The Regulation Of Cationic Amino Acid Transporter-1(CAT-1) On The FLS Proliferation In Promoting The Pathogenesis Of Rheumatoid Arthritis

BackgroundAbnormal proliferation of fibroblast-like synoviocytes(FLS)in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis(RA).Currently,the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation.However,little is known regarding the relationship between amino acid metabolism and RA.In this study,in vivo and in vitro experiments were employed to clarify the role and mechanism of cationic amino acid transporter-1(CAT-1)in regulating the proliferation of FLS and promoting RA.Methods1.The concentrations of amino acids and cytokines in the synovial fluid of RA(n=9)and osteoarthritis(OA,n=9)were detected by LC-MS/MS and CBA assay,respectively.2.The m RNA and protein expression of CAT-1 were determined in FLSs isolated from RA and OA patients by real-time PCR,western blotting and immunohistochemical technique.3.CCK8 assay,cell cycle,apoptosis,invasion and cytokine secretion were determined in FLS knocked down of CAT-1 using si RNA or treated with D-arginine under normoxic and hypoxic culture conditions.4.Western blot was used to detect the influence on signal pathway in FLS overexpressed of CAT-1 by plasmid or knocked down of CAT-1 using si RNA.Rapamycin was used as m TOR inhibitor and its influence on cell growth was detected by CCK8 assay and cell apoptosis was detected by flow cytometry.5.A mouse collagen-induced arthritis(CIA)model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo.Results1.L-arginine was upregulated in the synovial fluid of RA patients and was positively correlated with elevation of the cytokines IL-1β,IL-6 and IL-8.2.Further examination demonstrated that cationic amino acid transporter-1(CAT-1)was the primary transporter for L-arginine and was overexpressed on RA FLS compared to OA FLS.3.Moreover,knockdown of CAT-1 using si RNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism,cell proliferation,migration and cytokine secretion in RA FLS under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner.4.CAT-1 promotes RA FLS cell proliferation mainly by activating m TOR signaling pathway.By blocking m TOR signaling pathway with Rapamycin,CAT-1mediated FLS cell proliferation was inhibited and apoptosis was increased.5.Meanwhile,in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice,accompanied by the decreasing of activated B cells,memory B cells,plasma cells and Tfh cells in peripheral blood,bone marrow,spleen,lymph nodes and thymus.ConclusionCAT-1 is upregulated and promotes FLS proliferation by activating the m TOR signaling pathway,thereby promoting RA progression.