Project PXD012121

Summary

Title

The auophagy receptor SQSTM1/p62 mediates anti-inflammatory actions of the selective NR3C1/glucocorticoid receptor modulator compound A (CpdA) in macrophages

Description

Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy.

Sample Processing Protocol

Mouse bone marrow-derived macrophages were isolated from 8- to 12-week-old male wild-type C57BL/6 mice. Leg bones were washed with 70% ethanol and twice with ice-cold, sterile phosphate-buffered saline (PBS; Gibco, 14,190–094). After washing, bones were gently smashed using a pestle in a mortar containing 10 ml ice-cold, sterile PBS. Bone marrow was filtered through a 70-μm cell strainer and centrifuged (400 g, 7 min, 4°C). Cells were resuspended in 2 ml of RBC osmotic lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3) for 2 min followed by washing with PBS. After centrifugation under the same condition (400 g, 7 min, 4°C), cells were filtered again and seeded for 7 days differentiation in DMEM (Gibco, 41,966–029) supplemented with 10% fetal calf serum, 0.55% gentamycin and 50 ng/ml recombinant CSF1/M-CSF (VIB Protein Service Facility, L00810). The medium was refreshed every 2–3 days. Cells were maintained in a 5% CO2 humidified atmosphere at 37°C. Approximately 600,000 BMDM cells from each treatment were pelleted by centrifugation and washed with cold PBS. The cell pellets were resuspended in lysis buffer and lysed using a microtip sonicator. The lysates were cleared by centrifugation. S-reduction and S-alkylation of the Cys residues was performed by adding DTT and), respectively. The protein concentration was determined by the Bradford assay and 250 μg of protein material was isolated from each sample, which was then diluted 2-fold (to 4 M urea) and pre-digested with 2.5 μg endoproteinase Lys-C before diluting further to 2 M urea for overnight digestion with 5 μg. The resulting peptide mixtures were purified on Sampli-Q C18 columns (Agilent, 5982–1111), dried completely and then resuspended in 20 μL of loading solvent A (0.1% TFA in water:acetonitrile, 2:98 [v:v]), from which 2 μL was used to determine the peptide concentration (Lunatic, Unchained Labs). An aliquot of the peptide material (3 μg) from each sample was introduced into an LC-MS/MS system through a tandem configured Ultimate 3000 RSLC nano LC (Thermo Scientific, Germany) in-line connected to an LTQ Orbitrap Elite (Thermo Fisher Scientific, Germany). The sample mixture was first loaded on a trapping column (made inhouse, 100-μm internal diameter x 20-mm length, 5-μm beads C18 Reprosil-HD, Dr. Maisch). After flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75-μm internal diameter x 400-mm length, 1.9-μm beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with loading solvent A and were separated with a non-linear gradient from 98% solvent A (0.1% formic acid in water) to 56% solvent B (0.1% formic acid in water:ACN 20:80 [v:v]) at a flow rate of 250 nl/min followed by a 10 min wash reaching 99% solvent B. The mass spectrometer was operated in a data-dependent mode, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 20 most abundant peaks in a given MS spectrum. The source voltage was 3.5 kV, and the capillary temperature was 275°C. In the LTQ-Orbitrap Elite, full scan MS spectra were acquired in the Orbitrap (m/z 300−2 000, AGC target 3,000,000 ions, maximum ion injection time 100 ms) with a resolution of 60,000 (at 400 m/z). The 20 most intense ions fulfilling predefined selection criteria (AGC target 5,000 ions, maximum ion injection time 20 ms, spectrum data type: centroid, exclusion of unassigned and 1 positively charged precursors, dynamic exclusion time 20 sec) were then isolated in the linear ion trap and fragmented in the high-pressure cell of the ion trap. The CID collision energy was set to 35 V and the polydimethylcyclosiloxane background ion at 445.120028 Da was used for internal calibration (lock mass). Column temperature was kept constant at 50°C.

Data Processing Protocol

Data searching was done with the MaxQuant software (v1.6.0.16) using the Andromeda search engine with default search settings including a false-discovery rate (FDR) set at 1% on both the peptide and protein level. Spectra were searched against the mouse proteins in the UniProt database (September 2017 database release, www.uniprot.org). The mass tolerance for precursor and fragment ions was set to 20 ppm and 4.5 ppm, respectively, during the main search. Enzyme specificity was set to C-terminal of Arg and Lys residues, even when they were followed by a Pro residue, with a maximum of 2 missed cleavages. Variable modifications were set to oxidation of methionine residues and protein N-terminal acetylation. A minimum of 1 peptide was required for identification, and matching between runs was allowed using a 2-min time window and a 20-min alignment window. Proteins were quantified by the MaxLFQ algorithm integrated in the software and a minimum ratio count of 2 unique or razor peptides was required for quantification. Downstream data analysis was performed with the Perseus software (v.1.6.0.7) using the ProteinGroup.txt file from a MaxQuant database search. Proteins that were only identified by site and reversed database hits were removed as well as potential contaminants. The protein LFQ intensities were log2 transformed to obtain a normal distribution, and the replicate samples were grouped by their treatment. For each comparison the concerning groups were isolated and filtered for at least 3 valid values in at least 1 group. The missing values were input with values from the lower part of the normal log2 transformed LFQ distribution, representing the detection limit. Multiple 2-sample t-tests were performed, each with a permutation-based FDR of 0.05 and S0 of 0.1 for truncation with a total of 1,000 randomizations. The results were visualized as volcano plots.

Contact

Jarne Pauwels, VIB-Ugent
Karolien De Bosscher, Receptor Research Laboratories, Nuclear Receptor Lab, Ghent University, Ghent, Belgium; cDepartment of Biochemistry, VIB-UGent Center for Medical Biotechnology, Ghent, Belgium; Department of Biochemistry, Ghent University, Ghent, Belgium ( lab head )

Submission Date

19/12/2018

Publication Date

11/06/2019

Publication

    Mylka V, Deckers J, Ratman D, De Cauwer L, Thommis J, De Rycke R, Impens F, Libert C, Tavernier J, Vanden Berghe W, Gevaert K, De Bosscher K. The autophagy receptor SQSTM1/p62 mediates anti-inflammatory actions of the selective NR3C1/glucocorticoid receptor modulator compound A (CpdA) in macrophages. Autophagy. 2018 14(12):2049-2064 PubMed: 30215534