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. 2015 Jan;29(1):208-15.
doi: 10.1096/fj.14-259663. Epub 2014 Oct 17.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses

Valerie F Curtis  1 , Stefan F Ehrentraut  2 , Eric L Campbell  1 , Louise E Glover  1 , Amanda Bayless  1 , Caleb J Kelly  1 , Douglas J Kominsky  3 , Sean P Colgan  4
Affiliations

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses

Valerie F Curtis et al. FASEB J. 2015 Jan.

Abstract

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization VSports手机版. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4. 7 nM) stabilized HIF-1α, activated HIF promoter activity by 2. 5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0. 5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease. .

Keywords: hypoxia; inflammation; intestinal epithelia. V体育安卓版.

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Figures

Figure 1.
Figure 1.
MLN4924 deneddylates Cul-2 and stabilizes HIF-1α in HeLa cells. A) Western blotting of HeLa cells treated with vehicle control (veh) or 330 nM MLN4924 for 1 h or increasing doses of MLN4924 indicates deneddylation of Cul-2 and stabilization of HIF-1α in the nuclear fraction. B) Densitometry of the ratio of neddylated:unneddylated Cul-2 protein in HeLa cells treated with vehicle control or 330 nM MLN4924 for 1 h (n = 3 independent experiments). *P < 0.01 C) Western blotting of HeLa cells treated with vehicle control (veh) or increasing doses of MLN4924 for 1 h indicates deneddylation of Cul-2 and stabilization of HIF-1α in the nuclear fraction. D) Western blotting of HeLa cells with 330 nM MLN4924 over a time course demonstrates a loss of NEDD8 conjugated to cullin proteins at T = 5, and a stabilization of HIF-1α and HIF-1α-OH at T = 15 in the nuclear fraction. In all cases, n = 3 independent experiments.
Figure 2.
Figure 2.
HIF-1α stabilization via MLN4924 can promote HIF target gene transcription. A) Treatment of HeLa cells transfected with an HRE-containing luciferase reporter plasmid with MLN4924 increased luciferase expression at 330 nM concentration. B) RT-PCR analysis of HeLa cells treated with 330 nM MLN4924 reveals significant up-regulation of BNIP3L and Eno1 mRNA at T = 60 and T = 120 compared to T = 0. In all cases, n = 3 independent experiments. *P < 0.05, as determined by Student’s t test. Error bars represent sem.
Figure 3.
Figure 3.
Lentiviral KD of DEN-1 in T84 cells. A) Lentiviral shRNA-mediated KD of DEN-1 in T84 intestinal epithelial cells results in diminished DEN-1 mRNA relative to shNTC. B) DEN-1 KD results in loss of DEN-1 protein in cytoplasmic fraction and Cul-2 neddylation in nuclear fraction (n = 3 independent experiments). C) Western blotting of T84 shNTC and DEN-1 KD cells after increasing doses of MLN4924 for 1 h demonstrating increased HIF-1α stabilization in nuclear fraction (n = 3). D) Relative mRNA expression of HIF targets Glut and Eno-1 in shNTC and DEN-1 KD cells after 2 h treatment with 10 μM AKB4924 (n = 2). *P < 0.05, as determined by Student’s t test. Error bars represent sem.
Figure 4.
Figure 4.
Inhibition of the neddylation pathway enhances barrier function of intestinal epithelial cells. A) TER measurements reveal a significant increase in transcellular barrier kinetics over time in DEN-1 KD T84 cells compared to shNTC. B) FITC-dextran flux assay of DEN-1 KD T84 cells reveals significant decrease in paracellular permeability compared to shNTC on day 3 of barrier formation. C) shNTC and DEN-1 KD T84 cells were subjected to Ca2+ switch, and TER was monitored over time. D) T84 cells were subjected to Ca2+ switch, recovered in vehicle- or 330 nM MLN4924-containing buffers, and TER was monitored over time. In all cases, n = 3 independent experiments. *P < 0.05, unless otherwise indicated, as determined by Student’s t test. Error bars represent sem.
Figure 5.
Figure 5.
MLN4924 attenuates inflammation in a mouse model of colitis. A) Weight loss curve for C57BL/6 mice receiving water or 2.5% DSS. Results indicate that pretreatment with 0.1 MLN4924 mg/kg per day significantly decreases weight loss and increases weight recovery (n = 5 mice per group). B) After euthanasia, colon length measurements indicate that MLN4924 treatment in mice receiving DSS significantly prevented colon shortening compared to vehicle-treated DSS mice. C) Representative H&E colon sections from each of the 4 treatment groups. D) MLN4924 treatment in mice receiving DSS significantly decreased the histologic injury score compared to vehicle-treated DSS mice. Results are presented as the mean ± sem. *P < 0.05.
Figure 6.
Figure 6.
Neddylation and the HIF pathway. Under normoxic conditions, HIF-1α is hydroxylated by PHDs. In its hydroxylated form, HIF-1α is recognized by the Cul-2-Nedd8-pVHL complex, leading to polyubiquitination and degradation by the proteasome. Pharmacologic inhibition of Cul-2 neddylation using MLN4924 stabilizes cellular HIF-1α levels, leading to increased transcription of proresolving HIF target genes and increased barrier function. Loss of DEN-1 also positively influences barrier function of intestinal epithelial cells.
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