The online version of this article (doi:10.1186/s13075-017-1283-3) contains supplementary material, which is available to authorized users.
The recent findings of cancer-specific metabolic changes, including increased glucose and glutamine consumption, have provided new therapeutic targets for consideration. Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients exhibit several tumor cell-like characteristics; however, the role of glucose and glutamine metabolism in the aberrant proliferation of these cells is unclear. Here, we evaluated the role of these metabolic pathways in RA-FLS proliferation and in autoimmune arthritis in SKG mice.
The expression of glycolysis- or glutaminolysis-related enzymes was evaluated by real-time polymerase chain reaction (PCR) and Western blotting, and the intracellular metabolites were evaluated by metabolomic analyses. The effects of glucose or glutamine on RA-FLS cell growth were investigated using glucose- or glutamine-free medium. Glutaminase (GLS)1 small interfering RNA (siRNA) and the GLS1 inhibitor compound 968 were used to inhibit GLS1 in RA-FLS, and compound 968 was used to study the effect of GLS1 inhibition in zymosan A-injected SKG mice.
GLS1 expression was increased in RA-FLS, and metabolomic analyses revealed that glutamine metabolism was increased in RA-FLS. RA-FLS proliferation was reduced under glutamine-deprived, but not glucose-deprived, conditions. Cell growth of RA-FLS was inhibited by GLS1 siRNA transfection or GLS1 inhibitor treatment. Treating RA-FLS with either interleukin-17 or platelet-derived growth factor resulted in increased GLS1 levels. Compound 968 ameliorated the autoimmune arthritis and decreased the number of Ki-67-positive synovial cells in SKG mice.
Our results suggested that glutamine metabolism is involved in the pathogenesis of RA and that GLS1 plays an important role in regulating RA-FLS proliferation, and may be a novel therapeutic target for RA.
Additional file 1: Table S1. Primer sequences used in this study. All primer sets were used for measuring gene expression by real-time PCR using SYBR® green chemistry. (DOCX 29 kb)13075_2017_1283_MOESM1_ESM.docx
Additional file 2: Figure S1. GLS1 plays a major role in glutamine metabolism in FLS. GLS1 and GLS2 mRNAs were examined in 12 OA-FLS and 19 RA-FLS by real-time PCR, and the levels were normalized to that of GAPDH mRNA. Each experiment was performed in triplicate. Bars indicate mean ± SEM. (TIF 2028 kb)13075_2017_1283_MOESM2_ESM.tif
Additional file 3: Figure S2. Glutamine/glutamate ratio was not significantly different between OA-FLS and RA-FLS. Intracellular glutamine/glutamate ratio in 7 OA-FLS and 11 RA-FLS were analyzed by GC/MS, and in 3 OA-FLS and 3 RA-FLS were analyzed by CE-MS. Bars indicate mean ± SEM. (TIF 2028 kb)13075_2017_1283_MOESM3_ESM.tif
Additional file 4: Figure S3. siRNA efficiency of HK2, MCT4, GLS1, and PDK1 in RA-FLS. After transfection with HK2, MCT4, PDK1, GLS1, or control siRNA, mRNA levels were examined by real-time PCR in RA-FLS (n = 3 for HK2, MCT4, and GLS1, n = 4 for PDK1). Each experiment was performed in triplicate. Bars indicate mean ± SEM. *P < 0.05, **P < 0.01. (TIF 2028 kb)13075_2017_1283_MOESM4_ESM.tif
Additional file 5: Figure S4. Silencing of MCT4, PDK1, or GLS1 did not significantly affect IL-6 or MMP-3 production in supernatants. After silencing of MCT4, PDK1, or GLS1, IL-6 and MMP-3 levels in culture supernatants of RA-FLS were examined by ELISA (n = 4). Each experiment was performed in duplicated. Bars indicate mean ± SEM. *P < 0.05. (TIF 2028 kb)13075_2017_1283_MOESM5_ESM.tif
Additional file 6: Figure S5. PDGF stimulation did not enhance the proliferation of RA-FLS under the glutamine-deprived medium condition. RA-FLS, culturing in medium with or without glutamine (Gln), were stimulated with or without PDGF (10 ng/ml). RA-FLS proliferation was determined using BrdU assay 48 h after stimulation (n = 4). Each experiment was performed in quintuplicate. Bars indicate mean ± SEM. **P < 0.01. (TIF 2028 kb)13075_2017_1283_MOESM6_ESM.tif
Additional file 7: Figure S6. IL-17 or PDGF stimulation did not significantly change the intracellular glutamine and glutamate levels in RA-FLS. Relative levels of intracellular glutamine and glutamate in 3 RA-FLS after IL-17 or PDGF stimulation were analyzed by GC/MS. Bars indicate mean ± SEM. (TIF 677 kb)13075_2017_1283_MOESM7_ESM.tif
Additional file 8: Figure S7. Influence of GLS1 inhibition on immune cells was minimal in arthritic SKG mice. The number of RORγt-expressing CD4+ Th17 cells and FoxP3-expressing CD4+ regulatory T (Treg) cells in the spleens from compound 968-treated and DMSO-treated SKG mice at day 42 after ZyA injection were analyzed by flow cytometry (n = 5 for Th17 cells in DMSO group, n = 4 for Treg cells in DMSO group, and n = 4 for Th17 cells and Treg cells in compound 968 group). (TIF 2028 kb)13075_2017_1283_MOESM8_ESM.tif
Additional file 9: Figure S8. c-MYC expression in RA-FLS. c-MYC protein levels in 5 OA-FLS and 5 RA-FLS were determined by immunoblotting. Protein levels were calculated by phosphorimager analysis. Bars indicate mean ± SEM. (TIF 677 kb)13075_2017_1283_MOESM9_ESM.tif
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