#METABOLOMICS WORKBENCH kevin.cho@wustl.edu_20241126_081352 DATATRACK_ID:5407 STUDY_ID:ST003598 ANALYSIS_ID:AN005910 PROJECT_ID:PR002226 VERSION 1 CREATED_ON November 27, 2024, 7:47 am #PROJECT PR:PROJECT_TITLE Nuclear Factor I A (NFIA) regulates articular chondrocyte fatty acid metabolism PR:PROJECT_TITLE and joint homeostasis PR:PROJECT_SUMMARY Osteoarthritis (OA) has recently been recognized as a joint disease with an PR:PROJECT_SUMMARY etiology partially rooted in metabolic dysfunction, yet the underlying PR:PROJECT_SUMMARY mechanism(s) in this context are not determined, limiting opportunities to PR:PROJECT_SUMMARY develop therapeutic treatments. In this study, we utilized a multi-omics PR:PROJECT_SUMMARY approach combining RNA-seq, ATAC-seq, MRE-seq, and metabolomics to reveal that PR:PROJECT_SUMMARY OA articular chondrocytes induced by imbalanced Transforming Growth Factor β PR:PROJECT_SUMMARY 1/Bone Morphogenetic Protein 2 (TGF-β/BMP) signaling, possess increased fatty PR:PROJECT_SUMMARY acid synthesis and oxidation processes regulated by Nuclear Factor I A (NFIA) PR:PROJECT_SUMMARY upregulation PR:INSTITUTE Washington University in St. Louis PR:DEPARTMENT Orthopaedic Surgery, Chemistry, Medicine PR:LABORATORY Shen Laboratory and Patti Laboratory PR:LAST_NAME Cho PR:FIRST_NAME Kevin PR:ADDRESS 1 Brookings Drive PR:EMAIL kevin.cho@wustl.edu PR:PHONE 314-935-8813 #STUDY ST:STUDY_TITLE Nuclear Factor I A (NFIA) Regulates Articular Chondrocyte Fatty Acid Metabolism ST:STUDY_TITLE and Joint Homeostasis ST:STUDY_SUMMARY Osteoarthritis (OA) has recently been recognized as a joint disease with an ST:STUDY_SUMMARY etiology partially rooted in metabolic dysfunction, yet the underlying ST:STUDY_SUMMARY mechanism(s) in this context are not determined, limiting opportunities to ST:STUDY_SUMMARY develop therapeutic treatments. In this study, we utilized a multi-omics ST:STUDY_SUMMARY approach combining RNA-seq, ATAC-seq, MRE-seq, and metabolomics to reveal that ST:STUDY_SUMMARY OA articular chondrocytes induced by imbalanced Transforming Growth Factor β ST:STUDY_SUMMARY 1/Bone Morphogenetic Protein 2 (TGF-β/BMP) signaling, possess increased fatty ST:STUDY_SUMMARY acid synthesis and oxidation processes regulated by Nuclear Factor I A (NFIA) ST:STUDY_SUMMARY upregulation ST:INSTITUTE Washington University in St. Louis ST:DEPARTMENT Orthopaedic Surgery, Chemistry, Medicine ST:LABORATORY Shen Laboratory and Patti Laboratory ST:LAST_NAME Cho ST:FIRST_NAME Kevin ST:ADDRESS 1 Brookings Drive ST:EMAIL kevin.cho@wustl.edu ST:PHONE 314-935-8813 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus; Homo sapiens SU:TAXONOMY_ID 10090; 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS M-Chon-01 M_TGFb_Gln_1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=M-Chon-01.mzML SUBJECT_SAMPLE_FACTORS M-Chon-02 M_TGFb_Gln_2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=M-Chon-02.mzML SUBJECT_SAMPLE_FACTORS M-Chon-03 M_TGFb_Gln_3 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=M-Chon-03.mzML SUBJECT_SAMPLE_FACTORS M-Chon-04 M_BMP_Gln_1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=M-Chon-04.mzML SUBJECT_SAMPLE_FACTORS M-Chon-05 M_BMP_Gln_2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=M-Chon-05.mzML SUBJECT_SAMPLE_FACTORS M-Chon-06 M_BMP_Gln_3 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=M-Chon-06.mzML SUBJECT_SAMPLE_FACTORS M-Chon-07 M_TGFb_PA_1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=M-Chon-07.mzML SUBJECT_SAMPLE_FACTORS M-Chon-08 M_TGFb_PA_2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=M-Chon-08.mzML SUBJECT_SAMPLE_FACTORS M-Chon-09 M_TGFb_PA_3 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=M-Chon-09.mzML SUBJECT_SAMPLE_FACTORS M-Chon-10 M_BMP_PA_1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=M-Chon-10.mzML SUBJECT_SAMPLE_FACTORS M-Chon-11 M_BMP_PA_2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=M-Chon-11.mzML SUBJECT_SAMPLE_FACTORS M-Chon-12 M_BMP_PA_3 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=M-Chon-12.mzML SUBJECT_SAMPLE_FACTORS M-Chon-13 M_TGFb_OA_1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=M-Chon-13.mzML SUBJECT_SAMPLE_FACTORS M-Chon-14 M_TGFb_OA_2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=M-Chon-14.mzML SUBJECT_SAMPLE_FACTORS M-Chon-15 M_TGFb_OA_3 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=M-Chon-15.mzML SUBJECT_SAMPLE_FACTORS M-Chon-16 M_BMP_OA_1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=M-Chon-16.mzML SUBJECT_SAMPLE_FACTORS M-Chon-17 M_BMP_OA_2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=M-Chon-17.mzML SUBJECT_SAMPLE_FACTORS M-Chon-18 M_BMP_OA_3 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=M-Chon-18.mzML SUBJECT_SAMPLE_FACTORS H-Chon-01 H_Healthy_Gln_1 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-01.mzML SUBJECT_SAMPLE_FACTORS H-Chon-02 H_Healthy_Gln_2 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-02.mzML SUBJECT_SAMPLE_FACTORS H-Chon-03 H_Healthy_Gln_3 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-03.mzML SUBJECT_SAMPLE_FACTORS H-Chon-04 H_OA_Gln_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-04.mzML SUBJECT_SAMPLE_FACTORS H-Chon-05 H_OA_Gln_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-05.mzML SUBJECT_SAMPLE_FACTORS H-Chon-06 H_OA_Gln_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-06.mzML SUBJECT_SAMPLE_FACTORS H-Chon-07 H_Healthy_PA_1 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-07.mzML SUBJECT_SAMPLE_FACTORS H-Chon-08 H_Healthy_PA_2 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-08.mzML SUBJECT_SAMPLE_FACTORS H-Chon-09 H_Healthy_PA_3 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-09.mzML SUBJECT_SAMPLE_FACTORS H-Chon-10 H_OA_PA_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-10.mzML SUBJECT_SAMPLE_FACTORS H-Chon-11 H_OA_PA_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-11.mzML SUBJECT_SAMPLE_FACTORS H-Chon-12 H_OA_PA_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-12.mzML SUBJECT_SAMPLE_FACTORS H-Chon-13 H_Healthy_OA_1 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=H-Chon-13.mzML SUBJECT_SAMPLE_FACTORS H-Chon-14 H_Healthy_OA_2 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=H-Chon-14.mzML SUBJECT_SAMPLE_FACTORS H-Chon-15 H_Healthy_OA_3 Sample source:Human Chondrocytes | Genotype:Healthy | Treatment:N/A Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=H-Chon-15.mzML SUBJECT_SAMPLE_FACTORS H-Chon-16 H_OA_OA_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=H-Chon-16.mzML SUBJECT_SAMPLE_FACTORS H-Chon-17 H_OA_OA_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=H-Chon-17.mzML SUBJECT_SAMPLE_FACTORS H-Chon-18 H_OA_OA_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:N/A Label=U-13C-Oleate; RAW_FILE_NAME(Raw file name)=H-Chon-18.mzML SUBJECT_SAMPLE_FACTORS H-Chon-19 H_Ctrl_Gln_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-GFP Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-19.mzML SUBJECT_SAMPLE_FACTORS H-Chon-20 H_Ctrl_Gln_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-GFP Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-20.mzML SUBJECT_SAMPLE_FACTORS H-Chon-21 H_Ctrl_Gln_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-GFP Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-21.mzML SUBJECT_SAMPLE_FACTORS H-Chon-22 H_Lenti_Gln_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-shNFIA Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-22.mzML SUBJECT_SAMPLE_FACTORS H-Chon-23 H_Lenti_Gln_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-shNFIA Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-23.mzML SUBJECT_SAMPLE_FACTORS H-Chon-24 H_Lenti_Gln_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-shNFIA Label=U-13C-Glutamine; RAW_FILE_NAME(Raw file name)=H-Chon-24.mzML SUBJECT_SAMPLE_FACTORS H-Chon-25 H_Ctrl_PA_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-GFP Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-25.mzML SUBJECT_SAMPLE_FACTORS H-Chon-26 H_Ctrl_PA_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-GFP Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-26.mzML SUBJECT_SAMPLE_FACTORS H-Chon-27 H_Ctrl_PA_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-GFP Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-27.mzML SUBJECT_SAMPLE_FACTORS H-Chon-28 H_Lenti_PA_1 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-shNFIA Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-28.mzML SUBJECT_SAMPLE_FACTORS H-Chon-29 H_Lenti_PA_2 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-shNFIA Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-29.mzML SUBJECT_SAMPLE_FACTORS H-Chon-30 H_Lenti_PA_3 Sample source:Human Chondrocytes | Genotype:OA | Treatment:Lenti-shNFIA Label=U-13C-Palmitate; RAW_FILE_NAME(Raw file name)=H-Chon-30.mzML #COLLECTION CO:COLLECTION_SUMMARY Human articular chondrocytes were isolated from the femoral condyle and tibia CO:COLLECTION_SUMMARY plateau specimens obtained from healthy donors and OA patients. Following with CO:COLLECTION_SUMMARY 90 minutes digestion at 37 °C by 0.4% pronase and 14 hours digestion by 0.035% CO:COLLECTION_SUMMARY collagenase P, human articular chondrocytes were filtered through the 70 μm CO:COLLECTION_SUMMARY cell strainer and seeded at the density of 105 cells/cm2 for experiments. Murine CO:COLLECTION_SUMMARY articular chondrocytes were isolated from femoral heads of 3-week-old wildtype CO:COLLECTION_SUMMARY (C57BL/6J) pups. Following with 6 hours digestion at 37°C by 0.05% collagenase CO:COLLECTION_SUMMARY P, murine chondrocytes were harvested and plated at the density of 105 cells/cm2 CO:COLLECTION_SUMMARY for experiments. Both human and murine articular chondrocytes were cultured in CO:COLLECTION_SUMMARY the Dulbecco’s modified Eagle’s medium supplemented with 25 mM Glucose, 2 mM CO:COLLECTION_SUMMARY L-Glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin. The CO:COLLECTION_SUMMARY chondrocytes were treated with vehicle, TGF-β1 (5 ng/ml) and BMP2 (100 ng/ml) CO:COLLECTION_SUMMARY for 24-72 hours to examine the alterations of gene expression and metabolites. CO:COLLECTION_SUMMARY For NFIA/Nfia knockdown experiments, the chondrocytes were transfected with CO:COLLECTION_SUMMARY lentivirus expressing shRNA against NFIA and Nfia gene at a multiplicity of 10 CO:COLLECTION_SUMMARY in the presence of polybrene (10 μg/ml) in viral infection media (DMEM medium CO:COLLECTION_SUMMARY supplemented with 25 mM Glucose, 2 mM L-Glutamine and 2% FBS) for 24 hours. CO:COLLECTION_SUMMARY Lentivirus expressing shRNA against scramble sequences was used as control. For CO:COLLECTION_SUMMARY Nfia overexpression, the primary murine articular chondrocytes were transfected CO:COLLECTION_SUMMARY with lentivirus expressing murine Nfia gene at a multiplicity of 5 in the CO:COLLECTION_SUMMARY presence of polybrene for 48 hours. Lentivirus expression Gfp gene was used as CO:COLLECTION_SUMMARY control. CO:SAMPLE_TYPE Chondrocytes #TREATMENT TR:TREATMENT_SUMMARY The chondrocytes were treated with vehicle, TGF-β1 (5 ng/ml) and BMP2 (100 TR:TREATMENT_SUMMARY ng/ml). For isotope labeling, Articular chondrocytes were cultured in DMEM TR:TREATMENT_SUMMARY containing stable isotopically nutrient to assess the lipid metabolism in vitro. TR:TREATMENT_SUMMARY To assess lipids synthesis, articular chondrocytes were cultured in glutamine TR:TREATMENT_SUMMARY free DMEM supplemented with 10% dialyzed FBS, and 4 mM U-13C glutamine for 72 TR:TREATMENT_SUMMARY hours or cells were cultured in glucose free DMEM supplemented with 10% dialyzed TR:TREATMENT_SUMMARY FBS, and 25 mM U-13C glucose for 72 hours. To assess lipids oxidation, articular TR:TREATMENT_SUMMARY chondrocytes were cultured with 100 mM U-13C palmitic acid conjugated to BSA for TR:TREATMENT_SUMMARY 24 hours or 400 uM U-13C oleic acid. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cell pellets were treated with a methanol/acetonitrile/water (2:2:1) solution SP:SAMPLEPREP_SUMMARY and reconstituted in 40 μL acetonitrile/water (1:1) per milligram dry weight. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1260 CH:COLUMN_NAME Phenomenex Luna NH2 (150 x 2mm,3um) CH:SOLVENT_A 95% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water CH:FLOW_GRADIENT 100% to 0% B from 0–45 min and 0% B from 45–50 min CH:FLOW_RATE 50 uL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6540 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data were collected with the following MS source settings: gas temperature, MS:MS_COMMENTS 300°C; gas flow, 9 l/min; sheath gas temperature, 350°C; sheath gas flow, 11 MS:MS_COMMENTS l/min; nebulizer, 35 psi; Vcap, 3.5kV; nozzle voltage, 1 kV. Skyline software MS:MS_COMMENTS was used for data processing. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples M_TGFb_PA_1 M_TGFb_PA_2 M_TGFb_PA_3 M_BMP_PA_1 M_BMP_PA_2 M_BMP_PA_3 Factors Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:TGFb1 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Sample source:Mice Chondrocytes | Genotype:WT | Treatment:BMP2 Citrate 809139.4771 796525.7111 1107266.616 1203795.57 1628554.689 2158584.928 Fumarate 112063.1296 102631.9003 137926.7466 148819.488 154910.2336 175655.9606 Glutamate 19746839.1 19132993.88 22726236.4 26051072.34 26108739.13 27434165.05 Aspartate 1217025.491 1429359.76 1695545.944 1987413.989 1931851.658 2254840.996 ATP 14266167.87 15352677.65 19980281.48 15255755.06 17470578.84 20831070.57 NAD+ 1639254.523 1656076.695 1918094.417 1973685.137 2046508.491 2068327.05 NADH 86797.20375 97541.64033 111929.8746 116182.9808 122309.4475 125782.237 NADP+ 174591.6164 139270.5613 176223.4689 147608.3494 163587.98 165293.8494 dGTP 13970630.85 15164164.56 19497678.93 15022223.5 17344714.21 20413674.81 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention time quantified m/z KEGG ID Citrate 37.2 191 C00158 Fumarate 24.9 115 C00122 Glutamate 24.8 146 C00025 Aspartate 25 132 C16433 ATP 46.2 506 C00002 NAD+ 25.1 662 C00003 NADH 25.5 664 C00004 NADP+ 37.2 742 C00006 dGTP 46.2 506 C00286 METABOLITES_END #END