ER stress depresses NF-kappaB activation in mesangial cells through preferential induction of C/EBP beta

Abstract

Modest induction of endoplasmic reticulum (ER) stress confers resistance to inflammation in glomeruli. Recently, we found that ER stress leads to mesangial insensitivity to cytokine-induced activation of NF-kappaB, but the underlying mechanisms are incompletely understood. ER stress can trigger expression of CCAAT/enhancer-binding proteins (C/EBPs), which interact with transcription factors including NF-kappaB. Here, we investigated a role for C/EBPs in the ER stress-induced resistance to cytokines. Mesangial cells preferentially induced C/EBPbeta after exposure to thapsigargin or tunicamycin; induction of C/EBPdelta was modest and transient, and expression of C/EBPalpha was absent. The induction of C/EBPbeta correlated with accumulation of C/EBPbeta protein and enhanced transcriptional activity of C/EBP. Overexpression of C/EBPbeta markedly suppressed TNF-alpha-induced activation of NF-kappaB, independent of its transacting potential. Knockdown of C/EBPbeta by small interfering RNA reversed the suppressive effect of ER stress on NF-kappaB. In vivo, preconditioning of mice with ER stress induced renal C/EBPbeta and suppressed NF-kappaB-dependent gene expression in response to LPS. Using dominant negative mutants and null mutants for individual branches of the unfolded protein response, we identified the RNA-dependent protein kinase-like ER kinase (PERK) and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathways as the unfolded protein response responsible for ER stress-induced C/EBPbeta. These results suggest that ER stress blunts cytokine-triggered activation of NF-kappaB, in part through PERK- and IRE1-mediated preferential induction of C/EBPbeta.

Figure 1.

Preferential induction of C/EBPβ by ER stress is shown. (A) Mesangial cells were treated with thapsigargin (Tg; 100 nM) or tunicamycin (Tm; 10 μg/ml) for up to 12 h, and expression of C/EBPα, C/EBPβ, and C/EBPδ was examined by Northern blot analysis. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown at the bottom as a loading control. (B) Densitometric analysis of A. The levels of C/EBPs were normalized by the levels of GAPDH. ●, C/EBPα; ■, C/EBPβ; ▴, C/EBPδ. (C) Mesangial cells were treated with Tg or Tm for 9 or 24 h and subjected to Western blot analysis of C/EBPβ. The level of β-actin is shown at the bottom as a loading control. (D) Mesangial cells were transiently transfected with pC/EBP-Luc, stimulated with Tg or Tm for 8 h, and subjected to luciferase assay. Assays were performed in quadruplicate, and data are means ± SEM. *Statistically significant differences versus untreated controls (P < 0.05). RLU, relative light unit.

Figure 2.

Suppression of cytokine-triggered NF-κB activation by C/EBP is shown. (A and B) Mesangial cells were transiently transfected with pNFκB-Luc together with empty vector, pCMV-C/EBPβ, or pCMV-C/EBPδ; treated with TNF-α (10 ng/ml; A) or IL-1β (1 ng/ml; B) for 8 h; and subjected to luciferase assay. (C and D) Cells were transfected with pNFκB-Luc together with empty vector, pCMV-C/EBPβ, or pCMV-A-C/EBP (C/EBP-DN); treated with TNF-α (C) or IL-1β (D) for 8 h; and subjected to luciferase assay. Assays were performed in quadruplicate, and data are means ± SEM. *Statistically significant differences versus vector controls (P < 0.05).

Figure 3.

Involvement of C/EBPβ in the suppression of NF-κB by ER stress is shown. (A) Mesangial cells were transiently co-transfected with pNFκB-Luc and pRNA-U6.1-Neo (vector) or pRNA-U6.1-siC/EBPβ (siC/EBPβ). After the transfection, the cells were pretreated with or without Tg for 6 h, exposed to TNF-α for 18 h, and subjected to luciferase assay. Assays were performed in quadruplicate, and data are means ± SEM. (B) Mesangial cells were stably transfected with pRNA-U6.1-Neo or pRNA-U6.1-siC/EBPβ, and SM/U6.1-Neo cells (Mock) and SM/siC/EBPβ cells (siC/EBPβ) were established. The level of basal C/EBPβ protein was examined by Western blot analysis. (C) SM/U6.1-Neo cells and SM/siC/EBPβ cells were pretreated with Tg, treated with TNF-α for 30 min, and subjected to Western blot analysis of IκBα. (D) Densitometric analysis of C. The levels of IκBα were normalized by the levels of β-actin. (E) Mice were administered Tg (1 mg/kg) intraperitoneally, and after 16 h, kidneys were subjected to Northern blot analysis of GRP78 and C/EBPβ. (F) Mice were administered an intraperitoneal injection of LPS (200 μg/mouse) at 16 h after the Tg injection, and after 12 h, kidneys were subjected to Northern blot analysis of MCP-1.

Figure 4.

Induction of C/EBPβ by the PERK and IRE1 branches of UPR is shown. (A) Mesangial cells were treated with Tg for up to 8 h, and induction of ATF4 was examined by Northern blot analysis. (B) Mesangial cells were transiently transfected with pC/EBP-Luc together with empty vector, pcDNA3-heIF2αS51A (eIF2α-DN), pcDNA3.1-ATF6α(171 to 373)ΔAD (ATF6-DN), pCAG-hIRE1αK699A (IRE1α-DN), or pcDNA3.1-dnXBP1 (XBP1-DN); pretreated with Tg for 8 h; and subjected to luciferase assay. *Significant suppression, †significant upregulation versus vector controls. (C) Wild-type MEF (wt), PERK^−/− MEF, siATF6α-expressing MEF (iATF6α), IRE1α/β^−/− MEF, and XBP1^−/− MEF were treated with Tg (100 or 1000 nM) for 6 h, and Northern blot analysis of C/EBPβ was performed. (D) Densitometric analysis of C. The levels of C/EBPβ were normalized by the levels of GAPDH, and relative percentages are shown. (E) Wild-type MEF, PERK^−/− MEF, and IRE1α/β^−/− MEF were treated with 100 nM Tg for 24 h and subjected to Northern blot analysis of MCP-1.