Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May;29(5):1869-78.
doi: 10.1096/fj.14-258533. Epub 2015 Feb 9.

A20 suppresses vascular inflammation by recruiting proinflammatory signaling molecules to intracellular aggresomes

Karine Enesa  1 , Herwig P Moll  1 , Le Luong  1 , Christiane Ferran  1 , Paul C Evans  2
Affiliations

A20 suppresses vascular inflammation by recruiting proinflammatory signaling molecules to intracellular aggresomes

Karine Enesa et al. FASEB J. 2015 May.

Abstract

A20 protects against pathologic vascular remodeling by inhibiting the inflammatory transcription factor NF-κB. A20's function has been attributed to ubiquitin editing of receptor-interacting protein 1 (RIP1) to influence activity/stability. The validity of this mechanism was tested using a murine model of transplant vasculopathy and human cells. Mouse C57BL/6 aortae transduced with adenoviruses containing A20 (or β-galactosidase as a control) were allografted into major histocompatibility complex-mismatched BALB/c mice. Primary endothelial cells, smooth muscle cells, or transformed epithelial cells (all human) were transfected with wild-type A20 or with catalytically inactive mutants as a control. NF-κB activity and intracellular localization of RIP1 was monitored by reporter gene assay, immunofluorescent staining, and Western blotting. Native and catalytically inactive versions of A20 had similar inhibitory effects on NF-κB activity (-70% vs. -76%; P > 0.05). A20 promoted localization of RIP1 to insoluble aggresomes in murine vascular allografts and in human cells (53% vs. 0%) without altering RIP1 expression, and this process was increased by the assembly of polyubiquitin chains (87% vs. 28%; P < 0.05). A20 captures polyubiquitinated signaling intermediaries in insoluble aggresomes, thus reducing their bioavailability for downstream NF-κB signaling. This novel mechanism contributes to protection from vasculopathy in transplanted organs treated with exogenous A20.

Keywords: NF-κB; molecular mechanism; receptor interacting protein.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
A20 did not alter RIP1 expression in vascular allografts and cultured cells. A–C) Vascular allografts were treated with rAd.A20 or rAd.βgal at the time of transplant and harvested 48 hours later (n = 3/group). A) Quantitative RT-PCR was used to measure levels of A20 (left) and VCAM-1 (right) mRNA. Results are expressed as mean ± sd. B) Expression levels of RIP1, A20, and GAPDH were assessed by Western blotting using total protein lysates. RIP1 levels in vascular allografts treated with rAd.A20 or rAd.βgal were normalized to those of housekeeping gene GAPDH and quantified by densitometry. RIP1 over GAPDH ratios are expressed as means ± sd. C) HCAECs or HCASMCs were transduced with rAd.A20 or rAd.βgal or were left nontransduced (NT). After 48 hours, expression levels of RIP1, A20, and β-actin were determined by Western blotting. Representative blots are shown (left). Expression levels of RIP1 were quantified by densitometry and normalized by measuring β-actin. The ratio RIP1:β-actin was calculated for each sample, and mean values with sd are shown (right) (n = 3 for HCAECs; n = 4 for HCASMC). D) HEK293 cells were cotransfected with plasmids containing E-tagged RIP1 and with varying quantities of pHM6-A20 wild-type (WT) or catalytically inactive A20 (pHM6-C103S/C624627A). Cell lysates were analyzed by Western blotting using anti-HA epitope (A20) or anti–E-tag (RIP1) antibodies. Data shown are representative of 12 independent experiments.
Figure 2.
Figure 2.
NF-κB suppression by A20 does not require ubiquitin editing. A) A20−/− MEFs were cotransfected with varying amounts of pHM6-A20 wild-type (WT) or with A20 versions mutated at C103S, C624627A, or C103S/C624627A and with fixed amounts of NF-κB reporter gene (pGL2) and Renilla luciferase control (pRL-TK) before stimulation with TNF-α or IL-1 for 16 hours. Cell lysates were analyzed to determine ratio firefly/renilla luciferase, which is a measure of NF-κB activity normalized for transfection efficiency. Mean values calculated from triplicate wells were pooled from 4 experiments and are shown with sd. B) HeLa cells were transfected with pEGFP-A20 wild type (WT, green) or with versions mutated at C103S, C624627A, or C103S/C624627A (green). Transfected cultures (or untransfected controls) were stimulated with IL-1 (20 ng/ml) for 15 minutes. The intracellular localization of RelA (red) was assessed by immunofluorescence staining and confocal microscopy. Representative images and the proportion of RelA that localized to the nucleus (mean nuclear/cytoplasmic ratio ± sem) are shown.
Figure 3.
Figure 3.
The punctate localization of A20 is required for NF-κB suppression. A) HeLa cells were transfected with pEGFP-A20 and with various amounts of FLAG-14-3-3. After 48 hours, cells were fixed before analysis of GFP-A20 (green) localization by laser scanning confocal microscopy. Representative images are shown. The proportion of cells containing punctate A20 were calculated and pooled from 3 experiments, and mean values are shown with sd. B) HeLa cells were transfected with pEGFP-A20 (green) and were cotransfected with FLAG-14-3-3 (or with empty vector as a control). The intracellular localization of RelA (red) was assessed by immunofluorescence staining and confocal microscopy. Representative images and the proportion of RelA that localized to the nucleus (mean nuclear/cytoplasmic ratio ± sem) are shown.
Figure 4.
Figure 4.
A20 localizes to aggresomes. A) HeLa cells were transfected with pEGFP-A20 (green) or pHM6-A20 (red). The localization of A20 in transfected HeLa cells was determined by immunofluorescence staining and confocal microscopy (HA-A20; red; right) or confocal microscopy (GFP-A20; green; left). B) HeLa cells transfected with pEGFP-A20 were stained using anti-GFP antibodies conjugated to gold and analyzed by scanning electronic microscopy. Representative images are shown, and aggresomes are indicated (arrows). C) HeLa cells transfected with 1 μg of GFP-A20 (green) were incubated for 1 hour with lysotracker probe (red) under growth conditions. The localization of lysosomes and GFP-A20 was then monitored in live cells by confocal laser scanning microscopy. Representative images are shown. Structures in the boxed region (labeled A, B, C) were tracked over 175 seconds to assess localization dynamics (right). D) HeLa cells were transfected with GFP-A20 (green). Intracellular localization of vimentin was assessed by immunofluorescent staining (red) and confocal microscopy. Colocalization images are representative of 2 independent experiments.
Figure 5.
Figure 5.
A20 targets RIP1 to punctate structures in vitro and in vascular allografts in vivo. A) HEK293 cells were transfected with E-tagged RIP (red) and cotransfected either with an empty vector GFP (green; middle) or GFP-A20 (green; right). The intracellular localization of RIP1 was assessed by immunostaining using anti–E-tag antibodies (red) followed by confocal microscopy. Images shown are representative of those from 3 similar experiments. B) HUVECs were exposed to hypoxia (4 hours) followed by reoxygenation (4 hours; H/R) or were treated with TNF-α (10 ng/ml for 4 hours) or remained untreated. Triton-soluble (left) or -insoluble fractions (right) were tested by Western blotting using anti-A20, anti-RIP1, and by using anti-PDHX antibodies to assess total protein levels. Representative blots (upper) and results from densitometry analysis of 3 experiments (lower) are shown.
Figure 6.
Figure 6.
A20 and RIP1 colocalize at punctate bodies in vascular allografts. Vascular allografts treated with rAd.A20 or rAd.βgal were harvested 2 days later (n = 3/group). Cross-sections were costained for RIP1 (red), A20 (green), and nuclei (DAPI; blue). A) Representative images are shown using ×40 magnification, and RIP1/A20 costaining is shown at higher magnification in the lower panels. Note that the majority of green signal in rAd.βgal-treated vessels is auto-fluorescence from the internal elastic lamina. B) Representative images are shown using ×40 magnification. Punctate A20/RIP1 colocalization appears yellow and is indicated (arrows).
See this image and copyright information in PMC

Comment in

  • Transplant Arteriosclerosis.
    Nadig SN. Nadig SN. Transplantation. 2016 Nov;100(11):2249-2250. doi: 10.1097/TP.0000000000001440. Transplantation. 2016. PMID: 27495767 No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kunter U., Floege J., von Jürgensonn A. S., Stojanovic T., Merkel S., Gröne H. J., Ferran C. (2003) Expression of A20 in the vessel wall of rat-kidney allografts correlates with protection from transplant arteriosclerosis. Transplantation 75, 3–9 - PubMed
    1. Siracuse J. J., Fisher M. D., da Silva C. G., Peterson C. R., Csizmadia E., Moll H. P., Damrauer S. M., Studer P., Choi L. Y., Essayagh S., Kaczmarek E., Maccariello E. R., Lee A., Daniel S., Ferran C. (2012) A20-mediated modulation of inflammatory and immune responses in aortic allografts and development of transplant arteriosclerosis. Transplantation 93, 373–382 - PMC - PubMed
    1. Patel V. I., Daniel S., Longo C. R., Shrikhande G. V., Scali S. T., Czismadia E., Groft C. M., Shukri T., Motley-Dore C., Ramsey H. E., Fisher M. D., Grey S. T., Arvelo M. B., Ferran C. (2006) A20, a modulator of smooth muscle cell proliferation and apoptosis, prevents and induces regression of neointimal hyperplasia. FASEB J. 20, 1418–1430 - PubMed
    1. Xu M. Q., Yan L. N., Gou X. H., Li D. H., Huang Y. C., Hu H. Y., Wang L. Y., Han L. (2009) Zinc finger protein A20 promotes regeneration of small-for-size liver allograft and suppresses rejection and results in a longer survival in recipient rats. J. Surg. Res. 152, 35–45 - PubMed
    1. Grey S. T., Arvelo M. B., Hasenkamp W., Bach F. H., Ferran C. (1999) A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets. J. Exp. Med. 190, 1135–1146 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources