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. 2011 Feb 11;286(6):4912-21.
doi: 10.1074/jbc.M110.199729. Epub 2010 Dec 7.

Preconditioning with endoplasmic reticulum stress mitigates retinal endothelial inflammation via activation of X-box binding protein 1

Jingming Li  1 , Joshua J Wang, Sarah X Zhang
Affiliations

Preconditioning with endoplasmic reticulum stress mitigates retinal endothelial inflammation via activation of X-box binding protein 1

Jingming Li et al. J Biol Chem. .

Abstract

Endoplasmic reticulum (ER) stress is widely implicated in various pathological conditions such as diabetes. Previously, we reported that enhanced ER stress contributes to inflammation and vascular damage in diabetic and ischemia-induced retinopathy. However, the exact role of the signaling pathways activated by ER stress in vascular inflammation remains poorly understood. In the present study, we investigated the role of X-box binding protein 1 (XBP1) in retinal adhesion molecule expression, leukostasis, and vascular leakage. Exposure of human retinal endothelial cells to low dose ER stress inducers resulted in a robust activation of XBP1 but did not affect inflammatory gene expression. However, ER stress preconditioning almost completely abolished TNF-α-elicited NF-κB activation and adhesion molecule ICAM-1 and VCAM-1 expression. Pharmaceutical inhibition of XBP1 activation or knockdown of XBP1 by siRNA markedly attenuated the effects of preconditioning on inflammation. Moreover, loss of XBP1 led to an increase in ICAM-1 and VCAM-1 expression. Conversely, overexpression of spliced XBP1 attenuated TNF-α-induced phosphorylation of IKK, IκBα, and NF-κB p65, accompanied by decreased NF-κB activity and reduced adhesion molecule expression. Finally, in vivo studies show that activation of XBP1 by ER stress preconditioning prevents TNF-α-induced ICAM-1 and VCAM-1 expression, leukostasis, and vascular leakage in mouse retinas. These results collectively indicate a protective effect of ER stress preconditioning against retinal endothelial inflammation, which is likely through activation of XBP1-mediated unfolded protein response (UPR) and inhibition of NF-κB activation.

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Figures

FIGURE 1.
FIGURE 1.
ER stress preconditioning suppressed TNF-α-induced ICAM-1 and VCAM-1 expression in HREC. HREC were preincubated with 0.1 μg/ml tunicamycin (TM; A–G) or 10 nm thapsigargin (TG; H–J) for 8 h followed by exposure to 10 ng/ml TNF-α for 4h or 24 h. A, the mRNA level of ICAM-1 and VCAM-1 was measured by real-time RT-PCR in tunicamycin-preconditioned HREC after TNF-α treatment for 4 h. B and C, expression of ICAM-1 (B) and VCAM-1 (C) was analyzed by immunocytochemistry in tunicamycin-preconditioned HREC after TNF-α treatment for 24 h. Results show that ICAM-1 (B, a) and VCAM-1 (C, a) were expressed at low levels in unstimulated HREC but robustly increased after TNF-α treatment (B, b and C, b). Pretreatment with TM markedly inhibited TNF-α-induced ICAM-1 (B, c) and VCAM-1 (C, c) expression. D–G, expression of ICAM-1 (D) and VCAM-1 (F) was determined by Western blot analysis in tunicamycin-preconditioned HREC after TNF-α treatment for 4 and 24 h and semiquantified by densitometry (E and G). H–J, expression of ICAM-1 and VCAM-1 in thapsigargin-preconditioned HREC were detected by Western blot analysis after TNF-α treatment for 24 h and semiquantified by densitometry (I and J). **, p < 0.01 versus control and ‡, p < 0.01 versus TNF-α. Ctrl, control.
FIGURE 2.
FIGURE 2.
ER stress preconditioning alleviated inducible nitric oxide synthase expression and inhibited NF-κB activation in HREC. A, HREC was pretreated with 0.1 μg/ml tunicamycin (TM) for 8 h followed by exposure to 10 ng/ml TNF-α for 4 or 24 h. inducible nitric oxide synthase (iNOS) expression were determined by Western blot analysis. B, nuclear translocation of NF-κB detected by immunocytochemistry in HREC after TNF-α treatment for 1 h. B, a–b, Control; B, c–d, TNF-α; B, e–f, TNF-α+TM. C, phosphorylation of NF-κB p65 subunit at Ser536 was determined by Western blot analysis. *, p < 0.05; **, p < 0.01 versus control; †, p < 0.05, ‡, p < 0.01 versus TNF-α. D and E, XBP1 splicing determined by RT-PCR (D) and Western blot analysis (E) in HREC after 0.1 μg/ml tunicamycin treatment for the indicated period. XBP1U, unspliced XBP1; XBP1S, spliced XBP1. *, p < 0.05; **, p < 0.01 versus control.
FIGURE 3.
FIGURE 3.
Pharmaceutical and genetic inhibition of XBP1 splicing attenuated the protective effect of ER stress preconditioning in HREC. A and B, HREC were incubated with 0.1 μg/ml tunicamycin (TM) in presence or absence of quinotrierixin (QT) (0.001–1 μm) for 8 h. A, spliced XBP1 was determined by RT-PCR (upper panel) and Western blot analysis (lower panel) using specific antibody against spliced form of XBP1 (XBP1S). B, protein level of XBP1S was semiquantified by densitometry. **, p < 0.01 versus control; ‡, p < 0.01 versus tunicamycin. C and D, HREC were preincubated with 0.1 μg/ml tunicamycin with or without 0.1 μm quinotrierixin for 8 h followed by treatment with 10 ng/ml TNF-α for 24 h. Expression of ICAM-1 (C) and VCAM-1 (D) were determined by Western blot analysis and semiquantified by densitometry. **, p < 0.01 versus control; ‡, p < 0.01 versus TNF-α; #, p < 0.05 versus tunicamycin+TNF-α. E, in vitro leukocyte adhesion assay. HREC were preincubated with 0.1 μg/ml tunicamycin with or without 0.1 μm quinotrierixin for 8 h followed by treatment with 10 ng/ml TNF-α for 4 h, and then THP-1 monocytes were added and co-cultured for 3 h. Adherent monocytes were counted per visual fields and expressed as mean ± S.D. E, a, Control; E, b, TNF-α; E, c, TNF-α+TM; E, d, TNF-α+TM+QT. **, p < 0.01 versus control; ‡, p < 0.01 versus TNF-α; #, p < 0.05 versus tunicamycin+TNF-α. F, HREC were pretreated with 0.1 μg/ml tunicamycin with or without 0.1 μm quinotrierixin for 8 h followed by TNF-α treatment for 4 h. Phosphorylation of NF-κB p65 subunit (Ser536) was determined by Western blot analysis. G–J, HREC were transfected with XBP1 siRNA for 48 h. The knockdown efficiency was determined by protein level of XBP1S in the presence of MG-132 (10 μm) (G). Expression of ICAM-1 (H) and VCAM-1 (I) were determined by Western blot analysis. J, XBP1 siRNA-tranfected cells were pretreated with 0.1 μg tunicamycin for 8 h, followed by TNF-α treatment for 4 h. Phosphorylation of NF-κB p65 (Ser536) was determined. Ctrli, control siRNA; XBP1i, XBP1 siRNA.
FIGURE 4.
FIGURE 4.
Overexpression of spliced XBP1 inhibited TNF-α-induced ICAM-1 and VCAM-1 and leukocyte adhesion via suppression of NF-κB activation in HREC. Overexpression of spliced XBP1 was achieved by infection of HREC with adenovirus encoding with the spliced form of XBP1 (XBP1S; Ad-XBP1S) at a multiplicity of infection of 20 for 48 h. Adenovirus encoding GFP (Ad-GFP) were used as control. After infection with Ad-GFP and Ad-XBP1S, HREC were treated with 10 ng/ml TNF-α for 0–24 h. Cells were harvested for biochemical assays (A, B, D, E, and F) or used for leukocyte adhesion assay (C). A and B, expression of ICAM-1 (A) and VCAM-1 (B) was determined by Western blot (IB) analysis and semiquantified by densitometry. C, THP-1 monocytes were co-cultured with HREC for 3 h, and adherent monocytes were counted from three different visual fields and expressed at mean ± S.D. C, a, Ad-GFP; C, b, Ad-GFP+TNF-α; C, c, Ad-XBP1S; C, d, Ad-XBP1S+TNF-α. D, phosphorylation NF-κB p65 (Ser536) was determined in HREC after treatment with TNF-α for 0.5–24 h. E, agarose oligonucleotide pulldown assay. Nuclear extract from cells was incubated with agarose beads coated by the NF-κB consensus sequence. After intensive wash, bound NF-κB was determined by Western blot analysis. Level of nuclear membrane protein P62 in the input was used as loading control. F, the transcriptional activity of NF-κB was quantified using ELISA-based TransAM NF-κB activity assay. *, p < 0.05; **, p < 0.01 versus Ad-GFP; †, p < 0.05; ‡, p < 0.01 versus Ad-GFP+TNF-α.
FIGURE 5.
FIGURE 5.
Spliced XBP1 blocked TNF-α induced IKK activation through inhibition of IRE phosphorylation and up-regulation of GRP78. A, HREC were infected with Ad-XBP1S or Ad-GFP for 48 h, followed by treatment with 10 ng/ml TNF-α for up to 60 min. Phosphorylation of IKKα/β at Ser176/180, IκB-α at Ser32, and NF-κB at Ser536 were detected by Western blot analysis. Total IKKα, IKKβ, IκB-α, and NF-κB were also determined. p, phosphorylated; t, total. Representative results were from three independent experiments. B–D, HREC were infected with Ad-XBP1S or Ad-GFP as described and treated with 10 ng/ml TNF-α for indicated time periods. Expression of IκB-β (B), total and phospho-IRE1α (C), and GRP78 (D) were determined by Western blot analysis. E, HREC were pretreated with tunicamycin (TM) followed by exposure to TNF-α for 4 and 24 h. Expression of GRP78 were determined by Western blot analysis. **, p < 0.01 versus Ad-GFP (D) or control (E).
FIGURE 6.
FIGURE 6.
Preconditioning with ER stress reduces adhesion molecule expression, ameliorates leukostasis, and alleviates vascular leakage in mouse retinas. Eight-week-old C57BL/6J mice received a periocular injection of tunicamycin (TM, 10 ng/eye) in one eye and vehicle in the contralateral eye. Twelve hours later, mice were given an intravitreal injection of TNF-α (1 pmol/eye) or PBS as control in both eyes. Retinal adhesion molecule expression, leukostasis, and vascular permeability were measured 24 h after TNF-α injection. A–D, retinal expression of VCAM-1, ICAM-1, and XBP1S was determined by Western blot analysis and semiquantified by densitometry. **, p < 0.01 versus control; ‡, p < 0.01 versus TNF-α (n = 4 in all groups). E, representative images of retinal leukostasis analyzed by concanavalin A-lectin staining. E, a, control; E, b, TNF-α; E, c, TNF-α with tunicamycin preconditioning. F, quantification of adherent leukocytes per retina (mean ± S.D.). *, p < 0.05 versus control; †, p < 0.05 versus TNF-α (n = 3 in all groups). G, retinal vascular leakage was measured by Evan's blue-albumin method. Results were expressed microgram of extravascular Evan's blue per milligram of total retinal protein. **, p < 0.01 versus control; †, p < 0.05 versus TNF-α (n = 5 in control group, n = 10 in groups of TNF-α or TNF-α with tunicamycin preconditioning).
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