Skip to main page content (V体育官网)
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2013 Sep 19;122(12):2062-73.
doi: 10.1182/blood-2013-02-486373. Epub 2013 Jul 17.

Neddylation plays an important role in the regulation of murine and human dendritic cell function

Nathan Mathewson  1 , Tomomi Toubai, Steven Kapeles, Yaping Sun, Katherine Oravecz-Wilson, Hiroya Tamaki, Ying Wang, Guoqing Hou, Yi Sun, Pavan Reddy
Affiliations

Neddylation plays an important role in the regulation of murine and human dendritic cell function

Nathan Mathewson et al. Blood. .

Abstract

Posttranslational protein modifications (PTMs) are necessary for cells to function properly. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple but complementary approaches, we determined the role of an important but less understood type of PTM, namely, neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to Toll-like receptor, nucleotide oligomerization domain-like receptor, and noninfectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and noncanonical nuclear factor-κB (NF-κB) activity. Neddylation inhibition prevented the degradation of inhibitor-κB and thus reduced the translocation and activation of NF-κB, but without perturbation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease processes. VSports手机版.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Neddylation inhibition attenuates LPS-induced TNF-α and IL-6 release and gene expression in BMDC. Protein analysis by western blot of (A, top panel) neddylated Cullin proteins and (A, middle panel) Cul1 protein using whole cell lysate of BMDC stimulated with LPS in the presence or absence of MLN4924 or dexamethasone at the indicated doses. Cells receiving treatment were preincubated with MLN4924 or dexamethasone for 2 hours followed by concurrent LPS stimulation (0.5 µg/mL) for 4 hours. α-tubulin protein levels were analyzed as an equal loading control. Quantification via ELISA of cytokines TNF-α (B) and IL-6 (C) and qPCR analysis of TNF-α (D) and IL-6 (E) transcripts in BMDC stimulated with LPS in the presence or absence of MLN4924 or dexamethasone at the indicated doses. Cells receiving treatment were preincubated with MLN4924 or dexamethasone for 2 hours followed by concurrent LPS stimulation (0.5 µg/mL) for 4 hours. One representative experiment of 3 is shown. ***P < .0001.
Figure 2
Figure 2
Neddylation inhibition in BMDC mitigates release of non-TLR4–stimulated cytokines in vitro. ELISA quantification of TNF-α release in Pam3CSK4 (300 ng/mL) stimulated (A), PGN (5 μg/mL) stimulated (B), and CD40L (1 μg/mL) stimulated (C) BMDC in the presence or absence of vehicle, MLN4924, or dexamethasone at the indicated doses. (D) Comparison of TNF-α release via ELISA of cells treated with vehicle, MLN4924, dexamethasone, or bortezomib followed by concurrent stimulation with LPS (0.5 µg/mL). Cells receiving treatment were preincubated with vehicle, MLN4924, dexamethasone, or bortezomib for 2 hours followed by concurrent stimulation for 4 hours. One representative experiment of 3 is shown in cells treated in triplicate. *P < .05; **P < .01; ***P < .0001.
Figure 3
Figure 3
Neddylation inhibition mitigates allogeneic T-cell proliferation. BALB/C T cells were cultured with C57BL/6 irradiated (30 Gy) BMDC at 40:1 and 100:1 ratios in the presence or absence of MLN4924 for 72 hours (A) and 96 hours (B). Where indicated, BMDC were preincubated in the presence or absence of vehicle or MLN4924 at specified dosage for 6 hours. One representative experiment of 3 is shown of groups treated in triplicate. *P < .05; **P < .01; ***P < .0001.
Figure 4
Figure 4
Neddylation blockade regulates DC-mediated T-cell activation. (A) Survival of BALB/C animals lethally irradiated and transplanted with BM and CD90.2+ T cells from either syngeneic BALB/C or allogeneic C57BL/6 donors. Following lethal irradiation (8 Gy) on day 1, MLN4924 (20 mg/kg) was administered in 5 daily doses, day 1 to day 3 relative to BMT. Data are combined from 2 independent experiments (n = 10 to 12 animals in the allogeneic groups). (B-C) Abb animals were lethally irradiated (10 Gy) on day 1 and WT B6 BMDC (10 × 106) pretreated with vehicle or MLN4924 overnight and subsequently transferred in 2 doses separated by 24 hours (day 1 and day 0). CD90.2+ T cells (2 × 106) from syngeneic WT-C57BL/6 or allogeneic bm12 animals were transferred on day 0. Following sacrifice on day 6, spleens were analyzed for CD69 (B) and Tbet (C). (D) Quantification via ELISA of TNF-α (left) and IL-6 (right) released from human moDCs stimulated with LPS in the presence or absence of MLN4924 or dexamethasone at the indicated doses. Cells receiving treatment were preincubated with vehicle, MLN4924, or dexamethasone for 2 hours followed by concurrent LPS stimulation (0.5 µg/mL) for 4 hours. One representative experiment of 3 is shown of groups treated in triplicate. (E) Human PBMCs (1 × 105/well) and moDCs were obtained from 2 healthy donors and cocultured at 1:1 and 10:1 ratios in an MLR in the presence or absence of MLN4924 for 96 and 120 hours.
Figure 5
Figure 5
siRNA-mediated neddylation inhibition inhibits LPS-induced TNF-α production in JAWSII cells. (A) qPCR analysis of SAG expression in JAWSII cells transfected with indicated siRNA (3 μg). Scramble transfection performed as control. (B) qPCR analysis of TNF-α expression in JAWSII cells transfected with indicated siRNA and stimulated with LPS (0.5 µg/mL). Scramble transfection performed as control. One representative experiment of 3 is shown. (C-E) JAWSII cells transfected with siRNA for βTrCP, Cul5, or scramble as described in Materials and methods. Expression of βTrCP (C) and Cul5 (D) mRNA transcripts in JAWSII cells receiving indicated siRNA-mediated knockdown. (E) Expression of TNF-α mRNA transcripts in cells transfected with indicated siRNA and cultured in the presence or absence of LPS (0.5 µg/mL) for 4 hours. *P < .05; **P < .01; ***P < .0001.
Figure 6
Figure 6
Neddylation inhibition and dexamethasone differentially affect NF-κB translocation in BMDC. (A) Protein analysis by western blot (left) of p65 isoform of NF-κB protein using nuclear (N) and cytosolic (C) fractions of BMDC in the presence or absence of 500 nm MLN4924 or 500 nm dexamethasone and stimulated concurrently with LPS (0.5 µg/mL) for 30 minutes. Plot (right) shows densitometric and statistical analysis of p65 NF-κB presence in the nuclear fraction and cytosolic fraction. Lamin A/C and LDH proteins were analyzed to demonstrate the presence of nuclear and cytosolic fractions, respectively. One representative experiment of 3 is shown. (B) Immunocytochemistry analysis of p65 NF-κB (column 3) localization in BMDC cultured in the presence or absence of MLN4924 and stimulated with LPS (0.5 µg/mL) for 1 hour. Cell nuclei were stained using DAPI (column 1). Cytoplasmic actin was stained with phalloidin (column 2). Merged images of staining (column 4). One representative experiment of 3 is shown. **P < .01.
Figure 7
Figure 7
Neddylation inhibition prevents IκB degradation in BMDC without perturbation of the MAPK/ERK pathway. Protein analysis via western blot of phosphorylated IκBα protein (A-B, top panels) and total IκBα (A-B, middle panels) using whole cell lysate of BMDC stimulated with LPS (0.5 µg/mL) for 6 hours in the presence of vehicle (A) or 500 nm MLN4924 (B). Densitometric and statistical analysis of total IκB (C, top) or pIκB (C, bottom) of vehicle or MLN4924-treated cells. α-tubulin protein levels were analyzed as an equal loading control. One representative experiment of 3 is shown. Protein analysis via western blot of phosphorylated ERK protein (D-E, top panels) and total ERK (D-E, middle panels) using whole cell lysate of BMDC stimulated with LPS for 6 hours in the presence of vehicle (D) or 500 nm MLN4924 (E). Densitometric analysis of vehicle-treated cells (D, right) or 500 nm MLN4924-treated cells (E, right) of phosphorylated ERK and total ERK protein. α-tubulin protein levels were analyzed as an equal loading control. One representative experiment of 3 is shown. *P < .05.
See this image and copyright information in PMC

Comment in

  • The path(way) less traveled.
    Abedi M, Murphy WJ. Abedi M, et al. Blood. 2013 Sep 19;122(12):1996-7. doi: 10.1182/blood-2013-07-516856. Blood. 2013. PMID: 24052539

References

    1. Cloos PAC, Christgau S. Post-translational modifications of proteins: implications for aging, antigen recognition, and autoimmunity. Biogerontology. 2004;5(3):139–158. - PubMed
    1. Sun Y, Chin YE, Weisiger E, et al. Cutting edge: Negative regulation of dendritic cells through acetylation of the nonhistone protein STAT-3. J Immunol. 2009;182(10):5899–5903. - PMC - PubMed
    1. Magnani M, Crinelli R, Bianchi M, Antonelli A. The ubiquitin-dependent proteolytic system and other potential targets for the modulation of nuclear factor-kB (NF-kB). Curr Drug Targets. 2000;1(4):387–399. - PubMed
    1. Satpathy AT, Wu X, Albring JC, Murphy KM. Re(de)fining the dendritic cell lineage. Nat Immunol. 2012;13(12):1145–1154. - PMC - PubMed
    1. Hespel C, Moser M. Role of inflammatory dendritic cells in innate and adaptive immunity. Eur J Immunol. 2012;42(10):2535–2543. - V体育官网入口 - PubMed

"VSports" Publication types

MeSH terms

Substances

LinkOut - more resources