Dual role of ERK2/NF-κB signaling in TRAIL sensitivity

Abstract

Targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling is a promising approach in cancer treatment. Although ERK and/or NF-κB signaling is involved in the expression of TRAIL receptors (TRAIL-R), the exact underlying mechanisms remain unknown. In this study, we evaluated the role of ERK2 and NF-κB in the cytotoxicity of TRAIL during cisplatin treatment. Cisplatin treatment of neuroepithelioma cells (SK-N-MC) significantly induced ERK2 activation and increased TRAIL cytotoxicity via the upregulation of death receptor 5 (DR5) expression. In partial ERK2 knockdown cell lines that maintained only basal levels of ERK2 activity, cisplatin treatment did not increase ERK2 activity or DR5 expression. These findings indicate that induced (rather than basal) ERK2 activity enhances TRAIL susceptibility via DR5 expression. In complete ERK2 knockdown cell lines with no basal ERK2 activity, DR4, DR5, and DcRs expression levels were increased, and additional treatment with cisplatin did not further increase TRAIL-R expression. Chemical inhibition of ERK2 also enhanced TRAIL cytotoxicity by upregulating DR4 and DR5 expression. These findings indicate that basal ERK2 activity suppresses TRAIL-R expression. Both basal and inducible ERK2 activities regulate TRAIL-R expression via the NF-κB signaling pathway. Overall, our findings suggest that the ERK2/NF-κB signaling pathway has a dual role in TRAIL susceptibility by differentially regulating TRAIL-R expression in the same cellular system.

Keywords: ERK2; NF-κB; TRAIL; cisplatin; death receptor.

Figure 1

Cisplatin treatment induces cell death via the induction of TRAIL-R2 expression in the neuroepithelioma cell line. A and B. SK-N-MC cells were seeded in 96-well plates and treated with cisplatin or TRAIL for the indicated times. Thereafter, cell viability was assessed using an AlamarBlue assay. C. Surface expression of DR4, DR5, DcR1, and DcR2 in cells treated with cisplatin was analyzed using flow cytometry with receptor-specific antibodies. D. The percentage of TRAIL-R positive cells at each exposure time was determined using flow cytometric analyses. E. Cell viability was assessed after treatment with cisplatin or TRAIL combined with the DR5 antibody (DR5: FC). F. Representative images of Hoechst 33258-positive cells after treatment with cisplatin, TRAIL, or DR5 antibody.

Figure 2

Cisplatin-induced elevation of TRAIL cytotoxicity is mediated by ERK activation. A. Phosphorylated ERK1/2 level was determined using western blotting after treatment with cisplatin or TRAIL. B. Phosphorylated ERK1/2 levels were determined using immunocytochemistry with phosphorylated ERK-specific antibody. C. Western blotting was performed after U0126 treatment to detect ERK1/2 phosphorylation. D. Surface expression of DR4, DR5, DcR1, and DcR2 in U0126 treated cells were analyzed using flow cytometry with receptor-specific antibodies. E. TRAIL-R positive cells at each exposure time were determined using flow cytometric analyses. F. Cell viability was assessed after treatment with cisplatin or TRAIL combined with U0126. G. Surface expression of DR4, DR5, DcR1, and DcR2 in cells treated with cisplatin was analyzed using flow cytometry with receptor-specific antibodies. H. Cell viability was assessed after treatment with U0126, cisplatin, or TRAIL combined with the DR5 antibody (DR5: FC). I. Cell viability assessment was carried out after treatment with U0126 and TRAIL.

Figure 3

Knockdown of ERK2 changed TRAIL-R expression levels. (A) Western blotting demonstrated ERK1 and ERK2 knockdown using their specific siRNA. (B) Flow cytometry revealed DR5 positivity after ERK1- or ERK2-specific siRNA treatment. (C) ERK2 knockdown cells were screened using western blotting with phosphorylated ERK antibody. (D) ERK2 knockdown was determined using immunocytochemistry with phosphorylated ERK antibody. (E-G) Effects of ERK2 knockdown on the changes in TRAIL-R expression using flow cytometry in the control (E), clone 8 (F), and clone 4 (G).

Figure 4

ERK2 phosphorylation does not respond to cisplatin treatment in ERK2 knockdown cells. A. ERK2 phosphorylation induced by cisplatin treatment in ERK2 knockdown cells as determined using western blotting. B. Immunocytochemical analysis of ERK2 phosphorylation in partial (clone 8) and complete (clone 4) ERK2 knockdown cells. C. Immunoprecipitation assay to demonstrate ERK2 level. D. Surface expression of DR5 in partial (clone 8) and complete (clone 4) ERK2 knockdown cells after cisplatin or PMA treatment.

Figure 5

Inactivation of ERK2 elevated TRAIL-R expression levels. A. Inhibition of ERK2 by U0126 in partial (clones 3 and 8) and complete (clones 2 and 4) ERK2 knockdown cells. B. Immunocytochemistry analysis of ERK2 phosphorylation in partial (clone 8) and complete (clone 4) ERK2 knockdown cells after ERK inhibitor treatment. C. Immunoprecipitation assay using anti-ERK2 antibody for detecting the ERK2 band. D. Flow cytometric analysis to determine TRAIL-R expression levels in ERK2 knockdown cells after U0126 treatment.

Figure 6

TRAIL-R expression levels were elevated by cisplatin treatment or ERK1/2 inhibition. Changes in TRAIL-R expression determined using flow cytometry in (A) IMR-32, a neuroblastoma cell line, (B) SK-N-BE, a neuroblastoma cell line, (C) C6, a glioma cell line, and (D) T98G, a glioblastoma cell line, after treatment with cisplatin and U0126.

Figure 7

DR5 expression was regulated by NF-κB. A. Western blot analysis to determine ERK1/2 activation and IκBα inactivation induced by cisplatin treatment for the indicated times. B. NF-κB activity assay after cisplatin, IKK inhibitor, or competitor treatment. C. Effect of cotreatment with IKK inhibitor and TRAIL on cell viability. D. Western blotting to determine p65 levels in an NF-κB knockdown cell line using specific shRNA. E. Activity of NF-κB in NF-κB knockdown cells (clone 10) after treatment with U0126, cisplatin, or NF-κB inhibitor. F. Activity of NF-κB in NF-κB knockdown cells (clone 14) after treatment with U0126, cisplatin, or NF-κB inhibitor. G. Flow cytometric analysis to determine TRAIL-R expression in an NF-κB knockdown clone (clone 10).

Figure 8

Induction of ERK2 and NF-κB activity by cisplatin directs DR5 expression. A. Western blotting demonstrating the induction of ERK1/2 phosphorylation by cisplatin in the presence of U0126. B. NF-κB activity assay after U0126 and/or cisplatin treatment. C. Flow cytometric analysis to determine the effect of U0126 and cisplatin treatment. D. NF-κB activity in ERK2 knockdown cell lines (clones 4 and 8) after U0126 and cisplatin treatment.

Figure 9

Schematic of ERK2, NF-κB, and DR5. A. Under normal conditions, basal (constitutively activated) levels of both ERK2 and NF-κB suppress TRAIL-R expression, rendering cells resistant to TRAIL. B. During cisplatin stimulation, induced ERK2 activates NF-κB, which results in DR5 overexpression and TRAIL-mediated apoptotic cell death.