Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells

Abstract

To reveal the effects of cadmium exposure on the endoplasmic reticulum (ER) stress response, we examined the expression and function of 78-kDa glucose-regulated protein (Grp78) , an ER-resident molecular chaperone, in LLC-PK1 cells. In cells treated with 10 microM cadmium chloride, Grp78 protein levels increased after 6 hr and remained elevated at 24 hr. When cells were incubated with 1-20 microM CdCl2 for 6 hr, Grp78 increased in a dose-dependent manner. In addition, Grp78 mRNA levels were elevated in response to CdCl2 exposure. After exposure to 10 microM CdCl2, the levels of activating transcription factor 4 (ATF4) were increased at 2 hr, with a further enhancement after that ; this accumulation followed the transient but marked phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2(alpha)) on serine 51. Although ATF4 mRNA levels increased mildly by CdCl2 exposure, treatment with actinomycin D did not suppress CdCl2-induced accumulation of ATF4 protein, suggesting the involvement of posttranscriptional and, in part, transcriptional mechanisms. Compared with other heavy-metal compounds such as manganese chloride, zinc chloride, mercuric chloride, and lead chloride, CdCl2 could increase the levels of Grp78, ATF4, and the phosphorylated form of eIF2(alpha) more markedly without definite cellular damage. The silencing of Grp78 expression using short-interference RNA enhanced CdCl2-induced cellular damage. These results show that cadmium induces the expression of Grp78 probably via phosphorylation of eIF2(alpha) and resultant translation of ATF4, and this ER stress response plays a role in protection against cadmium cytotoxicity in this renal epithelial cell.

Figure 1

Time course (A,C) and dose effects (B,D) of CdCl₂-induced accumulation of Grp78 protein in LLC-PK1 cells incubated with 10 μM CdCl₂ for 2–24 hr (A,C) or with 0, 1, 2, 5, 10, or 20 μM CdCl₂ for 6 hr (B,D). (A,B) Cell lysates subjected to Western immunoblotting using Grp78 and actin antibodies. (C,D) Densitometric analyses of (A and B, respectively). Results shown are from representative analyses. Control values (0 hr or 0 μM) were set to 1; values are mean ± SD of four experiments. *p < 0.05 compared with control.

Figure 2

Induction of Grp78 gene expression in LLC-PK1 cells incubated with 10 μM CdCl₂ for 2–24 hr; total RNA was isolated and subjected to RT-PCR analysis using Grp78 and β-actin primers. (A) Agarose gel stained with ethidium bromide. (B) Grp78 gene expression shown by densitometric analysis of the gel in (A). The control value (0 hr) was set to 1. Values are mean ± SD of three experiments. *p < 0.05 compared with control.

Figure 3

Accumulation of ATF4 and phospho-eIF2α by CdCl₂ in LLC-PK1 cells incubated with 10 μM CdCl₂ for 1–12 hr; cell lysates were subjected to Western immunoblotting (A) using ATF4, phospho-eIF2α, total eIF2α, and actin antibodies. Densitometric analysis of the Western blot in (A) showing ATF4 (B) and phospho-eIF2α (C). Results shown are representative analyses. The control value (0 hr) was set to 1. Values are mean ± SD of three experiments. *p < 0.01 compared with control.

Figure 4

Posttranscriptional regulation of ATF4 expression. (A) LLC-PK1 cells were incubated with 10 μM CdCl₂ for 2–12 hr, and total RNA was isolated and subjected to RT-PCR analysis using ATF4 and β-actin primers. (B) Densitometric analysis of (A). The control value (0 hr) was set to 1; values are mean ± SD of three experiments. (C,D) LLC-PK1 cells were incubated for 6 hr with (C) 10 μM CdCl₂, 1 μg/mL actinomycin D (AcD), or 1 μg/mL AcD plus 10 μM CdCl₂, or (D) 10 μM CdCl₂, 10 μg/mL cycloheximide (CHX), or 10 μg/mL CHX plus 10μM CdCl₂. The untreated cells (control) were incubated with serum-free medium containing DMSO at the concentration used in the treated cells (0.05 or 0.1%). Cell lysates were subjected to Western immunoblotting using ATF4, Grp78, and actin antibodies. Results shown are representative of three experiments. *p < 0.01 compared with control.

Figure 5

Expression of Grp78, ATF4, and phospho-eIF2α proteins in LLC-PK1 cells incubated with 10 μM of each heavy-metal compound for 6 hr. (A) Representative immunoblot obtained with Grp78, ATF4, phospho-eIF2α, total eIF2α, and actin antibodies. Lane 1, control; lane 2, MnCl₂; lane 3, ZnCl₂; lane 4, CdCl₂; lane 5, HgCl₂; lane 6, PbCl₂. (B–D) Densitometric analysis showing (B) Grp78, (C) ATF4, and (D) phospho-eIF2α. The control value (without metals) was set to 1; values are mean + SD of three (for ATF4 and phospho-eIF2α) or four (for Grp78) experiments. *p < 0.05 compared with control.

Figure 6

siRNA-mediated knockdown of Grp78. (A) Western immunoblotting of cell lysates from LLC-PK1 cells transfected without (lanes 1–3) or with (lanes 4–6) siRNA against the Grp78 gene and incubated with 10μM CdCl₂ (lanes 2 and 5) or 1 μM thapsigargin (Tg) (lanes 3 and 6) for 12 hr. Results shown are representative immunoblots obtained with Grp78, HSP70, Grp94, and actin antibodies. (B) Densitometric analysis of Grp78 protein from the immunoblot shown in (A). The control value (untreated cells without siRNA transfection) was set to 1; values are mean + SD of three experiments. *p < 0.001 compared with corresponding treatment without siRNA transfection.

Figure 7

Effects of Grp78 knockdown on the cytotoxicity of CdCl₂. LDH leakage was determined in LLC-PK1 cells transfected without or with siRNA against the Grp78 gene and incubated with 10 μM CdCl₂ or 1 μM thapsigargin (Tg) for 12 hr. Values are mean + SD of four determinations; results shown are representative of three experiments. *p < 0.001 compared with corresponding treatment without siRNA transfection.