here we demonstrated that cadmium (Cd) activated the Fas/FasL apoptosis pathway in rat proximal tubular (rPT) cells; this was accompanied by simultaneous activation of autophagy resulted in reduced apoptosis. In this model, we induced autophagy through RAPA and further demonstrated that autophagy protects against activation of Fas/FasL signaling and apoptosis. The antiapoptotic effect of autophagy was blocked by 3-MA, an autophagy inhibitor. The interactions between Beclin-1 and Fas, FasL, FADD, caspase-8 and BID/tBID were relatively weak, with the exception of cleaved caspase-8, indicated that minimal interactions between these proteins and Beclin-1 are involved in maintaining the balance of autophagy and apoptosis. Beclin-1 precipitated with cleaved caspase-8 in a dose-dependent mannter, and the expression was increased by siRNA against Beclin-1. These data suggested that Beclin-1-mediated autophagy impairs the expression and function of cleaved caspase-8 to protect against Cd-induced activation of apopotosis through Fas/FasL signaling pathway.

Figure 1

Effects of Cd on Fas/FasL signaling apoptosis pathway activation in rPT cells, measured by flow cytometry and western blot. (A) Cells were incubated with 0, 2.5 and 5 μM Cd for 12 h, and apoptosis markers were assessed using flow cytometry. Data are mean ± SEM of three separate experiments and each one performed in triplicate (n = 9). (B) Fas, FasL, FADD, and cleaved caspase-8 protein levels were assessed in rPT cells by western blot analysis after 12 h Cd treatment. The quantitative analysis was performed on the western blot results of four independent experiments (mean ± SEM, n = 4), respectively. ^* P < 0.05, ^** P < 0.01, ^## P < 0.01, ^@ P < 0.05, ^@@ P < 0.01 and ^&& P < 0.01.

Figure 2

Effects of Cd on apoptosis of rPT cells. (A) Cells were incubated with 0, 2.5, and 5 μM Cd for 6 h and nuclear chromatin changes (apoptosis) were analyzed by confocal microscopy after DAPI staining. Changes of nuclei fragmentation with condensed chromatin are indicated by white arrows. The quantified results are expressed as mean ± SEM of three separate experiments, each performed in triplicate (n = 9). (B) rPT cells were treated with Cd for 6 h and apoptosus was assessed by flow cytometry. Data are presented as mean ± SEM of three separate experiments, each one performed in triplicate (n = 9). *P < 0.05 and **P < 0.01.

Figure 3

Effects of Cd treatment on activation of autophagy in rPT cells. (A) rPT cells were incubated with 0, 2.5, and 5 μM Cd for 6 h and autophagic vacuoles were analyzed by confocal microscopy after MDC staining. Fluorescence particles in the cytoplasm indicate autophagic vacuoles. The quantified analyses as assessed by MDC staining are expressed as mean ± SEM of three separate experiments; each experiment was performed in triplicate (n = 9). (B) rPT Cells were treated with Cd for 6 h and autophagy was assessed using flow cytometry. Data are mean ± SEM of three separate experiments, each experiment performed in triplicate (n = 9). (C) LC3II and Beclin-1 protein levels were assessed by western blot analysis after Cd treatment for 6 h. The quantitative analysis was performed on western blot images of four independent experiments (mean ± SEM, n = 4). ^** P < 0.01 and ^## P < 0.01.

Figure 4

Effects of RAPA and 3-MA on Cd-induced apoptosis and activation of the Fas/FasL signaling pathway. (A) rPT cells were incubated with 2.5 μM Cd and/or 5 μM RAPA for 6 h, and apoptosis was assessed by flow cytometry. Data are presented as mean ± SEM of three separate experiments, each one performed in triplicate (n = 9). Fas, FasL, FADD and cleaved caspase-8 protein levels were assessed by western blot analysis after 6 h treatment with Cd and/or RAPA (B) and Cd and/or 3-MA (C). The quantitative analysis was performed on western blot images of four independent experiments (mean ± SEM, n = 4). ^* P < 0.05, ^** P < 0.01, ^# P < 0.05, ^## P < 0.01, ^@ P < 0.05, ^@@ P < 0.01, ^& P < 0.05 and ^&& P < 0.01.

Figure 5

Interaction between Beclin-1 and cleaved caspase-8 after Cd treatment. (A) rPT cells were incubated with 0, 2.5, and 5 μM Cd for 6 h and the interaction between Beclin-1 and cleaved caspase-8 was analyzed by co-immunoprecipitation. The quantitative analysis was performed on western blot images of four independent experiments (mean ± SEM, n = 4). (B) Representative confocal images of co-localization of Beclin-1 and cleaved caspase-8; yellow fluorescence represent co-localization and the quantitative analysis performed with images of three independent experiments (mean ± SEM, n = 3). ^** P < 0.01.

Figure 6

Effect of Beclin-1 knockdown on cleaved caspase-8 expression after Cd treatment. Cleaved caspase-8 and Beclin-1 protein levels were assessed by western blot analysis after 2.5 μM Cd and/or 100 nM siRNA Beclin-1 treatment 6 h. The quantitative analysis performed on western blot images from four independent experiments (mean ± SEM, n = 4). ^* P < 0.05, ^** P < 0.01, ^# P < 0.05 and ^## P < 0.01.