Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO
- Washington University School of Medicine, United States
Abstract
Inflammatory osteolysis is governed by exacerbated osteoclastogenesis. Ample evidence points to central role of NF-kB in such pathologic responses, yet the precise mechanisms underpinning specificity of these responses remain unclear. We propose that motifs of the scaffold protein IKKg/NEMO partly facilitate such functions. As proof-of-principle, we used site-specific mutagenesis to examine the role of NEMO in mediating RANKL-induced signaling in mouse bone marrow macrophages, known as osteoclast precursors. We identified lysine (K)270 as a target regulating RANKL signaling as K270A substitution results in exuberant osteoclastogenesis in vitro and murine inflammatory osteolysis in vivo. Mechanistically, we discovered that K270A mutation disrupts autophagy, stabilizes NEMO, and elevates inflammatory burden. Specifically, K270A directly or indirectly hinders binding of NEMO to ISG15, a ubiquitin-like protein, which we show targets the modified proteins to autophagy-mediated lysosomal degradation. Taken together, our findings suggest that NEMO serves as a toolkit to fine-tune specific signals in physiologic and pathologic conditions.
Data availability
The following datasets and raw data will be available on Dryad: Proteomics dataset, All Western blot raw data, FACS dataset microCT raw dataset. https://doi.org/10.5061/dryad.tx95x69tn
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Source Dateset: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMODryad Digital Repository, doi:10.5061/dryad.tx95x69tn.
Article and author information
Author details
Funding
National Institutes of Health (AR049192)
- Yousef Abu-Amer
National Institutes of Health (AR068972)
- Gabriel Mbalaviele
National Institutes of Health (AR054326)
- Yousef Abu-Amer
National Institutes of Health (AR072623)
- Yousef Abu-Amer
National Institutes of Health (AR057235)
- Yousef Abu-Amer
Shriners Hospital For Children (86200)
- Yousef Abu-Amer
Shriners Hospital for Children (85160)
- Yousef Abu-Amer
National Institutes of Health (AR075860)
- Jie Shen
National Institutes of Health (AR077226)
- Jie Shen
National Institutes of Health (AR064755)
- Gabriel Mbalaviele
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States
Ethics
Animal experimentation: All the animals were housed at the Washington University School of Medicine barrier facility. All experimental protocols were carried out in accordance with the ethical guidelines approved by the Washington University School of Medicine Institutional Animal Care and Use Committee (approval protocol #20190002).
Version history
- Received: February 17, 2020
- Accepted: March 22, 2020
- Accepted Manuscript published: March 23, 2020 (version 1)
- Version of Record published: April 9, 2020 (version 2)
Copyright
© 2020, Adapala et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO
eLife 9:e56095.
https://doi.org/10.7554/eLife.56095
Further reading
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- Medicine
- Neuroscience
Background:
Ketamine has emerged as one of the most promising therapies for treatment-resistant depression. However, inter-individual variability in response to ketamine is still not well understood and it is unclear how ketamine’s molecular mechanisms connect to its neural and behavioral effects.
Methods:
We conducted a single-blind placebo-controlled study, with participants blinded to their treatment condition. 40 healthy participants received acute ketamine (initial bolus 0.23 mg/kg, continuous infusion 0.58 mg/kg/hr). We quantified resting-state functional connectivity via data-driven global brain connectivity and related it to individual ketamine-induced symptom variation and cortical gene expression targets.
Results:
We found that: (i) both the neural and behavioral effects of acute ketamine are multi-dimensional, reflecting robust inter-individual variability; (ii) ketamine’s data-driven principal neural gradient effect matched somatostatin (SST) and parvalbumin (PVALB) cortical gene expression patterns in humans, while the mean effect did not; and (iii) behavioral data-driven individual symptom variation mapped onto distinct neural gradients of ketamine, which were resolvable at the single-subject level.
Conclusions:
These results highlight the importance of considering individual behavioral and neural variation in response to ketamine. They also have implications for the development of individually precise pharmacological biomarkers for treatment selection in psychiatry.
Funding:
This study was supported by NIH grants DP5OD012109-01 (A.A.), 1U01MH121766 (A.A.), R01MH112746 (J.D.M.), 5R01MH112189 (A.A.), 5R01MH108590 (A.A.), NIAAA grant 2P50AA012870-11 (A.A.); NSF NeuroNex grant 2015276 (J.D.M.); Brain and Behavior Research Foundation Young Investigator Award (A.A.); SFARI Pilot Award (J.D.M., A.A.); Heffter Research Institute (Grant No. 1–190420) (FXV, KHP); Swiss Neuromatrix Foundation (Grant No. 2016–0111) (FXV, KHP); Swiss National Science Foundation under the framework of Neuron Cofund (Grant No. 01EW1908) (KHP); Usona Institute (2015 – 2056) (FXV).
Clinical trial number:
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- Medicine
The nuclear receptor, farnesoid X receptor (FXR/NR1H4), is increasingly recognized as a promising drug target for metabolic diseases, including nonalcoholic steatohepatitis (NASH). Protein-coding genes regulated by FXR are well known, but whether FXR also acts through regulation of long non-coding RNAs (lncRNAs), which vastly outnumber protein-coding genes, remains unknown. Utilizing RNA-seq and global run-on sequencing (GRO-seq) analyses in mouse liver, we found that FXR activation affects the expression of many RNA transcripts from chromatin regions bearing enhancer features. Among these we discovered a previously unannotated liver-enriched enhancer-derived lncRNA (eRNA), termed FXR-induced non-coding RNA (Fincor). We show that Fincor is specifically induced by the hammerhead-type FXR agonists, including GW4064 and tropifexor. CRISPR/Cas9-mediated liver-specific knockdown of Fincor in dietary NASH mice reduced the beneficial effects of tropifexor, an FXR agonist currently in clinical trials for NASH and primary biliary cholangitis (PBC), indicating that amelioration of liver fibrosis and inflammation in NASH treatment by tropifexor is mediated in part by Fincor. Overall, our findings highlight that pharmacological activation of FXR by hammerhead-type agonists induces a novel eRNA, Fincor, contributing to the amelioration of NASH in mice. Fincor may represent a new drug target for addressing metabolic disorders, including NASH.
