Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

Abstract

We examined the roles of Notch1 signaling and its cross-talk with other signaling pathways, including p53 and phosphatidylinositol-3-kinase (PI3K)/Akt, in cadmium-induced cellular damage in HK-2 human renal proximal tubular epithelial cells. Following exposure to cadmium chloride (CdCl2), the level of Notch intracellular domain (NICD), the cleaved form of the Notch1 receptor, was increased and accumulated in the nuclear fraction. Knockdown of Notch1 with siRNA or treatment with the γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester), prevented CdCl2-induced morphological change of HK-2 cells and reduction of cell viability. Knockdown of Jagged1 or Jagged2, the ligands of the Notch1 receptor, partially suppressed cadmium cytotoxicity. Inhibition of p53 activity with pifithrin-α or inhibition of PI3K with LY294002 suppressed CdCl2-induced cellular damage and elevation of Notch1-NICD. In addition, treatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, and the insulin-like growth factor-1 receptor inhibitor, PPP, suppressed both Notch1-NICD accumulation and Akt phosphorylation in HK-2 cells exposed to CdCl2. However, knockdown of Notch1 did not affect CdCl2-induced p53 accumulation and phosphorylation but suppressed phosphorylation of EGFR, Akt, and p70 S6 kinase. Depletion of Notch1 suppressed CdCl2-induced reduction of E-cadherin expression and elevation of Snail expression. Furthermore, treatment with SB216763, an inhibitor of glycogen synthase kinase-3, suppressed the potency of LY294002 treatment to reduce Snail expression in HK-2 cells exposed to CdCl2. Knockdown of Snail with siRNA partially prevented HK-2 cells from CdCl2-induced reduction of E-cadherin expression and cellular damage. These results suggest that cadmium exposure induces the activation of Notch1 signaling in renal proximal tubular cells with cooperative activation by the p53 and PI3K/Akt signaling pathways; the resultant expression of Snail, a repressor of E-cadherin expression, might lead to cellular damage by decreasing cell-cell adhesion.

Figure 1

Involvement of Notch1 signaling in CdCl₂-induced cellular damage in HK-2 cells. (a and b) Cells were incubated with 20 μM CdCl₂ (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Nohch1-NTM, and actin (a). Equal amounts of protein (20 μg) in nuclear and cytoplasmic extracts were subjected to western blotting using antibodies against Notch1-NICD, MEK1/2, lamin A/C, and actin. MEK1 and lamin A/C served as a loading control for cytoplasmic and nuclear extracts, respectively (b). (c–e) Cells transfected with control siRNA, Notch1 siRNA-1, or Notch1 siRNA-2 were incubated with or without 20 μM CdCl₂ (Cd) for 12 h (c), 24 h (d), or 30 h (e). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin (c). Phase-contrast micrographs were taken (d). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. *P<0.05, **P<0.01 versus CdCl₂-treated cells transfected with control siRNA (e). (f–h) Cells were incubated with 0.1% DMSO or 40 μM DAPT for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 12 h (f) or 30 h (g and h). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin (f). Phase-contrast micrographs were taken (g). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. **P<0.01 versus CdCl₂-treated cells incubated with DMSO (h). Immunoblots shown are representative of at least three independent experiments

Figure 2

Jagged1 and Jagged2 are involved in CdCl₂-induced cellular damage in HK-2 cells. (a) Cells were incubated with or without 20 μM CdCl₂ (Cd) for the indicated time. Cell lysates were subjected to western blotting using antibodies against Jagged1, Jagged2, and actin. (b–d) Cells transfected with control siRNA, Jagged1 siRNA, or Jagged2 siRNA were incubated with or without 20 μM CdCl₂ (Cd) for 12 h (b), 12 h (c), or 30 h (d). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Jagged1 (left), Jagged2 (right), and actin (b). Phase-contrast micrographs were taken (c). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. *P<0.05 versus CdCl₂-treated cells transfected with control siRNA (d). Immunoblots shown are representative of at least three independent experiments

Figure 3

Modulation of Notch1 signaling by p53 in HK-2 cells exposed to CdCl₂. (a and b) Cells were incubated with 20 μM CdCl₂ (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against p53, phopho-p53, and actin (a). Equal amounts of protein (20 μg) in nuclear and cytoplasmic extracts were subjected to western blotting using antibodies against p53, MEK1/2, lamin A/C, and actin. MEK1 and lamin A/C served as a loading control for cytoplasmic and nuclear extracts, respectively. Data are from the same experiment shown in Figure 1b (b). (c–e) Cells were incubated with 0.1% DMSO or 40 μM pifithrin-α for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 24 h (c), 30 h (d), or 12 h (e). Phase-contrast micrographs were taken (c). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. **P<0.01 versus CdCl₂-treated cells incubated with DMSO (d). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin (e). (f) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against p53, phospho-p53, Notch1-NICD, and actin. Immunoblots shown are representative of at least three independent experiments

Figure 4

Cross-talk between the Notch1 and PI3K/Akt signaling pathways in HK-2 cells exposed to CdCl₂. (a) Cells were incubated with 20 μM CdCl₂ (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against phospho-EGFR, total EGFR, phospho-Akt, total Akt, phospho-GSK-3α, phospho-GSK-3β, total GSK-3α/β, phospho-S6K, and actin. (b–e) Cells were incubated with 0.1% DMSO, 25 μM LY294002 (b and d), or 10 μM MK2206 (c and e) for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 24 h (b and c) or 30 h (d and e). Phase-contrast micrographs were taken (b and c). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. **P<0.01 versus CdCl₂-treated cells incubated with DMSO (d and e). (f) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against phospho-EGFR, total EGFR, phospho-Akt, total Akt, phospho-S6K, Notch1-NICD, and actin. Data are from the same experiment shown in Figure 3f. (g) Cells were incubated with 0.1% DMSO, 2.5 μM AG1478, 5 μM PPP, or 25 μM LY294002 for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, phospho-Akt, total Akt, and actin. Immunoblots shown are representative of at least three independent experiments

Figure 5

The Notch1 and PI3K/Akt signaling pathways are involved in the regulation of Snail and E-cadherin expression in HK-2 cells exposed to CdCl₂. (a) Cells were incubated with 20 μM CdCl₂ (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, and actin. Data are from the same experiment shown in Figure 4a. (b) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, Notch1-NICD, and actin. Data are from the same experiment shown in Figure 3f. (c) Cells were incubated with 0.1% DMSO or 25 μM LY294002 for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, phospho-Akt, total Akt, and actin. (d) Cells were incubated with 0.2% DMSO, 20 μM SB216763 (SB), 25 μM LY294002 (LY), or 20 μM SB216763 and 25 μM LY294002 (SB+LY) for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against Snail, Notch1-NICD, phospho-Akt, total Akt, and actin. Immunoblots shown are representative of at least three independent experiments

Figure 6

Effects of Snail knockdown on CdCl₂-induced cellular damage in HK-2 cells. Cells transfected with control siRNA, SNAI1 siRNA-1, or SNAI1 siRNA-2 were incubated with or without 20 μM CdCl₂ (Cd) for 12 h (a and b) or 30 h (c). Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, and actin. Immunoblots shown are representative of at least three independent experiments (a). Phase-contrast micrographs were taken (b). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. **P<0.01 versus CdCl₂-treated cells transfected with control siRNA (c)